CN118696045A - BRM selective degradation agent compounds and their applications - Google Patents

Abstract

The present disclosure relates to protein degradation targeting chimera (PROTAC) molecules, and further to BRM selective degradation agent compounds, specifically providing a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the compound has a substituent and structural features described in the present application. The present application also describes a pharmaceutical composition comprising a compound of formula (I) or a pharmaceutically acceptable salt thereof, and the use of the compound or a pharmaceutically acceptable salt thereof in medicine.

Description

BRM selective degradation agent compounds and their applications

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit and priority of the following three Chinese invention patent applications, the entire contents of which are hereby incorporated by reference in their entirety:

Patent application No. 202111395500.6 filed with the State Intellectual Property Office of China on November 23, 2021;

Patent application No. 202210192251.9 filed with the China National Intellectual Property Administration on February 28, 2022; and

Patent application No. 202210784815.8 filed with the State Intellectual Property Office of China on July 5, 2022.

Technical Field

The present disclosure relates to compounds or pharmaceutically acceptable salts thereof as selective degraders of BRMs, methods for preparing the same, pharmaceutical compositions containing the same, and uses of the same in preventing or treating diseases or conditions mediated by BRMs.

Background Art

SWI/SNF is an important nucleosome remodeling complex that can hydrolyze ATP to generate energy and use it to break the interaction between DNA and histones in nucleosomes, thereby regulating gene expression and repairing gene damage. Epigenetic abnormalities have been shown to be an important cause of the occurrence and development of many chronic diseases (such as cancer), and mutations in SWI/SNF complex components have been found in about 20% of cancers. BRM (SMARCA2) and BRG1 (SMARCA4) are both ATPase subunits of the SWI/SNF complex and are its core components. Their main function is to hydrolyze ATP to provide energy for mobilizing nucleosomes to regulate gene transcription, DNA replication, and DNA damage repair. The two proteins have the same function in the complex but are mutually exclusive. They do not exist in the same SWI/SNF complex at the same time. Simultaneous inhibition of the functions of BRM and BRG1 proteins will lead to cell death, that is, synthetic lethality. BRG1 deficiency occurs in a variety of tumors, including lung cancer, gastric cancer, pancreatic cancer and other highly malignant tumors with limited treatment options. For example, the proportion of BRG1 mutations in non-small cell lung cancer (NSCLC) is 5-10%, and it is mutually exclusive with the most common driver genes in NSCLC (such as EGFR, ALK, MET, ROS1 and RET). Tumor cells with BRG1 mutations or functional loss are highly dependent on BRM, and are therefore more sensitive to BRM inhibitors, producing a synergistic lethal effect, while normal cells are well tolerated by BRM inhibitors, so BRM is a potential specific tumor target.

Proteolysis-Targeting Chimeras (PROTACs) are an emerging technology with good prospects, which is expected to turn many "undruggable" potential targets into "druggable". Traditional small molecule drugs are often powerless against proteins without enzyme functions, which account for about 80% of human proteins, because these drugs usually need to bind to enzymes or receptors to work. PROTACs consist of three parts: a ligand (anchor) that recruits E3 ubiquitin ligases, a ligand molecule (warhead) that binds to the target protein (protein of interest, POI), and a linker that connects the two parts. Traditional small molecules need to exert pharmacological effects by occupying key sites of the target protein (i.e., occupancy-driven), so a certain drug concentration in the body needs to be maintained, and the requirements for small molecule binding sites are high. PROTACs achieve pharmacodynamic effects by degrading the target protein, and theoretically do not require very high drug concentrations.

BRM and BRG1 proteins have two main functional regions, namely the ATPase region and the bromodomain. The homology of amino acid residues in the corresponding functional regions of these two proteins is greater than 90%. It is difficult for small molecule inhibitors to achieve selectivity for BRM and BRG1, so they also have strong killing ability against normal cells. Clinically, there is an urgent need for BRM protein degradation PROTAC molecules with excellent stability and activity and good selectivity for BRG1.

Summary of the invention

In one aspect, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof is provided.

TL-Linker-DIM

(I)

in:

The DIM is a ligand compound capable of binding to E3 ubiquitin ligase;

The Linker is a linking group that covalently binds at least one TL and at least one DIM;

The TL is a group as shown below:

in:

Selected from: C=CN, wherein X ⁴ is N; or NC=C, wherein X ⁴ is C;

X ³ is selected from CR ³ or N;

Y ¹ , Y ² , Y ³ , Y ⁴ , and X ¹ are selected from one of the following five situations:

(1) ^Y1 is CR, ^Y2 is selected from ^CR2a or N, ^Y3 is selected from ^CR3a or N, ^Y4 is selected from ^CR4a or N, and ^X1 is selected from ^CR1 or N;

(2) ^Y1 is selected from ^CR1a or N, ^Y2 is CR, ^Y3 is selected from ^CR3a or N, ^Y4 is selected from ^CR4a or N, and ^X1 is selected from ^CR1 or N;

(3) ^Y1 is selected from ^CR1a or N, ^Y2 is selected from ^CR2a or N, ^Y3 is CR, ^Y4 is selected from ^CR4a or N, and ^X1 is selected from ^CR1 or N;

(4) ^Y1 is selected from ^CR1a or N, ^Y2 is selected from ^CR2a or N, ^Y3 is selected from ^CR3a or N, ^Y4 is CR, and ^X1 is selected from ^CR1 or N;

(5) ^Y1 is selected from ^CR1a or N, ^Y2 is selected from ^CR2a or N, ^Y3 is selected from ^CR3a or N, ^Y4 is selected from ^CR4a or N, and ^X1 is CR;

The R is wherein n is selected from 0, 1, 2, 3 or 4; ^Ra is selected from halogen, OH, CN, _NO2 , _C1 - _C6 alkyl, _C1 - _C6 alkoxy, _C3 - _C6 cycloalkyl, _NH2 , NH( _C1 - _C6 alkyl) or N( _C1 - _C6 alkyl) ₂ , wherein said _C1 - _C6 alkyl, _C3 - _C6 cycloalkyl, _C1 - _C6 alkoxy is optionally substituted by ^Rb ; ^Rb is selected from halogen, OH, =O, _NH2 , CN or COOH;

Said R ^1a , R ^2a , R ^3a , R ^4a , R ¹ are independently selected from H, halogen, NH ₂ , NO ₂ , CN, COOH, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , NH(C ₃ -C ₆ cycloalkyl), NH-4-8 membered heterocyclyl, NH-5-10 membered heteroaryl, NH-(C ₆ -C ₁₀ aryl), NHC(O)-4-8 membered heterocyclyl, NHC(O)(C ₁ -C ₆ alkyl), NHC(O)-O(C ₁ -C 6 alkyl), N(C ₁ -C ₆ alkyl)C(O)-O(C 1 -C ₆ alkyl) _C ( _O ) _NH2 , C(O)NH( _{C1 - C6} alkyl), C(O)N( _C1 - _C6 alkyl) ₂ , C(O)NH( _C3 -C6 cycloalkyl), C(O)NH-( _C6 - _{C10 aryl} ), C(O)NH-5-10 membered heteroaryl or C(O)NH- _{4-8 membered heterocyclyl, wherein the C1-C6 alkyl , C1} - _C6 alkoxy, _{C3 - C6} cycloalkyl, _{C6 - C10} aryl, C _{( O} ) _-5-10 membered heteroaryl _10- membered aryl, 5-10-membered heteroaryl, 4-8-membered heterocyclyl are optionally substituted by R ⁵ ;

Said R ³ and R ⁴ are independently selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, C ₃ -C ₆ cycloalkyloxy, NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , NH(C ₃ -C ₆ cycloalkyl), NHC(O)(C ₁ -C ₆ alkyl), NHC(O)(C ₃ -C ₆ cycloalkyl), NHC(O)-(C ₆ -C ₁₀ aryl), NHC(O)-5-10 membered heteroaryl, NHC(O)-4-8 membered heterocyclyl, C(O)NH(C ₁ -C R ₆ alkyl), C(O)NH(C ₃ -C ₆ cycloalkyl), C(O)NH-(C ₆ -C ₁₀ aryl), C(O)NH-5-10 membered heteroaryl or C(O)NH-4-8 membered heterocyclyl, wherein the C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl or C ₃ -C ₆ cycloalkyloxy is optionally substituted with R ⁶ ;

Alternatively, R ^1a and R ³ and the atoms to which they are attached together form a C ₅ -C ₁₀ cycloalkenyl group or a 5-14-membered heterocyclic group, wherein the C ₅ -C ₁₀ cycloalkenyl group or the 5-14-membered heterocyclic group is optionally substituted by R ⁷ ;

Alternatively, R ^4a and R ⁴ and the atoms to which they are attached together form a C ₅ -C ₁₀ cycloalkenyl or a 5-14 membered heterocyclyl, wherein the C ₅ -C ₁₀ cycloalkenyl or the 5-14 membered heterocyclyl is optionally substituted by R ⁸ ;

Alternatively, R ^2a and R ^3a and the atoms to which they are attached together form a C ₅ -C ₁₀ cycloalkenyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 5-14 membered heterocyclyl, wherein the C ₅ -C ₁₀ cycloalkenyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 5-14 membered heterocyclyl is optionally substituted by R ⁹ ;

Alternatively, R ¹ and R ⁴ and the atoms to which they are attached together form a C ₅ -C ₁₀ cycloalkenyl group or a 5-14-membered heterocyclic group, and the C ₅ -C ₁₀ cycloalkenyl group or the 5-14-membered heterocyclic group is optionally substituted by R ¹⁰ ;

R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ are independently selected from deuterium, halogen, OH, =O, NH ₂ , NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , CN, C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl, wherein the C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl is further optionally substituted by R ^5a ;

The R ^5a is selected from halogen, OH, NH ₂ , ═O or C ₁ -C ₃ alkyl;

Provided that: at least one of R ^1a , R ^2a , R ^3a , R ^4a , R ¹ , R ³ , and R ⁴ is not H; and when R ⁴ is H, R ³ is not optionally substituted or optionally substituted

In another aspect, a pharmaceutical composition is provided, comprising the compound described herein or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

In another aspect, provided is a use of a compound described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, in the preparation of a medicament for preventing or treating a BRM-mediated disease, wherein the BRM-mediated disease is preferably a tumor.

In another aspect, provided is a compound described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein for use in preventing or treating a BRM-mediated disease.

In another aspect, provided is a use of a compound described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein, in preventing or treating a BRM-mediated disease.

In another aspect, provided is a method for treating a disease mediated by a BRM, comprising administering to a subject in need thereof a therapeutically effective amount of a compound described herein or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a graph showing the tumor growth curves of mice receiving the test compounds in the A549 subcutaneous tumor model.

DETAILED DESCRIPTION

The present disclosure is described in detail below by specific embodiments, but it is not intended to limit the present disclosure in any way. Various specific embodiments of the present disclosure have been described in detail herein, and it will be apparent to those skilled in the art that various changes and modifications may be made to the specific embodiments of the present disclosure without departing from the spirit and scope of the present disclosure.

The present disclosure relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof:

TL-Linker-DIM

(I)

in:

The DIM is a ligand compound capable of binding to E3 ubiquitin ligase;

The Linker is a linking group that covalently binds at least one TL and at least one DIM;

The TL is a group as shown below:

in:

Selected from: C=CN, wherein X ⁴ is N; or NC=C, wherein X ⁴ is C;

X ³ is selected from CR ³ or N;

Y ¹ , Y ² , Y ³ , Y ⁴ , and X ¹ are selected from one of the following five situations:

(1) ^Y1 is CR, ^Y2 is selected from ^CR2a or N, ^Y3 is selected from ^CR3a or N, ^Y4 is selected from ^CR4a or N, and ^X1 is selected from ^CR1 or N;

(2) ^Y1 is selected from ^CR1a or N, ^Y2 is CR, ^Y3 is selected from ^CR3a or N, ^Y4 is selected from ^CR4a or N, and ^X1 is selected from ^CR1 or N;

(3) ^Y1 is selected from ^CR1a or N, ^Y2 is selected from ^CR2a or N, ^Y3 is CR, ^Y4 is selected from ^CR4a or N, and ^X1 is selected from ^CR1 or N;

(4) ^Y1 is selected from ^CR1a or N, ^Y2 is selected from ^CR2a or N, ^Y3 is selected from ^CR3a or N, ^Y4 is CR, and ^X1 is selected from ^CR1 or N;

(5) ^Y1 is selected from ^CR1a or N, ^Y2 is selected from ^CR2a or N, ^Y3 is selected from ^CR3a or N, ^Y4 is selected from ^CR4a or N, and ^X1 is CR;

The R is wherein n is selected from 0, 1, 2, 3 or 4; ^Ra is selected from halogen, OH, CN, _NO2 , _C1 - _C6 alkyl, _C1 - _C6 alkoxy, _C3 - _C6 cycloalkyl, _NH2 , NH( _C1 - _C6 alkyl) or N( _C1 - _C6 alkyl) ₂ , wherein said _C1 - _C6 alkyl, _C3 - _C6 cycloalkyl, _C1 - _C6 alkoxy is optionally substituted by ^Rb ; ^Rb is selected from halogen, OH, =O, _NH2 , CN or COOH;

Said R ^1a , R ^2a , R ^3a , R ^4a , R ¹ are independently selected from H, halogen, NH ₂ , NO ₂ , CN, COOH, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , NH(C ₃ -C ₆ cycloalkyl), NH-4-8 membered heterocyclyl, NH-5-10 membered heteroaryl, NH-(C ₆ -C ₁₀ aryl), NHC(O)-4-8 membered heterocyclyl, NHC(O)(C ₁ -C ₆ alkyl), NHC(O)-O(C ₁ -C 6 alkyl), N(C ₁ -C ₆ alkyl)C(O)-O(C 1 -C ₆ alkyl) _C ( _O ) _NH2 , C(O)NH( _{C1 - C6} alkyl), C(O)N( _C1 - _C6 alkyl) ₂ , C(O)NH( _C3 -C6 cycloalkyl), C(O)NH-( _C6 - _{C10 aryl} ), C(O)NH-5-10 membered heteroaryl or C(O)NH- _{4-8 membered heterocyclyl, wherein the C1-C6 alkyl , C1} - _C6 alkoxy, _{C3 - C6} cycloalkyl, _{C6 - C10} aryl, C _{( O} ) _-5-10 membered heteroaryl _10- membered aryl, 5-10-membered heteroaryl, 4-8-membered heterocyclyl are optionally substituted by R ⁵ ;

Said R ³ and R ⁴ are independently selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, C ₃ -C ₆ cycloalkyloxy, NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , NH(C ₃ -C ₆ cycloalkyl), NHC(O)(C ₁ -C ₆ alkyl), NHC(O)(C ₃ -C ₆ cycloalkyl), NHC(O)-(C ₆ -C ₁₀ aryl), NHC(O)-5-10 membered heteroaryl, NHC(O)-4-8 membered heterocyclyl, C(O)NH(C ₁ -C _{R 6} is substituted with R 6 in the following aspects: C (O) NH (C ₃ -C ₆ alkyl), C (O) NH (C 3 -C 6 cycloalkyl), C (O) NH- (C ₆ -C ₁₀ aryl), C (O) NH-5-10 membered heteroaryl or C (O) NH-4-8 membered heterocyclyl, wherein the C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl or C ₃ -C ₆ cycloalkyloxy is optionally substituted with R ⁶ ;

Alternatively, R ^1a and R ³ and the atoms to which they are attached together form a C ₅ -C ₁₀ cycloalkenyl group or a 5-14-membered heterocyclic group, wherein the C ₅ -C ₁₀ cycloalkenyl group or the 5-14-membered heterocyclic group is optionally substituted by R ⁷ ;

Alternatively, R ^4a and R ⁴ and the atoms to which they are attached together form a C ₅ -C ₁₀ cycloalkenyl or a 5-14 membered heterocyclyl, wherein the C ₅ -C ₁₀ cycloalkenyl or the 5-14 membered heterocyclyl is optionally substituted by R ⁸ ;

Alternatively, R ^2a and R ^3a and the atoms to which they are attached together form a C ₅ -C ₁₀ cycloalkenyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 5-14 membered heterocyclyl, wherein the C ₅ -C ₁₀ cycloalkenyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 5-14 membered heterocyclyl is optionally substituted by R ⁹ ;

Alternatively, R ¹ and R ⁴ and the atoms to which they are attached together form a C ₅ -C ₁₀ cycloalkenyl group or a 5-14-membered heterocyclic group, and the C ₅ -C ₁₀ cycloalkenyl group or the 5-14-membered heterocyclic group is optionally substituted by R ¹⁰ ;

R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ are independently selected from deuterium, halogen, OH, =O, NH ₂ , NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , CN, C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl, wherein the C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl is further optionally substituted by R ^5a ;

The R ^5a is selected from halogen, OH, NH ₂ , ═O or C ₁ -C ₃ alkyl;

Provided that: at least one of R ^1a , R ^2a , R ^3a , R ^4a , R ¹ , R ³ , and R ⁴ is not H; and when R ⁴ is H, R ³ is not optionally substituted or optionally substituted

In some embodiments, R ⁵ , R ⁶ , R ⁷ , R ⁸ , R ⁹ , R ¹⁰ are independently selected from halogen, OH, =O, NH ₂ , NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , CN, C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, or 4-8 membered heterocyclyl, and the C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, or 4-8 membered heterocyclyl is further optionally substituted with R ^5a , and the R ^5a is selected from halogen, OH, NH ₂ , =O, or C ₁ -C ₃ alkyl.

In some embodiments, Selected from C=CN, wherein X ⁴ is N.

In some embodiments, Selected from NC=C, wherein ^X4 is C.

In some embodiments, X ³ is CR ³ .

In some embodiments, ^X3 is N.

In some embodiments, R ^1a , R ^2a , R ^3a , R ^4a , R ¹ are independently selected from H, halogen, NH ₂ , CN, C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , NH(C ₃ -C ₆ cycloalkyl), NH-4-8 membered heterocyclyl, NH-5-10 membered heteroaryl, NH-(C ₆ -C ₁₀ aryl), NHC(O)(C ₃ -C ₆ cycloalkyl), NHC(O)-4-8 membered heterocyclyl, NHC(O)-(C ₆ -C ₁₀ aryl), NHC(O)-5-10 membered heteroaryl, C(O)NH(C R5 is an alkyl group selected from the group consisting of C(O)NH-(C ₃ -C ₆ cycloalkyl), C(O)NH-(C ₆ -C ₁₀ aryl), C(O)NH-5-10 membered heteroaryl or C(O)NH-4-8 membered heterocyclyl, wherein the C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl is optionally substituted by ^R5 .

In some embodiments, R ^1a is selected from H, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl, wherein said C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl is optionally substituted with R ⁵ .

In some embodiments, R ^1a is selected from H, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, NH(C ₁ -C ₆ alkyl) or N(C ₁ -C ₆ alkyl) ₂ , wherein the C ₁ -C ₆ alkyl is optionally substituted with R ⁵ .

In some embodiments, R ^2a is selected from H, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , NHC(O)(C ₃ -C ₆ cycloalkyl), NHC(O)-4-8 membered heterocyclyl, NHC(O)-(C ₆ -C ₁₀ aryl), NHC(O)-5-10 membered heteroaryl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl, wherein the C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl is optionally substituted with R ⁵ .

In some embodiments, R ^2a is selected from H, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl, wherein the C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl is optionally substituted with R ⁵ .

In some embodiments, R ^3a is selected from H, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , C ₃ -C 6 cycloalkyl, C _{6 -C 10 aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, NH(C 3 -C 6 cycloalkyl), NH-4-8 membered heterocyclyl, NH-5-10 membered heteroaryl, NH-(C 6 -C 10 aryl), NHC(O)(C 3 -C 6 cycloalkyl), NHC(O)-4-8 membered heterocyclyl, NHC(O)-(C 6 -C 10 aryl ) or} NHC(O)-5-10 membered heteroaryl, wherein the C ₁ -C 6 alkyl, C 1 -C 6 alkoxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, NH(C ₃ -C ₆ cycloalkyl), NH-4-8 membered heterocyclyl, NH-5-10 membered heteroaryl, NH-(C 6 -C 10 aryl), NHC(O)(C ₃ -C ₆ cycloalkyl), NHC(O)-4-8 membered heterocyclyl, NHC(O)-(C ₆ -C ₁₀ aryl) or NHC(O) _-5-10 membered heteroaryl -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclic group is optionally substituted by R ⁵ .

In some embodiments, R ^3a is selected from H, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , C _{3 -C 6} cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, NH(C ₃ -C ₆ cycloalkyl), NH-4-8 membered heterocyclyl, NH-5-10 membered heteroaryl or NH-(C ₆ -C ₁₀ aryl), wherein the C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl is optionally substituted with R ⁵ .

In some embodiments, R ^3a is selected from H, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl, and the C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl is optionally substituted with R ⁵ .

In some embodiments, R ^3a is selected from H or 4-8 membered heterocyclyl optionally substituted with R ⁵ .

In some embodiments, R ^3a is selected from H or piperazinyl optionally substituted with R ⁵ .

In some embodiments, R ^4a is selected from H, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, NHC(O)(C ₃ -C 6 cycloalkyl), NHC(O)-4-8 membered heterocyclyl, NHC(O)-(C ₆ -C ₁₀ aryl), NHC(O)-5-10 membered heteroaryl, C(O)NH(C ₃ -C ₆ cycloalkyl), C(O)NH-(C 6 -C ₁₀ aryl), C(O)NH-5-10 membered heteroaryl or C(O)NH-4-8 membered heterocyclyl, wherein the C ₁ -C ₆ alkyl, C ₁ -C 6 cycloalkyl, C ₆ -C ₁₀ aryl, C(O)NH-5-10 membered heteroaryl or C(O)NH-4-8 membered heterocyclyl R ⁵ is optionally substituted by _{C 6} alkoxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl.

In some embodiments, R ^4a is selected from H, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, NHC(O)(C ₃ -C ₆ cycloalkyl), NHC(O)-4-8 membered heterocyclyl, NHC(O)-(C ₆ -C ₁₀ aryl), NHC(O)-5-10 membered heteroaryl, C(O)NH(C ₃ -C ₆ cycloalkyl), C(O)NH-(C ₆ -C ₁₀ aryl), C(O)NH-5-10 membered heteroaryl or C(O)NH-4-8 membered heterocyclyl, wherein the C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl is optionally substituted with R ⁵ .

In some embodiments, R ^4a is selected from H, NH ₂ , C ₁ -C ₆ alkoxy or NHC(O)(C ₃ -C ₆ cycloalkyl), wherein the C ₁ -C ₆ alkoxy or C ₃ -C ₆ cycloalkyl is optionally substituted with R ⁵ .

In some embodiments, R ¹ is selected from H, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , NHC(O)(C ₃ -C ₆ cycloalkyl), NHC(O)-4-8 membered heterocyclyl, NHC(O)-(C ₆ -C ₁₀ aryl), NHC(O)-5-10 membered heteroaryl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl, wherein the C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl is optionally substituted with R ⁵ .

In some embodiments, R ¹ is selected from H, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl, wherein the C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl is optionally substituted with R ⁵ .

In some embodiments, R ⁵ is selected from halogen, OH, =O, NH ₂ , CN, C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl, wherein the C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl is optionally substituted with R ^5a .

In some embodiments _{, R} ⁵ is selected from halogen, OH, =0, _{NH 2} , CN, C ₁ -C ₆ alkyl, C ₆ -C ₁₀ aryl, or _5-10 membered heteroaryl, which is optionally substituted with R ^5a .

In some embodiments, R ⁵ is selected from phenyl.

In some embodiments, R ^1a is selected from H.

In some embodiments, R ^2a is selected from H or NH ₂ .

In some embodiments, R ^2a is selected from NH ₂ .

In some embodiments, R ^3a is selected from H or

In some embodiments, R ^4a is selected from H, NH ₂ , or OCH ₃ .

In some embodiments, R ^4a is selected from H.

In some embodiments, R ¹ is selected from H or NH ₂ .

In some embodiments, ^R3 is selected from H, OH, halogen, _NH2 , _C1 - _C6 alkyl, _C2 - _C6 alkenyl, _C2 - _C6 alkynyl, _C1 - _C6 alkoxy, C3- _{C6 cycloalkyl, C3-C6 cycloalkyloxy , NH} ( _C1 - _C6 alkyl), N( _C1 - _C6 alkyl) ₂ , NH( _C3 - _C6 cycloalkyl), _C6 - _C10 aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, NHC(O)( _C1 - _C6 alkyl), NHC(O)( _C3 - _C6 cycloalkyl), NHC(O)-( _C6 - _C10 aryl), NHC(O)-5-10 membered heteroaryl, C(O)NH( _C1 - _C6 alkyl), C(O)NH( _C3 -C6 cycloalkyl), _R6 is an alkyl group selected from the group consisting of _C1 - _{C6 alkyl, C2-C6} alkenyl, _C2 - _C6 alkynyl, _C1 - _C6 alkoxy, _C3 - _C6 cycloalkyloxy, _{C3 - C6} ^cycloalkyl , _{C6 - C10} aryl, 5-10 membered heteroaryl or _4-8 membered _heterocyclyl .

In some embodiments, R ³ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C ₆ alkynyl, C ₁ -C ₆ alkoxy, NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , NH(C ₃ -C ₆ cycloalkyl), NHC(O)(C ₃ -C ₆ cycloalkyl), C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, NHC(O)(C ₁ -C ₆ alkyl), NHC(O)-(C ₆ -C ₁₀ aryl), NHC(O)-5-10 membered heteroaryl, C(O)NH(C ₁ -C ₆ alkyl), C(O)NH-(C ₆ -C ₁₀ aryl) or C(O)NH-5-10 membered heteroaryl, wherein the C ₁ -C R ₆ is optionally substituted by C ₂ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₂ -C 6 alkynyl, C ₁ -C ₆ alkoxy, C 3 -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or ^4-8 membered heterocyclyl.

In some embodiments, R ³ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C 2 -C _{6 alkenyl, C 2 -C 6 alkynyl , C 1} -C ₆ alkoxy, NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, NHC(O)(C 1 -C 6 alkyl), NHC(O)-(C ₆ -C _{10 aryl), NHC(O)-5-10 membered heteroaryl, C(O)NH(C 1 -C 6} alkyl), C(O)NH-(C ₆ -C ₁₀ aryl) or C(O)NH-5-10 membered heteroaryl, wherein the C 1 -C 6 alkyl, C ₂ -C ₆ alkenyl, C 2 -C 6 alkynyl, C 1 -C 6 alkoxy, NH(C 1 -C 6 alkyl), N(C ₁ -C ₆ alkyl) 2 , C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, NHC(O)(C 1 -C ₆ alkyl), NHC(O)-(C ₆ -C ₁₀ aryl), _NHC (O) _-5-10 membered heteroaryl R ⁶ is optionally substituted by C ₆ -C 10 alkoxy, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl.

In some embodiments, ^R3 is selected from H, _NH2 , NH( _C1 - _C3 alkyl), NH( _C3 - _C6 cycloalkyl), NHC(O)( _C3 - _C6 cycloalkyl), _C2 - _C3 alkenyl, _C2 - _C3 alkynyl or C(O)NH( _C1 - _C3 alkyl), wherein the C2 _{- C3} alkenyl, _C2 - _C3 alkynyl, _C1 - _C3 alkyl or _C3 - _C6 cycloalkyl is optionally substituted with ^R6 .

In some embodiments, R ³ is selected from H, NH ₂ , NH (C ₁ -C ₃ alkyl), C ₂ -C ₃ alkenyl, C ₂ -C ₃ alkynyl or C (O) NH (C ₁ -C ₃ alkyl), wherein the C ₂ -C ₃ alkenyl, C ₂ -C ₃ alkynyl or C ₁ -C ₃ alkyl is optionally substituted with R ^6. In some embodiments, R ³ is selected from H, NH ₂ , C ₂ -C ₃ alkenyl, C ₂ -C ₃ alkynyl or C (O) NH (C ₁ -C ₃ alkyl), wherein the C ₂ -C ₃ alkenyl, C ₂ -C ₃ alkynyl or C ₁ -C ₃ alkyl is optionally substituted with R ⁶ .

In some embodiments, ^R4 is selected from H, OH, halogen, _NH2 , _C1 - _C6 alkyl, _C1 - _C6 alkoxy, NH( _C1 - _C6 alkyl), N( _C1 - _C6 alkyl) ₂ , _C3 - _C6 cycloalkyl, C6- _C10 aryl, _5-10 membered heteroaryl, 4-8 membered heterocyclyl, NHC(O)( _C1 - _C6 alkyl), NHC(O)-( _C6 - _C10 aryl), NHC(O)-5-10 membered heteroaryl, C(O)NH(C1-C6 alkyl), C(O)NH-( _C6 -C10 aryl) or C(O)NH-5-10 membered heteroaryl, wherein the C1-C6 alkyl, C1- _C6 alkoxy, C3-C6 cycloalkyl, C6-C10 aryl, 5-10 membered heteroaryl, _4-8 membered heterocyclyl, NHC(O)( _C1 - _C6 alkyl), NHC(O)-(C6- _{C10 aryl} ), NHC(O)-5-10 membered heteroaryl, C(O)NH( _C1 - _C6 alkyl), C(O)NH-(C6-C10 aryl) or C( _O )NH- _5-10 membered heteroaryl R ⁶ is optionally substituted with _10- membered aryl, 5-10-membered heteroaryl or 4-8-membered heterocyclyl.

In some embodiments, R ⁴ is selected from H, OH, halogen, NH ₂ , C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, C(O)NH(C ₁ -C ₆ alkyl), C(O)NH-(C ₆ -C ₁₀ aryl) or C(O)NH-5-10 membered heteroaryl, wherein the C ₁ -C ₆ alkyl, C ₁ -C ₆ alkoxy, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl is optionally substituted with R ⁶ .

In some embodiments, R ⁴ is selected from H, C ₁ -C ₃ alkyl, 4-8 membered heterocyclyl, C(O)NH(C ₁ -C ₃ alkyl) or C(O)NH-(C ₆ -C ₁₀ aryl), wherein the C ₁ -C ₃ alkyl, 4-8 membered heterocyclyl or C ₆ -C ₁₀ aryl is optionally substituted with R ⁶ .

In some embodiments, R ⁴ is selected from H, 4-8 membered heterocyclyl, C(O)NH(C ₁ -C ₃ alkyl) or C(O)NH-(C ₆ -C ₁₀ aryl), wherein the C ₁ -C ₃ alkyl, 4-8 membered heterocyclyl or C ₆ -C ₁₀ aryl is optionally substituted with R ⁶ .

In some embodiments, R ⁴ is selected from H, CH ₃ , C(O)NH-phenyl, C(O)NH(C ₁ -C ₃ alkyl), azetidinyl, pyrrolidinyl, piperidinyl, or 8-azabicyclo[3.2.1]octanyl, and the methyl, phenyl, C ₁ -C ₃ alkyl, azetidinyl, pyrrolidinyl, piperidinyl, or 8-azabicyclo[3.2.1]octanyl is optionally substituted with R ⁶ .

In some embodiments, R ⁴ is selected from H, CH ₃ , C(O)NH-phenyl, C(O)NH(C ₁ -C ₃ alkyl), azetidinyl, or piperidinyl, and the methyl, phenyl, C ₁ -C ₃ alkyl, azetidinyl, or piperidinyl is optionally substituted with R ⁶ .

In some embodiments, R ⁴ is selected from H, C(O)NH-phenyl, C(O)NH(C ₁ -C ₃ alkyl), azetidinyl, or piperidinyl, said phenyl, C ₁ -C ₃ alkyl, azetidinyl, or piperidinyl being optionally substituted with R ⁶ .

In some embodiments, R ⁶ is selected from deuterium, halogen, OH, NH ₂ , NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , CN, C ₁ -C ₆ alkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, or 4-8 membered heterocyclyl, and the C ₁ -C ₆ alkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, or 4-8 membered heterocyclyl is optionally substituted with R ^5a .

In some embodiments, R ⁶ is selected from halogen, OH, NH ₂ , NH(C ₁ -C ₆ alkyl), N(C ₁ -C ₆ alkyl) ₂ , CN, _{C 1} -C ₆ alkyl, C ₆ -C 10 aryl, 5-10 membered heteroaryl, or 4-8 membered heterocyclyl, wherein the C ₁ -C ₆ alkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, or 4-8 membered heterocyclyl is optionally substituted with R ^5a .

In some embodiments, R ⁶ is selected from deuterium, C ₁ -C ₆ alkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, or 4-8 membered heterocyclyl, wherein the C ₁ -C ₆ alkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, or 4-8 membered heterocyclyl is optionally substituted with R ^5a .

In some embodiments, _R ⁶ is selected from C ₆ -C ₁₀ aryl, _5-10 membered heteroaryl, or 4-8 membered heterocyclyl, which is optionally substituted with R ^5a .

In some embodiments, R ⁶ is selected from _{C 6} -C ₁₀ aryl or _5-10 membered heteroaryl, which is optionally substituted with R ^5a .

In some embodiments, R ⁶ is selected from deuterium, OH, halogen, C ₁ -C ₆ alkyl, pyridinyl, pyrimidinyl, piperidinyl, azetidinyl or phenyl, wherein the C ₁ -C ₆ alkyl, pyridinyl, pyrimidinyl, piperidinyl, azetidinyl or phenyl is optionally substituted with R ^5a .

In some embodiments, R ⁶ is selected from deuterium, halogen, C ₁ -C ₆ alkyl, pyridinyl, pyrimidinyl, piperidinyl, azetidinyl or phenyl, wherein the C ₁ -C ₆ alkyl, pyridinyl, pyrimidinyl, piperidinyl, azetidinyl or phenyl is optionally substituted with R ^5a .

In some embodiments, R ⁶ is selected from pyridinyl, phenyl, or piperidinyl optionally substituted with R ^5a .

In some embodiments, R ⁶ is selected from pyridinyl or phenyl optionally substituted with R ^5a .

In some embodiments, ^R6 is selected from deuterium, OH, fluorine, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, pyrimidin-2-yl, pyridin-4-yl, pyridin-2-yl, piperidinyl, azetidinyl, or phenyl.

In some embodiments, ^R6 is selected from deuterium, fluorine, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, pyrimidin-2-yl, pyridin-4-yl, pyridin-2-yl, piperidinyl, azetidinyl, or phenyl.

In some embodiments, R ⁶ is selected from pyridin-4-yl, phenyl, or piperidinyl.

In some embodiments, R ⁶ is selected from pyridin-4-yl or phenyl.

In some embodiments, R ⁶ is selected from phenyl.

In some embodiments, R ⁶ is selected from piperidinyl.

In some embodiments, R ⁶ is selected from pyridin-2-yl or pyrimidin-2-yl.

In some embodiments, R ³ is selected from H, NH ₂ , NHCH ₃ , NHCD ₃ , NHCH ₂ CH ₃ , NHCH ₂ CF ₃ , NHCH(CH ₃ ) ₂ , NHCH ₂ CH ₂ CH ₃ ,

In some embodiments, R ³ is selected from H, NH ₂ , NHCH ₃ , NHCD ₃ , NHCH ₂ CH ₃ , NHCH ₂ CF ₃ , NHCH(CH ₃ ) ₂ , NHCH ₂ CH ₂ CH ₃ ,

In some embodiments, R ³ is selected from H, NH ₂ , NHCH ₃ ,

In some embodiments, R ³ is selected from H, NH ₂ ,

In some embodiments, R ³ is selected from H, NH ₂ , NHCH ₃ , NHCD ₃ , NHCH ₂ CH ₃ , NHCH ₂ CF ₃ , NHCH(CH ₃ ) ₂ , NHCH ₂ CH ₂ CH ₃ ,

In some embodiments, R ³ is selected from H, NH ₂ or NHCH ₃ .

In some embodiments, ^R4 is selected from H,

In some embodiments, ^R4 is selected from H,

In some embodiments, ^R4 is selected from H,

In some embodiments, ^R4 is selected from

In some embodiments, ^R4 is selected from

In some embodiments, R ^1a and R ³ and the atoms to which they _are each attached together form a _{C 5} -C ₁₀ cycloalkenyl or a 5-14 membered heterocyclyl, which is optionally substituted with R ⁷ .

In some embodiments, R ^1a and R ³ and the atoms to which they are attached are taken together to form a 5-14 membered heterocyclyl optionally substituted with R ⁷ .

In some embodiments, R ^1a and R ³ and the atoms to which they are attached are taken together to form a 6-10 membered heterocyclyl optionally substituted with R ⁷ .

In some embodiments, R ^1a and R ³ and the atoms to which they are attached are taken together to form a 6-8 membered heterocyclyl optionally substituted with R ⁷ .

In some embodiments, R ^1a and R ³ and the atoms to which they are attached together form optionally substituted with R ⁷

In some embodiments _{, R} ⁷ is selected from OH, =O, NH ₂ , _{C 1} -C ₆ alkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, or 4-8 membered heterocyclyl, ^{which is optionally further substituted with R 5a} _.

In some embodiments, _R ⁷ is selected from OH, =0, NH ₂ , C _{1 -C 6} alkyl, or 4-8 membered heterocyclyl, which is optionally further substituted with R ^5a .

In some embodiments, R ⁷ is selected from =0 or a 4-8 membered heterocyclyl optionally substituted with R ^5a .

In some embodiments, R ⁷ is selected from ═O or piperazinyl optionally substituted with R ^5a .

In some embodiments, R ⁷ is selected from ＝O or

In some embodiments, R ^1a and R ³ and the atoms to which they are attached together form

In some embodiments, R ^4a and R ⁴ and the atoms to which they _are each attached together form a _{C 5} -C ₁₀ cycloalkenyl or a 5-14 membered heterocyclyl, which is optionally substituted with R ⁸ .

In some embodiments, R ^4a and R ⁴ and the atoms to which they are each attached are taken together to form a 5-14 membered heterocyclyl optionally substituted with R ⁸ .

In some embodiments, R ^4a and R ⁴ and the atoms to which they are each attached are taken together to form a 6-14 membered heterocyclyl optionally substituted with R ⁸ .

In some embodiments, R ^4a and R ⁴ and the atoms to which they are each attached are taken together to form a 6-11 membered heterocyclyl optionally substituted with R ⁸ .

In some embodiments, ^R4a and ^R4 and the atoms to which they are attached together form optionally substituted with ^R8

In some embodiments, R ⁸ is selected from OH, ═O, NH ₂ , _{C 1} -C ₆ alkyl, C _{3 -C 6} cycloalkyl, C _{6 -C 10} aryl, _5-10 membered heteroaryl, or 4-8 membered heterocyclyl _, which is optionally further substituted with R ^5a _.

In some embodiments _{, R} ⁸ is selected from OH _, =O, NH ₂ , C ₁ -C ₆ alkyl, C _{6 -C 10} aryl, or 4-8 membered heterocyclyl, which is optionally further substituted with R ^5a .

In some embodiments, R ⁸ is selected from =0 or a 4-8 membered heterocyclyl optionally substituted with R ^5a .

In some embodiments, R ⁸ is selected from ═O or piperazinyl optionally substituted with R ^5a .

In some embodiments, R ⁸ is selected from ＝O or

In some embodiments, R ^4a and R ⁴ and the atoms to which they are attached together form

In some embodiments, R ^4a and R ⁴ and the atoms to which they are attached together form And wherein ^X4 is N.

In some embodiments, R ^2a and R ^3a and the atoms to which they are each attached together form a C _{5 -C 10} cycloalkenyl, C ₆ -C ₁₀ aryl, _5-10 membered heteroaryl, or 5-14 membered heterocyclyl, which is optionally substituted with _{R 9} ^.

In some embodiments, R ^2a and R ^3a and the atoms to which they _are each attached together form a _{C 5} -C ₁₀ cycloalkenyl or a 5-14 membered heterocyclyl, which is optionally substituted with R ⁹ .

In some embodiments, R ^2a and R ^3a and the atoms to which they are each attached are taken together to form a 5-10 membered heterocyclyl optionally substituted with R ⁹ .

In some embodiments, R ^2a and R ^3a and the atoms to which they are each attached are taken together to form a 5-8 membered heterocyclyl optionally substituted with R ⁹ .

In some embodiments, R ^2a and R ^3a and the atoms to which they are attached together form optionally substituted with R ⁹

In some embodiments _, R ⁹ is selected from OH, =O, _{NH 2} , _{C 1} -C ₆ alkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, or 4-8 membered heterocyclyl, ^{which is optionally further substituted with R 5a} _.

In some embodiments, R ⁹ is selected from OH, =0, NH ₂ , or _{C 1 -C 6} alkyl, which is optionally further substituted with R ^5a .

In some embodiments, R ^2a and R ^3a and the atoms to which they are attached together form

In some embodiments, R ¹ and R ⁴ and the atoms _to which they are attached together form a C _{5 -C 10} cycloalkenyl or a 5-14 membered heterocyclyl, which is optionally substituted with R ¹⁰ .

In some embodiments, R ¹ and R ⁴ and the atoms to which they are each attached are taken together to form a 5-10 membered heterocyclyl optionally substituted with R ¹⁰ .

In some embodiments, R ¹ and R ⁴ and the atoms to which they are attached together form a 5-8 membered heterocyclyl optionally substituted with R ¹⁰ .

In some embodiments, R ¹ and R ⁴ and the atoms to which they are attached together form optionally substituted with R ¹⁰

In some embodiments, R ¹ and R ⁴ and the atoms to which they are each attached are taken together to form piperazinyl or piperidinyl optionally substituted with R ¹⁰ .

In some embodiments, R ¹⁰ is selected from OH, =O, NH ₂ , C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, or 4-8 membered heterocyclyl, wherein the C ₁ -C ₆ alkyl, C ₃ -C ₆ cycloalkyl, C ₆ -C ₁₀ aryl, 5-10 membered heteroaryl, or 4-8 membered heterocyclyl is optionally further substituted with R ^5a .

In some embodiments _{, R} ¹⁰ is selected from OH, =O, _{NH 2} , C ₁ -C ₆ alkyl, C _{6 -C 10} aryl, or 4-8 membered heterocyclyl, which is optionally further substituted with R ^5a .

In some embodiments, R ¹⁰ is selected from =0 or 4-8 membered heterocyclyl optionally substituted with R ^5a .

In some embodiments, R ¹⁰ is selected from =0, azetidinyl, or piperazinyl, which is optionally substituted with R ^5a .

In some embodiments, R ¹⁰ is selected from ＝O,

In some embodiments, R ¹ and R ⁴ and the atoms to which they are attached together form

In some embodiments, R ¹ and R ⁴ and the atoms to which they are attached together form

In some embodiments, ^Ra is selected from halogen, OH, _NH2 , CN, _C1 - _C6 alkyl or _C1 - _C6 alkoxy, wherein the _C1 - _C6 alkyl or _C1 - _C6 alkoxy is optionally substituted with ^Rb .

In some embodiments, ^Ra is selected from halogen, OH, _NH2 , or _C1 - _C6 alkyl optionally substituted with ^Rb .

In some embodiments, R ^b is selected from halogen, OH, or =0.

In some embodiments, ^Ra is selected from halogen, OH, _NH2 , or halogenated _C1 - _C6 alkyl.

In some embodiments, ^Ra is selected from F or Cl.

In some embodiments, ^Ra is selected from F.

In some embodiments, n is selected from 0, 1 or 2.

In some embodiments, n is selected from 0 or 1.

In some embodiments, n is selected from 0.

In some embodiments, R is selected from

In some embodiments, R is selected from

In some embodiments, R is selected from

In some embodiments, R is selected from

In some embodiments, R is selected from

In some embodiments, Y ¹ is CR, Y ² is selected from CR ^2a or N, Y ³ is selected from CR ^3a or N, Y ⁴ is selected from CR ^4a or N, and X ¹ is selected from CR ¹ or N.

In some embodiments, Y ¹ is CR, Y ² is N, Y ³ is N, Y ⁴ is CR ^4a , and X ¹ is CR ¹ .

In some embodiments, Y ¹ is CR, R is ^Y2 is N, ^Y3 is N, ^Y4 is ^CR4a , ^R4a is -NH2 _or ^X1 is CH.

In some embodiments, Y ¹ is CR, R is ^Y2 is N, ^Y3 is N, ^Y4 is ^CR4a , and ^R4a and ^R4 and the atoms to which they are attached together form an optionally substituted ^{R8 X1} is CH.

In some embodiments, Y ¹ is selected from CR ^1a or N, Y ² is CR, Y ³ is selected from CR ^3a or N, Y ⁴ is selected from CR ^4a or N, and X ¹ is selected from CR ¹ or N.

In some embodiments, Y ¹ is selected from CR ^1a or N, Y ² is CR, Y ³ is CH or N, Y ⁴ is CR ^4a or N, and X ¹ is selected from CR ¹ .

In some embodiments, Y ¹ is selected from CH or N, Y ² is CR, and R is Y ³ is selected from CR ^3a or N, Y ⁴ is selected from CH, C-OCH ₃ or N, and X ¹ is selected from CH or C-NH ₂ .

In some embodiments, Y ¹ is selected from CH or N, Y ² is CR, and R is ^Y3 is CH, ^Y4 is CH, ^X1 is ^CR1 , and ^R1 and ^R4 and the atoms to which they are attached together form an optionally substituted ^R10

In some embodiments, Y ¹ is selected from CR ^1a or N, Y ² is selected from CR ^2a or N, Y ³ is CR, Y ⁴ is selected from CR ^4a or N, and X ¹ is selected from CR ¹ or N.

In some embodiments, Y ¹ is N, Y ² is N, Y ³ is CR, Y ⁴ is CR ^4a , and X ¹ is selected from CR ¹ or N.

In some embodiments, ^Y1 is N, ^Y2 is N, ^Y3 is CR, and R is ^Y4 is CH, and ^X1 is selected from CH or N.

In some embodiments, ^Y1 is N, ^Y2 is N, ^Y3 is CR, and R is ^Y4 is CH, and ^X1 is selected from CH or N.

In some embodiments, ^Y1 is N, ^Y2 is N, ^Y3 is CR, and R is ^Y4 is CH, and ^X1 is selected from N.

In some embodiments, ^Y1 is N, ^Y2 is N, ^Y3 is CR, and R is ^Y4 is CH, and ^X1 is selected from N.

In some embodiments, Y ¹ is selected from CR ^1a or N, Y ² is selected from CR ^2a or N, Y ³ is selected from CR ^3a or N, Y ⁴ is CR, and X ¹ is selected from CR ¹ or N.

In some embodiments, Y ¹ is CR ^1a , Y ² is N, Y ³ is N, Y ⁴ is CR, and X ¹ is CR ¹ .

In some embodiments, ^Y1 is ^CR1a , ^Y2 is N, ^Y3 is N, ^Y4 is CR, and R is ^X1 is CH, and ^R1a and ^R3 and the atoms to which they are attached together form an optionally substituted ^R7

In some embodiments, ^Y1 is selected from ^CR1a or N, ^Y2 is selected from ^CR2a or N, ^Y3 is selected from ^CR3a or N, ^Y4 is selected from ^CR4a or N, and ^X1 is CR.

In some embodiments, ^Y1 is N, ^Y2 is ^CR2a , ^Y3 is selected from ^CR3a , ^Y4 is ^CR4a , and ^X1 is CR.

In some embodiments, Y ¹ is N, Y ² is C-NH ₂ , Y ³ is selected from CH or CR ^3a , and R ^3a is Y ⁴ is CH, X ¹ is CR, R is

In some embodiments, ^Y1 is N, ^Y2 is ^CR2a , ^Y3 is selected from ^CR3a , ^Y4 is CH, and ^R2a and ^R3a and the atoms to which they are attached together form an optionally substituted ^R9 . ^X1 is CR, R is

In some embodiments, the TL is connected to the Linker via R ⁴ , that is, the TL is

In some embodiments, the TL is connected to the Linker via R ³ , that is, the TL is

In some embodiments, the TL is connected to the Linker via R ^3a , that is, the TL is

In some embodiments, the TL is connected to the Linker via R ^4a , that is, the TL is

In some embodiments, the TL is connected to the Linker through a ring atom of the ring formed by R ^4a and R ⁴ and the atoms to which they are respectively connected, or is connected to the Linker through a substituent R ⁸ on the ring.

In some embodiments, the TL is connected to the Linker via ^R8 .

In some embodiments, the TL is connected to the Linker through a ring atom of the ring formed by R ¹ and R ⁴ and the atoms to which they are respectively connected, or is connected to the Linker through a substituent R ¹⁰ on the ring.

In some embodiments, the TL is connected to the Linker via ^R10 .

In some embodiments, the TL is connected to the Linker through a ring atom of the ring formed by R ^1a and R ³ and the atoms to which they are respectively connected, or is connected to the Linker through a substituent R ⁷ on the ring.

In some embodiments, the TL is connected to the Linker through a ring atom of the ring formed by R ^2a and R ^3a and the atoms to which they are respectively connected, or is connected to the Linker through a substituent R ⁹ on the ring.

In some embodiments, the TL is connected to the Linker through a ring atom of the ring formed by R ^2a and R ^3a and the atoms to which they are respectively connected.

In some embodiments, the TL is further selected from the group shown below:

Among them: ^Ra , Y ² , Y ³ , Y ⁴ , X ¹ , X ³ , R ⁴ and n are as defined above. In some embodiments, the TL is further selected from the group shown below:

Among them: ^Ra , Y ¹ , Y ³ , Y ⁴ , X ¹ , X ³ , R ⁴ and n are as defined above. In some embodiments, the TL is further selected from the group shown below:

Among them: ^Ra , Y ¹ , Y ² , Y ⁴ , X ¹ , X ³ , R ⁴ and n are as defined above. In some embodiments, the TL is further selected from the group shown below:

Among them: ^Ra , Y ¹ , Y ² , Y ³ , X ¹ , X ³ , R ⁴ and n are as defined above. In some embodiments, the TL is further selected from the group shown below:

Among them: ^Ra , Y ¹ , Y ² , Y ³ , Y ⁴ , X ³ , R ⁴ and n are as defined above. In some embodiments, the TL is selected from the following structures:

In some embodiments, the TL is selected from the following structures

In some embodiments, the TL is selected from

In some embodiments, the Linker is a linking group that covalently binds a TL and a DIM.

In some embodiments, the linker is selected from -L ^A -, -L ^B -, -R ^1L -, -R ^2L -, -Q ¹ -, -Q ² -,

wherein: ^-LA- , ^-LB- are independently selected from a bond, -O-, -S-, ^-NR3'- , ^-CR4'R5'- , -C(O ⁾ -, -S(O)-, -S(O) _2- , -C(S)-, -C(O)O- or -C(O) ^NR6'- ;

R ^1L and R ^2L are independently selected from a bond, alkylene, heteroalkylene, alkenylene and alkynylene, wherein said alkylene, heteroalkylene, alkenylene and alkynylene are optionally substituted with a group selected from the group consisting of halogen, alkyl, alkoxy, haloalkyl, OH, hydroxyalkyl, CN, NH ₂ , ═O, cycloalkyl, heterocyclyl, aryl, heteroaryl;

Q ¹ , Q ² , Q ³ and Q ⁴ are independently selected from cycloalkyl, heterocyclyl, aryl, heteroaryl or cycloalkenyl, wherein the cycloalkyl, heterocyclyl, aryl, heteroaryl and cycloalkenyl are each independently optionally substituted by a group selected from the following: halogen, alkyl, alkoxy, haloalkyl, OH, hydroxyalkyl, CN, NH ₂ , ＝O, cycloalkyl, heterocyclyl, aryl, heteroaryl;

R ^3′ is selected from H, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl;

R ^4′ and R ^5′ are each independently selected from H, halogen, alkyl, alkoxy, haloalkyl, OH, hydroxyalkyl, CN, NH ₂ , ═O, cycloalkyl, heterocyclyl, aryl or heteroaryl;

R ^6′ is selected from H, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl.

In some embodiments, the linker is selected from the following structures:

In some embodiments, the linker is selected from: a bond, -O-, -C(O)-, -CH ₂ -,

In some embodiments, the Linker is selected from:

In some embodiments, the Linker is selected from

In some embodiments, the DIM is selected from a VHL ligand, ie, a binding moiety that binds a Von Hippel-Lindau type E3 ubiquitin ligase; or a CRBN ligand, ie, a binding moiety that binds a cereblon type E3 ubiquitin ligase.

In some embodiments, the DIM is selected from CRBN ligands.

In some embodiments, the DIM is selected from the structure shown in formula (DIM-1) or (DIM-2):

in:

Selected from

Y is a bond, or Y is selected from _YA , O, NH, NR _E , C(O)O, C(O)NR _E ', NR _E'C (O), YA _-NH , YA _- NR _E , YA _- C(O), YA _- C(O)O, YA _- OC(O), YA _-C (O)NR _E ' or YA- _{NR E'C} (O), wherein _YA is selected from _C1 - _C6 alkylene, _C2 - _C6 alkenylene or _C2 - _C6 alkynylene;

X is selected from C(O) or C( _RA ) ₂ ; XA _{- XB} is selected from C( _RA )=N or C( _RA ) ₂ -C( _RA ) ₂ ;

Each _RA is independently selected from H or C ₁ -C ₃ alkyl, wherein the C ₁ -C ₃ alkyl is optionally substituted with C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl;

Each _RA ' is independently selected from _C1 - _C3 alkyl;

Each _RB is independently selected from H or _C1 - _C3 alkyl, or two _RBs together with the atoms to which they are attached form C(O), _C3 - _C6 cycloalkyl, _C3 - _C6 cycloalkenyl or 4-6 membered heterocyclyl;

R _C is selected from H, halogen or C ₁ -C ₃ alkyl;

Each R _D is independently selected from halogen, NO ₂ , NH ₂ , OH, COOH, C ₁ -C ₆ alkyl or C ₁ -C ₆ alkoxy;

Each _RE is independently selected from C1 _{- C6} alkyl, _C2 - _C6 alkenyl, _C3 - _C8 cycloalkyl, 3-8 membered heterocycloalkyl, C(O) _-C1 - _C6 alkyl, C(O) _-C2 - _C6 alkenyl, C(O)-C3 _{-C8 cycloalkyl} or C(O)-3-8 membered heterocycloalkyl, said _RE is optionally substituted by a group selected from halogen, N(R _a ) ₂ , NHC(O)R _a , NHC(O)OR _a , OR _b , _C3 - _C8 cycloalkyl, _{3-8 membered heterocycloalkyl, C6-C10 aryl} or 5-10 membered heteroaryl, wherein said C3 _{- C8} cycloalkyl, 3-8 membered heterocycloalkyl, C6 _{- C10} aryl or 5-10 membered heteroaryl is optionally further substituted by a group selected from halogen, NH ₂ , CN, NO ₂ , OH, COOH, C ₁ -C ₆ alkyl, C ₁ -C ₆ haloalkyl, C ₁ -C ₆ alkoxy or C ₁ -C ₆ haloalkoxy;

R _E 'is selected from H, C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₃ -C ₈ cycloalkyl or 3-8 membered heterocycloalkyl, wherein the C ₁ -C ₆ alkyl, C ₂ -C ₆ alkenyl, C ₃ -C ₈ cycloalkyl or 3-8 membered heterocycloalkyl is optionally substituted by a group selected from the group consisting of halogen, N(R _a ) ₂ , NHC(O)R _a , NHC(O)OR _a , OR _b , C ₃ -C ₈ cycloalkyl, 3-8 membered heterocycloalkyl, C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl, wherein the C ₃ -C 8 cycloalkyl, 3-8 membered heterocycloalkyl, C ₆ -C ₁₀ aryl or 5-10 membered heteroaryl is optionally further substituted by a group selected from the group consisting of halogen, NH ₂ , CN, NO ₂ , OH, COOH, C ₁ -C ₆ alkyl, C ₁ -C 6 alkenyl, C ₃ -C 8 cycloalkyl or 3-8 membered heterocycloalkyl C 1 -C ₆ haloalkyl, C ₁ -C ₆ alkoxy or C ₁ -C ₆ haloalkoxy;

Each _Ra is independently selected from H or _C1 - _C6 alkyl;

R _b is selected from H or p-toluenesulfonyl;

t is selected from 0 or 1;

m is selected from 0, 1, 2 or 3;

p is selected from 0, 1 or 2.

In some embodiments, the DIM is further selected from the structure shown in formula (DIM-3) or (DIM-4):

wherein the ring A, Y, _RA , _RA ', _RB , _RC , _RD , m and p are as defined above.

In some embodiments, the DIM is further selected from the structures shown in formula (DIM-5), (DIM-6), (DIM-7) or (DIM-8):

wherein Y, X, XA- _{XB , RA} , _RA ', _RB , _RC , _RD , m and p are as defined above.

In some embodiments, the DIM is further selected from the structure shown in formula (DIM-9) or (DIM-10):

wherein Y, X, XA- _{XB , RA} , _RA ', _RB , _RC , _RD , m and p are as defined above.

In some embodiments, the DIM is selected from the structure shown in formula (DIM-11):

in:

_XC is selected from a bond, _-CH2- , -CHCF3-, _-SO2- , _-S (O)-, -P(O)R'-, -P(O)OR'-, -P(O) _NR'2- , -C(O)-, -C(S)- or

_XD is selected from C, N or Si;

X _E is selected from a bond, -CR' ₂ -, -NR'-, -O-, -S- or -SiR' ₂ -;

_RF is absent or is selected from H, deuterium, halogen, CN, -OR'-, -SR'-, -S(O)R'-, -S(O)2R'- _{, -NR'2-, -P(O)(OR')2 ,} -P(O)( _NR'2 )OR'-, -P(O)( _NR'2 ) ₂ , -Si(OH) _2R ', _-Si (OH) _R'2 , _-SiR'3 , or _C1 - _C4 alkyl;

Each R _G is independently selected from H, deuterium, _RH , halogen, CN, -NO ₂ , -OR', -SR', -NR' ₂ , -SiR' ₃ , -S(O) ₂ R', -S(O) ₂ NR' ₂ , -S(O)R' , -C(O)R' , -C(O)OR' , -C(O)NR' ₂ , -C(O)N( R')OR', -C(R') ₂ N(R')C(O)R', -C(R') ₂ N(R')C(O)NR' ₂ , -OC(O) R', -OC(O)NR' ₂ , -OP(O)R' ₂ , -OP(O)(OR') ₂ , -OP(O)(OR')NR' ₂ , -OP(O) (NR' ₂ ) ₂ , -N(R')C(O)OR', -N(R')C(O)R', -N(R')C(O)NR' ₂ , -N(R')S(O ) ₂ R', -NP(O)R' ₂ , -N(R')P(O)(OR') ₂ , -N(R')P(O)(OR')NR' ₂ or -N (R')P(O)(NR' ₂ ) ₂ ;

Each _RH is independently selected from _C1 - _C6 alkyl, phenyl, 4-7 membered heterocyclyl or 5-6 membered heteroaryl;

Ring E, Ring F, Ring G are independently selected from phenyl, 6-membered heteroaryl, C ₅ -C ₇ cycloalkyl, C ₅ -C ₇ cycloalkenyl, 5-7-membered heterocyclyl or 5-6-membered heteroaryl, wherein Ring E, Ring F and Ring G are each optionally further substituted by =O;

L ¹ is selected from a bond, C ₁ -C ₃ alkylene, C ₂ -C ₃ alkenylene or C ₂ -C ₃ alkynylene, wherein any one or two methylene groups in the C ₁ -C ₃ alkylene, C ₂ -C ₃ alkenylene or C ₂ -C ₃ alkynylene are optionally replaced by the following groups: -O-, -C(O)-, -C(S)-, -C(R') ₂ -, -CH(R')-, -C(F) ₂ -, -N(R')-, -S- or -S(O) ₂ -;

Each R' is independently selected from H, C ₁ -C ₆ alkyl, phenyl, 4-7 membered heterocyclyl or 5-6 membered heteroaryl, or two R' and the atoms to which they are attached together form a 4-7 membered heterocyclyl or 5-6 membered heteroaryl;

q is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.

In some embodiments, the DIM is selected from the structure shown in formula (DIM-11'):

wherein X _C , _RF , RG , _q , ring E, ring F and ring G are as defined in formula (DIM-11).

In some embodiments, the DIM is selected from the structure shown in formula (DIM-12):

wherein: ring H is selected from C ₅ -C ₉ cycloalkyl, C ₅ -C ₉ cycloalkenyl or 5-9 membered heterocyclyl, said C ₅ -C ₉ cycloalkyl, C ₅ -C ₉ cycloalkenyl or 5-9 membered heterocyclyl is optionally substituted by =O; k is selected from 0, 1, 2, 3 or 4; X _C , X _D , X _E , _RF , _RG , L ¹ and ring E are as defined in formula (DIM-11).

In some embodiments, the DIM is selected from the structure shown in (DIM-12'):

wherein X _C , _RF , _RG , k, ring E and ring H are as defined in formula (DIM-12).

In some embodiments, the DIM is selected from VHL ligands.

In some embodiments, the DIM is selected from the structures shown in formula (DIM-13), (DIM-14), (DIM-15), (DIM-16) or (DIM-17):

in:

R _J is selected from optionally substituted C ₁ -C ₆ alkyl, optionally substituted -(CH ₂ ) _j OH, optionally substituted -(CH ₂ ) _j SH, optionally substituted -(CH ₂ ) _j -O-(C ₁ -C ₆ )alkyl, optionally substituted -(CH ₂ ) _j -CH ₂ OCH ₂ -(C ₀ -C ₆ )alkyl, optionally substituted -(CH ₂ ) _j COOH, optionally substituted -(CH ₂ ) _j C(O)-(C ₁ -C ₆ alkyl), optionally substituted -(CH ₂ ) _j NR _c R _d , optionally substituted -(CH ₂ ) _j NHC(O)-R _c , optionally substituted -(CH ₂ ) _j C(O)-NR _c R _d , optionally substituted -(CH ₂ ) _j OC(O)-NR _c R _d , -(CH ₂ O) _j H, optionally substituted -(CH ₂ ) _j OC(O)-(C ₁ -C ₆ alkyl), optionally substituted -(CH ₂ ) _j C(O)-O-(C ₁ -C ₆ alkyl), optionally substituted -(CH ₂ O) _j COOH, optionally substituted -(OCH ₂ ) _j O-(C ₁ -C ₆ alkyl), optionally substituted -(CH ₂ O) _j C(O)-(C ₁ -C ₆ alkyl), optionally substituted -(OCH ₂ ) _j NHC(O)-R _c , optionally substituted -(CH ₂ O) _j C(O)-NR _c R _d , -(CH ₂ CH ₂ O) _j H, optionally substituted -(CH ₂ CH ₂ O) _j COOH, optionally substituted -(OCH ₂ CH ₂ ) _j O-(C ₁ -C ₆ alkyl), optionally substituted -(CH ₂ CH ₂ O) _j C(O)-(C ₁ -C 6 alkyl), optionally substituted -( ₆ alkyl), optionally substituted -(OCH ₂ CH ₂ ) _j NHC(O)-R _c , optionally substituted -(CH ₂ CH ₂ O) _j C(O)-NR _c R _d , optionally substituted -SO ₂ R _s , optionally substituted -S(O)R _s , NO ₂ , CN or halogen;

R _c and R _d are independently selected from H or C ₁ -C ₆ alkyl, wherein the C ₁ -C ₆ alkyl is optionally substituted by OH or halogen;

R _s is selected from C ₁ -C ₆ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl or -(CH ₂ ) _g NR _c R _d ;

_XF and _XG are independently selected from C=O, C=S, S(O) or S(O) ₂ ;

_RL is selected from optionally substituted -( _CH2 ) _j- (C=O) _u ( _NRc ) _v ( _SO2 ) _w ( _C1 - _C6 )alkyl, optionally substituted -( _CH2 ) _j- (C=O) _u ( _NRc )v( _{SO2 )wNR1NR2N ,} optionally substituted -( _CH2 ) _j- (C=O)u(NRc) _v ( _SO2 ) _{w -} aryl, optionally substituted -( _CH2 ) _j- (C=O) _u ( _NRc ) _v ( _SO2 ) _w -heteroaryl, optionally substituted -(CH2) _j- (C=O)vNRc(SO2)w-heterocyclyl, optionally substituted -NRe-( _CH2 ) _j -C(O)u( _NRc ) _v ( _{SO2 ) w} -C1-C6alkyl, optionally substituted _-NRe- ( _CH2 ) _j -C(O) _u (NRc) _v ( _SO2 ) _{w - C1} - _C6alkyl , optionally substituted _-NRe- ( _CH2) j-C=O)u(NRc)v( _SO2 )w- wherein the substituted _-NRe- (CH2) _j -C( _O ) _u (NRc) _v ( _{SO2 ) w} - _NR1NR2N , optionally substituted -NRe-(CH2) _j - _C (O) _u ( _NRc ) _v ( _SO2 ) _{w- NRcC} (O) _R1N , optionally substituted _-NRe- ( _CH2 ) _j- (C＝O) _u ( _NRc ) _v ( _SO2 ) _w -aryl, optionally substituted _-NRe- ( _CH2 ) _j- (C＝O) _u ( _NRc ) _v ( _SO2 ) _w -heteroaryl, or optionally substituted _-NRe- ( _CH2 ) _j- (C＝O) _vNRc ( _SO2 ) _{w -} heterocyclyl, optionally substituted ^-XR2' - _C1 - _C6alkyl , optionally substituted ^-XR2' -aryl, optionally substituted ^-XR2' -heteroaryl, or optionally substituted ^-XR2' -heterocyclic group;

R _M is selected from optionally substituted C ₁ -C ₆ alkyl, optionally substituted -(CH ₂ ) _j -C(O) _u (NR _c ) _v (SO ₂ ) _w -C ₁ -C ₆ alkyl, optionally substituted -(CH ₂ ) _j -C(O) _u (NR _c ) _v (SO ₂ ) _w -N _1N R _2N , optionally substituted -(CH ₂ ) _j -C(O) _u (NR _c ) _v (SO ₂ ) _w -NR _c C(O)R _1N , optionally substituted -(CH ₂ ) _j -C(O) _u (NR _c ) _v (SO ₂ ) _w -C(O)NR _c R _d , optionally substituted -(CH ₂ ) _j -C(O) _u (NR _c ) _v (SO ₂ ) _w -aryl, optionally substituted -(CH ₂ ) _j -C(O) _u (NR _c ) _v (SO ₂ ) _w -heteroaryl, optionally substituted -(CH ₂ ) _j -C(O) _u (NR _c ) _v (SO ₂ ) _w -heterocycle, optionally substituted -NR _e -(CH ₂ ) _j -C(O) _u (NR _c ) _v (SO ₂ ) _w -C ₁ -C ₆ alkyl, optionally substituted -NR _e -(CH ₂ ) _j -C(O) _u (NR _c ) _v (SO _{2 ) w -NRR2N} , optionally substituted -NR _e -(CH ₂ ) _j -C(O) _u (NR _c ) _v (SO ₂ ) _w -NR _c C(O) _R1N , optionally substituted -NR _e -(CH ₂ ) _j -C(O) _u (NR _c ) _v (SO ₂ ) _w -aryl, optionally substituted -NR _e -(CH ₂ ) _j -C(O) _u (NR _c ) _v (SO ₂ ) _w -heteroaryl, optionally substituted -NR _e -(CH ₂ ) _j -C(O) _u (NR _c ) _v (SO ₂ ) _w -heterocycle, optionally substituted -O-(CH ₂ ) _j -(C═O) _u (NR _c ) _v (SO ₂ ) _w -C ₁ -C ₆ alkyl, optionally substituted -O-(CH ₂ ) _j -(C═O) _u (NR _c ) _v (SO ₂ ) _w -NR _1N R _2N , optionally substituted -O-(CH ₂ ) _j -(C═O) _u (NR _c ) _v (SO ₂ ) _w -NR _c C(O)R _1N , optionally substituted -O-(CH ₂ ) _j -(C═O) _u (NR _c ) _v (SO ₂ ) _w -aryl, optionally substituted -O-(CH ₂ ) _j -(C═O) _u (NR _c ) _v (SO ₂ ) _w -heteroaryl or optionally substituted -O-(CH ₂ ) _j -(C═O) _u (NR _c ) _v (SO ₂ ) _w -heterocycle, optionally substituted -(CH ₂ ) _j -(V) _n′ -(CH ₂ ) _j -(V) _n′ -C ₁ -C ₆ alkyl, optionally substituted -(CH ₂ ) _j -(V) _n′ -(CH ₂ ) _n -(V) _n′ -aryl, optionally substituted -(CH ₂ ) _j -(V) _n′ -(CH _{2 ) j -(V) n′-heteroaryl, optionally substituted -(CH 2} ) _j -(V) _{n′ -} (CH ₂ ) _j -(V) _n′ -heterocyclyl, optionally substituted _- (CH ₂ ) _j -N(R _1′ )(C═O) _m′ -(V) _n′ -C ₁ -C ₆ alkyl, optionally substituted -(CH ₂ ) _j -(V) n′ -(CH _{2 )} n -(V) n′-aryl -N( _Re )(C=O) _m' -(V) _n'- aryl, optionally substituted -( _CH2 ) _j -N( _Re )(C=O) _m' -(V) _n' -heteroaryl, optionally substituted -( _CH2 ) _j -N( _Re )(C=O) _m' -(V) _n' -heterocyclyl, optionally substituted ^-XR3' - _C1 - _C6 alkyl, optionally substituted ^-XR3' -aryl, optionally substituted ^-XR3' -heteroaryl or optionally substituted ^-XR3' -heterocyclyl;

wherein R _1N and R _2N are each independently H, C ₁ -C ₆ alkyl optionally substituted by OH and halogen, or optionally substituted -(CH ₂ ) _j -aryl, -(CH ₂ ) _j -heteroaryl or -(CH ₂ ) _j -heterocyclyl;

V is O, S or NR _c ;

R _e is independently selected from H or C ₁ -C ₃ alkyl;

^XR2' and ^XR3' are each independently selected from optionally substituted -( _CH2 ) _j- , -( _CH2 ) _j -C( ^Xv )=C( ^Xv )-, -( _CH2 ) _j -C≡C-, -( _CH2CH2O ) _j- or _C3 - _C6 cycloalkyl, wherein ^Xv is H, halogen or optionally substituted _C1 - _C3 alkyl _;

g is independently 0, 1, 2, 3, 4, 5, 6;

j is independently 0, 1, 2, 3, 4, 5 or 6;

m' is independently 0 or 1;

n' is independently 0 or 1;

u is independently 0 or 1;

v is independently 0 or 1;

w is independently 0 or 1.

In some embodiments, R _J in formula (DIM-14), (DIM-15), (DIM-16) or (DIM-17) is OH.

In some embodiments, _XF and _XG in the formula (DIM-13), (DIM-14) or (DIM-17) are both C=O.

In some embodiments, the DIM is selected from the structure represented by formula (DIM-13), (DIM-14) or (DIM-17), and _XF and _XG are both C=O.

In some embodiments, the DIM is selected from the structure shown in formula (DIM-14), R _J is OH, and X _F and X _G are both C=O.

In some embodiments, the DIM is selected from the following structures:

In some embodiments, the DIM is selected from the following structures:

In some embodiments, the DIM is selected from

In some embodiments, the DIM is selected from

In some embodiments, the DIM is selected from

In some embodiments, the compound of formula (I) of the present disclosure is selected from the following compounds:

On the other hand, the present disclosure provides a pharmaceutical composition comprising a compound represented by any of the above general formulae of the present disclosure or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

On the other hand, the present disclosure provides a method for treating a disease mediated by BRM in a mammal, comprising administering a therapeutically effective amount of a compound represented by any of the above general formulas of the present disclosure or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to a mammal, preferably a human, in need of such treatment.

On the other hand, the present disclosure provides a method for treating tumors in mammals, comprising administering a therapeutically effective amount of a compound represented by any of the above general formulas or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof to a mammal, preferably a human, in need of such treatment.

On the other hand, the present disclosure provides use of a compound represented by any of the above general formulae or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a medicament for preventing or treating a BRM-mediated disease.

On the other hand, the present disclosure provides use of a compound represented by any of the above general formulae or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof, in the preparation of a drug for preventing or treating tumors.

On the other hand, the present disclosure provides the use of a compound represented by any of the above general formulas or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in preventing or treating a BRM-mediated disease.

On the other hand, the present disclosure provides the use of a compound represented by any of the above general formulas or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof in preventing or treating tumors.

In another aspect, the present disclosure provides a compound of any of the above general formulas or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for preventing or treating a BRM-mediated disease.

In another aspect, the present disclosure provides a compound of any of the above general formulas or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition thereof for preventing or treating tumors.

In some embodiments, the BRM-mediated disease is selected from a tumor.

In some embodiments, the tumor is selected from a cancer.

Definitions and explanations of terms

Unless otherwise indicated, the terms used in this disclosure have the following meanings, and the groups and term definitions recorded in this disclosure, including their definitions as examples, exemplary definitions, preferred definitions, definitions recorded in tables, definitions of specific compounds in the examples, etc., can be arbitrarily combined and combined with each other. A particular term should not be considered as uncertain or unclear in the absence of a special definition, but should be understood according to the common meaning in the art. When a trade name appears in this article, it is intended to refer to its corresponding commodity or its active ingredient.

The carbon atom marked with "*" herein represents a chiral carbon atom, which means that its absolute configuration is one of (S)-configuration or (R)-configuration.

In this article In this article, of when When not connected to a fixed ring or atom, it means that it can be connected to a group after losing the hydrogen atom at any position in the molecule within "[]" containing a replaceable hydrogen atom (including a hydrogen atom directly connected to a ring atom, a hydrogen atom on a non-hydrogen substituent of a ring atom, and a hydrogen atom in a further substituent on the substituent), for example middle The connection position of includes but is not limited to Y ¹ , R ⁴ and the substituent on R ⁴ and the like.

The term "capable of binding" means capable of measurably binding to a target (eg, a ligand of an E3 ubiquitin ligase is capable of forming a covalent bond with a cysteine of an E3 ubiquitin ligase, etc.).

The term "ubiquitin ligase" refers to a family of proteins that facilitate the transfer of ubiquitin to a specific substrate protein to target the substrate protein for degradation. E3 ubiquitin ligases, alone or in complex with E2 ubiquitin ligases, are responsible for transferring ubiquitin to target proteins. Typically, ubiquitin ligases participate in polyubiquitination, such that a second ubiquitin is attached to a first ubiquitin; a third ubiquitin is attached to a second ubiquitin, and so on. Polyubiquitination marks proteins for degradation by the proteasome. However, there are some ubiquitination events that are limited to monoubiquitination, in which ubiquitin ligases only add a single ubiquitin to a substrate molecule. Monoubiquitinated proteins are not targeted to the proteasome for degradation, but can change their cellular location or function, for example, by binding to other proteins with domains that can bind ubiquitin. To complicate matters further, E3 ubiquitin ligases can target different lysines on ubiquitin to make chains.

The term "target protein" refers to proteins and peptides having any biological function or activity, including structural, regulatory, hormonal, enzymatic, genetic, immune, contractile, storage, transport, and signal transduction. In some embodiments, a target protein refers to a protein or polypeptide that binds to a compound of the disclosure and can be degraded.

The graphic representations of racemates or enantiomerically pure compounds herein are from Maehr, J. Chem. Ed. 1985, 62: 114-120. Unless otherwise indicated, the wedge and dashed wedge keys are used. To indicate the absolute configuration of a stereocenter, use black real and imaginary bonds. Indicates the relative configuration of a stereocenter (such as the cis-trans configuration of an alicyclic compound).

The term "tautomer" refers to functional group isomers resulting from the rapid movement of an atom in a molecule between two positions. Compounds of the present disclosure may exhibit tautomerism. Tautomeric compounds may exist in two or more interconvertible species. Tautomers generally exist in equilibrium, and attempts to separate a single tautomer generally result in a mixture whose physical and chemical properties are consistent with a mixture of compounds. The position of equilibrium depends on the chemical properties within the molecule. For example, in many aliphatic aldehydes and ketones such as acetaldehyde, the keto form predominates; whereas in phenols, the enol form predominates. The present disclosure encompasses all tautomeric forms of the compounds.

The term "stereoisomer" refers to isomers resulting from different spatial arrangements of atoms in a molecule, including cis-trans isomers, enantiomers and diastereomers.

The compounds of the present disclosure may have asymmetric atoms such as carbon atoms, sulfur atoms, nitrogen atoms, phosphorus atoms or asymmetric double bonds, so the compounds of the present disclosure may exist in specific geometric or stereoisomeric forms. Specific geometric or stereoisomeric forms may be cis and trans isomers, E-type and Z-type geometric isomers, (-)- and (+)-enantiomers, (R)- and (S)-enantiomers, diastereomers, (D)-isomers, (L)-isomers, and racemic mixtures or other mixtures thereof, such as mixtures enriched in enantiomers or diastereomers, all of which are within the definition of the compounds of the present disclosure and their mixtures. Additional asymmetric carbon atoms, asymmetric sulfur atoms, asymmetric nitrogen atoms or asymmetric phosphorus atoms may be present in substituents such as alkyl, and all of these isomers and their mixtures involved in all substituents are also within the definition of the compounds of the present disclosure. Compounds of the present disclosure containing an asymmetric atom can be isolated in optically pure or racemic forms, which can be resolved from racemic mixtures or synthesized by using chiral starting materials or chiral reagents.

The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which may include deuterium and hydrogen variants, as long as the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxo (i.e., =O), it means that two hydrogen atoms are replaced, and oxo will not occur on an aromatic group.

The term "optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes both the occurrence and non-occurrence of the event or circumstance. For example, an ethyl group is "optionally" substituted with a halogen, which means that the ethyl group may be unsubstituted (CH ₂ CH ₃ ), monosubstituted (CH ₂ CH ₂ F, CH ₂ CH ₂ Cl, etc.), polysubstituted (CHFCH ₂ F, CH ₂ CHF ₂ , CHFCH ₂ Cl, CH ₂ CHCl ₂ , etc.) or fully substituted (CF ₂ CF ₃ , CF ₂ CCl ₃ , CCl ₂ CCl ₃ , etc.). It will be understood by those skilled in the art that for any group containing one or more substituents, no substitution or substitution pattern that is sterically impossible to exist and/or cannot be synthesized will be introduced.

The term "optionally substituted" means that the compound may be substituted or unsubstituted, and unless otherwise specified, the type and amount of the substituents may be any on the basis of what is chemically feasible.

The term "replaced" means that a specific atom or group can be replaced by another specified atom or group. For example, CH ₂ in -CH ₂ CH ₂ CH ₂ - can be replaced by O, S or NH to obtain -CH ₂ OCH ₂ -, -OCH ₂ CH ₂ -, -CH ₂ SCH ₂ -, -SCH ₂ CH ₂ -, -CH ₂ NHCH ₂ - or -NHCH ₂ CH ₂ -.

When any variable (e.g., ^Ra , ^Rb ) occurs more than once in the composition or structure of a compound, its definition in each case is independent. For example, if a group is substituted by 2 ^Rb , each ^Rb has independent options; for the group N( _C1 - _C6 alkyl) ₂ , when _C1 - _C6 alkyl is substituted by ^Rb , the two _C1 - _C6 alkyls have independent ^Rb options.

When the number of a linking group is 0, such as -(CH ₂ ) ₀ -, it means that the linking group is a bond.

When one of the variables is selected from a chemical bond or does not exist, it means that the two groups it connects are directly connected. For example, when L in A-L-Z represents a bond, it means that the structure is actually A-Z.

When the linking group mentioned in this article does not specify its connection direction, its connection direction is arbitrary. When L ¹ is selected from "C ₁ -C ₃ alkylene-O", L ¹ can connect ring Q and R ¹ from left to right to form "ring QC ₁ -C ₃ alkylene-OR ¹ ", or connect ring Q and R ¹ from right to left to form "ring QOC ₁ -C ₃ alkylene-R ¹ ".

In TL, "at least one of R ^1a , R ^2a , R ^3a , R ^4a , R ¹ , R ³ , and R ⁴ is not H" means that when these groups are contained in the molecule, at least one of them is not H. For example, when Y ¹ is CR, Y ² is selected from CR ^2a , Y ³ is selected from N, Y ⁴ is selected from CR ^4a , and X ¹ is selected from N, then at least one of R ^2a , R ^4a , R ³ , and R ⁴ is not H.

When a substituent's bond crosses two atoms in a ring, the substituent may be bonded to any atom in the ring. It means that ^Ra can remove OH and The substitution occurs at any position other than the attachment site.

_Cm - _Cn herein refers to an integer number of carbon atoms in the range of mn or m to n. For example, " _C1 - _C10 " means that the group may have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, 6 carbon atoms, 7 carbon atoms, 8 carbon atoms, 9 carbon atoms or 10 carbon atoms. Similarly, m-membered to n-membered means that the number of ring atoms is m to n, for example, 5-14-membered ring includes 5-membered ring, 6-membered ring, 7-membered ring, 8-membered ring, 9-membered ring, 10-membered ring, 11-membered ring, 12-membered ring, 13-membered ring and 14-membered ring, and also includes any range from n to m, for example, 5-14-membered ring includes 6-14-membered ring, 6-11-membered ring, 5-10-membered ring, 6-10-membered ring, 6-8-membered ring, etc.

The term "alkyl" refers to a hydrocarbon group of the general formula _{CnH2n +1} , which is a straight or branched group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 (e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, and more preferably an alkyl group containing 1 to 6 carbon atoms. The term " _C1 - _C10 alkyl" is understood to mean a straight or branched saturated monovalent hydrocarbon group having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. Specific examples of the alkyl group include, but are not limited to, methyl, ethyl, propyl, butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, tert-butyl, isopentyl, 2-methylbutyl, 1-methylbutyl, 1-ethylpropyl, 1,2-dimethylpropyl, neopentyl, 1,1-dimethylpropyl, 4-methylpentyl, 3-methylpentyl, 2-methylpentyl, 1-methylpentyl, 2-ethylbutyl, 1-ethylbutyl, 3,3-dimethylbutyl, 2,2-dimethylbutyl, 1,1-dimethylbutyl, 2,3-dimethylbutyl, 1,3-dimethylbutyl or 1,2-dimethylbutyl, etc.; the term "C 1 -C 1 -C 1 -C 1 -C 1 -C ₁ -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C 1 -C The term "C 1 _{-C 4} alkyl" may be understood to mean an alkyl group having 1 to 6 carbon atoms, specific examples of which include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, neopentyl, hexyl, 2-methylpentyl, and the like. The term "C ₁ -C ₄ alkyl" may be understood to mean a straight-chain or branched saturated monovalent hydrocarbon group having 1 to 4 carbon atoms. The term "C ₁ -C ₃ alkyl" may be understood to mean a straight-chain or branched saturated monovalent hydrocarbon group having 1 to 3 carbon atoms. The “C ₁ -C ₁₀ alkyl” may include “C ₁ -C ₆ alkyl”, “C ₁ -C ₄ alkyl” or “C ₁ -C ₃ alkyl”, and the “C ₁ -C ₆ alkyl” may further include “C ₁ -C ₄ alkyl” or “C ₁ -C ₃ alkyl”, and the “C ₁ -C ₄ alkyl” may further include “C ₁ -C ₃ alkyl”.

The term "heteroalkyl" refers to an alkyl group in which one or more _-CH2- are replaced by a heteroatom selected from NH, O and S, or one or more -CH- are replaced by N; wherein the alkyl group is as defined above.

The term "haloalkyl" refers to a group obtained by further replacing the alkyl group with halogen, such as "C ₁ -C ₆ haloalkyl" refers to a C ₁ -C ₆ alkyl group further replaced with halogen. The term "hydroxyalkyl" refers to a group obtained by further replacing the alkyl group with OH.

The term "alkylene" refers to a saturated straight or branched aliphatic hydrocarbon group having two residues derived from the same carbon atom or two different carbon atoms of a parent alkane by removing two hydrogen atoms, and is a straight or branched group containing 1 to 20 carbon atoms, preferably an alkylene group containing 1 to 12 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 and 12) carbon atoms, and more preferably an alkylene group containing 1 to 6 carbon atoms. Non-limiting examples of alkylene groups include, but are not limited to, methylene, -CH(CH ₃ )-, -CH ₂ CH ₂ -, -CH(CH ₂ CH ₃ )-, -CH ₂ CH(CH ₃ )-, -CH ₂ CH ₂ CH ₂ -, -CH ₂ CH _{2 CH 2} -, and the like. The term "C ₁ -C ₆ alkylene" is understood to mean an alkylene group having 1 to 6 carbon atoms. The term "C ₁ -C ₃ alkylene" is understood to mean an alkylene group having 1 to 3 carbon atoms. Preferably, "C ₁ -C ₆ alkylene" may include "C ₁ -C ₃ alkylene". The term "heteroalkylene" means that one or more -CH ₂ - in the alkylene group is substituted by a heteroatom selected from N, O and S; wherein the alkylene group is as defined above.

The term "alkoxy" refers to a monovalent group generated by the loss of a hydrogen atom from a hydroxyl group of a straight or branched alcohol, and can be understood as "alkyloxy" or "alkyl-O-", wherein the definition of alkyl is as described above. The term "C ₁ -C ₁₀ alkoxy" can be understood as "C ₁ -C ₁₀ alkyloxy" or "C ₁ -C ₁₀ alkyl-O-"; the term "C ₁ -C ₆ alkoxy" can be understood as "C ₁ -C ₆ alkyloxy" or "C ₁ -C ₆ alkyl-O-". The "C ₁ -C ₁₀ alkoxy" can include "C ₁ -C ₆ alkoxy" and "C ₁ -C ₃ alkoxy" and the like, and the "C ₁ -C ₆ alkoxy" can further include "C ₁ -C ₃ alkoxy".

The term "haloalkoxy" refers to a group obtained by further substituted alkoxy with halogen, such as "C ₁ -C ₆ haloalkoxy" refers to a C ₁ -C ₆ alkoxy further substituted with halogen.

The term "alkenyl" refers to an unsaturated aliphatic hydrocarbon group consisting of carbon atoms and hydrogen atoms, which is straight or branched and has 2 to 20 carbon atoms and has at least one double bond. The term "C ₂ -C ₁₀ alkenyl" is understood to mean a straight or branched unsaturated monovalent hydrocarbon group, which contains one or more double bonds and has 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms, and the term "C ₂ -C ₆ alkenyl" is understood to mean a straight or branched unsaturated monovalent hydrocarbon group, which contains one or more double bonds and has 2, 3, 4, 5 or 6 carbon atoms. "C ₂ -C ₁₀ alkenyl" is preferably "C ₂ -C ₆ alkenyl" or "C ₂ -C ₄ alkenyl", "C ₂ -C ₆ alkenyl" is further preferably "C ₂ -C ₄ alkenyl", and further preferably C ₂ or C ₃ alkenyl. It should be understood that in the case where the alkenyl group contains more than one double bond, the double bonds may be separated or conjugated from each other. Specific examples of the alkenyl group include, but are not limited to, vinyl, allyl, (E)-2-methylvinyl, (Z)-2-methylvinyl, (E)-but-2-enyl, (Z)-but-2-enyl, (E)-but-1-enyl, (Z)-but-1-enyl, isopropenyl, 2-methylprop-2-enyl, 1-methylprop-2-enyl, 2-methylprop-1-enyl, (E)-1-methylprop-1-enyl or (Z)-1-methylprop-1-enyl, etc.

The term "alkenylene" refers to a residue derived from the same carbon atom or two different carbon atoms of a parent alkene by removing two hydrogen atoms, wherein alkenyl is as defined above. The term "C ₂ -C ₆ alkenylene" is understood as an alkenylene having 2 to 6 carbon atoms. The term "C ₂ -C ₃ alkenylene" is understood as an alkenylene having 2 or 3 carbon atoms. Preferably, "C ₂ -C ₆ alkenylene" includes "C ₂ -C ₃ alkenylene".

The term "alkynyl" refers to an unsaturated aliphatic hydrocarbon group having at least one triple bond, which is composed of carbon atoms and hydrogen atoms and contains 2 to 20 carbon atoms, which is straight or branched. The term " _C2 - _C10alkynyl " is understood to mean a straight or branched unsaturated monovalent hydrocarbon group, which contains one or more triple bonds and has 2, 3, 4, 5, 6, 7, 8, 9 or 10 carbon atoms. The term " _C2 - _C6alkynyl " is understood to mean a straight or branched unsaturated monovalent hydrocarbon group, which contains one or more triple bonds and has 2, 3, 4, 5 or 6 carbon atoms. Examples of " _C2 - _C6alkynyl " include, but are not limited to, ethynyl (-C≡CH3), propynyl (-C≡CCH3 _{, -CH2C≡CH3} ), but-1-ynyl, but-2-ynyl or but-3-ynyl. “C ₂ -C ₁₀ alkynyl” may include “C ₂ -C ₆ alkynyl” or “C ₂ -C ₃ alkynyl”, and “C ₂ -C ₆ alkynyl” may include “C ₂ -C ₃ alkynyl”. Examples of “C ₂ -C ₃ alkynyl” include ethynyl (—C≡CH), prop-1-ynyl (—C≡CCH ₃ ) or prop-2-ynyl (propargyl).

The term "alkynylene" refers to a residue derived from the same carbon atom or two different carbon atoms of a parent alkyne by removing two hydrogen atoms, wherein the definition of alkynyl is as shown above. The term "C ₂ -C ₆ alkynylene" is to be understood as an alkynylene having 2 to 6 carbon atoms. The term "C ₂ -C ₃ alkynylene" is to be understood as an alkynylene having 2 or 3 carbon atoms. Preferably, "C ₂ -C ₆ alkynylene" includes "C ₂ -C ₃ alkynylene".

The term "cycloalkyl" refers to a fully saturated carbocyclic ring that exists in the form of a monocyclic ring, a cyclic ring, a bridged ring, or a spirocyclic ring. Unless otherwise indicated, the carbocyclic ring is generally a 3- to 10-membered ring. The term "C ₃ -C ₁₀ cycloalkyl" is understood to mean a saturated monovalent monocyclic ring, a cyclic ring, a spirocyclic ring, or a bridged ring having 3 to 10 carbon atoms. The term "C ₃ -C ₈ cycloalkyl" is understood to mean a saturated monovalent monocyclic ring, a cyclic ring, a spirocyclic ring, or a bridged ring having 3 to 8 carbon atoms. The term "C ₃ -C ₆ cycloalkyl" is understood to mean a saturated monovalent monocyclic ring, a cyclic ring, a spirocyclic ring, or a bridged ring having 3 to 6 carbon atoms, and specific examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl. The term "C ₅ -C ₉ cycloalkyl" is understood to mean a saturated monovalent monocyclic ring, a cyclic ring, a spirocyclic ring, or a bridged ring having 5 to 9 carbon atoms. The term " _C5 - _C7 cycloalkyl" is understood to mean a saturated monovalent monocyclic, cyclopentyl, spirocyclic or bridged ring having 5 to 7 carbon atoms. Specific examples of the cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclononyl, cyclodecyl, norbornyl (bicyclo[2.2.1]heptyl), bicyclo[2.2.2]octyl, adamantyl, spiro[4.5]decyl, etc. The term " _C3 - _C10 cycloalkyl" may include " _C3 - _C8 cycloalkyl", " _C3 - _C6 cycloalkyl", " _C5 - _C9 cycloalkyl" or " _C5 - _C7 cycloalkyl", the term " _C3 - _C8 cycloalkyl" may include " _C3 - _C6 cycloalkyl" or " _C5 - _C7 cycloalkyl", the term " _C5 - _C9 cycloalkyl" may include " _C5 - _C7 cycloalkyl". 。

The term "cycloalkyloxy" may be understood as "cycloalkyl-O-". For example, "C ₃ -C ₆ cycloalkyloxy" may be understood as "C ₃ -C ₆ cycloalkyl-O-".

The term "cycloalkenyl" refers to a non-aromatic carbocyclic ring that is not fully saturated and exists in the form of a monocyclic ring, a cyclic ring, a bridged ring or a spirocyclic ring. Unless otherwise indicated, the carbocyclic ring is generally a 3-10-membered ring. Specific examples of the cycloalkenyl include, but are not limited to, cyclopentenyl, cyclopentadienyl, cyclohexenyl, cyclohexadienyl, cycloheptenyl or cycloheptadienyl, etc. The term "C ₅ -C ₁₀ cycloalkenyl" refers to a non-aromatic carbocyclic ring that is not fully saturated and exists in the form of a monocyclic ring, a cyclic ring, a bridged ring or a spirocyclic ring, and has 5-10 carbon atoms. The term "C ₅ -C ₉ cycloalkenyl" refers to a non-aromatic carbocyclic ring that is not fully saturated and exists in the form of a monocyclic ring, a cyclic ring, a bridged ring or a spirocyclic ring, and has 5-9 carbon atoms. The term "C ₅ -C ₇ cycloalkenyl" refers to a non-aromatic carbocyclic ring that is not fully saturated and exists in the form of a monocyclic ring, a cyclic ring, a bridged ring or a spirocyclic ring, and has 5-7 carbon atoms. The term "C ₃ -C ₆ cycloalkenyl" refers to a non-aromatic carbon ring which is not fully saturated and exists in the form of a monocyclic, fused, bridged or spirocyclic ring, and has 3 to 6 carbon atoms. The term "C ₅ -C ₁₀ cycloalkenyl" may include "C ₅ -C ₉ cycloalkenyl" or "C ₅ -C ₇ cycloalkenyl", and the term "C ₅ -C ₉ cycloalkenyl" may include "C ₅ -C ₇ cycloalkenyl".

The term "heterocyclyl" refers to a fully saturated or partially saturated (not a heteroaromatic group having aromaticity as a whole) monovalent monocyclic, fused, spiro or bridged ring group, which contains 1 to 5 heteroatoms or heteroatomic groups (i.e., heteroatom-containing atomic groups) in the ring atoms, wherein the "heteroatoms or heteroatomic groups" include, but are not limited to, nitrogen atom (N), oxygen atom (O), sulfur atom (S), phosphorus atom (P), boron atom (B), -S(=O) _2- , -S(=O)- and optionally substituted -NH-, -S(=O)(=NH)-, -C(=O)NH-, -C(=NH)-, -S(=O) _2NH- , S(=O)NH- or -NHC(=O)NH-, etc., which usually contain 3 to 20 ring atoms. The term "5-14 membered heterocyclyl" refers to a heterocyclyl having 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 ring atoms, and the ring atoms thereof contain 1 to 5 heteroatoms or heteroatom groups independently selected from the above. "5-14 membered heterocyclyl" may include "6-14 membered heterocyclyl", "6-11 membered heterocyclyl", "6-10 membered heterocyclyl", "6-8 membered heterocyclyl", "5-10 membered heterocyclyl", "5-9 membered heterocyclyl", "5-8 membered heterocyclyl" or "5-7 membered heterocyclyl". The term "5-10 membered heterocyclyl" may include "5-9 membered heterocyclyl", "5-8 membered heterocyclyl", "5-7 membered heterocyclyl", "6-10 membered heterocyclyl" or "6-8 membered heterocyclyl". The term "4-10 membered heterocyclyl" refers to a heterocyclyl having 4, 5, 6, 7, 8, 9 or 10 ring atoms, and the ring atoms contain 1-5 heteroatoms or heteroatom groups independently selected from the above. "4-10 membered heterocyclyl" includes "4-7 membered heterocyclyl", wherein specific examples of 4 membered heterocyclyl include but are not limited to azetidinyl or oxetanyl; specific examples of 5 membered heterocyclyl include but are not limited to tetrahydrofuranyl, dioxolyl alkyl, pyrrolidinyl, imidazolidinyl, pyrazolidinyl, pyrrolinyl, 4,5-dihydrooxazolyl or 2,5-dihydro-1H-pyrrolyl; specific examples of 6-membered heterocyclic groups include, but are not limited to, tetrahydropyranyl, piperidinyl, morpholinyl, dithianyl, thiomorpholinyl, piperazinyl, trithianyl, tetrahydropyridinyl or 4H-[1,3,4]thiadiazinyl; specific examples of 7-membered heterocyclic groups include, but are not limited to, diazepanyl. The heterocyclic group may also be a bicyclic group, wherein specific examples of 5,5-membered bicyclic groups include, but are not limited to, hexahydrocyclopenta[c]pyrrol-2(1H)-yl; specific examples of 5,6-membered bicyclic groups include, but are not limited to, hexahydropyrrolo[1 ,2-a]pyrazine-2(1H)-yl, 5,6,7,8-tetrahydro-[1,2,4]triazolo[4,3-a]pyrazinyl or 5,6,7,8-tetrahydroimidazo[1,5-a]pyrazinyl. Optionally, the heterocyclic group may be a benzo-fused ring group of the above-mentioned 4-7 membered heterocyclic group, and specific examples include but are not limited to dihydroisoquinolinyl and the like. "4-10 membered heterocyclic group" may include "5-10 membered heterocyclic group", "5-9 membered heterocyclic group", "5-8 membered heterocyclic group", "5-7 membered heterocyclic group", "5-6 membered heterocyclic group", "6-10 membered heterocyclic group", "6-8 membered heterocyclic group", "4-8 membered heterocyclic group", "4- "4-7 membered heterocyclyl", "4-6 membered heterocyclyl", "4-10 membered heterocycloalkyl", "5-10 membered heterocycloalkyl", "4-7 membered heterocycloalkyl", "5-6 membered heterocycloalkyl", "6-8 membered heterocycloalkyl" and the like, "4-7 membered heterocyclyl" may further include "4-6 membered heterocyclyl", "5-7 membered heterocyclyl", "5-6 membered heterocyclyl", "4-7 membered heterocycloalkyl", "4-6 membered heterocycloalkyl", "5-7 membered heterocycloalkyl", "5-6 membered heterocycloalkyl" and the like. Although some bicyclic heterocyclyls in the present disclosure partially contain a benzene ring or a heteroaromatic ring, the heterocyclyl as a whole is still non-aromatic.

The term "heterocyclyloxy" may be read as "heterocyclyl-O-".

The term "heterocycloalkyl" refers to a fully saturated monovalent cyclic group in the form of a monocyclic, fused, bridged or spirocyclic ring, wherein the ring atoms of the ring contain 1 to 5 heteroatoms or heteroatomic groups (i.e., heteroatomic groups containing heteroatoms), wherein the "heteroatoms or heteroatomic groups" include, but are not limited to, nitrogen atom (N), oxygen atom (O), sulfur atom (S), phosphorus atom (P), boron atom (B), -S(＝O) ₂ -, -S(＝O)- and optionally substituted -NH-, -S(＝O)(＝NH)-, -C(＝O)NH-, -C(＝NH)-, -S(＝O) ₂ NH-, S(＝O)NH- or -NHC(＝O)NH-, and the like, which usually contain 3 to 20 ring atoms. The term "3-10 membered heterocycloalkyl" refers to a heterocycloalkyl group having 3, 4, 5, 6, 7, 8, 9 or 10 ring atoms, and containing 1 to 5 heteroatoms or heteroatom groups independently selected from the above-mentioned heteroatoms. “3-10 membered heterocycloalkyl” includes “3-8 membered heterocycloalkyl”, wherein specific examples of 4 membered heterocycloalkyl include but are not limited to azetidinyl, oxetanyl or thietanyl; specific examples of 5 membered heterocycloalkyl include but are not limited to tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, isoxazolidinyl, oxazolidinyl, isothiazolidinyl, thiazolidinyl, imidazolidinyl or tetrahydropyrazolyl; specific examples of 6 membered heterocycloalkyl include but are not limited to piperidinyl, tetrahydropyranyl, tetrahydrothiopyranyl, morpholinyl, piperazinyl, 1,4-thioxanyl, 1,4-dioxane, thiomorpholinyl, 1,3-dithianyl or 1,4-dithianyl; specific examples of 7 membered heterocycloalkyl include but are not limited to azepanyl, oxetanyl or thiepanyl.

The term "heterocycloalkyloxy" may be read as "heterocycloalkyl-O-".

The term "aryl" refers to an all-carbon monocyclic or fused polycyclic aromatic ring group with a conjugated π electron system. The aryl group may have 6-20 carbon atoms, 6-14 carbon atoms or 6-12 carbon atoms. The term "C ₆ -C ₂₀ aryl" is understood to mean a monovalent aromatic monocyclic, bicyclic or tricyclic hydrocarbon ring having 6 to 20 carbon atoms. In particular, it is a ring having 6 carbon atoms ("C ₆ aryl"), such as phenyl; or a ring having 9 carbon atoms ("C ₉ aryl"), such as indanyl or indenyl; or a ring having 10 carbon atoms ("C ₁₀ aryl"), such as tetrahydronaphthyl, dihydronaphthyl or naphthyl; or a ring having 13 carbon atoms ("C ₁₃ aryl"), such as fluorenyl; or a ring having 14 carbon atoms ("C ₁₄ aryl"), such as anthracenyl. The term "C ₆ -C ₁₀ aryl" is understood to mean a monovalent aromatic all-carbon monocyclic or bicyclic group having 6 to 10 carbon atoms, in particular a ring having 6 carbon atoms ("C ₆ aryl"), such as phenyl; or a ring having 9 carbon atoms ("C ₉ aryl"), such as indenyl; or a ring having 10 carbon atoms ("C ₁₀ aryl"), such as naphthyl.

The term "aryloxy" may be understood as "aryl-O-".

The term "heteroaryl" refers to a monocyclic or fused polycyclic system with aromaticity, which contains at least one, preferably 1-4, ring atoms selected from N, O, S, and the remaining ring atoms are carbon 5-14 membered aromatic ring radicals. Heteroaryl is preferably 5-10 membered, more preferably 5- or 6-membered heteroaryl. The term "5-10 membered heteroaryl" is understood to include such monovalent monocyclic or bicyclic aromatic ring systems: it has 5, 6, 7, 8, 9 or 10 ring atoms, in particular 5 or 6 or 9 or 10 ring atoms, and it contains 1-5, preferably 1-3 heteroatoms independently selected from N, O and S. In particular, the heteroaryl group is selected from thienyl, furanyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, oxadiazolyl, triazolyl or thiadiazolyl, and the like, and benzo derivatives thereof, such as benzofuranyl, benzothienyl, benzothiazolyl, benzoxazolyl, benzisoxazolyl, benzimidazolyl, benzotriazolyl, indazolyl, indolyl or isoindolyl, and the like; or pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl or triazinyl, and the like, and benzo derivatives thereof, such as quinolyl, quinazolinyl or isoquinolyl, and the like; or azinyl, indolizinyl, purinyl, and the like, and benzo derivatives thereof; or cinnolinyl, phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl, carbazolyl, acridinyl, phenazinyl, phenothiazinyl or phenoxazinyl, and the like. The term "5-6 membered heteroaryl" refers to an aromatic ring system having 5 or 6 ring atoms, and containing 1-3, preferably 1-2 heteroatoms independently selected from N, O and S. The term "6 membered heteroaryl" refers to an aromatic ring system having 6 ring atoms, and containing 1-3, preferably 1-2 heteroatoms independently selected from N, O and S. The term "5-10 membered heteroaryl" may include "5-6 membered heteroaryl" or "6 membered heteroaryl", and the term "5-6 membered heteroaryl" may include "6 membered heteroaryl".

The term "halo" or "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "hydroxy" refers to an -OH group.

The term "cyano" refers to a -CN group.

The term "amino" refers to a _-NH2 group.

The term "nitro" refers to the _-NO2 group.

The term "therapeutically effective amount" means an amount of a compound of the present disclosure that (i) treats or prevents a particular disease, condition, or disorder, (ii) alleviates, ameliorates, or eliminates one or more symptoms of a particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of a particular disease, condition, or disorder described herein. The amount of a compound of the present disclosure that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the age of the mammal to be treated, but can be routinely determined by those skilled in the art based on their own knowledge and the present disclosure.

The term "pharmaceutically acceptable" refers to those compounds, materials, compositions and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response or other problems or complications, commensurate with a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" refers to a salt of a pharmaceutically acceptable acid or base, including a salt formed between a compound and an inorganic acid or an organic acid, and a salt formed between a compound and an inorganic base or an organic base.

The term "pharmaceutical composition" refers to a mixture of one or more compounds of the present disclosure or their salts and pharmaceutically acceptable excipients. The purpose of a pharmaceutical composition is to facilitate administration of the compounds of the present disclosure to an organism.

The term "pharmaceutically acceptable excipients" refers to those excipients that have no significant irritation to the organism and do not impair the biological activity and performance of the active compound. Suitable excipients are well known to those skilled in the art, such as carbohydrates, waxes, water-soluble and/or water-swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, etc.

The word "comprise" or "comprises" and its English variations such as comprises or comprising should be construed in an open and non-exclusive sense, ie, "including but not limited to".

The present disclosure also includes isotopically labeled compounds of the present disclosure that are identical to those described herein, but in which one or more atoms are replaced by atoms having an atomic mass or mass number different from that normally found in nature. Examples of isotopes that can be incorporated into compounds of the present disclosure include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, iodine, and chlorine, such as ² H, ³ H, ¹¹ C, 13 C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ³¹ P, ³² P, ³⁵ S, ¹⁸ F, ¹²³ I, ¹²⁵ I, and ³⁶ Cl ^, etc., respectively.

Certain isotopically labeled compounds of the disclosure (e.g., labeled with ³ H and ¹⁴ C) are useful in compound and/or substrate tissue distribution assays. Tritiated (i.e., ³ H) and carbon-14 (i.e., ¹⁴ C) isotopes are particularly preferred for their ease of preparation and detectability. Positron emitting isotopes, such as ¹⁵ O, ¹³ N, ¹¹ C, and ¹⁸ F, can be used in positron emission tomography (PET) studies to determine substrate occupancy. Isotopically labeled compounds of the disclosure can generally be prepared by the following procedures, similar to those disclosed in the schemes and/or examples below, by substituting an isotopically labeled reagent for an unlabeled reagent.

The pharmaceutical compositions of the present disclosure can be prepared by combining the compounds of the present disclosure with suitable pharmaceutically acceptable excipients, for example, they can be formulated into solid, semi-solid, liquid or gaseous preparations, such as tablets, pills, capsules, powders, granules, ointments, emulsions, suspensions, suppositories, injections, inhalants, gels, microspheres and aerosols.

Typical routes of administration of the compounds of the present disclosure or pharmaceutically acceptable salts thereof or pharmaceutical compositions thereof include, but are not limited to, oral, rectal, topical, inhalation, parenteral, sublingual, intravaginal, intranasal, intraocular, intraperitoneal, intramuscular, subcutaneous, intravenous administration.

The pharmaceutical composition of the present disclosure can be manufactured by methods well known in the art, such as conventional mixing methods, dissolution methods, granulation methods, emulsification methods, freeze-drying methods, and the like.

In some embodiments, the pharmaceutical composition is in oral form. For oral administration, the pharmaceutical composition can be formulated by mixing the active compound with pharmaceutically acceptable excipients well known in the art. These excipients enable the compounds of the present disclosure to be formulated into tablets, pills, lozenges, dragees, capsules, liquids, gels, slurries, suspensions, etc., for oral administration to patients.

Solid oral compositions can be prepared by conventional mixing, filling or tableting methods. For example, they can be obtained by mixing the active compound with a solid excipient, optionally grinding the resulting mixture, adding other suitable excipients if necessary, and then processing the mixture into particles to obtain a tablet or sugar-coated core. Suitable excipients include, but are not limited to, adhesives, diluents, disintegrants, lubricants, glidants or flavoring agents, etc.

The pharmaceutical composition may also be suitable for parenteral administration, such as sterile solutions, suspensions or lyophilized products in appropriate unit dosage forms.

The dosage of the compound or composition used in the treatment methods of the present disclosure will generally vary with the severity of the disease, the patient's weight and the relative efficacy of the compound, however, as a general guide, a suitable daily dosage of the compound of formula (I) described herein is 0.01 mg/kg to 1000 mg/kg.

The compounds of the present disclosure can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present disclosure.

The chemical reactions of the specific embodiments of the present disclosure are performed in a suitable solvent, which must be suitable for the chemical changes of the present disclosure and the reagents and materials required. In order to obtain the compounds of the present disclosure, it is sometimes necessary for those skilled in the art to modify or select the synthesis steps or reaction processes based on the existing embodiments.

All reagents used in this disclosure were commercially available and used without further purification.

Unless otherwise specified, the ratios expressed for mixed solvents are volume mixing ratios. Unless otherwise specified, % means wt%.

The structure of the compound is determined by nuclear magnetic resonance (NMR) and/or mass spectrometry (MS). The unit of NMR shift is 10 ^-6 (ppm). The solvent for NMR measurement is deuterated dimethyl sulfoxide, deuterated chloroform, deuterated methanol, etc., and the internal standard is tetramethylsilane (TMS); "IC ₅₀ " refers to the half inhibitory concentration, which refers to the concentration at which half of the maximum inhibitory effect is achieved.

Explanation of terms or abbreviations:

THP: tetrahydro-2H-pyran-2-yl; Pd(OAc) ₂ : palladium acetate; t-BuOH: tert-butyl alcohol; t-BuOK: potassium tert-butylate; t-BuONa: sodium tert-butylate; DPPP: 1,3-bis(diphenylphosphino)propane; TEA: triethylamine; THF: tetrahydrofuran; Pd/C: palladium on carbon; TsOH: p-toluenesulfonic acid; MeOH: methanol; EtOH: ethanol; PPh ₃ : triphenylphosphine; DIAD: diisopropyl azodicarboxylate; DCM: dichloromethane; DCE: dichloroethane; DMF: N,N-dimethylformamide; DIEA: N,N-diisopropylethylamine; HATU: O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate; Boc: tert-butyloxycarbonyl; Boc ₂ O: di-tert-butyl dicarbonate; Ruphos Pd G ₃ : methanesulfonic acid (2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl) palladium(II); NaBH(OAc) ₃ : sodium triacetylborohydride; HOAc: acetic acid; EtOAc: ethyl acetate; NH ₄ OAc: ammonium acetate; NaOAc: sodium acetate; SFC: supercritical fluid chromatography; TFA: trifluoroacetic acid; PCC: pyridinium chlorochromate; MeCN: acetonitrile; Pd(PPh ₃ ) ₂ Cl ₂ : bistriphenylphosphinepalladium dichloride; Pd(dppf)Cl ₂ : 1,1-bis(diphenylphosphino)ferrocenepalladium chloride; Pd(dppf)Cl ₂ ·DCM: 1,1-bis(diphenylphosphino)ferrocenepalladium chloride dichloromethane complex; Py: pyridine; Cu(OAc) ₂ : copper acetate; DMAP: 4-dimethylaminopyridine; t-AmOH: tert-amyl alcohol; TMSCHN ₂ : trimethylsilyldiazomethane; MW: microwave; Bn: benzyl; Ts: p-toluenesulfonyl; TsCl: p-toluenesulfonyl chloride; Ms: methanesulfonyl; Ms ₂ O: methanesulfonic anhydride; BnBr: benzyl bromide; PPTS: pyridine p-toluenesulfonate; TMS: trimethylsilyl; MOM: methoxymethyl; OTs: p-toluenesulfonyloxy; TsCl/TosCl: p-toluenesulfonyl chloride; OBn: benzyloxy; FA: formic acid; PMBCl: p-methoxybenzyl chloride; PMBNH ₂ : p-methoxybenzylamine; NIS: N-iodosuccinimide; SEMCl: 2-(trimethylsilyl)ethoxymethyl chloride; Pd ₂ (dba) ₃ : tris(dibenzylideneacetone)dipalladium; Xphos: 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl; T ₃ P: tri-n-propyl cyclophosphoric anhydride; Tf ₂ O: trifluoromethanesulfonic anhydride; TfOH: trifluoromethanesulfonic acid; NaHMDS: sodium bis(trimethylsilyl)amide; dioxane: 1,4-dioxane; water: water; M: mole/liter; L-proline: L-proline; DMSO: dimethyl sulfoxide; tert-butyl nitrite: tert-butyl nitrite; Pd(PPh ₃ ) ₄ : tetrakis(triphenylphosphine)palladium; MOMO: methoxymethoxy; toluene: toluene; ammonia: ammonia; MeMgCl: methylmagnesium chloride; STAB: sodium triacetoxyborohydride; CbzCl: benzyl chloroformate; MsCl: methanesulfonyl chloride; t-BuBrettphos Pd G ₃ : (2-di-tert-butylphosphino-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) methanesulfonate; Oxone: potassium peroxymonosulfonate; Acetone: acetone; BINAP: (R)-(+)-2,2-bis(diphenylphosphino)-1,1-binaphthyl; EDC: 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide; Xantphos: 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene; m-CPBA: m-chloroperoxybenzoic acid; IBX: 2-iodoacylbenzoic acid; PhNTf ₂ : N-phenylbis(trifluoromethanesulfonyl)imide; KHMDS: potassium bis(trimethylsilyl)amide; PPTS: pyridine p-toluenesulfonate; TES: triethylsilane; DMP: Dess-Martin periodinane; B ₂ Pin ₂ : bispinacol borate; Alloc: allyloxycarbonyl.

The eluent mentioned below can be a mixed eluent formed by two or more solvents, and the ratio is the volume ratio of each solvent. For example, "0-10% methanol/dichloromethane" means that during the gradient elution process, the volume ratio of methanol to dichloromethane in the mixed eluent is 0:100-10:100.

Example 1: Synthesis of 2-[5-(azetidin-3-yl)pyrrolo[3,2-c]pyridazin-3-yl]phenol (1a)

Step 1: Synthesis of tert-butyl N-(3,6-dichloropyridazin-4-yl)carbamate

4-Amino-3,6-dichloropyridazine (15.0 g, 91.47 mmol) was dissolved in dichloromethane (250 mL), and Boc anhydride (22.2 g, 101.5 mmol) and 4-dimethylaminopyridine (1.12 g, 9.15 mmol) were added, and the reaction solution was stirred at 25 ° C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure and dried, and column chromatography (silica, petroleum ether/ethyl acetate = 100/7) was used to obtain tert-butyl N-(3,6-dichloropyridazine-4-yl)carbamate (21 g).

MS m/z(ESI):207.8[M+H-56] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.62 (s, 1H), 8.22 (s, 1H), 1.51 (s, 9H).

Step 2: Synthesis of tert-butyl N-[6-chloro-3-(2-trimethylsilylethynyl)pyridazin-4-yl]carbamate

Dissolve tert-butyl N-(3,6-dichloropyridazine-4-yl)carbamate (11.0 g, 41.7 mmol) in toluene (250 mL), add trimethylsilyl acetylene (4.50 g, 45.8 mmol), triethylamine (10.5 g, 104 mmol), cuprous iodide (793 mg, 4.16 mmol) and bistriphenylphosphine palladium dichloride (2.92 g, 4.16 mmol) under nitrogen atmosphere. The reaction solution was stirred at 50 ° C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, the organic phase was washed twice with saturated brine (200 mL), and the aqueous phase was extracted three times with ethyl acetate (200 mL). The organic layer was collected, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (silica, petroleum ether/ethyl acetate = 100/8) to give the product N-[6-chloro-3-(2-trimethylsilylethynyl)pyridazin-4-yl]carbamic acid tert-butyl ester (7 g).

MS m/z (ESI): 325.9 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 7.89 (s, 1H), 7.31 (s, 1H), 1.68 (s, 9H), 0.38 (s, 9H).

Step 3: Synthesis of tert-butyl 3-chloropyrrolo[3,2-c]pyridazine-5-carboxylate

Tert-butyl N-[6-chloro-3-(2-trimethylsilylethynyl)pyridazine-4-yl]carbamate (7.00 g, 21.5 mmol) was dissolved in N,N-dimethylformamide (130 mL), and potassium carbonate (6.53 g, 47.3 mmol) was added. The reaction solution was stirred at 60 ° C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was diluted with ethyl acetate (300 mL), the organic phase was washed twice with water (300 mL), and the aqueous phase was extracted three times with ethyl acetate (300 mL). The collected organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and column chromatography (silicon dioxide, petroleum ether/ethyl acetate = 100/20) was used to obtain the product tert-butyl 3-chloropyrrolo[3,2-c]pyridazine-5-carboxylate (1.6 g).

MS m/z(ESI):153.8[M+H-100] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 8.18 (d, J = 3.9 Hz, 1H), 8.03 (s, 1H), 7.15 (dd, J = 0.6, 4.0 Hz, 1H), 1.64 (s, 9H).

Step 4: Synthesis of 3-[2-(methoxymethoxy)phenyl]-5H-pyrrolo[3,2-c]pyridazine

3-Chloropyrrolo[3,2-c]pyridazine-5-carboxylic acid tert-butyl ester (50.0 mg, 197 μmol) and 2-[2-(methoxymethoxy)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (67.7 mg, 256 μmol) were dissolved in dioxane (2 mL) and water (0.5 mL), and potassium carbonate (81.7 mg, 591 μmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (16.1 mg, 19.7 μmol) were added under nitrogen atmosphere. The reaction solution was stirred at 90° C. for 16 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, diluted with methanol (2 mL), and purified by thin layer chromatography (silica, petroleum ether/tetrahydrofuran = 1/2, 0.1% ammonia methanol as the developing solvent) to give the product 3-[2-(methoxymethoxy)phenyl]-5H-pyrrolo[3,2-c]pyridazine (36 mg).

MS m/z(ESI):256.0[M+H] ⁺ .

Step 5: Synthesis of tert-butyl 3-[3-[2-(methoxymethoxy)phenyl]pyrrolo[3,2-c]pyridazin-5-yl]azetidine-1-carboxylate

3-[2-(Methoxymethoxy)phenyl]-5H-pyrrolo[3,2-c]pyridazine (31.0 mg, 121 μmol) and 1-Boc-3-iodoazetidine (68.8 mg, 243 μmol) were dissolved in N,N-dimethylformamide (3 mL), and cesium carbonate (119 mg, 364 μmol) was added. The reaction solution was stirred at 60 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was diluted with water (10 mL), the organic phase was washed three times with water (10 mL), and the aqueous phase was extracted three times with ethyl acetate (10 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by thin layer chromatography (silica, petroleum ether/tetrahydrofuran = 1/2) to give the product 3-[3-[2-(methoxymethoxy)phenyl]pyrrolo[3,2-c]pyridazin-5-yl]azetidine-1-carboxylic acid tert-butyl ester (40 mg).

MS m/z(ESI):411.1[M+H] ⁺ .

Step 6: Synthesis of 2-[5-(azetidin-3-yl)pyrrolo[3,2-c]pyridazin-3-yl]phenol

3-[3-[2-(methoxymethoxy)phenyl]pyrrolo[3,2-c]pyridazin-5-yl]azetidine-1-carboxylic acid tert-butyl ester (40.0 mg, 97.5 μmol) was dissolved in dichloromethane (0.5 mL), and trifluoroacetic acid (0.5 mL) was added at 0°C. The reaction solution was stirred at 25°C for 1 hour. LCMS showed that the reaction was complete. Ammonia methanol (7 M) was added, the pH of the reaction solution was adjusted to 7, and then concentrated under reduced pressure and purified by preparative liquid chromatography (YMC Triart C18, 5 μm silica, 25 mm diameter, 150 mm length; a mixture of water (containing ammonia) and acetonitrile (acetonitrile ratio: 5%-45%) was used as the eluent) to obtain the product 2-[5-(azetidine-3-yl)pyrrolo[3,2-c]pyridazin-3-yl]phenol (1a) (1.02 mg).

MS m/z(ESI):267.1[M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d4) δ = 8.56 (s, 1H), 8.38 (d, J = 2.3Hz, 1H), 7.86 (dd, J = 1.7, 8.1Hz, 1H), 7.41-7.26 (m ,1H),7.09-6.94(m,2H),6.71(d,J＝1.3Hz,1H),6.01(m,J＝7.3Hz,1H),4.50-4.40(m,2H),4.23-4.15( m,2H).

Example 2: Synthesis of 2-[7-amino-5-(4-piperidinyl)pyrrolo[3,2-c]pyridazin-3-yl]phenol (2a)

Step 1: Synthesis of 3-chloro-5H-pyrrolo[3,2-c]pyridazine

3-Chloropyrrolo[3,2-c]pyridazine-5-carboxylic acid tert-butyl ester (1.5 g, 5.91 mmol) was dissolved in dioxane (15 mL), and dioxane hydrochloride (30 mL, 4 M) was added. The reaction solution was stirred at 25 ° C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure to dryness to obtain the product 3-chloro-5H-pyrrolo[3,2-c]pyridazine (1 g, crude product).

MS m/z (ESI): 153.8 [M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d4) δ = 8.51 (d, J = 0.6 Hz, 1H), 8.42 (d, J = 3.5 Hz, 1H), 7.12 (d, J = 2.9 Hz, 1H).

Step 2: Synthesis of tert-butyl 4-(3-chloro-5H-pyrrolo[3,2-c]pyridazin-5-yl)piperidine-1-carboxylate

3-Chloro-5H-pyrrolo[3,2-c]pyridazine (1.50 g, 7.10 mmol) and tert-butyl 4-(p-toluenesulfonyloxy)piperidine-1-carboxylate (5.05 g, 14.2 mmol) were dissolved in N,N-dimethylformamide (40 mL), and cesium carbonate (6.94 g, 21.3 mmol) was added. The reaction solution was stirred at 100 ° C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was diluted with ethyl acetate (80 mL) and water (80 mL), the organic phase was washed twice with saturated brine (80 mL), and the aqueous phase was extracted twice with ethyl acetate (80 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 100/35) to give the product 4-(3-chloropyrrolo[3,2-c]pyridazine-5-yl)piperidine-1-carboxylic acid tert-butyl ester (1.1 g).

MS m/z(ESI):337.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 8.19 (s, 1H), 8.15 (d, J = 3.5Hz, 1H), 6.95 (d, J = 3.5Hz, 1H), 4.74-4.60 (m, 1H), 4.13 (d, J = 12.4Hz, 2H), 3.00-2.89 (m, 2H), 1.99-1.81 (m, 4H), 1.43 (s, 9H).

Step 3: Synthesis of 3-chloro-7-nitro-5-(4-piperidinyl)pyrrolo[3,2-c]pyridazine

Tert-butyl 4-(3-chloro-5H-pyrrolo[3,2-c]pyridazine-5-yl)piperidine-1-carboxylate (1.10 g, 3.27 mmol) was dissolved in sulfuric acid (concentration 98%, 10 mL), and nitric acid (concentration 68%, 1.40 g, 22.2 mmol) was slowly added dropwise under an ice bath at 0°C, and the reaction solution was stirred at 25°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was slowly added dropwise to ice water (20 mL) to quench, and then a saturated sodium carbonate solution was added dropwise to adjust the pH to 7-8. Concentrate under reduced pressure and add methanol (10 mL) and dichloromethane (100 mL). Filter, and the filtrate was concentrated under reduced pressure to obtain the product 3-chloro-7-nitro-5-(4-piperidinyl)pyrrolo[3,2-c]pyridazine (460 mg).

MS m/z (ESI): 281.9 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.28 (s, 1H), 8.58 (s, 1H), 4.76-4.53 (m, 1H), 3.07 (d, J = 12.4Hz, 2H), 2.71- 2.58(m,2H),1.96-1.87(m,4H).

Step 4: Synthesis of tert-butyl 4-(3-chloro-7-nitro-pyrrolo[3,2-c]pyridazin-5-yl)piperidine-1-carboxylate

3-Chloro-7-nitro-5-(4-piperidinyl)pyrrolo[3,2-c]pyridazine (460 mg, 1.63 mmol) was dissolved in dichloromethane (10 mL), Boc anhydride (428 g, 1.96 mmol) and 4-dimethylaminopyridine (19.95 mg, 163 μmol) were added, and the reaction solution was stirred at 25 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was diluted with dichloromethane (10 mL) and water (10 mL), the organic phase was washed once with saturated brine (10 mL) and then washed three times with saturated sodium bicarbonate solution (10 mL), and the aqueous phase was extracted twice with dichloromethane (10 mL). The collected organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the product 4-(3-chloro-7-nitro-pyrrolo[3,2-c]pyridazine-5-yl)piperidine-1-carboxylic acid tert-butyl ester (650 mg).

MS m/z(ESI):382.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.49-9.28 (m, 1H), 8.68-8.49 (m, 1H), 4.84-4.75 (m, 1H), 4.14 (d, J = 12.0Hz, 2H ),2.96-2.79(m,2H),2.06-1.92(m,4H),1.44(s,9H).

Step 5: Synthesis of tert-butyl 4-[3-(2-hydroxyphenyl)-7-nitro-pyrrolo[3,2-c]pyridazin-5-yl]piperidine-1-carboxylate

4-(3-chloro-7-nitro-pyrrolo[3,2-c]pyridazine-5-yl)piperidine-1-carboxylic acid tert-butyl ester (200 mg, 524 μmol) and 2-hydroxyphenylboronic acid (93.9 mg, 681 μmol) were dissolved in dioxane (4 mL) and water (1 mL), and potassium carbonate (217 mg, 1.57 mmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (42.8 mg, 52.4 μmol) were added under nitrogen atmosphere. The reaction solution was stirred at 90 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silica/petroleum ether/tetrahydrofuran = 100/80) to give the product 4-[3-(2-hydroxyphenyl)-7-nitro-pyrrolo[3,2-c]pyridazin-5-yl]piperidine-1-carboxylic acid tert-butyl ester (170 mg).

MS m/z(ESI):440.1[M+H] ⁺ .

Step 6: Synthesis of tert-butyl 4-[7-amino-3-(2-hydroxyphenyl)pyrrolo[3,2-c]pyridazin-5-yl]piperidine-1-carboxylate

4-[3-(2-hydroxyphenyl)-7-nitro-pyrrolo[3,2-c]pyridazine-5-yl]piperidine-1-carboxylic acid tert-butyl ester (70 mg, 159 μmol) was dissolved in ethanol (1.6 mL) and water (0.2 mL), and iron powder (44.5 mg, 796 μmol) and ammonium chloride (51.1 mg, 956 μmol) were added. The reaction solution was stirred at 90 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain the product 4-[7-amino-3-(2-hydroxyphenyl)pyrrolo[3,2-c]pyridazine-5-yl]piperidine-1-carboxylic acid tert-butyl ester (50 mg, crude product).

MS m/z(ESI):410.3[M+H] ⁺ .

Step 7: Synthesis of 2-[7-amino-5-(4-piperidinyl)pyrrolo[3,2-c]pyridazin-3-yl]phenol

Tert-butyl 4-[7-amino-3-(2-hydroxyphenyl)pyrrolo[3,2-c]pyridazin-5-yl]piperidine-1-carboxylate (50.0 mg, 122 μmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (0.25 mL) was added at 0°C. The reaction solution was stirred at 25°C for 2 hours. LCMS showed that the reaction was complete. Ammonia methanol (7 M) was added, the pH of the reaction solution was adjusted to 7-8, and then concentrated under reduced pressure and purified by preparative liquid chromatography (Phenomenex C18, 5 μm silica, 30 mm diameter, 75 mm length; a mixture of water (containing ammonia) and acetonitrile (acetonitrile ratio: 21%-61%) was used as the eluent) to obtain the product 2-[7-amino-5-(4-piperidinyl)pyrrolo[3,2-c]pyridazin-3-yl]phenol (2a) (1.07 mg).

MS m/z (ESI): 310.1 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.75 (s, 1H), 8.51 (s, 1H), 8.15 (d, J = 7.3Hz, 1H), 7.34-7.26 (m, 1H), 7.19 ( s,1H),6.99-6.94(m,2H),4.75(s,2H),4.63-4.62(m,1H),3.14-3.11(m,1H),2.74-2.72(m,2H),2.44- 2.41(m,2H),1.84-1.82(m,4H).

Example 3: Synthesis of 2-(3-amino-1-(azetidin-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (3a)

Step 1: Synthesis of 4,6-dihydroxypyridazine-3-carboxamide

Dissolve 4,6-dihydroxypyridazine-3-carboxylic acid ethyl ester (10 g, 54.30 mmol) in 7M ammonia methanol (100 mL). The reaction solution was reacted at 20°C for 16 hours, and the reaction was completed by thin layer chromatography (petroleum ether/tetrahydrofuran = 1/2). The reaction solution was filtered, and the filter cake was added to petroleum ether/ethyl acetate (1/1, 100 mL), stirred at 25°C for 30 minutes, filtered, and the filter cake was dried to obtain 4,6-dihydroxypyridazine-3-carboxamide (9 g).

¹ H NMR (DMSO-d ₆ , 400MHz): δ = 10.50 (br s, 1H), 8.52 (br s, 1H), 7.39 (br s, 2H), 5.21ppm (s, 1H).

Step 2: Synthesis of 4,6-dichloropyridazine-3-carbonitrile

4,6-dihydroxypyridazine-3-carboxamide (5g, 32.23mmol) was dissolved in phosphorus oxychloride (50mL). The reaction solution was reacted at 90°C for 7 hours, and the reaction was completed by LCMS. The reaction solution was cooled to room temperature and slowly added dropwise to cold water, stirred at room temperature for 30 minutes, extracted three times with dichloromethane (100mL), the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated, and purified by column chromatography to obtain 4,6-dichloropyridazine-3-carbonitrile (4.43g).

MS m/z(ESI):212.0[M+K] ⁺ .

Step 3: Synthesis of 6-chloro-1H-pyrazolo[4,3-c]pyridazin-3-amine

4,6-Dichloropyridazine-3-carbonitrile (4.7 g, 27.01 mmol) was dissolved in ethanol (50 mL), and 85% hydrazine hydrate (7.95 g, 135.07 mmol) was added. The reaction solution was reacted at 100 ° C for 3 hours, and the reaction was completed by LCMS. The reaction solution was cooled to room temperature, filtered, and the filter cake was added to methanol (50 mL), stirred at 25 ° C for 30 minutes, filtered, and the filter cake was dried to obtain 6-chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (5 g).

MS m/z(ESI):169.8[M+H] ⁺ .

Step 4: Synthesis of tert-butyl 3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-carboxylate

Dissolve 6-chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (4.3 g, 25.36 mmol) in dichloromethane (80 mL), add Boc anhydride (126.79 mmol, 29.13 mL) and triethylamine (126.79 mmol, 17.65 mL). The reaction solution was stirred at 20 ° C for 16 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silica, dichloromethane/methanol = 10/1) to obtain 3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-carboxylic acid tert-butyl ester (2.10 g).

MS m/z(ESI):270.2[M+H] ⁺ .

Step 5: Synthesis of tert-butyl 3-amino-6-(2-(benzyloxy)phenyl)-1H-pyrazolo[4,3-c]pyridazine-1-carboxylate

3-Amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-carboxylic acid tert-butyl ester (1.4 g, 5.19 mmol), 2-benzyloxyphenylboronic acid (1.66 g, 7.27 mmol) and potassium carbonate (1.43 g, 10.38 mmol) were dissolved in dioxane (5.00 mL) and water (1.00 mL), and tetrakistriphenylphosphine palladium (599.87 mg, 519.12 μmol) was added under nitrogen. The reaction solution was stirred at 100°C for 3 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silicon dioxide, petroleum ether/tetrahydrofuran = 2/1) to obtain 3-amino-6-(2-(benzyloxy)phenyl)-1H-pyrazolo[4,3-c]pyridazine-1-carboxylic acid tert-butyl ester (0.50 g).

MS m/z(ESI):318.2[M+H-100] ⁺ .

Step 6: Synthesis of 6-(2-(benzyloxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-3-amine

The reactant tert-butyl 3-amino-6-(2-(benzyloxy)phenyl)-1H-pyrazolo[4,3-c]pyridazine-1-carboxylate (500mg, 1.20mmol) was dissolved in anhydrous dichloromethane (10mL), and trifluoroacetic acid (27.55mmol, 2mL) was added. The reaction solution was stirred at 20°C for 3 hours. LCMS detected that the reaction was complete. Dichloromethane (15mL) was added to the reaction solution, and the pH was adjusted to 7-8 with sodium bicarbonate. After adding ethyl acetate (20mL) and anhydrous sodium sulfate, it was stirred at room temperature for 30 minutes, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 6-(2-(benzyloxy)phenyl)-1H-pyrazolo[4,3-c]pyridazine-3-amine (300mg).

MS m/z(ESI):318.1[M+H] ⁺ .

Step 7: Synthesis of tert-butyl 3-(3-amino-6-(2-(benzyloxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)azetidine-1-carboxylate

6-(2-(Benzyloxy)phenyl)-1H-pyrazolo[4,3-c]pyridazine-3-amine (300.00 mg, 567.21 μmol) and 1-Boc-3-iodo-azetidine (208.75 mg, 737.37 μmol) were dissolved in N,N-dimethylformamide (0.2 mL), and potassium carbonate (235.17 mg, 1.70 mmol) was added. The reaction solution was stirred at 100 ° C for 16 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by thin layer chromatography (silicon dioxide, petroleum ether/tetrahydrofuran = 2/1) to obtain tert-butyl 3-(3-amino-6-(2-(benzyloxy)phenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl)azetidine-1-carboxylate (180 mg).

MS m/z(ESI):473.2[M+H] ⁺ .

Step 8: Synthesis of tert-butyl 3-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]azetidine-1-carboxylate

3-(3-amino-6-(2-(benzyloxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)azetidine-1-carboxylic acid tert-butyl ester (170 mg, 251.83 μmol) was dissolved in tetrahydrofuran (10 mL), wet palladium carbon (134.00 mg, 10% purity), wet palladium hydroxide (176.83 mg, 10% purity) and formic acid (24.20 mg, 503.66 μmol, 2.00 eq) were added, and the reaction solution was stirred for 16 hours under a hydrogen atmosphere at 20°C. LCMS detected that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain a crude product of 3-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]azetidine-1-carboxylic acid tert-butyl ester (70 mg).

MS m/z(ESI):383.2[M+H] ⁺ .

Step 9: Synthesis of 2-(3-amino-1-(azetidin-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

3-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazine-1-yl]azetidine-1-carboxylic acid tert-butyl ester (60 mg, 156.90 μmol) was dissolved in dichloromethane (1.0 mL), and trifluoroacetic acid (232 μL, 3.14 mmol) was added. Under nitrogen protection, the reaction solution was stirred at 20 ° C for 3 hours. LCMS detected that the reaction was complete. The reaction solution was dried with nitrogen, and dichloromethane (5 mL) and water (2 mL) were added to the reaction solution. Sodium carbonate was added to adjust the pH to 7, and extracted twice with dichloromethane (15 mL), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness under reduced pressure. Purification by preparative liquid chromatography (Boston Prime C18, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing aqueous ammonia) and acetonitrile (acetonitrile ratio: 23%-43%) as eluent) gave 2-(3-amino-1-(azetidin-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (3a) (2.29 mg).

MS m/z(ESI):283.1[M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d4) δ = 8.25 (s, 1H), 8.00 (dd, J = 1.5, 8.3Hz, 1H), 7.42-7.32 (m, 1H), 7.12-6.96 (m, 2H) ,5.66-5.58(m,1H),4.42-4.27(m,2H),4.08-3.98(m,2H).

Example 4: Synthesis of 2-(3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (4a)

Step 1: Synthesis of tert-butyl 4-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate

6-Chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (4g, 23.59mmol) was dissolved in N,N-dimethylformamide (100mL), and tert-butyl 4-iodopiperidine-1-carboxylate (14.68g, 47.18mmol) and cesium carbonate (23.06g, 70.77mmol) were added. The reaction solution was reacted at 60°C for 3 hours, and the reaction was completed by LCMS detection. The reaction solution was cooled to room temperature, filtered, and the filtrate was diluted with distilled water (100mL), extracted three times with ethyl acetate (100mL), and the organic phases were combined, dried, concentrated, and purified by column chromatography (silicon dioxide, petroleum ether/ethyl acetate = 1/1) to obtain tert-butyl 4-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylate (1.5g).

MS m/z(ESI):353.1[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate

4-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylic acid tert-butyl ester (1.4 g, 3.97 mmol) was dissolved in dioxane (15 mL) and distilled water (5 mL), and 2-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol (1.31 g, 5.95 mmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (324.05 mg, 396.80 μmol) and potassium carbonate (1.10 g, 7.94 mmol) were added. The reaction solution was reacted at 90° C. under nitrogen for 16 hours, and the reaction was completed by LCMS detection. The reaction solution was cooled to room temperature and filtered. The filtrate was diluted with distilled water (15 mL), extracted three times with ethyl acetate (50 mL), and the organic phases were combined, dried, concentrated, and purified by column chromatography to give tert-butyl 4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate (900 mg).

MS m/z(ESI):411.2[M+H] ⁺ .

Step 3: Synthesis of 2-(3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

Dissolve tert-butyl 4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylate (900 mg, 2.19 mmol) in dichloromethane (10 mL), and add trifluoroacetic acid (5.00 g, 43.85 mmol). The reaction solution was reacted at 20°C for 3 hours, and the reaction was completed by LCMS. The reaction solution was concentrated, diluted with distilled water (10 mL), and solid sodium carbonate was added to adjust the pH to 7-8. The mixture was extracted three times with ethyl acetate/tetrahydrofuran (1/1, 10 mL), the organic phases were combined, dried, and concentrated to give a crude product (700 mg). The crude product (30 mg) was purified by preparative liquid chromatography (chromatographic column: Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; mobile phase: [water (containing 0.225% FA)-ACN]; acetonitrile ratio: 0%-32%, 9 min) to give 2-(3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (4a) (2.09 mg).

MS m/z (ESI): 311.1 [M+H] ⁺ ;

¹ H NMR (DMSO-d ₆ , 400MHz): δ = 13.88 (br s, 1H), 8.48 (s, 1H), 8.10 (dd, J = 8.3, 1.4Hz, 1H), 7.33-7.42 (m, 1H) ),6.96-7.06(m,2H),6.47(s,2H),4.71-4.65(m,1H),3.21(d,J＝11.6Hz,1H),2.82-2.78(m,4H),1.98- 2.13(m,2H),1.91-1.89(m,2H).

Example 5: Synthesis of 2-(7-amino-1H-pyrrolo[2,3-d]pyridazin-4-yl)phenol (5a)

Step 1: Synthesis of 4,6-dichloropyridazin-3(2H)-one and 5,6-dichloropyridazin-3(2H)-one

3,4,6-trichloropyridazine (100 g, 545.19 mmol) was dissolved in acetic acid (400 mL). The reaction solution was reacted at 110 ° C for 16 hours. TLC detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. Petroleum ether (300 mL) was used for slurrying at 25 ° C for 16 hours to obtain a mixture of 4,6-dichloropyridazine-3 (2H) -one and 5,6-dichloropyridazine-3 (2H) -one (81.2 g).

¹ H NMR (400MHz, DMSO-d ₆ ) δ 13.61 (s, 1H), 13.43 (s, 1H), 8.04 (s, 1H), 7.43 (s, 1H).

Step 2: Synthesis of 4,6-dichloro-2-(4-methoxybenzyl)pyridazin-3(2H)-one and 5,6-dichloro-2-(4-methoxybenzyl)pyridazin-3(2H)-one

A mixture of 4,6-dichloropyridazine-3(2H)-one and 5,6-dichloropyridazine-3(2H)-one (40 g, 20.14 μmol) was dissolved in N,N-dimethylformamide (500 mL), and sodium hydrogen (19.39 g, 484.91 mmol) was slowly added at 0°C. The reaction solution was stirred at 0°C for 1 hour. p-Methoxybenzyl chloride (56.96 g, 363.69 mmol) was slowly added at 0°C, and the reaction solution was stirred at 25°C for 2 hours. TLC detected that the reaction was complete. Water (300 mL) was added to the reaction solution and extracted three times with dichloromethane (600 mL), the organic phase was washed three times with water (600 mL), dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Purification by column chromatography (silica, petroleum ether/ethyl acetate = 1/0 to 2/1) gave a mixture of 4,6-dichloro-2-(4-methoxybenzyl)pyridazin-3(2H)-one and 5,6-dichloro-2-(4-methoxybenzyl)pyridazin-3(2H)-one (29 g).

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.10-7.49(m,1H),7.32-7.21(m,2H),6.94-6.87(m,2H),5.19-5.08(m,2H),3.73 (s,3H).

Step 3: Synthesis of 4-amino-6-chloro-2-(4-methoxybenzyl)pyridazin-3(2H)-one and 5-amino-6-chloro-2-(4-methoxybenzyl)pyridazin-3(2H)-one

A mixture of 4,6-dichloro-2-(4-methoxybenzyl)pyridazine-3(2H)-one and 5,6-dichloro-2-(4-methoxybenzyl)pyridazine-3(2H)-one (29 g, 20.14 μmol) was dissolved in aqueous ammonia (200 mL). The reaction solution was reacted at 140° C. for 2 hours. The reaction was completed by LCMS. Water (500 mL) was added to the reaction solution and extracted three times with ethyl acetate (900 mL). The organic phase was washed three times with water (900 mL) and dried over anhydrous sodium sulfate, and then concentrated to dryness under reduced pressure. Purification by column chromatography (silica, petroleum ether/ethyl acetate = 1/0 to 0/1) gave a mixture of 4-amino-6-chloro-2-(4-methoxybenzyl)pyridazine-3(2H)-one and 5-amino-6-chloro-2-(4-methoxybenzyl)pyridazine-3(2H)-one (9.24 g).

MS m/z(ESI):266.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ7.24 (d, J = 8.5 Hz, 2H), 7.14-6.81 (m, 4H), 6.21 (s, 1H), 5.08 (s, 2H), 3.73 ( s,3H).

Step 4: Synthesis of 4-amino-6-chloro-5-iodo-2-(4-methoxybenzyl)pyridazin-3(2H)-one

A mixture of 4-amino-6-chloro-2-(4-methoxybenzyl)pyridazine-3(2H)-one and 5-amino-6-chloro-2-(4-methoxybenzyl)pyridazine-3(2H)-one (4.24 g, 15.96 mmol) was dissolved in acetonitrile (80 mL), and N-iodosuccinimide (5.39 g, 23.94 mmol) was added. The reaction solution was reacted at 80 ° C for 16 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, water (70 mL) was added and extracted three times with ethyl acetate (150 mL), the organic phase was washed three times with water (150 mL), and dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Purification by column chromatography (silicon dioxide, petroleum ether/ethyl acetate = 1/0 to 2/1) gave 4-amino-6-chloro-5-iodo-2-(4-methoxybenzyl)pyridazine-3(2H)-one (6.7 g).

MS m/z (ESI): 391.9 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ7.23 (d, J = 8.8 Hz, 2H), 7.14-6.93 (m, 2H), 6.89 (d, J = 8.6 Hz, 2H), 5.08 (s, 2H),3.72(s,3H).

Step 5: Synthesis of (E)-4-amino-6-chloro-5-(2-ethoxyvinyl)-2-(4-methoxybenzyl)pyridazin-3(2H)-one

4-Amino-6-chloro-5-iodo-2-(4-methoxybenzyl)pyridazine-3(2H)-one (6.7 g, 17.11 mmol) was dissolved in 1,4-dioxane (60 mL) and water (15 mL), and (E)-1-ethoxyvinyl-2-boronic acid pinacol ester (6.78 g, 34.22 mmol), potassium phosphate (10.90 g, 51.33 mmol) and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (1.25 g, 1.71 mmol) were added. After nitrogen replacement three times, the reaction solution was reacted at 90°C for 16 hours. LCMS detected that the reaction was complete. Water (50 mL) was added to the reaction solution and extracted three times with ethyl acetate (90 mL). The organic phase was washed three times with water (90 mL) and dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Purification by column chromatography (silica, petroleum ether/ethyl acetate = 1/0 to 1/1) gave (E)-4-amino-6-chloro-5-(2-ethoxyvinyl)-2-(4-methoxybenzyl)pyridazin-3(2H)-one (3.38 g).

MS m/z(ESI):335.9[M+H] ⁺ .

Step 6: Synthesis of 4-chloro-6-(4-methoxybenzyl)-1H-pyrrolo[2,3-d]pyridazin-7(6H)-one

(E)-4-amino-6-chloro-5-(2-ethoxyvinyl)-2-(4-methoxybenzyl)pyridazine-3(2H)-one (3.38 g, 10.07 mmol) was dissolved in ethanol (60 mL), and concentrated hydrochloric acid (12.58 mL, 12 M) was added. The reaction solution was reacted at 80°C for 4 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain 4-chloro-6-(4-methoxybenzyl)-1H-pyrrolo[2,3-d]pyridazine-7(6H)-one (3.18 g).

MS m/z (ESI): 289.9 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.10 (s, 1H), 7.57 (t, J = 2.8Hz, 1H), 7.25 (d, J = 8.6Hz, 2H), 6.89 (d, J = 8.8Hz, 2H), 6.52 (dd, J = 2.0, 2.6Hz, 1H), 5.23 (s, 2H), 3.71 (s, 3H); MS m/z (ESI): 289.9 [M+H] ⁺ . ¹ H NMR (400MHz, DMSO-d ₆ ) δ13.10 (s, 1H), 7.57 (t, J = 2.8Hz, 1H), 7.25 (d, J = 8.6Hz, 2H), 6.89 (d, J = 8.8Hz, 2H), 6.52 (dd, J = 2.0, 2.6Hz, 1H), 5.23 (s, 2H), 3.71 (s, 3H).

Step 7: Synthesis of 4-chloro-1H-pyrrolo[2,3-d]pyridazin-7(6H)-one

4-Chloro-6-(4-methoxybenzyl)-1H-pyrrolo[2,3-d]pyridazine-7(6H)-one (3.18 g, 10.98 mmol) was dissolved in trifluoroacetic acid (30 mL) and trifluoromethanesulfonic acid (8.11 g, 54.03 mmol) was added. The reaction solution was reacted at 25°C for 1 hour. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and slurried with methanol (20 mL) and water (40 mL) at 25°C for 1 hour. After filtration, 4-chloro-1H-pyrrolo[2,3-d]pyridazine-7(6H)-one (2.2 g) was obtained and used directly in the next step.

MS m/z(ESI):170.1[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.03 (s, 1H), 12.56 (s, 1H), 7.55 (t, J = 2.8Hz, 1H), 6.51 (dd, J = 2.0, 2.8Hz, 1H).

Step 8: Synthesis of 4,7-dichloro-1H-pyrrolo[2,3-d]pyridazine

4-Chloro-1H-pyrrolo[2,3-d]pyridazine-7(6H)-one (2.2 g, 12.97 mmol) was dissolved in phosphorus oxychloride (20 mL). The reaction solution was reacted at 100°C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, dissolved in tetrahydrofuran (10 mL) and methanol (20 mL) and the pH was adjusted to 8-9 with sodium hydroxide aqueous solution, and the solution was concentrated to dryness under reduced pressure. Purification by column chromatography (silica, dichloromethane/tetrahydrofuran = 1/0 to 10/1) gave 4,7-dichloro-1H-pyrrolo[2,3-d]pyridazine (1.8 g).

MS m/z(ESI):188.1[M+H] ⁺ .

Step 9: Synthesis of 4,7-dichloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-d]pyridazine

4,7-Dichloro-1H-pyrrolo[2,3-d]pyridazine (400 mg, 2.13 mmol) was dissolved in N,N-dimethylformamide (8 mL), sodium hydrogen (170.18 mg, 4.26 mmol) was added, the reaction solution was stirred at 25 ° C for 1 h, and 2-(trimethylsilyl)ethoxymethyl chloride (532.05 mg, 3.19 mmol) was added to the reaction solution. The reaction solution was reacted at 25 ° C for 1 hour. LCMS detected that there was residual raw material and product was generated. Water (10 mL) was added to the reaction solution to quench the reaction and extracted three times with dichloromethane (30 mL). The organic phase was washed three times with water (30 mL) and dried with anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure. Purification by column chromatography (silica, petroleum ether/ethyl acetate = 1/0 to 1/1) gave 4,7-dichloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-d]pyridazine (280 mg).

MS m/z (ESI): 318.1 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.17 (d, J＝3.3Hz, 1H), 6.89 (d, J＝3.1Hz, 1H), 5.87 (s, 2H), 3.53 (t, J＝ 7.9Hz, 3H), 0.82 (t, J = 7.9Hz, 2H), -0.11 (s, 9H).

Step 10: Synthesis of 4-chloro-N-(4-methoxybenzyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-d]pyridazin-7-amine

4,7-Dichloro-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-d]pyridazine (280 mg, 879.75 μmol) and p-methoxybenzylamine (120.68 mg, 879.75 μmol) were dissolved in toluene (10 mL), and sodium tert-butoxide (253.63 mg, 2.64 mmol) and methanesulfonic acid (2-dicyclohexylphosphine)-3,6-dimethoxy-2,4,6-triisopropyl-1,1-biphenyl)(2-amino-1,1-biphenyl-2-yl)palladium (II) (79.75 mg, 87.97 μmol) were added. After nitrogen replacement three times, the reaction solution was reacted at 100°C for 16 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. Purification by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 2/1) gave 4-chloro-N-(4-methoxybenzyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-d]pyridazin-7-amine (40 mg).

MS m/z(ESI):419.1[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ7.74 (d, J = 3.1 Hz, 1H), 7.35 (d, J = 8.6 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H), 6.68 (t,J＝5.2Hz,1H),6.54(d,J＝3.1Hz,1H),5.71(s,2H),4.67-4.58(m,2H),3.72(s,3H),3.52-3.44( m,2H),0.77-0.68(m,2H),-0.11(s,9H).

Step 11: Synthesis of 2-(7-((4-methoxybenzyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-d]pyridazin-4-yl)phenol

4-Chloro-N-(4-methoxybenzyl)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-d]pyridazin-7-amine (40 mg, 95.47 μmol) and 2-hydroxyphenylboronic acid (19.75 mg, 143.20 μmol) were dissolved in 1,4-dioxane (1.6 mL) and water (0.4 mL), and potassium phosphate (60.79 mg, 286.40 μmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (7.80 mg, 9.55 μmol) were added. The reaction solution was reacted at 100°C for 16 hours. LCMS detected that the reaction was complete. Water (10 mL) was added to the reaction solution and extracted three times with dichloromethane (15 mL). The organic phase was washed three times with water (15 mL), dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure. Purification by thin layer chromatography (silica, petroleum ether/ethyl acetate = 2/1) gave 2-(7-((4-methoxybenzyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-d]pyridazin-4-yl)phenol (25.5 mg).

MS m/z(ESI):477.1[M+H] ⁺ .

Step 12: Synthesis of 2-(7-amino-1H-pyrrolo[2,3-d]pyridazin-4-yl)phenol

2-(7-((4-methoxybenzyl)amino)-1-((2-(trimethylsilyl)ethoxy)methyl)-1H-pyrrolo[2,3-d]pyridazin-4-yl)phenol (25.5 mg, 53.50 μmol) was dissolved in trifluoroacetic acid (1 mL) and trifluoromethanesulfonic acid (0.1 mL). The reaction solution was reacted at 25°C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness and then ammonia water was added to adjust the pH to greater than 7. 2-(7-amino-1H-pyrrolo[2,3-d]pyridazin-4-yl)phenol (5a) (6.66 mg) was obtained by HPLC (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile ratio: 0%-34%) as eluent).

MS m/z (ESI): 226.9 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.67 (br.s, 1H), 12.23 (s, 1H), 7.86 (d, J = 3.1Hz, 1H), 7.81 (d, J = 7.0Hz, 1H),7.48-7.12(m,3H),7.07-6.94(m,2H),6.91(d,J=2.9Hz,1H).

Example 6: Synthesis of 2-(2-aminopyrrolo[1,2-b]pyridazin-6-yl)phenol (6a)

Step 1: Synthesis of 2-chloro-6-(2-methoxyphenyl)pyrrolo[1,2-b]pyridazine

3-Chloro-6-methylpyridazine (1 g, 7.78 mmol) and 2-bromo-1-(2-methoxyphenyl)ethanone (1.96 g, 8.56 mmol) were dissolved in ethanol (20 mL), sodium bicarbonate (1.96 g, 23.34 mmol) was added, and the reaction solution was stirred at 90 ° C for 12 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, and the concentrate was purified by column chromatography (silica, petroleum ether: ethyl acetate = 10: 1) to obtain the product 2-chloro-6-(2-methoxyphenyl)pyrrolo[1,2-b]pyridazine (140 mg).

MS m/z(ESI):259.1[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.35 (d, J = 1.5 Hz, 1H), 8.04 (d, J = 9.3 Hz, 1H), 7.74 (dd, J = 1.7, 7.6 Hz, 1H) ,7.30-7.29(m,1H),7.16-7.08(m,2H),7.06-6.98(m,1H),6.77(d,J＝9.3Hz,1H),3.91(s,3H).

Step 2: Synthesis of tert-butyl N-[6-(2-methoxyphenyl)pyrrolo[1,2-b]pyridazin-2-yl]carbamate

2-Chloro-6-(2-methoxyphenyl)pyrrolo[1,2-b]pyridazine (120 mg, 463.85 μmol) and tert-butyl carbamate (65.21 mg, 556.62 μmol) were dissolved in dioxane (6 mL), and cesium carbonate (302.27 mg, 927.71 μmol), 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl (44.23 mg, 92.77 μmol) and tris(dibenzylideneacetone)dipalladium (42.48 mg, 46.39 μmol) were added under nitrogen atmosphere, and the reaction solution was stirred at 80° C. for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, and the concentrate was purified by column chromatography (silica, petroleum ether:ethyl acetate=10:1) to obtain the product N-[6-(2-methoxyphenyl)pyrrolo[1,2-b]pyridazin-2-yl]carbamic acid tert-butyl ester (90 mg).

MS m/z (ESI): 340.1 [M+H] ⁺ ;

¹ H NMR (400MHz, CDCl ₃ ) δ8.04(d,J＝1.3Hz,1H),7.75(d,J＝15.9Hz,1H),7.68(d,J＝9.7Hz,1H),7.36(d ,J＝9.7Hz,1H),7.09(d,J＝16.0Hz,1H),7.05-6.97(m,2H),6.82(d,J＝1.6Hz,1H),3.96-3.90(m,3H) ,1.53(s,9H).

Step 3: Synthesis of 2-(2-aminopyrrolo[1,2-b]pyridazin-6-yl)phenol

Tert-butyl N-[6-(2-methoxyphenyl)pyrrolo[1,2-b]pyridazin-2-yl]carbamate (30 mg, 88.39 μmol) was dissolved in dichloromethane (1 mL), and boron tribromide (88.58 mg, 353.58 μmol) was added dropwise at 0° C. The reaction solution was stirred at 25° C. for 2 hours. LCMS showed that the reaction was complete. The reaction solution was poured into water (2 mL), the pH value was adjusted to 8 with aqueous sodium bicarbonate solution, extracted with dichloromethane (2 mL) three times, the organic phase was concentrated to dryness under reduced pressure, and the concentrate was purified by preparative liquid chromatography (Boston Prime C18, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.225% formic acid) and acetonitrile (acetonitrile ratio: 25%-45%) as eluent) to obtain the product 2-(2-aminopyrrolo[1,2-b]pyridazin-6-yl)phenol (6a) (1.41 mg).

MS m/z(ESI):226.1[M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d4) δ7.93 (d, J＝1.5Hz, 1H), 7.59-7.51 (m, 2H), 7.04-6.98 (m, 1H), 6.85 (d, J＝7.8Hz ,2H), 6.72(d,J＝1.8Hz,1H), 6.17(d,J＝9.3Hz,1H).

Example 7: Synthesis of 5-(2-hydroxyphenyl)-3,6,7,9-tetraazatricyclo[6.4.1.04,13]trideca-1,4(13),5,7,11-penten-10-one (7a)

Step 1: Synthesis of 4-hydroxy-1,6-dihydropyrrolo[2,3-d]pyridazin-7-one

1H-pyrrole-2,3-dicarboxylic acid dimethyl ester (10 g, 54.60 mmol) was dissolved in methanol (100 mL), hydrazine hydrate (14.43 g, 245.02 mmol, 85% purity) was added, and the reaction solution was stirred at 70°C for 2 hours. TLC showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain 4-hydroxy-1,6-dihydropyrrolo[2,3-d]pyridazin-7-one (8 g).

Step 2: Synthesis of 4,7-dichloro-1H-pyrrolo[2,3-d]pyridazine

4-Hydroxy-1,6-dihydropyrrolo[2,3-d]pyridazine-7-one (8 g, 52.94 mmol) was dissolved in phosphorus oxychloride (80 mL), and the reaction solution was stirred at 100°C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the concentrate was purified by column chromatography (silicon dioxide, petroleum ether: tetrahydrofuran = 3:2) to obtain the product 4,7-dichloro-1H-pyrrolo[2,3-d]pyridazine (1.5 g).

MS m/z (ESI): 187.8 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ 13.23 (br s, 1H), 7.97 (d, J = 3.1 Hz, 1H), 6.83 (d, J = 2.9 Hz, 1H).

Step 3: Synthesis of 4-chloro-7-(2-methoxyphenyl)-1H-pyrrolo[2,3-d]pyridazine

4,7-Dichloro-1H-pyrrolo[2,3-d]pyridazine (1 g, 5.32 mmol) and 2-methoxyphenylboronic acid (727.56 mg, 4.79 mmol) were dissolved in dioxane (20 mL) and water (4 mL), potassium phosphate (2.26 g, 10.64 mmol) and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride dichloromethane mixture (217.18 mg, 266.00 μmol) were added under nitrogen atmosphere, and the reaction solution was stirred at 80°C for 12 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, and the concentrate was purified by column chromatography (silicon dioxide, petroleum ether: tetrahydrofuran = 2:1) to obtain the product 4-chloro-7-(2-methoxyphenyl)-1H-pyrrolo[2,3-d]pyridazine (700 mg).

MS m/z(ESI):259.9[M+H] ⁺ .

Step 4: Synthesis of 4-chloro-3-iodo-7-(2-methoxyphenyl)-1H-pyrrolo[2,3-d]pyridazine

4-Chloro-7-(2-methoxyphenyl)-1H-pyrrolo[2,3-d]pyridazine (700 mg, 2.70 mmol) was dissolved in N,N-dimethylformamide (10 mL), and N-iodosuccinimide (909.67 mg, 4.04 mmol) was added. The reaction solution was stirred at 25°C for 2 hours. Thin layer chromatography showed that the reaction was complete. The reaction solution was extracted three times with water (8 mL) and dichloromethane (6 mL), and the organic phase was washed with saturated brine (10 mL) and concentrated. The concentrate was purified by column chromatography (silica, petroleum ether: ethyl acetate = 3:2) to obtain the product 4-chloro-3-iodo-7-(2-methoxyphenyl)-1H-pyrrolo[2,3-d]pyridazine (700 mg).

¹ H NMR (400MHz, DMSO-d ₆ ) δ12.57 (br s, 1H), 7.98-7.95 (m, 1H), 7.62-7.54 (m, 1H), 7.50 (dd, J＝1.7, 7.5Hz, 1H), 7.23 (d, J = 8.3Hz, 1H), 7.18-7.12 (m, 1H), 3.74 (s, 3H).

Step 5: Synthesis of ethyl (E)-3-[4-chloro-7-(2-methoxyphenyl)-1H-pyrrolo[2,3-d]pyridazin-3-yl]prop-2-enoate

4-Chloro-3-iodo-7-(2-methoxyphenyl)-1H-pyrrolo[2,3-d]pyridazine (700 mg, 1.82 mmol) and 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)acryloylethyl ester (451.46 mg, 2.00 mmol) were dissolved in dioxane (8 mL) and water (2 mL), and sodium carbonate (577.25 mg, 5.45 mmol) and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride dichloromethane mixture (148.25 mg, 181.54 μmol) were added under nitrogen atmosphere, and the reaction solution was stirred at 65° C. for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, and the concentrate was purified by column chromatography (silica, petroleum ether:ethyl acetate=3:2) to give the product (E)-3-[4-chloro-7-(2-methoxyphenyl)-1H-pyrrolo[2,3-d]pyridazin-3-yl]prop-2-enoic acid ethyl ester (600 mg).

MS m/z(ESI):357.9[M+H] ⁺ .

Step 6: Synthesis of ethyl (E)-3-[7-(2-methoxyphenyl)-4-[(4-methoxyphenyl)methylamino]-1H-pyrrolo[2,3-d]pyridazin-3-yl]prop-2-enoate

(E)-3-[4-chloro-7-(2-methoxyphenyl)-1H-pyrrolo[2,3-d]pyridazin-3-yl]prop-2-enoic acid ethyl ester (600 mg, 1.68 mmol) and p-methoxybenzylamine (460.09 mg, 3.35 mmol) were dissolved in dioxane (10 mL), and sodium tert-butoxide (483.49 mg, 5.03 mmol), 2-dicyclohexylphospho-2,4,6-triisopropylbiphenyl (159.89 mg, 335.39 μmol), tris(dibenzylideneacetone)dipalladium (153.56 mg, 167.70 μmol) and 4A molecular sieves (600 mg) were added under nitrogen atmosphere. The reaction solution was stirred at 100 ° C for 4 hours. LCMS showed that the reaction was completed. The reaction solution was filtered and concentrated under reduced pressure. The concentrate was purified by thin layer chromatography (silica, petroleum ether:tetrahydrofuran = 1:1) to give the product (E)-3-[7-(2-methoxyphenyl)-4-[(4-methoxyphenyl)methylamino]-1H-pyrrolo[2,3-d]pyridazin-3-yl]prop-2-enoic acid ethyl ester (160 mg).

MS m/z(ESI):459.1[M+H] ⁺ .

Step 7: Synthesis of (E)-3-[4-amino-7-(2-methoxyphenyl)-1H-pyrrolo[2,3-d]pyridazin-3-yl]prop-2-enoic acid ethyl ester

(E)-3-[7-(2-methoxyphenyl)-4-[(4-methoxyphenyl)methylamino]-1H-pyrrolo[2,3-d]pyridazin-3-yl]prop-2-enoic acid ethyl ester (160 mg, 348.96 μmol) was dissolved in dichloromethane (2 mL), trifluoroacetic acid (397.89 mg, 3.49 mmol) was added, and the reaction solution was stirred at 50 ° C for 2 hours. LCMS showed that the reaction was complete. Dichloromethane (10 mL) was added to the reaction solution, and then washed with saturated sodium bicarbonate aqueous solution (4 mL), and the organic phase was concentrated to dryness under reduced pressure to obtain the product (E)-3-[4-amino-7-(2-methoxyphenyl)-1H-pyrrolo[2,3-d]pyridazin-3-yl]prop-2-enoic acid ethyl ester (120 mg).

MS m/z(ESI):339.1[M+H] ⁺ .

Step 8: Synthesis of (E)-3-[4-amino-7-(2-methoxyphenyl)-1H-pyrrolo[2,3-d]pyridazin-3-yl]prop-2-enoic acid

(E)-3-[4-amino-7-(2-methoxyphenyl)-1H-pyrrolo[2,3-d]pyridazin-3-yl]prop-2-enoic acid ethyl ester (120 mg, 354.65 μmol) was dissolved in tetrahydrofuran (2 mL), sodium hydroxide (2M, 1.77 mL) was added, and the reaction solution was stirred at 40°C for 12 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure to remove tetrahydrofuran, and the pH value was adjusted to 5-6 with 1M hydrochloric acid solution, and concentrated under reduced pressure to dryness to obtain the product (E)-3-[4-amino-7-(2-methoxyphenyl)-1H-pyrrolo[2,3-d]pyridazin-3-yl]prop-2-enoic acid (50 mg).

MS m/z(ESI):311.1[M+H] ⁺ .

Step 9: Synthesis of 5-(2-methoxyphenyl)-3,6,7,9-tetraazatricyclo[6.4.1.04,13]trideca-1,4(13),5,7,11-penten-10-one

(E)-3-[4-Amino-7-(2-methoxyphenyl)1H-pyrrolo[2,3-d]pyridazin-3-yl]prop-2-enoic acid (40 mg, 128.90 μmol) was dissolved in dioxane (2 mL), and a solution of tri-n-propyl cyclophosphoric anhydride in ethyl acetate (1.64 g, 2.58 mmol, 50% purity) was added. The reaction solution was stirred at 110° C. for 12 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, then extracted with dichloromethane (2 mL×3) and aqueous sodium bicarbonate solution (4 mL), the organic phase was concentrated to dryness under reduced pressure, and the concentrate was purified by thin layer chromatography (silicon dioxide, petroleum ether:tetrahydrofuran = 1:2) to give the product 5-(2-methoxyphenyl)-3,6,7,9-tetraazatricyclo[6.4.1.04,13]trideca-1,4(13),5,7,11-penten-10-one (5 mg).

MS m/z(ESI):292.9[M+H] ⁺ .

Step 10: Synthesis of 5-(2-hydroxyphenyl)-3,6,7,9-tetraazatricyclo[6.4.1.04,13]trideca-1,4(13),5,7,11-penten-10-one

5-(2-Methoxyphenyl)-3,6,7,9-tetraazatricyclo[6.4.1.04,13]trideca-1,4(13),5,7,11-penten-10-one (5 mg, 17.11 μmol) was dissolved in dichloromethane (0.5 mL), and boron tribromide (12.86 mg, 51.32 μmol) was added dropwise at 0°C. The reaction solution was stirred at 25°C for 1 hour. LCMS showed that the reaction was completed. The reaction solution was diluted with dichloromethane (10 mL) and then extracted with sodium bicarbonate aqueous solution (2 mL) and washed twice. The organic phase was concentrated to dryness under reduced pressure, and the concentrate was purified by preparative liquid chromatography (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.225% formic acid) and acetonitrile (acetonitrile ratio: 5%-45%) as eluent) to obtain the product 5-(2-hydroxyphenyl)-3,6,7,9-tetraazatricyclo[6.4.1.04,13]trideca-1,4(13),5,7,11-pentene-10-one (7a) (0.82 mg).

MS m/z (ESI): 278.9 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ10.34(s,1H),7.95(s,1H),7.73(s,1H),7.36-7.27(m,1H),7.10-6.87(m,3H ),5.55(s,1H).

Example 8: Synthesis of 2-amino-6-(2-hydroxyphenyl)-4-methoxy-N-phenyl-pyrazolo[1,5-a]pyridine-3-carboxamide (8a)

Step 1: Synthesis of 3-bromo-5-methoxy-pyridin-1-ium-1-amine

O-(2,4-dinitrophenyl)hydroxylamine (5.30 g, 26.59 mmol) was dissolved in dichloromethane (100 mL), 3-bromo-5-methoxypyridine (5 g, 26.59 mmol) was added, and the reaction solution was stirred at 25°C for 16 hours. TLC showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the product 3-bromo-5-methoxy-pyridin-1-ium-1-amine (3.8 g).

Step 2: Synthesis of ethyl 2-amino-6-bromo-4-methoxy-pyrazolo[1,5-a]pyridine-3-carboxylate

3-Bromo-5-methoxy-pyridin-1-ium-1-amine (2.8 g, 6.86 mmol, 50% purity) and 3-ethoxy-3-iminopropionic acid ethyl ester (1.34 g, 6.86 mmol) were dissolved in ethanol (100 mL), potassium carbonate (3.79 g, 27.45 mmol) was added, the system was replaced with nitrogen three times, and the reaction solution was stirred at 25 ° C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated, and then ethyl acetate (50 mL) and water (60 mL) were added, and extracted three times with ethyl acetate (50 mL). The organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. The concentrate was purified by column chromatography (silicon dioxide, petroleum ether: ethyl acetate = 4: 1) to obtain the product 2-amino-6-bromo-4-methoxy-pyrazolo[1,5-a]pyridine-3-carboxylic acid ethyl ester (200 mg).

MS m/z(ESI):313.9,315.9[M+H] ⁺ ;

¹ H NMR (400MHz, CDCl ₃ ) δ = 8.02 (d, J = 1.3Hz, 1H), 6.70 (d, J = 1.3Hz, 1H), 5.29 (br s, 2H), 4.33 (q, J = 7.1 Hz, 2H), 3.93 (s, 3H), 1.39 (t, J = 7.1Hz, 3H).

Step 3: Synthesis of ethyl 2-[bis(tert-butoxycarbonyl)amino]-6-bromo-4-methoxy-pyrazolo[1,5-a]pyridine-3-carboxylate

2-Amino-6-bromo-4-methoxy-pyrazolo[1,5-a]pyridine-3-carboxylic acid ethyl ester (150 mg, 477.50 μmol) and di-tert-butyl dicarbonate (208.43 mg, 955.00 μmol) were dissolved in dichloromethane (2 mL), triethylamine (144.95 mg, 1.43 mmol) and 4-dimethylaminopyridine (5.83 mg, 47.75 μmol) were added, and the reaction solution was stirred at 25°C for 12 hours. The reaction solution was washed three times with water (2 mL) and concentrated. The concentrate was purified by column chromatography (silica, petroleum ether: ethyl acetate = 4:1) to obtain the product 2-[bis(tert-butoxycarbonyl)amino]-6-bromo-4-methoxy-pyrazolo[1,5-a]pyridine-3-carboxylic acid ethyl ester (190 mg).

Step 4: Synthesis of 6-bromo-2-(tert-butoxycarbonylamino)-4-methoxy-pyrazolo[1,5-a]pyridine-3-carboxylic acid

2-[bis(tert-butoxycarbonyl)amino]-6-bromo-4-methoxy-pyrazolo[1,5-a]pyridine-3-carboxylic acid ethyl ester (220 mg, 427.71 μmol) was dissolved in tetrahydrofuran (2 mL) and water (2 mL), sodium hydroxide (68.43 mg, 1.71 mmol) was added, and the reaction solution was stirred at 50°C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was washed with ethyl acetate (2 mL), the aqueous phase was adjusted to pH 5-6 with saturated citric acid aqueous solution, and then extracted three times with ethyl acetate (15 mL), and the organic phase was concentrated to dryness under reduced pressure to obtain the product 6-bromo-2-(tert-butoxycarbonylamino)-4-methoxy-pyrazolo[1,5-a]pyridine-3-carboxylic acid (140 mg).

MS m/z(ESI):408.0,410.0[M+Na] ⁺ .

Step 5: Synthesis of tert-butyl N-[6-bromo-4-methoxy-3-(phenylcarbamoyl)pyrazolo[1,5-a]pyridin-2-yl]carbamate

6-Bromo-2-(tert-butoxycarbonylamino)-4-methoxy-pyrazolo[1,5-a]pyridine-3-carboxylic acid (90 mg, 233.04 μmol) and aniline (32.55 mg, 349.56 μmol) were dissolved in N,N-dimethylformamide (2 mL), triethylamine (70.74 mg, 699.12 μmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (106.33 mg, 279.65 μmol) were added, and the reaction solution was stirred at 25° C. for 2 hours. LCMS showed that the reaction was complete. Ethyl acetate (4 mL) and water (6 mL) were added to the reaction solution, and then extracted three times with ethyl acetate (10 mL). The organic phase was concentrated to dryness, and the concentrate was purified by column chromatography (silica, petroleum ether = 4:1) to obtain the product N-[6-bromo-4-methoxy-3-(phenylcarbamoyl)pyrazolo[1,5-a]pyridin-2-yl]carbamic acid tert-butyl ester (55 mg).

MS m/z(ESI):484.9,486.9[M+Na] ⁺ .

Step 6: Synthesis of tert-butyl N-[6-(2-hydroxyphenyl)-4-methoxy-3-(phenylcarbamoyl)pyrazolo[1,5-a]pyridin-2-yl]carbamate

Tert-butyl N-[6-bromo-4-methoxy-3-(phenylcarbamoyl)pyrazolo[1,5-a]pyridin-2-yl]carbamate (55 mg, 119.23 μmol) and 2-hydroxyphenylboronic acid (19.73 mg, 143.07 μmol) were dissolved in dioxane (2 mL) and water (0.5 mL). Potassium phosphate (50.62 mg, 238.45 μmol) and a mixture of 1,1-bis(diphenylphosphino)ferrocenepalladium chloride and dichloromethane (9.74 mg, 11.92 μmol) were added under nitrogen atmosphere. The reaction solution was stirred at 80 ° C for 2 hours. LCMS showed that the reaction was completed. The reaction solution was concentrated to dryness under reduced pressure, and the concentrate was purified by thin layer chromatography (silica, petroleum ether:tetrahydrofuran = 2:1) to give the product N-[6-(2-hydroxyphenyl)-4-methoxy-3-(phenylcarbamoyl)pyrazolo[1,5-a]pyridin-2-yl]carbamic acid tert-butyl ester (15 mg).

MS m/z(ESI):475.2[M+H] ⁺ .

Step 7: Synthesis of 2-amino-6-(2-hydroxyphenyl)-4-methoxy-N-phenyl-pyrazolo[1,5-a]pyridine-3-carboxamide

Tert-butyl N-[6-(2-hydroxyphenyl)-4-methoxy-3-(phenylcarbamoyl)pyrazolo[1,5-a]pyridin-2-yl]carbamate (12 mg, 25.29 μmol) was dissolved in dichloromethane (1 mL), trifluoroacetic acid (57.67 mg, 505.79 μmol) was added, and the reaction solution was stirred at 25 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, and the concentrate was purified by preparative liquid chromatography (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.225% formic acid) and acetonitrile (acetonitrile ratio: 26%-66%) as eluent) to obtain the product 2-amino-6-(2-hydroxyphenyl)-4-methoxy-N-phenyl-pyrazolo[1,5-a]pyridine-3-carboxamide (8a) (1.00 mg).

MS m/z(ESI):375.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ10.12 (s, 1H), 9.89 (br s, 1H), 8.40 (d, J = 1.0Hz, 1H), 7.69 (d, J = 7.5Hz, 2H ),7.49(dd,J＝1.5,7.8Hz,1H),7.35(t,J＝7.9Hz,2H),7.26-7.18(m,2H),7.06(t,J＝7.4Hz,1H),6.99 (d, J = 7.5Hz, 1H), 6.93 (t, J = 7.3Hz, 1H), 6.45 (s, 2H), 4.17 (s, 3H).

Example 9: Synthesis of 2-[5-[2-(2-fluoro-4-pyridyl)ethynyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl]phenol (9a)

Step 1: Synthesis of 2-(2-fluoro-4-pyridyl)ethynyl-trimethylsilane

Dissolve 4-bromo-2-fluoropyridine (4g, 22.73mmol) in toluene (50mL), add trimethylsilyl acetylene (2.46g, 25.00mmol, 3.46mL), triethylamine (5.75g, 56.82mmol, 7.91mL), cuprous iodide (432.87mg, 2.27mmol) and bistriphenylphosphine palladium dichloride (1.60g, 2.27mmol). The reaction solution was stirred at 50°C under nitrogen protection for 16 hours. LCMS detected that the reaction was complete. The reaction solution was filtered, the filter cake was concentrated to dryness under reduced pressure, and purified by column chromatography (petroleum ether: ethyl acetate = 5:1) to obtain 2-(2-fluoro-4-pyridyl)ethynyl-trimethylsilane (4.39g).

MS m/z (ESI): 193.9 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 8.25 (d, J = 5.1 Hz, 1H), 7.42-7.36 (m, 1H), 7.28 (s, 1H), 0.28-0.22 (m, 9H).

Step 2: Synthesis of 4-ethynyl-2-fluoro-pyridine

Dissolve 2-(2-fluoro-4-pyridyl)ethynyl-trimethylsilane (1.38 g, 7.14 mmol) in anhydrous methanol (140 mL), add potassium fluoride (829.56 mg, 14.28 mmol, 334.50 μL). The reaction solution was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. Add saturated brine (140 mL) and extract three times with dichloromethane (140 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 4-ethynyl-2-fluoropyridine (654 mg).

MS m/z (ESI): 121.9 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 8.27 (d, J = 5.3Hz, 1H), 7.43 (d, J = 5.0Hz, 1H), 7.35-7.30 (m, 1H), 4.78 (s, 1H).

Step 3: Synthesis of 6-chloro-4-(2-trimethylsilylethynyl)pyridazin-3-amine

4-Bromo-6-chloro-pyridazine-3-amine (20.0 g, 96.0 mmol) was dissolved in toluene (400 mL), and trimethylsilyl acetylene (10.4 g, 106 mmol), triethylamine (24.3 g, 240 mmol), cuprous iodide (1.83 g, 9.59 mmol) and bistriphenylphosphine palladium dichloride (6.73 g, 9.59 mmol) were added under nitrogen atmosphere. The reaction solution was stirred at 25 ° C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure and purified by column chromatography (silicon dioxide, petroleum ether/ethyl acetate=100/15) to obtain the product 6-chloro-4-(2-trimethylsilylethynyl)pyridazine-3-amine (14.5 g).

MS m/z(ESI):226.2[M+H] ⁺ ;

¹ H NMR (400MHz, METHANOL-d ₄ ) δ = 7.46 (s, 1H), 0.31-0.28 (m, 9H).

Step 4: Synthesis of 3-chloro-7H-pyrrolo[2,3-c]pyridazine

6-Chloro-4-(2-trimethylsilylethynyl)pyridazine-3-amine (14.5 g, 64.2 mmol) was dissolved in N,N-dimethylformamide (150 mL), and potassium tert-butoxide (1 M, 109 mL) and dimethylamine hydrochloride (2 M, 32.1 mL) were added. The reaction solution was stirred at 25 ° C for 0.5 hours, and then the temperature was raised to 120 ° C for 2.5 hours. LCMS showed that the reaction was complete. The reaction solution was diluted with saturated ammonium chloride solution (50 mL), filtered, and the filtrate was concentrated under reduced pressure. Ethyl acetate (100 mL) and water (70 mL) were added to dilute, the organic phase was washed twice with water (70 mL), and the aqueous phase was extracted twice with ethyl acetate (100 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 100/25) to give the product 3-chloro-7H-pyrrolo[2,3-c]pyridazine (4 g).

MS m/z (ESI): 153.8 [M+H] ⁺ ;

¹ H NMR (400MHz, METHANOL-d ₄ ) δ = 7.91-7.81 (m, 2H), 6.54 (d, J = 3.3Hz, 1H).

Step 5: Synthesis of 3-[2-(methoxymethoxy)phenyl]-7H-pyrrolo[2,3-c]pyridazine

3-Chloro-7H-pyrrolo[2,3-c]pyridazine (1.5 g, 9.77 mmol) and 2-[2-(methoxymethoxy)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborane (5.53 g, 14.65 mmol, 70% purity) were dissolved in dioxane (64 mL) and water (16 mL), and potassium carbonate (4.05 g, 29.30 mmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (797.66 mg, 976.76 μmol) were added. The reaction solution was stirred at 90 ° C for 16 hours under nitrogen protection. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. Saturated brine (80 mL) was added and extracted three times with ethyl acetate (80 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain the product 3-[2-(methoxymethoxy)phenyl]-7H-pyrrolo[2,3-c]pyridazine (1.04 g).

MS m/z(ESI):256.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 12.39 (br s, 1H), 8.14 (s, 1H), 7.92-7.84 (m, 1H), 7.72 (dd, J = 1.8, 7.5Hz, 1H) ,7.43-7.35(m,1H),7.27(d,J＝7.5Hz,1H),7.18-7.12(m,1H),6.58(d,J＝2.3Hz,1H),5.21(s,2H), 3.29(s,3H).

Step 6: Synthesis of 5-iodo-3-[2-(methoxymethoxy)phenyl]-7H-pyrrole

3-[2-(methoxymethoxy)phenyl]-7H-pyrrolo[2,3-c]pyridazine (700mg, 2.74mmol) was dissolved in N,N-dimethylformamide (10mL), and N-iodosuccinimide (740.34mg, 3.29mmol) was added. The reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete. Saturated brine (10mL) was added and extracted three times with ethyl acetate (10mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether/ethyl acetate = 100/50) to obtain 5-iodo-3-[2-(methoxymethoxy)phenyl]-7H-pyrrole (653mg).

MS m/z(ESI):382.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 12.90 (br s, 1H), 8.16 (s, 1H), 7.88 (s, 1H), 7.78 (dd, J = 1.5, 7.5Hz, 1H), 7.46 -7.40(m,1H),7.27(d,J＝8.3Hz,1H),7.17(t,J＝7.3Hz,1H),5.25(s,2H),3.36(s,3H).

Step 7: Synthesis of 5-iodo-3-[2-(methoxymethoxy)phenyl]-7-(p-toluenesulfonyl)pyrrolo[2,3-c]pyridazine

Dissolve 5-iodo-3-[2-(methoxymethoxy)phenyl]-7H-pyrrole (200 mg, 524.70 μmol) in N,N-dimethylformamide (3 mL), add sodium hydrogen (37.78 mg, 944.47 μmol, 60% purity). The reaction solution was stirred at 0°C for 0.5 hours under nitrogen protection, and p-toluenesulfonyl chloride (180.06 mg, 944.47 μmol) was added. The reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete. Water (3 mL) was added at 0°C for quenching. Saturated brine (3 mL) was added and extracted three times with ethyl acetate (3 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether/ethyl acetate = 100/50) to give the product 5-iodo-3-[2-(methoxymethoxy)phenyl]-7-(p-toluenesulfonyl)pyrrolo[2,3-c]pyridazine (205 mg).

MS m/z(ESI):536.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 8.52 (s, 1H), 8.12 (d, J = 8.3Hz, 2H), 7.96 (s, 1H), 7.75 (dd, J = 1.6, 7.7Hz, 1H),7.49-7.43(m,3H),7.26(d,J＝8.0Hz,1H),7.20-7.14(m,1H),5.23(s,2H),3.33(s,3H),2.36(s ,3H).

Step 8: Synthesis of 5-[2-(2-fluoro-4-pyridinyl)ethynyl]-3-[2-(methoxymethoxy)phenyl]-7-(p-toluenesulfonyl)pyrrolo[2,3-c]pyridazine

5-iodo-3-[2-(methoxymethoxy)phenyl]-7-(p-toluenesulfonyl)pyrrolo[2,3-c]pyridazine (180 mg, 336 μmol) and 4-ethynyl-2-fluoro-pyridine (81.4 mg, 672 μmol) were dissolved in toluene (4 mL), and triethylamine (102 mg, 1.01 mmol), cuprous iodide (6.40 mg, 33.6 μmol) and bistriphenylphosphine palladium dichloride (23.6 mg, 33.6 μmol) were added under nitrogen atmosphere. The reaction solution was stirred at 25 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure and purified by column chromatography (silica, petroleum ether/ethyl acetate = 100/30) to give the product 5-[2-(2-fluoro-4-pyridyl)ethynyl]-3-[2-(methoxymethoxy)phenyl]-7-(p-toluenesulfonyl)pyrrolo[2,3-c]pyridazine (160 mg).

MS m/z(ESI):529.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 8.83 (s, 1H), 8.45 (s, 1H), 8.33 (d, J = 5.0Hz, 1H), 8.17 (d, J = 8.3Hz, 2H) ,7.70(dd,J＝1.6,7.7Hz,1H),7.52-7.44(m,5H),7.28(d,J＝8.0Hz,1H),7.18(t,J＝7.4Hz,1H),5.23( s,2H),3.29(s,3H),2.37(s,3H).

Step 9: Synthesis of 5-[2-(2-fluoro-4-pyridinyl)ethynyl]-3-[2-(methoxymethoxy)phenyl]-7H-pyrrolo[2,3-c]pyridazine

5-[2-(2-fluoro-4-pyridinyl)ethynyl]-3-[2-(methoxymethoxy)phenyl]-7-(p-toluenesulfonyl)pyrrolo[2,3-c]pyridazine (50 mg, 94.6 μmol) was dissolved in tert-butanol (1 mL), and potassium tert-butoxide (31.9 mg, 284 μmol) was added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS showed that the reaction was complete. Acetic acid was added to the reaction solution until the pH of the reaction solution reached 7, and the product 5-[2-(2-fluoro-4-pyridinyl)ethynyl]-3-[2-(methoxymethoxy)phenyl]-7H-pyrrolo[2,3-c]pyridazine (30 mg, crude product) was directly used in the next step.

MS m/z(ESI):375.1[M+H] ⁺ .

Step 10: Synthesis of 2-[5-[2-(2-fluoro-4-pyridinyl)ethynyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl]phenol

5-[2-(2-fluoro-4-pyridinyl)ethynyl]-3-[2-(methoxymethoxy)phenyl]-7H-pyrrolo[2,3-c]pyridazine (30.0 mg, 80.1 μmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (0.1 mL) was added. The reaction solution was stirred at 25° C. for 1 hour. LCMS showed that the reaction was complete. Ammonia methanol (7M) was added to adjust the pH of the reaction solution to 7-8, and then concentrated under reduced pressure and purified by preparative liquid chromatography (Phenomenex C18, 3μm silica, 30mm diameter, 75mm length; using a mixture of water (containing 0.225% formic acid) and acetonitrile (acetonitrile ratio: 27%-67%) as eluent) to obtain the product 2-[5-[2-(2-fluoro-4-pyridyl)ethynyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl]phenol (9a) (2.05mg).

MS m/z(ESI):331.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.60-13.20 (m, 2H), 8.80 (s, 1H), 8.60 (s, 1H), 8.32 (d, J = 5.1Hz, 1H), 8.20 ( d,J＝8.0Hz,1H),7.58(br d,J＝5.1Hz,1H),7.49(s,1H),7.38-7.30(m,1H),7.05-6.97(m,2H).

Example 10: Synthesis of 2-[5-[(Z)-2-(2-fluoro-4-pyridyl)vinyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl]phenol (10a)

Step 1: Synthesis of 5-[(Z)-2-(2-fluoro-4-pyridinyl)vinyl]-3-[2-(methoxymethoxy)phenyl]-7-(p-toluenesulfonyl)pyrrolo[2,3-c]pyridazine

5-[2-(2-fluoro-4-pyridinyl)ethynyl]-3-[2-(methoxymethoxy)phenyl]-7-(p-toluenesulfonyl)pyrrolo[2,3-c]pyridazine (60 mg, 114 μmol) was dissolved in methanol (4 mL) and Lindlar catalyst (234 mg, 1.14 mmol) was added. The mixture was reacted at 25° C. for 4 hours under H ₂ (15 psi) atmosphere. LCMS showed that the reaction was complete. The reaction solution was filtered and the filtrate was concentrated under reduced pressure to give the product 5-[(Z)-2-(2-fluoro-4-pyridinyl)ethynyl]-3-[2-(methoxymethoxy)phenyl]-7-(p-toluenesulfonyl)pyrrolo[2,3-c]pyridazine (67 mg, crude product), which was directly used in the next step.

MS m/z(ESI):531.0[M+H] ⁺ .

Step 2: Synthesis of 5-[(Z)-2-(2-fluoro-4-pyridinyl)vinyl]-3-[2-(methoxymethoxy)phenyl]-7H-pyrrolo[2,3-c]pyridazine

5-[(Z)-2-(2-fluoro-4-pyridinyl)vinyl]-3-[2-(methoxymethoxy)phenyl]-7-(p-toluenesulfonyl)pyrrolo[2,3-c]pyridazine (50 mg, 94.2 μmol) was dissolved in tert-butanol (1 mL), and potassium tert-butoxide (31.7 mg, 283 μmol) was added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS showed that the reaction was complete. Acetic acid was added to the reaction solution until the pH of the reaction solution reached 7, and the product 5-[(Z)-2-(2-fluoro-4-pyridinyl)vinyl]-3-[2-(methoxymethoxy)phenyl]-7H-pyrrolo[2,3-c]pyridazine (35 mg, crude product) was directly used in the next step.

MS m/z(ESI):376.1[M+H] ⁺ .

Step 3: Synthesis of 2-[5-[(Z)-2-(2-fluoro-4-pyridinyl)vinyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl]phenol

5-[(Z)-2-(2-fluoro-4-pyridinyl)vinyl]-3-[2-(methoxymethoxy)phenyl]-7H-pyrrolo[2,3-c]pyridazine (35.0 mg, 93.0 μmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (0.8 mL) was added. The reaction solution was stirred at 25° C. for 4 hours. LCMS showed that the reaction was complete. Ammonia methanol (7M) was added to adjust the pH of the reaction solution to 7-8, and then concentrated under reduced pressure and purified by preparative liquid chromatography (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.225% formic acid) and acetonitrile (acetonitrile ratio: 16%-56%) as eluent) to obtain the product 2-[5-[(Z)-2-(2-fluoro-4-pyridyl)vinyl]-7H-pyrrolo[2,3-c]pyridazin-3-yl]phenol (10a) (2.76 mg).

MS m/z(ESI):333.1[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.19 (br s, 1H), 8.23 (br s, 1H), 8.17-8.08 (m, 2H), 7.59 (d, J = 6.8Hz, 1H), 7.33(t,J＝7.2Hz,1H),7.26(d,J＝5.0Hz,1H),7.14-7.08(m,2H),7.00(d,J＝8.3Hz,1H),6.95(t,J =7.0 Hz, 1H), 6.71 (d, J = 12.0 Hz, 1H).

Example 11: Synthesis of 2-[3-(methylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (11a)

Step 1: Synthesis of tert-butyl 4-[3-amino-6-[2-(methoxymethoxy)phenyl]pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Tert-butyl 4-(3-amino-6-chloropyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylate (1.2 g, 3.40 mmol) and 2-[2-(methoxymethoxy)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.35 g, 5.10 mmol) were dissolved in anhydrous dioxane (24 mL) and water (6 mL), and cesium carbonate (2.22 g, 6.80 mmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (138.88 mg, 170.00 μmol) were added. The reaction solution was stirred at 90 ° C for 16 hours under a nitrogen atmosphere. The reaction was completed by LCMS detection. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether:tetrahydrofuran = 1:1) to give tert-butyl 4-[3-amino-6-[2-(methoxymethoxy)phenyl]pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate (490 mg).

MS m/z (ESI): 455.3 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 7.93 (s, 1H), 7.70 (dd, J = 1.6, 7.6Hz, 1H), 7.49-7.42 (m, 1H), 7.28 (d, J = 8.2 Hz,1H),7.18(t,J＝7.4Hz,1H),6.30(s,2H),5.21(s,2H),4.71-4.51(m,1H),4.07(d,J＝12.2Hz,2H ),3.30(s,3H),2.89(s,2H),1.94-1.80(m,4H),1.42(s,9H).

Step 2: Synthesis of tert-butyl 4-[6-[2-(methoxymethoxy)phenyl]-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Dissolve tert-butyl 4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylate (30 mg, 66.00 μmol) in anhydrous dioxane (1 mL), add pyridine (15.98 μL, 198.01 μmol) and copper acetate (23.98 mg, 132.01 μmol). The mixture was stirred at 25°C for 0.5 hours, and methylboric acid (3.16 mg, 52.80 μmol) was added. The reaction solution was stirred at 100°C for 5.5 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (Phenomenex Gemini C18-1 column: 5 μm silica, 30 mm diameter, 80 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 35%-55%) as eluent) to give tert-butyl 4-[6-[2-(methoxymethoxy)phenyl]-3-(methylamino)pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate (10 mg).

MS m/z(ESI):469.2[M+H] ⁺ .

Step 3: Synthesis of 2-[3-(methylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

Dissolve tert-butyl 4-[6-[2-(methoxymethoxy)phenyl]-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate (10 mg, 21.34 μmol) in anhydrous methanol (1 mL), and add methanolic hydrochloric acid (4 M, 1 mL). Stir the reaction solution at 25° C. for 3 hours. LCMS detection shows that the reaction is complete. The mixture was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (Phenomenex Gemini C18-1 column: 5 μm silica, 30 mm diameter, 80 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 8%-28%) as eluent) to give 2-[3-(methylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (11a) (7.39 mg).

MS m/z (ESI): 325.1 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.77 (br s, 1H), 8.45 (s, 1H), 8.08 (dd, J = 1.3, 8.3Hz, 1H), 7.46-7.29 (m, 1H) ,7.12(d,J＝4.8Hz,1H),7.04-6.96(m,2H),4.75(t,J＝11.0Hz,1H),3.29(d,J＝9.8Hz,2H),2.96(d, J＝4.8Hz,3H),2.87(br s,2H),2.27-2.08(m,2H),1.94(d,J＝10.8Hz,2H).

Example 12: Synthesis of 2-(3-amino-1-benzyl-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (12a)

Step 1: Synthesis of 1-benzyl-6-chloro-1H-pyrazolo[4,3-c]pyridazin-3-amine

6-Chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (100 mg, 589.72 μmol) was dissolved in N,N-dimethylformamide (2 mL), benzyl bromide (151.29 mg, 884.58 μmol) and anhydrous cesium carbonate (576.43 mg, 1.77 mmol) were added, and the reaction solution was heated to 80°C and stirred for 16 hours. LCMS detected that the reaction was complete. The reaction solution was cooled to 25°C, water (5 mL) was added, and it was extracted twice with ethyl acetate (15 mL), dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by thin layer chromatography (petroleum ether: ethyl acetate = 1: 1 (5/1000 ammonia methanol solution)) to obtain 1-benzyl-6-chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (110 mg).

MS m/z(ESI):260.0[M+H] ⁺ .

Step 2: Synthesis of 2-(3-amino-1-benzyl-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

Dissolve 1-benzyl-6-chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (100 mg, 385.07 μmol) in anhydrous dioxane (4 mL) and water (1 mL), add 2-hydroxyphenylboronic acid (79.69 mg, 577.60 μmol), anhydrous potassium phosphate (245.21 mg, 1.16 mmol), and add [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (31.45 mg, 38.51 μmol) under nitrogen protection. Heat the reaction solution to 100°C and stir for 16 hours. LCMS detection of the reaction is complete. Cool the reaction solution to 25°C. Water (5 mL) was added, extracted twice with ethyl acetate (15 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude residue was purified by preparative liquid chromatography (Phenomenex C18 column: 3 μm silica, 30 mm diameter, 75 mm length; using a decreasingly polar mixture of water (containing ammonia (0.05%)) and acetonitrile (acetonitrile content: 31%-71%) as eluent) to give 2-(3-amino-1-benzyl-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (12a) (11 mg).

MS m/z (ESI): 318.0 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 8.64 (s, 1H), 8.11 (br d, J = 8.1Hz, 1H), 7.42-7.25 (m, 6H), 7.08-6.97 (m, 2H) ,6.52(s,2H),5.46(s,2H).

Example 13: Synthesis of 2-[3-amino-1-(pyrrolidin-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (13a)

Step 1: Synthesis of tert-butyl 3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)pyrrolidine-1-carboxylate

6-Chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (500 mg, 2.95 mmol) was dissolved in N,N-dimethylformamide (15 mL), and 1-Boc-3-iodopyrrolidine (963.74 mg, 3.24 mmol) and cesium carbonate (2.88 g, 8.85 mmol) were added. The reaction solution was stirred at 80 ° C for 2 hours. The reaction was complete as monitored by LCMS. Saturated sodium sulfite aqueous solution (20 mL) was added to the reaction solution to quench the reaction, saturated brine (45 mL) was added to the reaction solution and extracted three times with ethyl acetate (15 mL), the organic phase was washed three times with water (15 mL), and dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure and then purified by column chromatography (petroleum ether/ethyl acetate=1/1) to give tert-butyl 3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)pyrrolidine-1-carboxylate (176 mg).

MS m/z(ESI):339.2[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]pyrrolidine-1-carboxylate

Tert-butyl 3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)pyrrolidine-1-carboxylate (176 mg, 519.49 μmol) and 2-hydroxyphenylboronic acid (107.48 mg, 779.24 μmol) were dissolved in dioxane (8 mL) and water (2 mL), and cesium carbonate (338.52 mg, 1.04 mmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (42.42 mg, 51.95 μmol) were added. The reaction solution was stirred at 90° C. for 2 hours under nitrogen atmosphere. The reaction was complete when monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (petroleum ether/tetrahydrofuran = 1/1) to give tert-butyl 3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]pyrrolidine-1-carboxylate (126 mg).

MS m/z (ESI): 397.2 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.82 (br s, 1H), 8.50 (s, 1H), 8.07 (d, J = 7.1Hz, 1H), 7.41-7.32 (m, 1H), 7.06 -6.94(m,2H),6.56(br s,2H),5.45-5.28(m,1H),3.82-3.33(m,6H),1.41(br d,J＝12.6Hz,9H).

Step 3: Synthesis of 2-[3-amino-1-(pyrrolidin-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]pyrrolidine-1-carboxylic acid tert-butyl ester (126 mg, 317.83 μmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (362.40 mg, 3.18 mmol) was added. The reaction solution was stirred at 25°C for 2 hours. LCMS monitored the reaction to be complete. The reaction solution was concentrated to dryness under reduced pressure to obtain 2-[3-amino-1-(pyrrolidin-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (13a) (125 mg).

MS m/z(ESI):296.9[M+H] ⁺ .

Example 14: Synthesis of 2-[3-amino-1-(piperidin-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (14a)

Step 1: Synthesis of tert-butyl 3-(methylsulfonyloxy)piperidine-1-carboxylate

3-Hydroxy-piperidine-1-carboxylic acid tert-butyl ester (2 g, 9.94 mmol) was dissolved in tetrahydrofuran (40 mL), pyridine (1.57 g, 19.87 mmol) and methanesulfonic anhydride (1.90 g, 10.93 mmol) were added, and the reaction solution was stirred at 25 ° C for 2 hours. Thin layer chromatography (petroleum ether: ethyl acetate = 3:1) showed that the reaction was completed. The reaction solution was extracted with ethyl acetate (50 mL) and water (60 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to give the product 3-(methylsulfonyloxy)piperidine-1-carboxylic acid tert-butyl ester (2.3 g).

Step 2: Synthesis of tert-butyl 3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate

6-Chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (100 mg, 589.72 μmol) and tert-butyl 3-(methylsulfonyloxy)-piperidine-1-carboxylate (247.11 mg, 884.58 μmol) were dissolved in N,N-dimethylformamide (6 mL), and cesium carbonate (576.43 mg, 1.77 mmol) and sodium iodide (176.79 mg, 1.18 mmol) were added. The reaction solution was reacted at 80 ° C for 12 hours. The reaction was complete by LCMS. Water (10 mL) was added to the reaction solution, extracted three times with ethyl acetate (10 mL), and the organic phase was concentrated to dryness under reduced pressure. Purification by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 3/2) gave tert-butyl 3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate (20 mg).

MS m/z(ESI):353.0[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylic acid tert-butyl ester (20 mg, 56.69 μmol) and 2-hydroxyphenylboronic acid (9.38 mg, 68.02 μmol) were dissolved in 1,4-dioxane (0.8 mL) and water (0.2 mL), and cesium carbonate (36.94 mg, 113.37 μmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (4.63 mg, 5.67 μmol) were added. After nitrogen replacement, the reaction solution was reacted at 100°C for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by thin layer chromatography (silica, petroleum ether/tetrahydrofuran = 2/1) to give tert-butyl 3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate (14 mg).

MS m/z(ESI):411.3[M+H] ⁺ .

Step 4: Synthesis of 2-[3-amino-1-(piperidin-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylic acid tert-butyl ester (14 mg, 34.11 μmol) was dissolved in dichloromethane (2 mL), and dioxane hydrochloride (4M, 85.27 μL) was added. The reaction solution was reacted at 25°C for 1 hour. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure to obtain the product 2-[3-amino-1-(piperidin-3-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (10 mg).

MS m/z(ESI):311.0[M+H] ⁺ .

Example 15: Synthesis of 2-[3-amino-1-(8-azabicyclo[3.2.1]octan-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (15a)

Step 1: Synthesis of tert-butyl 3-methylsulfonyloxy-8-azabicyclo[3.2.1]octane-8-carboxylate

3-Hydroxy-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (2g, 8.80mmol) was dissolved in tetrahydrofuran (30mL), pyridine (1.39g, 17.60mmol) and methanesulfonic anhydride (2.30g, 13.20mmol) were added, and the reaction solution was stirred at 25°C for 4 hours. Thin layer chromatography (petroleum ether: ethyl acetate = 3:1) showed that the reaction was complete. The reaction solution was extracted with ethyl acetate (50mL×3) and water (50mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated. The concentrate was purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 3/1) to obtain the product 3-methylsulfonyloxy-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (2.5g).

¹ H NMR (400MHz, CDCl ₃ ) δ5.11-4.99 (m, 1H), 4.40-4.10 (m, 2H), 3.00 (d, J = 1.3Hz, 3H), 2.32-1.75 (m, 7H), 1.71-1.63(m,1H),1.46(d,J＝7.1Hz,9H).

Step 2: Synthesis of tert-butyl 3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate

6-Chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (100 mg, 589.72 μmol) and tert-butyl 3-methylsulfonyloxy-8-azabicyclo[3.2.1]octane-8-carboxylate (216.11 mg, 707.67 μmol) were dissolved in N,N-dimethylformamide (6 mL), and cesium carbonate (576.43 mg, 1.77 mmol) and sodium iodide (132.59 mg, 884.58 μmol) were added. The reaction solution was reacted at 80°C for 12 hours. The reaction was complete as determined by LCMS. Water (10 mL) was added to the reaction solution, and the organic phase was concentrated to dryness under reduced pressure after extraction with ethyl acetate (10 mL) three times. Purification by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 3/2) gave tert-butyl 3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (60 mg).

MS m/z(ESI):379.2[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 3-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylate

3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (60 mg, 158.37 μmol) and 2-hydroxyphenylboronic acid (26.21 mg, 190.05 μmol) were dissolved in 1,4-dioxane (2 mL) and water (0.5 mL), and cesium carbonate (154.80 mg, 475.12 μmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (12.93 mg, 15.84 μmol) were added. After nitrogen replacement, the reaction solution was reacted at 100°C for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by thin layer chromatography (silica, petroleum ether/tetrahydrofuran = 1/1) to give tert-butyl 3-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylate (30 mg).

MS m/z(ESI):437.4[M+H] ⁺ .

Step 4: Synthesis of 2-[3-amino-1-(8-azabicyclo[3.2.1]octan-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

3-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (30 mg, 68.73 μmol) was dissolved in dichloromethane (2 mL), and dioxane hydrochloride (4M, 171.82 μL) was added. The reaction solution was reacted at 25° C. for 1 hour. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure to obtain the product 2-[3-amino-1-(8-azabicyclo[3.2.1]octane-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (15a) (30 mg).

MS m/z(ESI):337.2[M+H] ⁺ .

Example 16: Synthesis of 2-[3-amino-1-(piperidin-4-ylmethyl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (16a)

Step 1: Synthesis of tert-butyl 4-[(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)methyl]piperidine-1-carboxylate

6-Chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (100 mg, 589.72 μmol) and 1-tert-butyloxycarbonyl-4-bromomethylpiperidine (196.86 mg, 707.67 μmol) were dissolved in N,N-dimethylformamide (6 mL), and cesium carbonate (384.28 mg, 1.18 mmol) was added. The reaction solution was reacted at 80°C for 16 hours. The reaction was complete as determined by LCMS. Water (15 mL) was added to the reaction solution, and the mixture was extracted three times with ethyl acetate (10 mL). The organic phase was concentrated to dryness under reduced pressure and purified by column chromatography (silicon dioxide, petroleum ether/tetrahydrofuran = 1/0 to 3/2) to obtain tert-butyl 4-[(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)methyl]piperidine-1-carboxylate (50 mg).

MS m/z(ESI):367.1[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-[[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]methyl]piperidine-1-carboxylate

Tert-butyl 4-[(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)methyl]piperidine-1-carboxylate (50 mg, 136.30 μmol) and 2-hydroxyphenylboronic acid (22.56 mg, 163.56 μmol) were dissolved in 1,4-dioxane (2 mL) and water (0.4 mL), and cesium carbonate (88.82 mg, 272.59 μmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (11.13 mg, 13.63 μmol) were added. After nitrogen replacement, the reaction solution was reacted at 100°C for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by thin layer chromatography (silica, petroleum ether/tetrahydrofuran = 1/1) to give tert-butyl 4-[[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]methyl]piperidine-1-carboxylate (45 mg).

MS m/z(ESI):425.3[M+H] ⁺ .

Step 3: Synthesis of 2-[3-amino-1-(piperidin-4-ylmethyl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]methyl]piperidine-1-carboxylic acid tert-butyl ester (45 mg, 106.01 μmol) was dissolved in dichloromethane (2 mL), and dioxane hydrochloride (4M, 265.02 μL) was added. The reaction solution was reacted at 25° C. for 1 hour. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure to obtain the product 2-[3-amino-1-(piperidin-4-ylmethyl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (16a) (50 mg).

MS m/z(ESI):325.0[M+H] ⁺ .

Example 17: Synthesis of 2-[3-(methylamino)-1-(pyrrolidin-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (17a)

Step 1: Synthesis of tert-butyl 3-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]pyrrolidine-1-carboxylate

Tert-butyl 3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)pyrrolidine-1-carboxylate (200 mg, 590.33 μmol), 2-[2-(methoxymethoxy)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborane (187.10 mg, 708.40 μmol), 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (43.19 mg, 59.03 μmol) and potassium phosphate (375.92 mg, 1.77 mmol) were dissolved in dioxane (4 mL) and water (1 mL). The reaction solution was stirred at 100 ° C for 4 hours under a nitrogen atmosphere. The reaction was completed by LCMS. The reaction solution was concentrated to dryness and purified by column chromatography (silica, petroleum ether:tetrahydrofuran = 1:1) to give tert-butyl 3-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]pyrrolidine-1-carboxylate (100 mg).

MS m/z(ESI):441.2[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 3-[6-[2-(methoxymethoxy)phenyl]-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]pyrrolidine-1-carboxylate

3-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazine-1-yl]pyrrolidine-1-carboxylic acid tert-butyl ester (50 mg, 113.51 μmol), methylboronic acid (6.79 mg, 113.51 μmol), pyridine (26.94 mg, 340.53 μmol, 27.49 μL) and copper acetate (41.23 mg, 227.02 μmol) were dissolved in dioxane (4 mL). The reaction solution was stirred at 100 ° C under an oxygen atmosphere for 6 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by thin layer chromatography (silica, petroleum ether:tetrahydrofuran = 1:1) to give tert-butyl 3-[6-[2-(methoxymethoxy)phenyl]-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]pyrrolidine-1-carboxylate (30 mg).

MS m/z(ESI):455.2[M+H] ⁺ .

Step 3: Synthesis of 2-[3-(methylamino)-1-(pyrrolidin-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (17a)

3-[6-[2-(methoxymethoxy)phenyl]-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]pyrrolidine-1-carboxylic acid tert-butyl ester (30 mg, 66.00 μmol) was dissolved in anhydrous methanol (1 mL) and dioxane hydrochloride (0.5 mL, 4 M). The reaction solution was stirred at 25 ° C for 16 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness to obtain 2-[3-(methylamino)-1-(pyrrolidin-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (17a) (30 mg).

MS m/z(ESI):311.1[M+H] ⁺ .

Example 18: Synthesis of 2-[3-[(methyl-d ₃ )amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (18a)

Step 1: Synthesis of tert-butyl 4-(3-((tert-butyloxycarbonyl)amino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate

Tert-butyl 4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylate (300 mg, 660.04 μmol) was dissolved in dichloromethane (5 mL), and pyridine (57.43 mg, 726.04 μmol), 4-dimethylaminopyridine (8.06 mg, 66.00 μmol) and Boc anhydride (144.05 mg, 660.04 μmol) were added. The reaction solution was reacted at 25° C. for 12 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated and purified by column chromatography (silica, petroleum ether/ethyl acetate = 1/1) to give tert-butyl 4-(3-((tert-butoxycarbonyl)amino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate (170 mg).

MS m/z(ESI):555.4[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-[6-[2-(methoxymethoxy)phenyl]-3-[(methyl-d ₃ )amino]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

4-(3-((tert-butyloxycarbonyl)amino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylic acid tert-butyl ester (50 mg, 90.15 μmol) was dissolved in tetrahydrofuran (3 mL), sodium hydrogen sulfide (18.03 mg, 450.75 μmol, 60% purity) was added, the reaction solution was stirred at 25° C. for 0.5 hours, and deuterated iodomethane (65.34 mg, 450.75 μmol) was added. The reaction solution was reacted at 25° C. for 11.5 hours, and the reaction was completed by LCMS detection. The reaction solution was quenched with water (10 mL), extracted with ethyl acetate (15 mL×3), the organic phases were combined, dried over anhydrous sodium sulfate, concentrated, and purified by column chromatography (silica, petroleum ether/ethyl acetate=1/1) to give tert-butyl 4-[6-[2-(methoxymethoxy)phenyl]-3-[(methyl-d ₃ )amino]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate (30 mg).

MS m/z(ESI):572.2[M+H] ⁺ .

Step 3: Synthesis of 2-[3-[(methyl-d ₃ )amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

tert-Butyl 4-(6-(2-(methoxymethoxy)phenyl)-3-((methyl-d ₃ )amino)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate (30 mg, 26.24 μmol) was dissolved in methanol (1 mL), and hydrochloric acid/dioxane (65.60 μL, 262.38 μmol) was added. The reaction solution was reacted at 25° C. for 12 hours. The reaction was completed after LCMS detection. The reaction solution was concentrated and purified by preparative liquid chromatography (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 19%-59%) as eluent) to give 2-[3-[(methyl- _d3 )amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (18a) (1.02 mg).

MS m/z(ESI):328.1[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.96 (br s, 1H), 8.48 (s, 1H), 8.13 (d, J = 7.1Hz, 1H), 7.37 (t, J = 7.0Hz, 1H ),7.05-6.96(m,3H),4.63(br t,J＝11.7Hz,1H),3.11(br d,J＝13.1Hz,2H),2.72-2.62(m,2H),2.02(dt, J＝8.2,11.9Hz,2H),1.85-1.77(m,2H).

Example 19: Synthesis of 2-[3-(ethylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (19a)

Step 1: Synthesis of tert-butyl 4-[3-(ethylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylic acid tert-butyl ester (50 mg, 110.01 μmol) and acetaldehyde (14.54 mg, 330.02 μmol) were dissolved in anhydrous methanol (1 mL), and acetic acid (18.87 μL, 330.02 μmol) was added. After the reaction solution was stirred at 25°C for 3 hours, sodium cyanoborohydride (20.74 mg, 330.02 μmol) was added. The reaction solution was stirred at 25°C for 18 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether:tetrahydrofuran = 2:1) to give tert-butyl 4-[3-(ethylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate (40 mg).

MS m/z(ESI):483.3[M+H] ⁺ .

Step 2: Synthesis of 2-[3-(ethylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[3-(ethylamino)-6-[2-(methoxymethoxy)phenyl]pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylic acid tert-butyl ester (40 mg, 82.89 μmol) was dissolved in anhydrous methanol (1 mL), and dioxane hydrochloride (4M, 207.22 μL) was added. The reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain 2-[3-(ethylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (19a) (2.30 mg).

MS m/z (ESI): 339.2 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.28 (d, J = 8.3Hz, 1H), 8.90 (d, J = 9.5Hz, 1H), 8.46 (s, 1H), 7.78 (br s, 1H) ),7.45(t,J＝7.3Hz,1H),7.13(d,J＝8.3Hz,1H),7.05(t,J＝7.5Hz,1H),4.96(t,J＝10.8Hz,1H), 3.42(d,J＝7.0Hz,2H),3.40-3.37(m,2H),3.12-2.94(m,2H),2.41-2.26(m,2H),2.09(d,J＝11.8Hz,2H) ,1.27(t,J=7.2Hz,3H).

Example 20: Synthesis of 2-[1-(piperidin-4-yl)-3-(propylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (20a)

Step 1: Synthesis of tert-butyl 4-[6-[2-(methoxymethoxy)phenyl]-3-(propylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylic acid tert-butyl ester (50 mg, 110.01 μmol) and propionaldehyde (19.17 mg, 330.03 μmol) were dissolved in anhydrous methanol (2 mL), and acetic acid (18.88 μL, 330.03 μmol) was added. After the reaction solution was stirred at 25°C for 3 hours, sodium cyanoborohydride (20.74 mg, 330.03 μmol) was added. The reaction solution was stirred at 25°C for 18 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether:tetrahydrofuran = 2:1) to give tert-butyl 4-[6-[2-(methoxymethoxy)phenyl]-3-(propylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate (44 mg).

MS m/z(ESI):497.2[M+H] ⁺ .

Step 2: Synthesis of 2-[1-(piperidin-4-yl)-3-(propylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[6-[2-(methoxymethoxy)phenyl]-3-(propylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylic acid tert-butyl ester (44 mg, 88.60 μmol) was dissolved in anhydrous methanol (1 mL), and dioxane hydrochloride (4M, 221.51 μL) was added. The reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain 2-[1-(piperidin-4-yl)-3-(propylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (20a) (2.65 mg).

MS m/z(ESI):353.2[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.35 (d, J = 10.0Hz, 1H), 8.93 (d, J = 10.5Hz, 1H), 8.46 (s, 1H), 7.70 (d, J = 7.5Hz,1H),7.52-7.39(m,1H),7.17(d,J＝8.0Hz,1H),7.06(t,J＝7.3Hz,1H),4.98(t,J＝11 .0Hz,1H),3.45(d,J＝12.0Hz,2H),3.35(t,J＝7.0Hz,2H),3.02(q,J＝11.5Hz,2H),2.40-2.28(m,2H) ,2.09(d,J＝12.3Hz,2H),1.74-1.72(m,2H),0.96(t,J＝7.4Hz,3H).

Example 21: Synthesis of 2-[3-(isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (21a)

Step 1: Synthesis of tert-butyl 4-[3-(isopropylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylic acid tert-butyl ester (50 mg, 110.01 μmol) was dissolved in methanol (1 mL), acetone (19.17 mg, 330.02 μmol) and acetic acid (19.82 mg, 330.02 μmol) were added, and the reaction solution was stirred at 25° C. for 3 hours, and then sodium cyanoborohydride (20.74 mg, 330.02 μmol) was added. The reaction solution was reacted at 25° C. for 9 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated to give a crude product, which was purified by thin layer chromatography (silica, petroleum ether/tetrahydrofuran = 1/1) to give tert-butyl 4-[3-(isopropylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate (50 mg).

MS m/z(ESI):497.2[M+H] ⁺ .

Step 2: Synthesis of 2-[3-(isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[3-(Isopropylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylic acid tert-butyl ester (20 mg, 40.27 μmol) was dissolved in methanol (0.5 mL), and dioxane hydrochloride (100.68 μL, 402.74 μmol) was added. The reaction solution was reacted at 25° C. for 2 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated and purified by preparative liquid chromatography (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 32%-72%) as eluent) to give 2-[3-(isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (21a) (3.5 mg).

MS m/z(ESI):353.1[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.02 (br s, 1H), 8.49 (s, 1H), 8.13 (br d, J = 7.5Hz, 1H), 7.40-7.32 (m, 1H), 7.04-6.96(m,2H),6.91(d,J＝7.8Hz,1H),4.68-4.57(m,1H),4.02(qd,J＝6.6,13.5Hz,1H),3.10(br d,J ＝12.3Hz,2H),2.71-2.61(m,2H),2.56-2.54(m,1H),2.05-1.94(m,2H),1.79(br d,J＝10.6Hz,2H),1.28(d,J＝6.5Hz,6H).

Example 22: Synthesis of N-[6-(2-hydroxyphenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-yl]cyclopropanecarboxamide (22a)

Step 1: Synthesis of tert-butyl 4-[3-[bis(cyclopropanecarbonyl)amino]-6-[2-(methoxymethoxy)phenyl]pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Dissolve tert-butyl 4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylate (100 mg, 220.01 μmol) and triethylamine (66.79 mg, 660.04 μmol, 91.87 μL) in anhydrous dichloromethane (2 mL), and then add cyclopropylcarbonyl chloride (46.00 mg, 440.02 μmol, 40.00 μL). The reaction solution was stirred at 25°C for 2 hours. The reaction was completed by thin layer chromatography. Water (5 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (10 mL). The organic phase was concentrated under reduced pressure and purified by thin layer chromatography (silica, petroleum ether:tetrahydrofuran = 1:1) to give tert-butyl 4-[3-[bis(cyclopropanecarbonyl)amino]-6-[2-(methoxymethoxy)phenyl]pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate (100 mg).

Step 2: Synthesis of N-[6-(2-hydroxyphenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-yl]cyclopropanecarboxamide

4-[3-[bis(cyclopropanecarbonyl)amino]-6-[2-(methoxymethoxy)phenyl]pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylic acid tert-butyl ester (50 mg, 84.65 μmol) was dissolved in anhydrous methanol (1 mL), sodium hydroxide solution (4M, 84.65 μL) was added, the reaction solution was stirred at 25°C for 2 hours, and the reaction solution was concentrated to dryness under reduced pressure. Then anhydrous methanol (1 mL) and dioxane hydrochloride (0.5 mL, 4 M) were added. The reaction solution was stirred at 25°C for 2 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex Luna column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content 0%-39%) as eluent) gave N-[6-(2-hydroxyphenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-yl]cyclopropanecarboxamide (22a) (2.02 mg).

MS m/z(ESI):379.1[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.13 (s, 1H), 11.10 (s, 1H), 8.76 (s, 1H), 8.12 (d, J = 8.3Hz, 1H), 7.45-7.29 ( m,1H),7.07-7.02(m,2H),4.94(t,J＝11.0Hz,1H),3.25(d,J＝12.4Hz,2H),2.83(t,J＝11.2Hz,2H), 2.12-1.96(m,6H),0.93-0.84(m,4H).

Example 23: Synthesis of 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]-6-chlorophenol (23a)

Step 1: Synthesis of tert-butyl 4-[3-amino-6-(3-chloro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Tert-butyl 4-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylate (50 mg, 141.72 μmol) and 3-chloro-2-hydroxyphenylboronic acid (24.43 mg, 141.72 μmol) were dissolved in anhydrous dioxane (0.8 mL) and water (0.2 mL), and cesium carbonate (92.35 mg, 283.44 μmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (11.57 mg, 14.17 μmol) were added. The reaction solution was stirred at 90° C. for 2 hours under a nitrogen atmosphere. The reaction was completed by LCMS detection. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether:tetrahydrofuran = 2:1) to give tert-butyl 4-[3-amino-6-(3-chloro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate (35 mg).

MS m/z(ESI):445.1[M+H] ⁺ .

Step 2: Synthesis of 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]-6-chlorophenol

Dissolve tert-butyl 4-[3-amino-6-(3-chloro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylate (20 mg, 44.95 μmol) in anhydrous methanol (1 mL), and add dioxane hydrochloride (4 M, 112.38 μL). Stir the reaction solution at 25° C. for 3 hours. LCMS detection shows that the reaction is complete. The reaction solution was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (Phenomenex Gemini C18 column: 5 μm silica, 30 mm diameter, 80 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 30%-50%) as eluent) to give 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]-6-chlorophenol (23a) (2.41 mg).

MS m/z (ESI): 345.2 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 8.64 (s, 1H), 8.20-8.08 (m, 1H), 7.52 (dd, J = 1.3, 7.8Hz, 1H), 6.98 (t, J = 7.9 Hz,1H),6.61-6.44(m,2H),4.63(br s,1H),3.10(br s,2H),2.67(br d,J＝1.8Hz,2H),2.02-1.88(m,2H ),1.87-1.76(m,2H).

Example 24: Synthesis of N-[6-(3-chloro-2-hydroxyphenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-yl]cyclopropanecarboxamide (24a)

Dissolve tert-butyl 4-[3-amino-6-(3-chloro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylate (150 mg, 337.14 μmol), cyclopropylcarbonyl chloride (140.97 mg, 1.35 mmol, 122.59 μL) and triethylamine (102.35 mg, 1.01 mmol, 140.78 μL) in anhydrous dichloromethane (4 mL). The reaction solution was stirred at 25°C for 2 hours, and the reaction was complete after thin layer chromatography (petroleum ether: tetrahydrofuran = 1:1). The reaction solution was extracted with water (5 mL) and ethyl acetate (10 mL), and the organic phase was concentrated to dryness under reduced pressure. Then it was dissolved in anhydrous methanol (2 mL) and water (2 mL), and sodium hydroxide solution (4 M, 0.1 mL) was added. The reaction solution was stirred at 25°C for 2 hours, and the reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (37%-77%) as eluent) gave N-[6-(3-chloro-2-hydroxyphenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-yl]cyclopropanecarboxamide (24a) (50 mg).

MS m/z(ESI):413.1[M+H] ⁺ .

Example 25: Synthesis of 2-[3-(cyclopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (25a)

Step 1: Synthesis of tert-butyl 4-[3-iodo-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Tert-butyl 4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylate (100 mg, 220.01 μmol) was dissolved in dichloromethane (5 mL), iodine (139.60 mg, 550.03 μmol) was added, nitrogen was replaced, and tert-butyl nitrite (45.38 mg, 440.02 μmol) was added. The reaction solution was reacted at 25 ° C for 2 hours, and the reaction was completed by LCMS detection. The reaction solution was quenched with saturated sodium sulfite solution (2 mL), dichloromethane was removed in vacuo, and water (5 mL) was added for dilution. The reaction mixture was extracted with ethyl acetate (15 mL×3), and the organic phases were combined, dried, concentrated, and purified by column chromatography (silica, petroleum ether/ethyl acetate = 1/1) to give tert-butyl 4-[3-iodo-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate (120 mg).

MS m/z(ESI):566.0[M+H] ⁺ ;

Step 2: Synthesis of tert-butyl 4-(3-(cyclopropylamino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate

4-[3-iodo-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylic acid tert-butyl ester (110 mg, 194.55 μmol) was dissolved in dimethyl sulfoxide (6 mL), and cuprous iodide (18.53 mg, 97.28 μmol), potassium phosphate (103.24 mg, 486.38 μmol) and L-proline (22.40 mg, 194.55 μmol) were added, nitrogen was replaced, and cyclopropylamine (77.75 mg, 1.36 mmol) was added. The reaction solution was reacted at 80° C. for 2 hours, and the reaction was completed by LCMS detection. The reaction solution was cooled to room temperature, diluted with water (10 mL), extracted with ethyl acetate (15 mL×3), the organic phases were combined, dried, concentrated, and purified by column chromatography (silica, petroleum ether/ethyl acetate=1/1) to give tert-butyl 4-(3-(cyclopropylamino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate (80 mg).

MS m/z(ESI):495.1[M+H] ⁺ .

Step 3: Synthesis of 2-[3-(cyclopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-(3-(cyclopropylamino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylic acid tert-butyl ester (40 mg, 80.88 μmol) was dissolved in methanol (1 mL), and hydrochloric acid/dioxane (202.19 μL, 808.76 μmol) was added. The reaction solution was reacted at 25° C. for 12 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated and purified by preparative liquid chromatography (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 47%-87%) as eluent) to give 2-[3-(cyclopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (25a) (15 mg).

MS m/z(ESI):351.1[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.97 (br s, 1H), 8.50 (s, 1H), 8.13 (d, J = 7.3Hz, 1H), 7.42-7.33 (m, 2H), 7.04 -6.97(m,2H),4.70-4.58(m,1H),3.53-3.51(m,1H),3.10(br d,J＝12.4Hz,2H),2.80(qt,J＝3.4,6.8Hz, 1H),2.69-2.62(m,2H),2.03(dq,J＝4.1,12.1Hz,2H),1.81(br d,J＝9.8Hz,2H),0.78-0.69(m,2H),0.63-0.56(m,2H).

Example 26: Synthesis of 2-[1-(piperidin-4-yl)-3-[(2,2,2-trifluoroethyl)amino]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (26a)

Step 1: Synthesis of tert-butyl 4-(3-((tert-butyloxycarbonyl)(2,2,2-trifluoroethyl)amino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate

4-(3-((tert-butyloxycarbonyl)amino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylic acid tert-butyl ester (80 mg, 144.24 μmol) was dissolved in tetrahydrofuran (7 mL), sodium hydrogen sulfide (28.84 mg, 721.19 μmol, 60% purity) was added, the reaction solution was stirred at 25° C. for 0.5 hours, and trifluoroethyl trifluoromethanesulfonate (133.91 mg, 576.95 μmol) was added. The reaction solution was reacted at 25° C. for 11.5 hours, and the reaction was completed by LCMS detection. The reaction solution was quenched with water (10 mL), extracted with ethyl acetate (15 mL×3), the organic phases were combined, dried, concentrated, and purified by column chromatography (silica, petroleum ether/ethyl acetate=1/1) to give tert-butyl 4-(3-((tert-butoxycarbonyl)(2,2,2-trifluoroethyl)amino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate (75 mg).

MS m/z(ESI):637.2[M+H] ⁺ .

Step 2: Synthesis of 2-[1-(piperidin-4-yl)-3-[(2,2,2-trifluoroethyl)amino]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-(3-((tert-butyloxycarbonyl)(2,2,2-trifluoroethyl)amino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylic acid tert-butyl ester (75 mg, 117.80 μmol) was dissolved in methanol (2 mL), and 4M hydrochloric acid/dioxane (294.51 μL, 1.18 mmol) was added. The reaction solution was reacted at 25° C. for 12 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated and purified by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia water) and acetonitrile (acetonitrile content 33%-73%) as eluent) to give 2-[1-(piperidin-4-yl)-3-[(2,2,2-trifluoroethyl)amino]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (26a) (20 mg).

MS m/z(ESI):393.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.65 (br s, 1H), 8.56 (s, 1H), 8.13 (dd, J = 1.4, 8.3Hz, 1H), 7.83 (t, J = 6.7Hz ,1H),7.41-7.34(m,1H),7.05-6.99(m,2H),4.71-4.62(m,1H),4.32-4.20(m,2H),3.31-3.18(m,1H),3.09 (br d,J＝12.0Hz,2H),2.68-2.61(m,2H),1.97(dq,J＝3.9,12.0Hz,2H),1.80(br d,J＝11.0Hz,2H).

Example 27: Synthesis of 2-[1-(8-azabicyclo[3.2.1]octan-3-yl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (27a)

Step 1: Synthesis of tert-butyl 3-methylsulfonyloxy-8-azabicyclo[3.2.1]octane-8-carboxylate

Dissolve tert-butyl 3-hydroxy-8-azacycloheptene [3.2.1] octane-8-carboxylate (3g, 13.20mmol) and methanesulfonic anhydride (3.45g, 19.80mmol) in tetrahydrofuran (60mL), and add pyridine (2.09g, 26.40mmol). The reaction solution was stirred at 25°C for 2 hours. The reaction was monitored by thin layer chromatography (petroleum ether: tetrahydrofuran = 3:1). Water (50mL) was added to the reaction solution and extracted three times with ethyl acetate (50mL), and the organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure to obtain tert-butyl 3-methylsulfonyloxy-8-azabicyclo [3.2.1] octane-8-carboxylate (3.79g).

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 5.04-4.90 (m, 1H), 4.08 (br d, J = 15.8Hz, 2H), 3.19 (d, J = 17.0Hz, 3H), 2.06 (br s,2H),1.96(br s,4H),1.75-1.69(m,1H),1.67-1.54(m,1H),1.40(d,J=6.1Hz,9H).

Step 2: Synthesis of tert-butyl 3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate

3-Methylsulfonyloxy-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (4.9 g, 16.05 mmol) was dissolved in N,N-dimethylformamide (80 mL) and sodium iodide (3.01 g, 20.06 mmol) was added. The reaction solution was stirred at 25 ° C for 1.5 hours. 6-Chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (2.27 g, 13.37 mmol) and cesium carbonate (13.07 g, 40.11 mmol) were added to the reaction solution. The reaction solution was stirred at 80 ° C for 10.5 hours. LCMS monitored the reaction to be complete. Water (100 mL) was added to the reaction solution and extracted three times with ethyl acetate (100 mL), and the organic phase was dried over anhydrous sodium sulfate. Purification by column chromatography (petroleum ether:tetrahydrofuran=1:1) gave tert-butyl 3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (862 mg).

MS m/z(ESI):379.2[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 3-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylate

Tert-butyl 3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)-8-azabicyclo[3.2.1]octane-8-carboxylate (200 mg, 527.91 μmol) and 2-(methoxymethoxy)phenylboronic acid (115.28 mg, 633.49 μmol) were dissolved in dioxane (4 mL) and water (1 mL), and potassium carbonate (218.88 mg, 1.58 mmol) and tetrakis(triphenylphosphine)palladium (61.00 mg, 52.79 μmol) were added. The reaction solution was stirred at 100°C for 12 hours. The reaction was complete when monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure and then purified by column chromatography (silica: petroleum ether/tetrahydrofuran = 1/1) to give tert-butyl 3-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylate (210 mg).

MS m/z(ESI):481.5[M+H] ⁺ .

Step 4: Synthesis of tert-butyl 3-[6-[2-(methoxymethoxy)phenyl]-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylate

3-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazine-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (100 mg, 208.09 μmol) and methylboronic acid (9.97 mg, 166.47 μmol) were dissolved in anhydrous dioxane (5 mL), and then pyridine (49.38 mg, 624.27 μmol) and copper acetate (75.59 mg, 416.18 μmol) were added. The reaction solution was stirred at 100 ° C for 12 hours under oxygen protection. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by thin layer chromatography (silica: petroleum ether/tetrahydrofuran = 1/1) to give tert-butyl 3-[6-[2-(methoxymethoxy)phenyl]-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylate (20 mg).

MS m/z(ESI):495.8[M+H] ⁺ .

Step 5: Synthesis of 2-[1-(8-azabicyclo[3.2.1]octan-3-yl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

3-[6-[2-(methoxymethoxy)phenyl]-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (18 mg, 36.39 μmol) was dissolved in anhydrous dioxane (1 mL), and then dioxane hydrochloride (4M, 90.99 μL) was added. The reaction solution was stirred at 25°C for 12 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain 2-[1-(8-azabicyclo[3.2.1]octane-3-yl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (27a) (10 mg).

MS m/z(ESI):351.1[M+H] ⁺ .

Example 28: Synthesis of 2-chloro-6-[3-(isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (28a)

Step 1: Synthesis of tert-butyl 4-[3-amino-6-(3-chloro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Tert-butyl 4-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylate (978 mg, 2.27 mmol, 82% purity) and 3-chloro-2-hydroxyphenylboronic acid (470 mg, 2.73 mmol) were dissolved in dioxane (20 mL) and water (5 mL), and cesium carbonate (1.48 g, 4.54 mmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (186 mg, 227 μmol) were added under nitrogen atmosphere. The reaction solution was stirred at 100 ° C for 3 hours. LCMS monitored the completion of the reaction. The reaction solution was concentrated under reduced pressure, diluted with water (30 mL) and dichloromethane (30 mL), the aqueous phase was extracted twice with dichloromethane (30 mL), and the organic phase was washed twice with water (30 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography (silica, petroleum ether/ethyl acetate = 100/40) to give the title compound (320 mg).

MS m/z(ESI):445.2[M+H] ⁺ ;

Step 2: Synthesis of tert-butyl 4-[6-(3-chloro-2-hydroxyphenyl)-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

4-[3-amino-6-(3-chloro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylic acid tert-butyl ester (200 mg, 450 μmol) and acetone (78.3 mg, 1.35 mmol) were dissolved in methanol (4 mL), acetic acid (81.0 mg, 1.35 mmol) was added, and the reaction was stirred at 60 ° C for 2 hours, and then sodium cyanoborohydride (141 mg, 2.25 mmol) was added. The reaction was stirred at 60 ° C for 16 hours, and LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure and purified by column chromatography (silicon dioxide, petroleum ether/tetrahydrofuran = 100/17) to give the title compound (77 mg).

MS m/z(ESI):487.2[M+H] ⁺ .

Step 3: Synthesis of 2-chloro-6-[3-(isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[6-(3-chloro-2-hydroxyphenyl)-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylic acid tert-butyl ester (77 mg, 158 μmol) was dissolved in dichloromethane (2 mL), and a hydrochloric acid dioxane solution (4M, 791 μL) was added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure to give 2-chloro-6-[3-(isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (60 mg, crude product), which was used directly in the next step.

MS m/z(ESI):387.1[M+H] ⁺ .

Example 29: Synthesis of 2-fluoro-6-[3-(isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (29a)

Step 1: Synthesis of tert-butyl 4-[6-chloro-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Tert-butyl 4-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylate (1g, 2.83mmol) and acetone (1.65g, 28.34mmol) were dissolved in methanol (25mL), and acetic acid (510.62mg, 8.50mmol) and sodium cyanoborohydride (534.34mg, 8.50mmol) were added. The reaction solution was stirred at 25°C for 24 hours. The reaction was complete as monitored by LCMS. Water (25mL) was added to the reaction solution and extracted with ethyl acetate (25mL×3), and the organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure and then purified by column chromatography (petroleum ether/tetrahydrofuran=5/1) to obtain the title compound (524mg).

MS m/z(ESI):395.1[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-[6-(3-fluoro-2-hydroxyphenyl)-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Tert-butyl 4-[6-chloro-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylate (100 mg, 253.23 μmol) and 3-fluoro-2-hydroxyphenylboronic acid (47.38 mg, 303.88 μmol) were dissolved in dioxane (4 mL) and water (1 mL), and cesium carbonate (165.01 mg, 506.46 μmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (20.68 mg, 25.32 μmol) were added. The reaction solution was stirred at 90 ° C for 2 hours under a nitrogen atmosphere. The reaction was complete when monitored by LCMS. Water (5 mL) was added to the reaction solution and extracted with ethyl acetate (5 mL×3), and the organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure and purified by column chromatography (petroleum ether/tetrahydrofuran = 2/1) to give the title compound (98 mg).

MS m/z(ESI):471.2[M+H] ⁺ .

Step 3: Synthesis of 2-fluoro-6-[3-(isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[6-(3-fluoro-2-hydroxyphenyl)-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylic acid tert-butyl ester (98 mg, 208.27 μmol) was dissolved in dichloromethane (1 mL), and dioxane hydrochloride (4M, 520.68 μL) was added. The reaction solution was stirred at 25°C for 2 hours. LCMS monitored the reaction to be complete. The reaction solution was concentrated to dryness under reduced pressure to obtain 2-fluoro-6-[3-(isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (70 mg).

MS m/z(ESI):371.1[M+H] ⁺ .

Example 30: Synthesis of 4-fluoro-2-(3-(isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (30a)

Step 1: Synthesis of tert-butyl 4-[6-(5-fluoro-2-hydroxyphenyl)-3-(isopropylamino)pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Tert-butyl 4-[6-chloro-3-(isopropylamino)pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylate (100 mg, 253.23 μmol) and 5-fluoro-2-hydroxyphenylboronic acid (47.38 mg, 303.88 μmol) were dissolved in dioxane (4 mL) and water (1 mL), and cesium carbonate (165.01 mg, 506.46 μmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium(II) dichloromethane complex (20.68 mg, 25.32 μmol) were added. The reaction solution was stirred at 90°C for 2 hours under nitrogen atmosphere. The reaction was complete when monitored by LCMS. Water (5 mL) was added to the reaction solution and extracted three times with ethyl acetate (25 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated to dryness under reduced pressure, and purified by column chromatography (petroleum ether/tetrahydrofuran = 2/1) to give the title compound (77 mg).

MS m/z(ESI):471.2[M+H] ⁺ .

Step 2: Synthesis of 4-fluoro-2-(3-(isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

Dissolve 4-[6-(5-fluoro-2-hydroxy-phenyl)-3-(isopropylamino)pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylic acid tert-butyl ester (77 mg, 163.64 μmol) in dichloromethane (2 mL), add dioxane hydrochloride (4M, 409.11 μL). The reaction solution was stirred at 25°C for 2 hours. The reaction was monitored to be complete by thin layer chromatography (petroleum ether: tetrahydrofuran = 2:1). The reaction solution was concentrated to dryness under reduced pressure to obtain 4-fluoro-2-(3-(isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (60 mg).

MS m/z(ESI):371.2[M+H] ⁺ .

Example 31: Synthesis of 2-[3-[(2-hydroxy-2-methylpropyl)amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (31a)

Step 1: Synthesis of tert-butyl 4-[3-[(2-hydroxy-2-methylpropyl)amino]-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

4-[3-iodo-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylic acid tert-butyl ester (200 mg, 353.73 μmol) was dissolved in dimethyl sulfoxide (4 mL), and cuprous iodide (33.68 mg, 176.87 μmol), potassium phosphate (187.71 mg, 884.33 μmol), L-proline (40.72 mg, 353.73 mg) and 1-amino-2-methyl-2-propanol (189.18 mg, 2.12 mmol) were added. The reaction solution was stirred at 80 ° C for 12 hours under a nitrogen atmosphere. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silicon dioxide, petroleum ether: tetrahydrofuran = 1: 1) to obtain the title compound (200 mg).

MS m/z(ESI):527.2[M+H] ⁺ .

Step 2: Synthesis of 2-[3-[(2-hydroxy-2-methylpropyl)amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[3-[(2-hydroxy-2-methylpropyl)amino]-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylic acid tert-butyl ester (200 mg, 379.78 μmol) was dissolved in anhydrous methanol (3 mL), and dioxane hydrochloride (4M, 949.44 μL) was added. The reaction solution was stirred at 25° C. for 3 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated to dryness under reduced pressure to obtain 2-[3-[(2-hydroxy-2-methylpropyl)amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (200 mg).

MS m/z(ESI):383.1[M+H] ⁺ .

Example 32: Synthesis of 2-fluoro-6-[1-(piperidin-4-yl)-3-propylamino-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (32a)

Step 1: Synthesis of tert-butyl 4-[3-amino-6-(3-fluoro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Tert-butyl 4-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylate (373.37 mg, 1.06 mmol) and 3-fluoro-2-hydroxyphenylboronic acid (165 mg, 1.06 mmol) were dissolved in dioxane (12 mL) and water (3 mL), and cesium carbonate (689.59 mg, 2.12 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (86.42 mg, 105.82 μmol) were added. The reaction solution was stirred at 80°C under nitrogen for 12 hours. The reaction was complete by LCMS monitoring. Water (15 mL) was added to the reaction solution and extracted with ethyl acetate (15 mL×3), and the organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure and purified by column chromatography (petroleum ether/tetrahydrofuran = 1/1) to give the title compound (260 mg).

MS m/z(ESI):429.1[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-[6-(3-fluoro-2-hydroxyphenyl)-3-propylamino-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Tert-butyl 4-[3-amino-6-(3-fluoro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylate (140 mg, 326.75 μmol) was dissolved in methanol (1.5 mL), and acetic acid (58.87 mg, 980.26 μmol) was added. Propionaldehyde (1.65 g, 28.34 mmol) was dissolved in methanol (1.5 mL) and added to the reaction solution. The reaction solution was stirred at 25°C for 0.5 hours, and then sodium cyanoborohydride (61.60 mg, 980.26 μmol) was added. The reaction solution was stirred at 25°C for 12 hours. The reaction was complete as monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure, and then purified by column chromatography (petroleum ether/tetrahydrofuran = 1/1) to obtain the title compound (104 mg).

MS m/z(ESI):471.2[M+H] ⁺ .

Step 3: Synthesis of 2-fluoro-6-[1-(piperidin-4-yl)-3-propylamino-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[6-(3-fluoro-2-hydroxyphenyl)-3-propylamino-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylic acid tert-butyl ester (104 mg, 221.02 μmol) was dissolved in dichloromethane (1.5 mL), and dioxane hydrochloride (4M, 1.11 mL) was added. The reaction solution was stirred at 25°C for 2 hours. LCMS monitored the reaction to be complete. The reaction solution was concentrated to dryness under reduced pressure to obtain 2-fluoro-6-[1-(piperidin-4-yl)-3-propylamino-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (78 mg).

MS m/z(ESI):371.6[M+H] ⁺ .

Example 33: Synthesis of 2-fluoro-6-[3-[(2-hydroxy-2-methylpropyl)amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (33a)

Step 1: Synthesis of tert-butyl 4-[3-amino-6-(3-fluoro-2-methoxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Tert-butyl 4-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylate (943.67 mg, 2.67 mmol) and 3-fluoro-2-methoxyphenylboronic acid (500 mg, 2.9 mmol) were dissolved in dioxane (20 mL) and water (5 mL), and cesium carbonate (1.74 g, 5.35 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (218.42 mg, 267.47 μmol) were added. The reaction solution was stirred at 80 ° C for 12 hours under nitrogen environment. LCMS monitored the reaction to be complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (petroleum ether/tetrahydrofuran = 1/1) to obtain the title compound (98 mg).

MS m/z(ESI):443.2[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-(6-(3-fluoro-2-methoxyphenyl)-3-iodo-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate

Dissolve tert-butyl 4-[3-amino-6-(3-fluoro-2-methoxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylate (600 mg, 1.36 mmol) in dichloromethane (15 mL), add iodine (860.39 mg, 3.39 mmol), and add tert-butyl nitrite (279.66 mg, 2.71 mmol) at 0 ° C under nitrogen protection. The reaction solution was stirred at 25 ° C for 2 hours under nitrogen environment. LCMS monitored the reaction to be complete. Saturated sodium sulfite was added to the reaction solution, extracted with ethyl acetate (15 mL×3), and purified by column chromatography (petroleum ether/ethyl acetate=1/1) to obtain the title compound (612 mg).

MS m/z(ESI):554.1[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-[6-(3-fluoro-2-methoxyphenyl)-3-[(2-hydroxy-2-methylpropyl)amino]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Tert-butyl 4-(6-(3-fluoro-2-methoxyphenyl)-3-iodo-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylate (610 mg, 1.10 mmol) and 1-amino-2-methyl-2-propanol (589.55 mg, 6.61 mmol) were dissolved in dimethyl sulfoxide (12 mL), and cuprous iodide (104.97 mg, 551.17 μmol), potassium phosphate (584.97 mg, 2.76 mmol) and L-proline (126.91 mg, 1.10 mmol) were added. The reaction solution was stirred at 80° C. under nitrogen atmosphere for 12 hours. The reaction was complete when monitored by LCMS. Water (12 mL) was added to the reaction solution and extracted with ethyl acetate (12 mL×3). The organic phase was concentrated to dryness under reduced pressure and purified by column chromatography (petroleum ether/tetrahydrofuran=1/1) to give the title compound (465 mg).

MS m/z(ESI):515.2[M+H] ⁺ .

Step 4: Synthesis of 2-fluoro-6-[3-[(2-hydroxy-2-methylpropyl)amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[6-(3-fluoro-2-methoxyphenyl)-3-[(2-hydroxy-2-methylpropyl)amino]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylic acid tert-butyl ester (60 mg, 116.60 μmol) was dissolved in dichloromethane (0.5 mL), and boron tribromide (29.21 mg, 116.60 μmol) was added at 0°C. The reaction solution was stirred at 25°C for 2 hours. LCMS monitored the reaction to be complete. The mixture was quenched in an ice-water bath, saturated sodium bicarbonate was added to adjust the pH to alkaline, dichloromethane/methanol = 10/1 was added for extraction, the organic phase was concentrated to dryness under reduced pressure, and purified by thin layer chromatography (petroleum ether/tetrahydrofuran = 1/3) to give 2-fluoro-6-[3-[(2-hydroxy-2-methylpropyl)amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (25 mg).

MS m/z(ESI):401.1[M+H] ⁺ .

Example 34: Synthesis of 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]-6-fluorophenol (34a)

4-[3-amino-6-(3-fluoro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylic acid tert-butyl ester (300.00 mg, 700.18 μmol) was dissolved in anhydrous methanol (5 mL), and dioxane hydrochloride (4M, 1.75 mL) was added. The reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]-6-fluorophenol (278.4 mg).

MS m/z(ESI):329.1[M+H] ⁺ .

Example 35: Synthesis of 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]-4-fluorophenol (35a) hydrochloride

Synthesis route:

Step 1: Synthesis of tert-butyl 4-[3-amino-6-(5-fluoro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Tert-butyl 4-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylate (100 mg, 283.43 μmol) and 5-fluoro-2-hydroxyphenylboronic acid (48.61 mg, 311.78 μmol) were dissolved in dioxane (3 mL) and water (1 mL), and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (23.15 mg, 28.34 μmol) and cesium carbonate (184.70 mg, 566.86 μmol) were added. The reaction solution was stirred at 80°C under a nitrogen atmosphere for 2 hours. The reaction was completed by LCMS detection. Add water (6 mL), extract with dichloromethane (20 mL×3), dry the organic layer over anhydrous sodium sulfate, filter, concentrate the filtrate to dryness under reduced pressure, and purify by column chromatography (silica, petroleum ether:tetrahydrofuran=1:1) to obtain the title compound (61 mg).

MS m/z(ESI):429.1[M+H] ⁺ .

Step 2: Synthesis of 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]-4-fluorophenol hydrochloride

4-[3-amino-6-(5-fluoro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylic acid tert-butyl ester (60 mg, 140.04 μmol) was dissolved in anhydrous dichloromethane (2 mL), and dioxane hydrochloride (4M, 385.26 μL) was added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated under reduced pressure to dryness to obtain 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]-4-fluorophenol hydrochloride (56.9 mg, crude product).

MS m/z(ESI):329.0[M+H] ⁺ .

Example 36: Synthesis of 4-fluoro-2-[3-[(2-hydroxy-2-methylpropyl)amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (36a)

Step 1: Synthesis of tert-butyl 4-[3-amino-6-[5-fluoro-2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Tert-butyl 4-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate (300 mg, 850.30 μmol) and 5-fluoro-2-(methoxymethoxy)phenylboronic acid (204.04 mg, 1.02 mmol) were dissolved in anhydrous dioxane (4 mL) and water (1 mL), and cesium carbonate (554.09 mg, 1.70 mmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (69.44 mg, 85.03 μmol) were added. The reaction solution was stirred at 100° C. under a nitrogen atmosphere for 2 hours. The reaction was completed by LCMS monitoring. The reaction solution was diluted with water (10 mL) and extracted with ethyl acetate (30 mL×3). The organic phase was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether: ethyl acetate = 3:2) to give the title compound (365.5 mg).

MS m/z(ESI):473.1[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-[6-[5-fluoro-2-(methoxymethoxy)phenyl]-3-iodo-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Tert-butyl 4-[3-amino-6-[5-fluoro-2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylate (335 mg, 708.98 μmol) was dissolved in anhydrous dichloromethane (10 mL), and iodine (449.86 mg, 1.77 mmol, 357.03 μL) and tert-butyl nitrite (146.22 mg, 1.42 mmol, 168.65 μL) were added. The reaction solution was stirred at 25 ° C for 2 hours under a nitrogen atmosphere. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, diluted with water (15 mL) and extracted with ethyl acetate (45 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. Purification by column chromatography (silicon dioxide, petroleum ether: ethyl acetate = 3:2) gave the title compound (236.4 mg).

MS m/z(ESI):584.1[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-[6-[5-fluoro-2-(methoxymethoxy)phenyl]-3-[(2-hydroxy-2-methylpropyl)amino]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

4-[6-[5-fluoro-2-(methoxymethoxy)phenyl]-3-iodo-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylic acid tert-butyl ester (236.4 mg, 405.22 μmol) was dissolved in dimethyl sulfoxide (2 mL), and cuprous iodide (38.59 mg, 202.61 μmol), potassium phosphate (215.03 mg, 1.01 mmol), L-proline (46.65 mg, 405.22 μmol) and 1-amino-2-methyl-2-propanol (216.72 mg, 2.43 mmol) were added. The reaction solution was stirred at 80° C. under a nitrogen atmosphere for 16 hours. LCMS detected that the reaction was complete, and the reaction solution was diluted with water (15 mL) and extracted three times with dichloromethane (45 mL). The organic phase was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (silicon dioxide, petroleum ether:tetrahydrofuran = 4:1) to give the title compound (81.2 mg).

MS m/z(ESI):545.2[M+H] ⁺ .

Step 4: Synthesis of 4-fluoro-2-[3-[(2-hydroxy-2-methylpropyl)amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[6-[5-fluoro-2-(methoxymethoxy)phenyl]-3-[(2-hydroxy-2-methylpropyl)amino]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylic acid tert-butyl ester (81.2 mg, 124.94 μmol) was dissolved in anhydrous methanol (1 mL), and dioxane hydrochloride (4M, 312.36 μL) was added, and the reaction solution was stirred at 25°C for 2 hours. LCMS monitored the completion of the reaction. The reaction solution was concentrated to dryness under reduced pressure to obtain 4-fluoro-2-[3-[(2-hydroxy-2-methylpropyl)amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (91.3 mg).

MS m/z(ESI):401.2[M+H] ⁺ .

Example 37: Synthesis of 5-fluoro-2-(3-((2-hydroxy-2-methylpropyl)amino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (37a)

Step 1: Synthesis of benzyl 4-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate

Dissolve 6-chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (5 g, 24.27 mmol) in N,N-dimethylformamide (100 mL), add cesium carbonate (23.73 g, 72.80 mmol). After stirring for 20 minutes, add 4-methanesulfonyloxypiperidine-1-carboxylic acid benzyl ester (15.21 g, 48.54 mmol), and heat to 80 ° C for 4 hours. After the reaction is complete as monitored by LC-MS, add water (100 mL), extract with ethyl acetate (150 mL × 3), combine the organic phases, dry, filter, concentrate the filtrate, and purify by column chromatography (silica, petroleum ether/ethyl acetate: 1/1) to obtain the title compound (7.3 g).

MS m/z(ESI):387.10[M+H] ⁺ .

Step 2: Synthesis of benzyl 4-(6-chloro-3-iodopyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate

Benzyl 4-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate (4.6 g, 11.89 mmol) and iodine (2.11 g, 8.32 mmol) were dissolved in dichloromethane (150 mL); tert-butyl nitrite (2.45 g, 23.78 mmol) was slowly added under stirring, and the mixture was reacted at room temperature for 1 hour; after the reaction was completed as monitored by LC-MS, dichloromethane (50 mL) was added to dilute the solution, and the reaction was quenched with saturated sodium sulfite solution (50 mL); after separation, the organic phase was concentrated and purified by column chromatography (silica, petroleum ether/ethyl acetate: 1/1) to obtain the title compound (3 g).

MS m/z(ESI):498.00[M+H] ⁺ .

Step 3: Synthesis of benzyl 4-(6-(4-fluoro-2-hydroxyphenyl)-3-((2-hydroxy-2-methylpropyl)amino)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate

Dissolve benzyl 4-(6-chloro-3-iodopyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate (3 g, 6.03 mmol), cuprous iodide (573.97 mg, 3.01 mmol), L-proline (693.95 mg, 6.03 mmol) and potassium phosphate (3.19 g, 15.07 mmol) in ultra-dry dimethyl sulfoxide (120 mL). After replacing the reaction apparatus with nitrogen, 1-amino-2-methyl-2-propanol (3.22 g, 36.17 mmol) was added under stirring, and the temperature was raised to 80°C for reaction overnight; after the reaction was completed as monitored by LC-MS, water (80 mL) and ethyl acetate (120 mL) were added, and after separation, the aqueous phase was extracted with ethyl acetate (50 mL×3), and the organic phases were combined and washed with saturated sodium chloride solution (50 mL×3), and the organic phase was concentrated. The residue was purified by column chromatography (silica, dichloromethane/methanol: 20/1) to obtain the title compound (2.04 g).

MS m/z(ESI):459.10[M+H] ⁺ .

Step 4: Synthesis of benzyl 4-(6-(4-fluoro-2-hydroxyphenyl)-3-((2-hydroxy-2-methylpropyl)amino)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate

Benzyl 4-(6-chloro-3-((2-hydroxy-2-methylpropyl)amino)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate (250 mg, 0.55 mmol), 4-fluoro-2-hydroxyphenylboronic acid (343 mg, 2.2 mmol), potassium carbonate (700 mg, 5 mmol) and Pd(dppf)Cl ₂ (55 mg, 0.07 mmol) were added to dioxane (10 mL) and water (2 mL), replaced with nitrogen three times, reacted at 85° C. for 4 h, and then concentrated under reduced pressure. Dichloromethane (20 mL×3) and water (20 mL) were added to the residue for extraction. The organic phases were combined and concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA=100%-0%) to obtain the title compound (190 mg).

MS m/z(ESI):535.20[M+H] ⁺ .

Step 5: Synthesis of 5-fluoro-2-(3-((2-hydroxy-2-methylpropyl)amino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

4-(6-(4-Fluoro-2-hydroxyphenyl)-3-((2-hydroxy-2-methylpropyl)amino)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylic acid benzyl ester (190 g, 0.36 mmol) was dissolved in methanol (10 mL), followed by the addition of 10% palladium on carbon (20 mg), hydrogen exchange three times, and then the reaction was continued in a hydrogen atmosphere until the raw material was completely consumed. The reaction solution was filtered and the filtrate was concentrated to give 5-fluoro-2-(3-((2-hydroxy-2-methylpropyl)amino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (90 mg).

MS m/z(ESI):401.10[M+H] ⁺ .

Example 38: Synthesis of (2S,4R)-1-[(2S)-2-[[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)acetamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 001)

Step 1: Synthesis of (S)-[1-(4-bromophenyl)-ethyl]-carbamic acid tert-butyl ester

(S)-1-(4-bromophenyl)ethylamine (30 g, 149.94 mmol) and Boc anhydride (36.00 g, 164.94 mmol) were dissolved in dichloromethane (300 mL), and triethylamine (30.35 g, 299.89 mmol) was added. The reaction solution was stirred at 25 ° C for 16 hours. LCMS detected that the reaction was complete. The reaction solution was extracted with 500 mL of dichloromethane and 200 mL of water, and the organic phase was washed with a saturated sodium chloride solution and concentrated to obtain compound (S)-[1-(4-bromophenyl)-ethyl]-carbamic acid tert-butyl ester (50 g).

MS m/z(ESI):244.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 7.49 (d, J = 8.5Hz, 2H), 7.42 (d, J = 7.8Hz, 1H), 7.24 (d, J = 8.3Hz, 2H), 4.57 (t, J=7.2Hz, 1H), 1.35 (s, 9H), 1.27 (d, J=7.0Hz, 3H).

Step 2: Synthesis of tert-butyl N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamate

(S)-[1-(4-bromophenyl)-ethyl]-carbamic acid tert-butyl ester (39.5g, 131.58mmol) and 4-methylthiazole (22.82g, 230.17mmol) were dissolved in N,N-dimethylformamide (50mL), and potassium acetate (22.59g, 230.17mmol) and palladium acetate (1.29g, 5.75mmol) were added under nitrogen atmosphere. The reaction solution was stirred at 90°C for 16 hours. LCMS detected that the reaction was complete. The reaction solution was extracted with 500mL of dichloromethane and 200mL of water, and the organic phase was washed with saturated sodium chloride solution and concentrated. The organic phase was purified by column chromatography (silicon dioxide, petroleum ether/ethyl acetate = 3/1) to obtain compound N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamic acid tert-butyl ester (30g).

MS m/z (ESI): 319.2 [M+H] ⁺ ;

¹ H NMR (400MHz, METHANOL-d ₄ ) δ = 8.88 (s, 1H), 7.43 (d, J = 1.8Hz, 4H), 4.74 (s, 1H), 1.44 (s, 9H), 1.42 (d, J=7.0Hz,3H).

Step 3: Synthesis of (1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethylamine

Dissolve tert-butyl N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamate (30 g, 94.21 mmol) in 1,4-dioxane (300 mL), and add hydrogen chloride 1,4-dioxane solution (4M, 235.53 mL). The reaction solution was stirred at 25°C for 16 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain compound (1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethylamine (30 g, crude product).

MS m/z(ESI):219.0[M+H] ⁺ ;

¹ H NMR (400MHz, METHANOL-d ₄ ) δ = 10.11 (s, 1H), 7.84-7.68 (m, 4H), 4.63 (q, J = 6.8Hz, 1H), 2.69 (s, 3H), 1.73 ( d,J＝6.8Hz,3H).

Step 4: Synthesis of tert-butyl (2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidine-1-carboxylate

Under nitrogen atmosphere at 0°C, (1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethylamine (24 g, 94.20 mmol) and (2S,4R)-1-Boc-4-hydroxyproline (23.96 g, 103.62 mmol) were dissolved in N,N-dimethylformamide (200 mL), and N,N-diisopropylethylamine (36.52 g, 282.60 mmol) and O-(7-azabenzotriazole-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (53.73 g, 141.30 mmol) were added. The reaction solution was stirred at 25°C for 3 hours. LCMS detected that the reaction was complete. 200 mL of water was added to the reaction solution for dilution, and the mixture was extracted three times with 450 mL of dichloromethane. The organic phases were combined and concentrated. Then, the residue was purified by column chromatography (silica, petroleum ether/ethyl acetate = 1/1) to give the compound (2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidine-1-carboxylic acid tert-butyl ester (31 g).

MS m/z(ESI):432.2[M+H] ⁺ ;

¹ H NMR (400MHz, METHANOL-d ₄ ) δ = 8.89 (s, 1H), 7.47-7.43 (m, 4H), 5.12-5.09 (m, 1H), 4.42-4.34 (m, 2H), 3.61-3.44 (m,2H),2.27-2.19(m,1H),2.25(s,3H),1.98-1.94(m,1H),1.53(d,J＝7.0Hz,3H),1.46-1.40(m,9H ).

Step 5: Synthesis of (2S,4R)-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

Dissolve (2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidine-1-carboxylic acid tert-butyl ester (31 g, 71.83 mmol) in 1,4-dioxane (400 mL), and add hydrogen chloride-dioxane solution (4M, 179.59 mL). The reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain compound (2S,4R)-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (27 g, crude product).

MS m/z(ESI):332.1[M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ = 9.98 (s, 1H), 9.04 (br d, J = 7.3Hz, 1H), 7.61-7.57 (m, 2H), 7.56-7.51 (m, 2H) ,5.18-5.06(m,1H),4.59-4.49(m,2H),3.42-3.35(m,1H),3.30-3.27(m,1H),2.62(s,3H),2.56-2.39(m, 1H), 2.03-1.89 (m, 1H), 1.54 (d, J = 7.0Hz, 3H).

Step 6: Synthesis of tert-butyl N-[(1S)-1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidine-1-carbonyl]-2,2-dimethylpropyl]carbamate

(2S,4R)-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (27 g, 81.46 mmol) and N-Boc-L-tert-leucine (18.84 g, 81.46 mmol) were dissolved in DMF (200 mL), and O-(7-azabenzotriazole-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (30.98 g, 81.46 mmol) and N,N-diisopropylethylamine (31.59 g, 244.39 mmol) were added. The reaction solution was stirred at 25°C for 3 hours. The reaction was completed by LCMS. The reaction solution was quenched by adding 100 mL of water, and the filtrate was filtered and concentrated to dryness under reduced pressure to obtain the compound N-[(1S)-1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidine-1-carbonyl]-2,2-dimethylpropyl]carbamic acid tert-butyl ester (30 g, crude product).

MS m/z(ESI):445.2[M+H-100] ⁺ ;

¹ H NMR (400MHz, METHANOL-d ₄ ) δ = 8.88 (s, 1H), 8.59 (br d, J = 7.5Hz, 1H), 7.47-7.43 (m, 2H), 7.43-7.40 (m, 2H) ,5.05-4.95(m,1H),4.59(t,J＝8.3Hz,1H),4.44(br s,1H),3.95-3.88(m,1H),3.86(br d,J＝11.0Hz,1H ),3.78-3.70(m,1H),2.48(s,3H),2.08-1.88(m,2H),1.51(d,J＝7.0Hz,3H),1.44(s,9H),1.02(s, 9H).

Step 7: Synthesis of (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

Dissolve tert-butyl N-[(1S)-1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidine-1-carbonyl]-2,2-dimethyl-propyl]carbamate (300 mg, 550.76 umol) in anhydrous dioxane (3 mL), and add dioxane hydrochloride (4M, 1.38 mL). The reaction solution turns light yellow and is stirred at 25°C for 3 hours. LCMS detection shows that the reaction is complete. Concentrate under reduced pressure to dryness to obtain a light yellow solid (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (280 mg).

Step 8: Synthesis of tert-butyl 2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]acetate

2-[3-amino-1-(4-piperidinyl)pyrazolo[4,3-c]pyridazin-6-yl]phenol (50 mg, 161.11 μmol) was dissolved in anhydrous dioxane (5 mL), and tert-butyl bromoacetate (157.12 mg, 805.53 μmol) and N,N-diisopropylethylamine (28.06 μL, 161.11 μmol) were added. The reaction solution was heated to 80°C and stirred for 1 hour. LCMS detected that the reaction was complete. The reaction solution was cooled to 25°C. The crude residue was concentrated under reduced pressure and purified by column chromatography (silica, petroleum ether: ethyl acetate = 1:2) to obtain tert-butyl 2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]acetate (22 mg).

MS m/z(ESI):425.2[M+H] ⁺ .

Step 9: Synthesis of 2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]acetic acid

Tert-butyl 2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazine-1-yl]-1-piperidinyl]acetate (17 mg, 40.05 μmol) was dissolved in anhydrous dichloromethane (0.5 mL) and trifluoroacetic acid (250 μL). The reaction solution was stirred at 25 ° C for 16 hours. LCMS detected that the reaction was complete. Concentrate under reduced pressure to give 2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazine-1-yl]-1-piperidinyl]acetic acid (13 mg).

MS m/z(ESI):369.1[M+H] ⁺ .

Step 10: Synthesis of (2S,4R)-1-[(2S)-2-[[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)acetamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

2-[4-[3-Amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]acetic acid (13 mg, 150.02 μmol) was dissolved in N,N-dimethylformamide (2 mL), and (2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl))phenyl]ethyl]pyrrolidine-2-carboxamide (16.98 mg, 35.29 μmol), O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (16.10 mg, 42.35 μmol) and triethylamine (14.74 μL, 105.87 μmol) were added. The reaction solution was stirred at 25°C for 1 hour. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and the crude residue was purified by preparative liquid chromatography (Phenomenex C18 column: 3 μm silica, 30 mm diameter, 75 mm length; using water (containing 0.05% ammonia) and acetonitrile with decreasing polarity mixtures (acetonitrile ratio: 30%-70%) as eluent) to obtain (2S,4R)-1-[(2S)-2-[[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)acetamide)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Compound 001) (2.18 mg).

MS m/z (ESI): 795.4 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.98 (s, 1H), 8.99 (s, 1H), 8.55-8.45 (m, 2H), 8.11 (br d, J = 7.3Hz, 1H), 7.85 (br d,J＝9.3Hz,1H),7.47-7.42(m,2H),7.40-7.34(m,3H),7.04-6.97(m,2H),6.57(s,2H),5.15(d, J＝2.8Hz,1H),4.90(br t,J＝7.2Hz,1H),4.64(br s,1H),4.55(br d,J＝9.8Hz,1H),4.47(t,J＝8.3Hz ,1H),4.29(br s,1H),3.58(br s,4H),3.17-3.09(m,1H),3.06-2.93(m,3H),2.44-2.36(m,2H),2.33(br s,1H),2.23- 2.04(m,3H),1.98-1.83(m,2H),1.80-1.70(m,1H),1.41(br d,J＝6.8Hz,3H),0.97(s,9H).

Example 39: Synthesis of 3-(4-(3-(4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)piperidin-1-yl)propyl)-9H-pyrido[2,3-b]indol-9-yl)piperidine-2,6-dione (Compound 002)

Step 1: Synthesis of N-(4-methoxybenzyl)-5-oxotetrahydrofuran-2-carboxamide

Tetrahydro-5-oxo-2-furancarboxylic acid (5g, 38.43mmol) was dissolved in dichloromethane (20mL), and oxalyl chloride (5.85g, 46.12mmol) and N,N-dimethylformamide (140.45mg, 1.92mmol) were added at 0°C. The reaction solution was stirred at 25°C for 2 hours. The reaction solution was concentrated to dryness under reduced pressure and dissolved in dichloromethane (20mL), and a solution of p-methoxybenzylamine (5.01g, 36.51mmol) and triethylamine (7.78g, 76.86mmol) in dichloromethane (20mL) was added. The reaction solution was reacted at 25°C for 1 hour. The reaction was complete as detected by LCMS. Water (200mL) was added to the reaction solution and washed twice, and the organic phase was concentrated to dryness under reduced pressure. Purification by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 1/1) gave N-(4-methoxybenzyl)-5-oxotetrahydrofuran-2-carboxamide (5.7 g).

MS m/z (ESI): 250.1 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.65 (br t, J＝5.4Hz, 1H), 7.26-7.11 (m, 2H), 6.95-6.82 (m, 2H), 4.89 (dd, J＝ 5.7,7.9Hz,1H),4.23(d,J＝5.9Hz,2H),3.73(s,3H),2.56-2.51(m,2H),2.45-2.37(m,1H),2.16-2.02(m ,1H).

Step 2: Synthesis of 3-hydroxy-1-(4-methoxybenzyl)piperidine-2,6-dione

N-(4-methoxybenzyl)-5-oxotetrahydrofuran-2-carboxamide (3g, 12.04mmol) was dissolved in tetrahydrofuran (30mL), and potassium tert-butoxide solution (1M, 12.28mL) was added at -70°C under nitrogen atmosphere, and the reaction solution was reacted at -40°C for 2 hours. The reaction was completed by thin layer chromatography. The reaction solution was poured into a cold saturated aqueous ammonium chloride solution (50mL), extracted three times with ethyl acetate (120mL), the organic phase was dried over anhydrous sodium sulfate and filtered, the filtrate was concentrated to dryness under reduced pressure, and purified by column chromatography (silicon dioxide, petroleum ether/ethyl acetate = 1/0 to 2/1) to obtain 3-hydroxy-1-(4-methoxybenzyl)piperidine-2,6-dione (1.67g).

¹ H NMR (400MHz, DMSO-d ₆ ) δ7.15 (d, J = 8.5 Hz, 2H), 6.90-6.80 (m, 2H), 5.83 (d, J = 5.0 Hz, 1H), 4.79-4.65 ( m,2H),4.32-4.21(m,1H),3.1(s,3H),2.78-2.65(m,2H),2.03(m,1H),1.89-1.74(m,1H).

Step 3: Synthesis of 1-(4-methoxybenzyl)-2,6-dioxopiperidin-3-yl trifluoromethanesulfonate

3-Hydroxy-1-(4-methoxybenzyl)piperidine-2,6-dione (500 mg, 2.01 mmol) was dissolved in dichloromethane (10 mL), and N,N-diisopropylethylamine (648.13 mg, 5.01 mmol) and trifluoromethanesulfonic anhydride (679.14 mg, 2.41 mmol) were added at 0°C. The reaction solution was reacted at 10°C for 1.5 hours. The reaction was completed by TLC. The reaction solution was washed with saturated sodium bicarbonate aqueous solution (10 mL), and the organic phase was concentrated to dryness under reduced pressure. 1-(4-methoxybenzyl)-2,6-dioxopiperidin-3-yl trifluoromethanesulfonate (600 mg) was obtained by column chromatography purification (silica, petroleum ether/ethyl acetate = 1/0 to 4/1).

¹ H NMR (400MHz, CDCl ₃ ) δ7.35 (d, J = 8.6 Hz, 2H), 6.82 (d, J = 8.6 Hz, 2H), 5.33-5.24 (m, 1H), 4.89 (s, 2H) ,3.78(s,3H),2.99(m,1H),2.73(m,1H),2.46-2.25(m,2H).

Step 4: Synthesis of 3-(4-bromo-9H-pyrido[2,3-b]indol-9-yl)-1-(4-methoxybenzyl)piperidine-2,6-dione

4-Bromo-9H-pyrido[2,3-b]indole (200 mg, 809.42 μmol) and octadecacrown-6 (42.79 mg, 161.88 μmol) were dissolved in tetrahydrofuran (10 mL), and sodium di(trimethylsilyl)amide tetrahydrofuran solution (1M, 1.21 mL) was added at -30°C under nitrogen. The reaction solution was stirred at -30°C for 1 hour. 1-(4-methoxybenzyl)-2,6-dioxopiperidin-3-yl trifluoromethanesulfonate (462.98 mg, 1.21 mmol) was added to the reaction solution. The reaction solution was stirred at -30°C for 2 hours. LCMS detected that most of the raw materials were reacted. The reaction solution was heated to 25°C and quenched with water (20 mL), and extracted three times with ethyl acetate (60 mL). The organic phase was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether/ethyl acetate = 1/0 to 4/1) to give 3-(4-bromo-9H-pyrido[2,3-b]indol-9-yl)-1-(4-methoxybenzyl)piperidine-2,6-dione (430 mg).

MS m/z(ESI):478.1[M+H] ⁺ .

Step 5: Synthesis of 3-(4-bromo-9H-pyrido[2,3-b]indol-9-yl)piperidine-2,6-dione

3-(4-bromo-9H-pyrido[2,3-b]indol-9-yl)-1-(4-methoxybenzyl)piperidine-2,6-dione (400 mg, 694.07 μmol) was dissolved in trifluoroacetic acid (8 mL), and trifluoromethanesulfonic acid (2.27 g, 15.10 mmol) was added. The reaction solution was reacted at 50°C for 12 hours. The reaction was complete as detected by LCMS. The reaction solution was concentrated under reduced pressure and saturated sodium bicarbonate aqueous solution (10 mL) was added to quench the reaction, and the mixture was extracted three times with ethyl acetate (15 mL). The organic phase was concentrated under reduced pressure to dryness and purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 4/1) to obtain 3-(4-bromo-9H-pyrido[2,3-b]indol-9-yl)piperidine-2,6-dione (210 mg).

MS m/z (ESI): 357.8 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ11.19 (s, 1H), 8.57 (d, J = 7.9Hz, 1H), 8.30 (d, J = 5.3Hz, 1H), 7.72 (br s, 1H ),7.63(br t,J＝7.5Hz,1H),7.56(d,J＝5.3Hz,1H),7.41(t,J＝7.9Hz,1H),6.10(br s,1H),3.17-2.95 (m,2H),2.72(br d,J＝15.5Hz,1H),2.23-2.09(m,1H).

Step 6: Synthesis of 3-(9-(2,6-dioxopiperidin-3-yl)-9H-pyrido[2,3-b]indol-4-yl)propanal

3-(4-bromo-9H-pyrido[2,3-b]indol-9-yl)piperidine-2,6-dione (150 mg, 418.77 μmol) and allyl alcohol (1.4 g, 24.1 mmol) were dissolved in 1,4-dioxane (6 mL), and N,N-diisopropylethylamine (163.37 mg, 1.26 mmol), tri-tert-butylphosphine (254.17 mg, 125.53 μmol, 10% purity) and tris(dibenzylideneacetone)dipalladium (38.35 mg, 41.88 μmol) were added. After nitrogen replacement, the reaction solution was reacted at 80°C for 2 hours. The reaction was completed by LCMS. Water (10 mL) was added to the reaction solution and extracted three times with ethyl acetate (30 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. Purification by thin layer chromatography (silica, petroleum ether/ethyl acetate = 1/1) gave 3-(9-(2,6-dioxopiperidine)-3-yl)-9H-pyrido[2,3-b]indol-4-yl)propanal (70 mg).

MS m/z(ESI):336.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ11.15 (s, 1H), 9.83 (s, 1H), 8.33 (d, J = 5.0Hz, 1H), 8.14 (d, J = 7.8Hz, 1H) ,7.64(br s,1H),7.53(br t,J＝7.7Hz,1H),7.33(t,J＝7.7Hz,1H),7.12(d,J＝5.0Hz,1H),6.07(br s ,1H),3.47(t,J=7.4Hz,2H),3.19-2.97(m,4H),2.77-2.63(m,1H),2.11(m,1H).

Step 7: Synthesis of 2-(3-amino-1-(1-(5-bromopyrimidin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

2-(3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl)phenol (320 mg, 922.68 μmol) and 5-bromo-2-chloropyrimidine (214.17 mg, 1.11 mmol) were dissolved in N,N-dimethylformamide (8 mL), and N,N-diisopropylethylamine (477.00 mg, 3.69 mmol) was added. The reaction solution was reacted at 100°C for 2 hours. The reaction was complete as determined by LCMS. Water (15 mL) was added to the reaction solution, and the organic phase was concentrated to dryness under reduced pressure after extraction with dichloromethane (30 mL) three times. Purification by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 1/1) gave 2-(3-amino-1-(1-(5-bromopyrimidin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (200 mg).

MS m/z(ESI):467.0[M+H] ⁺ .

Step 8: Synthesis of tert-butyl 4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)-5,6-dihydropyridine-1(2H)-carboxylate

2-(3-amino-1-(1-(5-bromopyrimidin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (80 mg, 171.19 μmol) and tert-butyl 4-(3,3,4,4-tetramethyldioxaborolan-1-yl)-5,6-dihydropyridine-1(2H)-carboxylate (58.23 mg, 188.31 μmol) were dissolved in 1,4-dioxane (1.6 mL) and water (0.4 mL), and cesium carbonate (167.33 mg, 513.57 μmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (13.98 mg, 17.12 μmol) were added. After nitrogen replacement, the reaction solution was reacted at 80°C for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether/ethyl acetate = 1/0 to 1/1) to give tert-butyl 4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)-5,6-dihydropyridine-1(2H)-carboxylate (45 mg).

MS m/z(ESI):570.3[M+H] ⁺ .

Step 9: Synthesis of tert-butyl 4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)piperidine-1-carboxylate

Tert-butyl 4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)-5,6-dihydropyridine-1(2H)-carboxylate (20 mg, 35.11 μmol) was dissolved in methanol (1 mL), and wet palladium carbon (5 mg, 10% purity) was added. After hydrogen replacement, the reaction solution was reacted at 25° C. for 12 hours under a hydrogen (15 psi) environment. The reaction was complete as determined by LCMS. The reaction solution was filtered and then concentrated to dryness under reduced pressure to obtain tert-butyl 4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)piperidine-1-carboxylate (20 mg).

MS m/z(ESI):571.5[M+H] ⁺ .

Step 10: Synthesis of 2-(3-amino-1-(1-(5-(piperidin-4-yl)pyrimidin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

Tert-butyl 4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)piperidine-1-carboxylate (11 mg, 19.24 μmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (43.88 mg, 384.84 μmol) was added. The reaction solution was reacted at 25° C. for 1 hour. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure to obtain 2-(3-amino-1-(1-(5-(piperidin-4-yl)pyrimidin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (9 mg).

MS m/z(ESI):472.5[M+H] ⁺ .

Step 11: Synthesis of 3-(4-(3-(4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)piperidin-1-yl)propyl)-9H-pyrido[2,3-b]indol-9-yl)piperidine-2,6-dione

2-(3-amino-1-(1-(5-(piperidin-4-yl)pyrimidin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (9 mg, 19.09 μmol) and 3-(9-(2,6-dioxopiperidin)-3-yl)-9H-pyrido[2,3-b]indol-4-yl)propanal (6.40 mg, 19.09 μmol) were dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), and acetic acid (5.73 mg, 95.43 μmol) and sodium acetate borohydride (12.14 mg, 57.26 μmol) were added. The reaction solution was reacted at 25° C. for 3 hours. The reaction was completed by LCMS. Water (0.5 mL) was added to the reaction solution to quench the reaction. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile ratio: 25%-45%) as eluent) gave 3-(4-(3-(4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)piperidin-1-yl)propyl)-9H-pyrido[2,3-b]indol-9-yl)piperidine-2,6-dione (Compound 002) (1.22 mg).

MS m/z (ESI): 791.2 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.88 (br s, 1H), 11.16 (s, 1H), 8.53 (s, 1H), 8.41-8.27 (m, 3H), 8.22 (d, J＝ 7.8Hz,1H),8.10(d,J＝7.3Hz,1H),7.65(br s,1H),7.54(br t,J＝7.9Hz,1H),7.40-7.30(m,2H),7.16( d,J＝4.8Hz,1H),7.05-6.95(m,2H),6.44(s,2H),6.06(br s,1H),4.98-4.86(m,1H),4.80(br d,J＝ 13.3Hz,2H),3.28(br d,J＝9.0Hz,4H),3.17-2.95(m,6H),2.71(br d,J＝15.1Hz,2H),2.58(br s,2H),2.45(br s,1H),2.10( br s,2H),2.02-1.86(m,6H),1.77(br s,2H).

Example 40: Synthesis of 3-(4-(3-(4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)-5,6-dihydropyridin-1(2H)-yl)propyl)-9H-pyrido[2,3-b]indol-9-yl)piperidine-2,6-dione (Compound 003)

Step 1: Synthesis of 2-(3-amino-1-(1-(5-(1,2,3,6-tetrahydropyridin-4-yl)pyrimidin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

Tert-butyl 4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)-5,6-dihydropyridine-1(2H)-carboxylate (15 mg, 26.33 μmol) was dissolved in dichloromethane (0.5 mL), and trifluoroacetic acid (60.05 mg, 526.63 μmol) was added. The reaction solution was reacted at 25° C. for 1 hour. The reaction was complete as determined by LCMS. The reaction solution was concentrated to dryness under reduced pressure to obtain 2-(3-amino-1-(1-(5-(1,2,3,6-tetrahydropyridin-4-yl)pyrimidin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (12 mg).

MS m/z(ESI):470.5[M+H] ⁺ .

Step 2: Synthesis of 3-(4-(3-(4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)-5,6-dihydropyridin-1(2H)-yl)propyl)-9H-pyrido[2,3-b]indol-9-yl)piperidine-2,6-dione

2-(3-amino-1-(1-(5-(1,2,3,6-tetrahydropyridin-4-yl)pyrimidin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (10 mg, 21.30 μmol) and 3-(9-(2,6-dioxopiperidine)-3-yl)-9H-pyrido[2,3-b]indol-4-yl)propanal (7.14 mg, 21.30 μmol) were dissolved in N,N-dimethylformamide (0.3 mL) and tetrahydrofuran (0.3 mL), and acetic acid (6.39 mg, 106.49 μmol) and sodium acetate borohydride (13.54 mg, 63.89 μmol) were added. The reaction solution was reacted at 25° C. for 3 hours. The reaction was completed by LCMS. Water (0.5 mL) was added to the reaction solution to quench the reaction. Purification by HPLC (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 25%-45%) as eluent) gave 3-(4-(3-(4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)-5,6-dihydropyridin-1(2H)-yl)propyl)-9H-pyrido[2,3-b]indol-9-yl)piperidine-2,6-dione (Compound 003) (3.68 mg).

MS m/z(ESI):789.2[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.90 (br s, 1H), 11.15 (s, 1H), 8.56-8.50 (m, 3H), 8.33 (d, J = 5.0Hz, 1H), 8.21 (d,J＝7.8Hz,1H),8.16(s,1H),8.11(d,J＝7.8Hz,1H),7.65(br s,1H),7.52(br t,J＝7.9Hz,1H) ,7.40-7.34(m,1H),7.30(t,J＝7.8Hz,1H),7.14(d,J＝5.0Hz,1H),7.05-6.97(m,2H),6.44(s,2H), 6.14(br s,1H),6.08(brs,1H),4.98-4.80(m,3H),3.28-3.22(m,4H),3.16-2.95(m,6H),2.67(br d,J＝4.0Hz,2H ), 2.59 (br d, J = 6.5Hz, 2H), 2.46-2.42 (m, 1H), 2.10 (br s, 1H), 1.96 (m, 6H).

Examples 41 and 41-1: trans-(2S,4R)-1-((S)-2-4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)cyclohexanecarboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-yl Synthesis of carboxamide and cis-(2S,4R)-1-((S)-2-4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrrolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)cyclohexanecarboxamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

Step 1: Synthesis of ethyl 4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)cyclohex-3-ene-1-olate

2-[3-Amino-1-[1-(5-bromopyrimidin-2-yl)-4-piperidinyl]pyrazolo[4,3-c]pyridazin-6-yl]phenol (120 mg, 256.78 μmol) and 1-ethoxycarbonylcyclohex-3-ene-4-boronic acid pinacol ester (79.14 mg, 282.46 μmol) were dissolved in dioxane (1.6 mL) and water (0.4 mL). Cesium carbonate (106.47 mg, 326.77 μmol) and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (18.79 mg, 25.68 μmol) were added under nitrogen atmosphere. The reaction solution was stirred at 80°C for 2 hours. LCMS showed that the reaction was completed. The reaction solution was concentrated to dryness under reduced pressure, and the concentrate was purified by column chromatography (silica, petroleum ether:ethyl acetate=3:2) to give the product 4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)cyclohex-3-enoic acid ethyl ester (65 mg).

MS m/z(ESI):541.1[M+H] ⁺ .

Step 2: Synthesis of ethyl 4-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]pyrimidin-5-yl]cyclohexanecarboxylate

4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidin-1-yl)pyrimidin-5-yl)cyclohex-3-enoic acid ethyl ester (50 mg, 92.49 μmol) was dissolved in methanol (1 mL), palladium carbon (50 mg, 10% purity) was added under hydrogen atmosphere, and the reaction solution was reacted for 12 hours at 25° C. and 15 psi hydrogen atmosphere. LCMS showed that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the product 4-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazine-1-yl]-1-piperidinyl]pyrimidin-5-yl]cyclohexanecarboxylic acid ethyl ester (45 mg).

MS m/z(ESI):543.3[M+H] ⁺ .

Step 3: Synthesis of 4-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]pyrimidin-5-yl]cyclohexanecarboxylic acid

Ethyl 4-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]pyrimidin-5-yl]cyclohexanecarboxylate (45 mg, 82.93 μmol) was dissolved in tetrahydrofuran (1 mL) and water (1 mL), sodium hydroxide (13.27 mg, 331.72 μmol) was added, and the reaction solution was stirred at 25°C for 12 hours. LCMS showed that the reaction was complete. The reaction solution was adjusted to pH 5-6 with 1M hydrochloric acid and concentrated to dryness under reduced pressure to obtain the product 4-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]pyrimidin-5-yl]cyclohexanecarboxylic acid (40 mg).

MS m/z(ESI):515.3[M+H] ⁺ .

Step 4: trans-(2S,4R)-1-((S)-2-(4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)cyclohexanecarboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide and Synthesis of cis-(2S,4R)-1-((S)-2-(4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrrolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)cyclohexanecarboxamido)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazolyl-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

4-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]pyrimidin-5-yl]cyclohexanecarboxylic acid (10 mg, 19.43μmol) and (2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl))phenyl]ethyl]pyrrolidine-2-carboxamide (9.35mg, 19.43μmol) were dissolved in N,N-dimethylformamide (1mL), triethylamine (7.87mg, 77.73μmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (8.87mg, 23.32μmol) were added, and the reaction solution was stirred at 25°C for 1 hour. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the concentrate was purified by preparative liquid chromatography (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.225% formic acid) and acetonitrile (acetonitrile ratio: 27%-67%) as the eluent) to obtain two products with a mass of 1.02 mg and 1.06 mg, namely trans-(2S,4R)-1-((S)-2-(4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)cyclohexanecarboxamido)-3,3-dimethylbutanoyl) -4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide and cis-(2S,4R)-1-((S)-2-(4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrrolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)cyclohexanecarboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide. After further HPLC analysis (analysis method see the table below), it was determined that the retention time of one of the two was 4.386 min (this product was named compound 004), and the other was 4.405 min (this product was named compound 004-1).

HPLC analysis method:

Spectral data of compound 004:

MS m/z(ESI):941.3[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.90 (br s, 1H), 8.98 (s, 1H), 8.54 (s, 1H), 8.38 (d, J = 7.8Hz, 1H), 8.31 (s ,2H),8.11(d,J＝7.5Hz,1H),7.74(br d,J＝10.0Hz,1H),7.48-7.41(m,2H),7.41-7.33(m,3H),7.04-6.97 (m,2H),6.44(br s,2H),5.12(d,J＝3.4Hz,1H),4.99-4.86(m,2H),4.80(br d,J＝13.0Hz,2H),4.51(d,J＝8.9Hz,1H),4.43(t,J＝7.8Hz,1H),4.30(br s,1H),3.61(br s,2H), 3.07(br s,2H),2.46(br s,5H),2.06-1.91(m,6H),1.88-1.73(m,4H),1.55-1.41(m,4H),1.38(br d,J＝ 6.8Hz,3H),0.95(s,9H).

Spectral data of compound 004-1:

MS m/z (ESI): 941.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.98 (s, 1H), 8.54 (s, 1H), 8.38 (br d, J = 7.5Hz, 1H), 8.27 (s, 2H), 8.00 (br s,1H),7.63(br d,J＝9.0Hz,1H),7.47-7.35(m,5H),7.06-6.99(m,2H),4.92(br t,J＝7.2Hz,2H),4.79 (br d,J＝12.5Hz,2H),4.55(d,J＝9.0Hz,1H),4.43(t,J＝8.0Hz,1H),4.29(br s,1H),3.62(br s,2H ),3.06(br s,3H),2.45(s,3H),1.96(m,6H),1.87-1.73(m,4H),1.63(br s,3H),1.51(br d,J＝6.5Hz,2H),1.38 (d, J=6.8Hz, 3H), 0.95 (s, 9H).

Example 42: Synthesis of (2S,4R)-1-[(2S)-2-[[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]pyrimidin-5-yl]cyclohex-3-ene-1-carbonyl]amino]-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 005)

Step 1: Synthesis of 4-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]pyrimidin-5-yl]cyclohex-3-ene-1-carboxylic acid

Ethyl 4-(2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)cyclohex-3-enecarboxylate (12 mg, 22.20 μmol) was dissolved in tetrahydrofuran (0.5 mL) and water (0.5 mL), sodium hydroxide (2.79 mg, 69.86 μmol) was added, and the reaction solution was stirred at 25°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was adjusted to pH 5-6 with 1M hydrochloric acid and concentrated to dryness under reduced pressure to obtain the product 4-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]pyrimidin-5-yl]cyclohex-3-ene-1-carboxylic acid (11 mg).

MS m/z(ESI):513.3[M+H] ⁺ .

Step 2: Synthesis of (2S,4R)-1-[(2S)-2-[[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]pyrimidin-5-yl]cyclohex-3-ene-1-carbonyl]amino]-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

4-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]pyrimidin-5-yl]cyclohex-3-ene-1-carboxylic acid (9 mg, 17.56 μmol) and (2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazole-5-yl)]-1-carboxylic acid (9 mg, 17.56 μmol) were added. [0366] 2-[(7-( ... The reaction solution was concentrated under reduced pressure, and the concentrate was purified by preparative liquid chromatography (Boston Prime C18, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.225% formic acid) and acetonitrile (acetonitrile ratio: 37%-57%) as eluent) to give the product (2S,4R)-1-[(2S)-2-[[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]pyrimidin-5-yl]cyclohex-3-ene-1-carbonyl]amino]-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 005) (4.05 mg).

MS m/z(ESI):939.3[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.98 (s, 1H), 8.98 (s, 1H), 8.54 (s, 1H), 8.49 (s, 2H), 8.41 (d, J = 8.0Hz, 1H),8.09(br d,J＝7.3Hz,1H),7.93-7.83(m,1H),7.47-7.41(m,2H),7.41-7.34(m,3H),7.06-6.97(m,2H ),6.49(br s,2H),6.13(br s,1H),5.15(br s,1H),4.98-4.87(m,2H),4.82(d,J＝12.3Hz,2H),4.59-4.50(m,1H),4.44(t,J＝7.2Hz,1H),4.29(br s,1H),3.62(br s,2H),3.15-3.04(m,2H),2.66-2.55(m,1H),2.45(s,5H),2.38(br s,1H),2.28(br s ,1H),2.08-1.90(m,6H),1.79(m,1H),1.64(m,1H),1.38(d,J＝7.0Hz,3H),0.96(d,J＝3.8Hz,9H) .

Example 43: Synthesis of (2S,4R)-1-[2-[3-[9-[2-[[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-2-yl]oxy]ethyl]-3,9-diazaspiro[5.5]undec-3-yl]isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 007)

Step 1: Synthesis of tert-butyl 9-[5-(1-methoxy-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl]-3,9-diazaspiro[5.5]undecane-3-carboxylate

3-Methyl-2-[3-(1,1,2,2,3,3,4,4,4-nonafluorobutylsulfonyloxy)isoxazol-5-yl]butyric acid methyl ester (400 mg, 831.10 μmol) was dissolved in N,N-dimethylformamide (6 mL), and tert-butyl 3,9-diazaspiro[5.5]undecane-3-carboxylate (253.69 mg, 997.32 μmol) and N,N-diisopropylethylamine (322.24 mg, 2.49 mmol, 434.29 μL) were added. The reaction solution was stirred at 80°C for 12 hours. LCMS detected that the reaction was complete. The reaction solution was cooled to 25°C, water (8 mL) was added, and then extracted twice with ethyl acetate (12 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (petroleum ether: ethyl acetate = 3: 1) to give 9-[5-(1-methoxy-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl]-3,9-diazaspiro[5.5]undecane-3-carboxylic acid tert-butyl ester (150 mg).

MS m/z(ESI):436.2[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 6.24 (s, 1H), 3.65 (d, J = 2.3Hz, 3H), 3.33 (d, J = 2.1Hz, 4H), 3.20-3.15 (m, 4H),2.35-2.22(m,1H),1.49(br s,4H),1.40(br d,J＝2.0Hz,14H),0.93(br d,J＝6.6Hz,3H),0.87-0.83( m,3H).

Step 2: Synthesis of 2-[3-(9-tert-butoxycarbonyl-3,9-diazaspiro[5.5]undec-3-yl)isoxazol-5-yl]-3-methylbutanoic acid

9-[5-(1-methoxy-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl]-3,9-diazaspiro[5.5]undecane-3-carboxylic acid tert-butyl ester (150 mg, 344.39 μmol) was dissolved in methanol (0.5 mL) and water (0.5 mL), and sodium hydroxide (27.55 mg, 688.78 μmol) was added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, water (5 mL) and ethyl acetate (5 mL) were added, and then the pH was adjusted to 6 with saturated citric acid solution. The aqueous phase was extracted twice with ethyl acetate (10 mL), the organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give 2-[3-(9-tert-butoxycarbonyl-3,9-diazaspiro[5.5]undecane-3-yl)isoxazol-5-yl]-3-methylbutanoic acid (140 mg, crude product).

MS m/z(ESI):444.2[M+Na] ⁺ .

Step 3: Synthesis of tert-butyl 9-[5-[1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]isoxazol-3-yl]-3,9-diazaspiro[5.5]undecane-3-carboxylate

2-[3-(9-tert-Butyloxycarbonyl-3,9-diazaspiro[5.5]undec-3-yl)isoxazol-5-yl]-3-methylbutanoic acid (140 mg, 332.12 μmol) was dissolved in N,N-dimethylformamide (4 mL), and (2S,4R)-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrole-2-carboxamide (121.08 mg, 365.34 μmol), O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (151.54 mg, 398.55 μmol) and triethylamine (100.82 mg, 996.37 μmol, 138.68 μL) were added. The reaction solution was stirred at 25°C for 1 hour. The reaction was completed when LCMS was detected. Water (5 mL) was added, and then the aqueous phase was extracted twice with ethyl acetate (10 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The product was purified by thin layer chromatography (petroleum ether: tetrahydrofuran = 1:2) to give 9-[5-[1-[(2S, 4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]isoxazol-3-yl]-3,9-diazaspiro[5.5]undecane-3-carboxylic acid tert-butyl ester (70 mg).

MS m/z(ESI):735.4[M+H] ⁺ .

Step 4: Synthesis of (2S,4R)-1-[2-[3-(3,9-diazaspiro[5.5]undec-3-yl)isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

9-[5-[1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]isoxazol-3-yl]-3,9-diazaspiro[5.5]undecane-3-carboxylic acid tert-butyl ester (70 mg, 95.27 μmol) was dissolved in anhydrous dioxane (3 mL), and dioxane hydrochloride (4N, 0.5 mL) was added. The reaction solution was stirred at 25° C. for 16 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to give (2S,4R)-1-[2-[3-(3,9-diazaspiro[5.5]undec-3-yl)isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrole-2-carboxamide (70 mg, crude product).

MS m/z(ESI):635.6[M+H] ⁺ .

Step 5: Synthesis of 4-(3-amino-6-chloro-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylic acid benzyl ester

6-Chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (5 g, 29.49 mmol) was dissolved in N,N-dimethylformamide (200 mL), and 4-iodopiperidine-1-carboxylic acid benzyl ester (20.36 g, 58.97 mmol) and cesium carbonate (28.82 g, 58.97 mmol) were added. The reaction solution was stirred at 60°C for 3 hours. LCMS showed that the reaction was complete. Water (100 mL) was added, and then the aqueous phase was extracted twice with ethyl acetate (150 mL), and the organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (petroleum ether: tetrahydrofuran = 1:1) to obtain 4-(3-amino-6-chloro-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylic acid benzyl ester (1.5 g).

MS m/z(ESI):387.1[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 7.99 (s, 1H), 7.41-7.31 (m, 5H), 6.46 (s, 2H), 5.12 (s, 2H), 4.68-4.58 (m, 1H) ),3.08-2.90(m,4H),1.93-1.83(m,4H).

Step 6: Synthesis of benzyl 4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

4-(3-amino-6-chloro-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylic acid benzyl ester (1.5 g, 3.88 mmol) was dissolved in dioxane (40 mL) and water (10 mL), and 2-(2-(methoxymethoxy)phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.23 g, 4.65 mmol), potassium carbonate (1.61 g, 11.63 mmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (253.33 mg, 310.21 μmol) were added, and then replaced with nitrogen three times. The reaction solution was stirred at 90 ° C for 3 hours. LCMS showed that the reaction was complete. Water (50 mL) was added, and the aqueous phase was extracted twice with ethyl acetate (100 mL), and the organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (petroleum ether:tetrahydrofuran = 1:1) to give benzyl 4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate (910 mg).

MS m/z(ESI):489.2[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d6) δ = 7.91 (s, 1H), 7.68 (dd, J = 1.8, 7.6Hz, 1H), 7.43-7.41 (m, 1H), 7.37-7.32 (m, 4H) ,7.32-7.23(m,2H),7.16-7.14(m,1H),6.26(s,2H),5.18(s,2H),5.08(s,2H),4.63-4.61(m,1H),4.14 -4.11(m,2H),3.27(s,3H),3.00-2.98(m,2H),1.96-1.80(m,4H).

Step 7: Synthesis of benzyl 4-[3-[bis(tert-butyloxycarbonyl)amino]-6-[2-(methoxymethoxy)phenyl]pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carboxylate

Benzyl 4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylate (910 mg, 1.86 mmol) was dissolved in dichloromethane (20 mL), and di-tert-butyl dicarbonate (1.22 g, 5.59 mmol), 4-dimethylaminopyridine (455.13 mg, 3.73 mmol) and triethylamine (565.46 mg, 5.59 mmol) were added. The reaction solution was stirred at 25 ° C for 12 hours. LCMS showed that the reaction was complete. Water (20 mL) was added, and the aqueous phase was extracted twice with ethyl acetate (40 mL), and the organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (petroleum ether:tetrahydrofuran=1:1) to give benzyl 4-[3-[bis(tert-butyloxycarbonyl)amino]-6-[2-(methoxymethoxy)phenyl]pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylate (800 mg).

MS m/z (ESI): 689.3 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 8.40 (s, 1H), 7.78 (dd, J = 1.6, 7.6Hz, 1H), 7.54-7.47 (m, 1H), 7.38 (d, J = 4.4 Hz,4H),7.35-7.32(m,2H),7.25-7.20(m,1H),5.24(s,2H),5.12(s,2H),5.10-4.98(m,1H),4.18(br d ,J＝13.1Hz,2H),3.26(s,3H),3.08(br s,2H),2.05-1.94(m,4H),1.38(s,18H).

Step 8: Synthesis of tert-butyl N-tert-butyloxycarbonyl-N-[6-[2-(methoxymethoxy)phenyl]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-yl]carbamate

4-[3-[bis(tert-butyloxycarbonyl)amino]-6-[2-(methoxymethoxy)phenyl]pyrazolo[4,3-c]pyridazine-1-yl]piperidine-1-carboxylic acid benzyl ester (750mg, 1.09mmol) was dissolved in tetrahydrofuran (12mL), wet palladium carbon (350mg, 10%) was added, replaced with hydrogen three times, and stirred at 25°C for 16 hours under a hydrogen (15Psi) atmosphere. LCMS showed that the reaction was complete. The reaction solution was filtered through diatomaceous earth, the filtrate was collected, and it was concentrated to dryness under reduced pressure to obtain tert-butyl N-tert-butyloxycarbonyl-N-[6-[2-(methoxymethoxy)phenyl]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-3-yl]carbamate (450mg).

MS m/z(ESI):555.2[M+H] ⁺ .

Step 9: Synthesis of 4-bromo-2-(2,2-dimethoxyethoxy)pyridine

4-Bromo-2-fluoropyridine (3g, 17.05mmol) was dissolved in acetonitrile (130mL), and hydroxyacetaldehyde dimethyl acetal (2.71g, 25.57mmol) and cesium carbonate (16.66mg, 51.14mmol) were added. The reaction solution was stirred at 90°C for 16 hours. Thin layer chromatography (petroleum ether: ethyl acetate = 10:1) showed that the reaction was complete. The reaction solution was cooled to 25°C and concentrated to dryness under reduced pressure. Water (30mL) was added, and then the aqueous phase was extracted twice with ethyl acetate (60mL), and the organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (petroleum ether: ethyl acetate = 10:1) to obtain 4-bromo-2-(2,2-dimethoxyethoxy)pyridine (4.15g).

¹ H NMR (400MHz, DMSO-d6) δ = 8.07 (br d, J = 5.4Hz, 1H), 7.29-7.21 (m, 1H), 7.16 (s, 1H), 4.69 (br t, J = 5.3Hz ,1H),4.26(br d,J＝5.3Hz,2H),3.33(s,6H).

Step 10: Synthesis of tert-butyl N-tert-butyloxycarbonyl-N-[1-[1-[2-(2,2-dimethoxyethoxy)-pyridin-4-yl]-piperidin-4-yl]-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-3-yl]carbamate

4-Bromo-2-(2,2-dimethoxyethoxy)pyridine (99.24 mg, 378.63 μmol) was dissolved in tert-amyl alcohol (10 mL), and tert-butyl N-tert-butyloxycarbonyl-N-[6-[2-(methoxymethoxy)phenyl]-1-(4-piperidinyl)pyrazolo[4,3-c]pyridazin-3-yl]carbamate (140 mg, 252.42 μmol), sodium tert-butoxide (48.52 mg, 504.84 μmol) and methanesulfonic acid (2-dicyclohexylphosphine-2',6'-diisopropoxy-1,1'-biphenyl) (2'-amino-1,1'-biphenyl-2-yl) palladium (II) were added, and the gas was replaced with nitrogen three times. The reaction solution was stirred at 110°C for 3 hours. LCMS showed that the reaction was complete. The reaction solution was cooled to 25°C and concentrated to dryness under reduced pressure. Water (4 mL) was added, and the aqueous phase was extracted twice with ethyl acetate (15 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure and purified by thin layer chromatography (petroleum ether: tetrahydrofuran = 1: 1) to give N-tert-butyloxycarbonyl-N-[1-[1-[2-(2,2-dimethoxyethoxy)-pyridin-4-yl]-piperidin-4-yl]-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-3-yl]carbamic acid tert-butyl ester (70 mg).

MS m/z(ESI):636.5[M+H-Boc] ⁺ .

Step 11: Synthesis of 2-[[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-piperidin-1-yl]pyridin-2-yl]oxy]acetaldehyde

N-tert-Butyloxycarbonyl-N-[1-[1-[2-(2,2-dimethoxyethoxy)-pyridin-4-yl]-piperidin-4-yl]-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-3-yl]carbamic acid tert-butyl ester (70 mg, 95.13 μmol) was dissolved in tetrahydrofuran (3 mL), sulfuric acid solution (1.90 mL, 1N) was added, and the reaction solution was stirred at 70°C for 7 hours. LCMS showed that the reaction was complete. The reaction solution was cooled to 25°C, adjusted to pH = 9 with saturated sodium bicarbonate, water (5 mL) and ethyl acetate (5 mL) were added, and the aqueous phase was extracted twice with ethyl acetate (15 mL). The organic layer was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 2-[[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-piperidin-1-yl]pyridin-2-yl]oxy]acetaldehyde (50 mg).

MS m/z(ESI):446.2[M+H] ⁺ .

Step 12: Synthesis of (2S,4R)-1-[2-[3-[9-[2-[[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-piperidin-1-yl]-pyridin-2-yl]oxy]ethyl]-3,9-diazaspiro[5.5]undec-3-yl]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

((2S,4R)-1-[2-[3-(3,9-diazaspiro[5.5]undec-3-yl)isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (30 mg, 47.26 μmol) was dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), and 2-[[4-[4-[3-amino-6-(2-hydroxyphenyl)- 1H-pyrazolo[4,3-c]pyridazin-1-yl]-piperidin-1-yl]pyridin-2-yl]oxy]acetaldehyde (40 mg, 44.90 μmol) and acetic acid (134.81 μg, 2.25 μmol). The reaction solution was stirred at 25°C for 20 minutes. Sodium acetate borohydride (28.55 mg, 134.70 μmol) was added, and the reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (Phenomenex C18 column: 3 μm silica, 30 mm diameter, 75 mm length; using water (containing 0.05% ammonia) and acetonitrile with decreasing polarity (acetonitrile content: 46%-86%) as eluent) to purify (2S,4R)-1-[2-[3-[9-[2-[[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-piperidin-1-yl]-pyridin-2-yl]oxy]ethyl]-3,9-diazaspiro[5.5]undec-3-yl]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 007) (2.34 mg).

MS m/z(ESI):532.8[M+2H] ²⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ = 8.94-8.84 (m, 1H), 8.29 (s, 1H), 8.05-7.98 (m, 1H), 7.82 (d, J = 6.3Hz, 1H), 7.46-7.41(m,3H),7.40-7.33(m,2H),7.06-6.97(m,2H),6.65(br d,J＝4.0Hz,1H),6.28(s,1H),6.16-6.06 (m,1H),4.43(br d,J＝5.0Hz,4H),4.19-4.09(m,2H),3.89-3.68(m,2H),3.65-3.48(m,2H),3.27-3.13(m,7H),2.95(br s ,2H),2.74(br s,4H),2.53-2.45(m,3H),2.42-2.32(m,2H),2.30-2.18(m,3H),2.02(br d,J＝10.4Hz,3H ),1.67(br s,7H),1.56-1.49(m,3H),1.10-1.04(m,3H),0.91(m,3H).

Example 44: Synthesis of (2S,4R)-N-[(S)-3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-[1,4'-dipiperidinyl]-1'-yl]-1-[4-(4-methylthiazol-5-yl)phenyl]-3-oxopropyl]-4-hydroxy-1-[3-methyl-2-(3-methylisoxazol-5-yl)butanoyl]pyrrolidine-2-carboxamide (Compound 008)

Step 1: Synthesis of (3S)-3-(tert-butoxycarbonylamino)-3-[4-(4-methylthiazol-5-yl)phenyl]propanoic acid

Boc-(S)-3-amino-3-(4-bromophenyl)-propionic acid (5 g, 14.53 mmol) and 4-methylthiazole (2.88 g, 29.05 mmol) were dissolved in anhydrous N,N-dimethylformamide (100 mL), and potassium acetate (2.85 g, 29.05 mmol) and palladium acetate (326.13 mg, 1.45 mmol) were added. The reaction solution was stirred at 90 ° C for 3 hours under a nitrogen atmosphere. The reaction solution was extracted with ethyl acetate (30 mL) and water (60 mL), concentrated to dryness under reduced pressure, and purified by column chromatography (silicon dioxide, petroleum ether: tetrahydrofuran = 1: 1) to obtain (3S)-3-(tert-butoxycarbonylamino)-3-[4-(4-methylthiazol-5-yl)phenyl]propionic acid (1.9 g).

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 12.25 (br s, 1H), 8.99 (s, 1H), 7.51 (d, J = 8.5Hz, 1H), 7.47-7.42 (m, 2H), 7.42 -7.37(m,2H),4.93(q,J=7.7Hz,1H),2.71-2.58(m,2H),2.45(s,3H),1.36(s,9H).

Step 2: Synthesis of methyl (3S)-3-(tert-butoxycarbonylamino)-3-[4-(4-methylthiazol-5-yl)phenyl]propanoate

(3S)-3-(tert-butoxycarbonylamino)-3-[4-(4-methylthiazol-5-yl)phenyl]propionic acid (1.6 g, 4.41 mmol) was dissolved in anhydrous methanol (2 mL) and anhydrous dichloromethane (18 mL), and trimethylsilyldiazomethane n-hexane solution (2 M, 6.62 mL) was added dropwise under an ice bath (0°C). The reaction solution was stirred at 25°C for 4 hours. LCMS detected that the reaction was complete. Acetic acid (3 mL) was added to the reaction solution to quench it, and it was concentrated under reduced pressure to dryness to obtain (3S)-3-(tert-butoxycarbonylamino)-3-[4-(4-methylthiazol-5-yl)phenyl]propionic acid methyl ester (2.2 g).

MS m/z(ESI):377.1[M+H] ⁺ .

Step 3: Synthesis of methyl (3S)-3-amino-3-[4-(4-methylthiazol-5-yl)phenyl]propanoate

Methyl (3S)-3-(tert-butyloxycarbonylamino)-3-[4-(4-methylthiazol-5-yl)phenyl]propanoate (2.4 g, 6.38 mmol) was dissolved in anhydrous dichloromethane (20 mL), and trifluoroacetic acid (9.44 mL, 127.50 mmol) was added. The reaction solution was stirred at 25 ° C for 3 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain methyl (3S)-3-amino-3-[4-(4-methylthiazol-5-yl)phenyl]propanoate (4 g, crude product).

MS m/z(ESI):276.9[M+H] ⁺ .

Step 4: Synthesis of tert-butyl (2S,4R)-4-hydroxy-2-[[(1S)-3-methoxy-1-[4-(4-methylthiazol-5-yl)phenyl]-3-oxopropyl]carbamoyl]pyrrolidine-1-carboxylate

Methyl (3S)-3-amino-3-[4-(4-methylthiazol-5-yl)phenyl]propanoate (2 g, 5.12 mmol) and Boc-L-hydroxyproline (1.18 g, 5.12 mmol) were dissolved in anhydrous N,N-dimethylformamide (50 mL), and N,N-diisopropylethylamine (4.46 mL, 25.62 mmol) and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.92 g, 7.68 mmol) were added. The reaction solution was stirred at 25 ° C for 16 hours. LCMS detected that the reaction was complete. The reaction solution was extracted with ethyl acetate (20 mL) and water (60 mL), concentrated to dryness under reduced pressure, and purified by column chromatography (silica, petroleum ether:tetrahydrofuran = 1:2) to give (2S,4R)-4-hydroxy-2-[[(1S)-3-methoxy-1-[4-(4-methylthiazol-5-yl)phenyl]-3-oxopropyl]carbamoyl]pyrrolidine-1-carboxylic acid tert-butyl ester (1.7 g).

MS m/z(ESI):490.1[M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ = 8.89 (s, 1H), 7.49-7.44 (m, 4H), 5.52-5.34 (m, 1H), 4.41-4.24 (m, 2H), 3.65 (s ,3H),3.58-3.46(m,2H),3.05-2.98(m,1H),2.92-2.84(m,1H),2.48(s,3H),2.34-2.13(m,1H),1.98-1.90 (m,1H),1.48-1.31(m,9H).

Step 5: Synthesis of methyl (3S)-3-[[(2S,4R)-4-hydroxypyrrole-2-carbonyl]amino]-3-[4-(4-methylthiazol-5-yl)phenyl]propanoate

Dissolve (2S,4R)-4-hydroxy-2-[[(1S)-3-methoxy-1-[4-(4-methylthiazol-5-yl)phenyl]-3-oxo-propyl]carbamoyl]pyrrole-1-carboxylic acid tert-butyl ester (1.7 g, 3.47 mmol) in anhydrous dioxane (2 mL), and add dioxane hydrochloride solution (4M, 8.68 mL). The reaction solution was stirred at 25°C for 3 hours. LCMS detected that the reaction was complete. Concentrate under reduced pressure to dryness to obtain (3S)-3-[[(2S,4R)-4-hydroxypyrrolidine-2-carbonyl]amino]-3-[4-(4-methylthiazol-5-yl)phenyl]propionic acid methyl ester (1.4 g).

MS m/z (ESI): 390.1 [M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ = 9.94 (s, 1H), 7.62-7.58 (m, 2H), 7.58-7.54 (m, 2H), 5.51-5.38 (m, 1H), 4.62-4.46 (m,2H),3.72(d,J＝11.0Hz,2H),3.67(s,3H),3.04-2.98(m,1H),2.98-2.93(m,1H),2.61(s,3H), 2.54-2.46(m,1H),1.95-1.93(m,1H).

Step 6: Synthesis of methyl 2-(3-methylisoxazol-5-yl)acetate

2-(3-methylisoxazol-5-yl)acetic acid (5 g, 35.43 mmol) was dissolved in methanol (50 mL), and concentrated sulfuric acid (921.91 mg, 9.21 mmol) was added. The reaction solution was reacted at 70 ° C for 2 hours, and TLC (petroleum ether/ethyl acetate = 1/1) detected that the reaction was complete. The reaction solution was cooled to room temperature, concentrated, diluted with distilled water (50 mL), and the pH was adjusted to 7-8 with solid sodium carbonate. Then, it was extracted three times with ethyl acetate (30 mL), and the organic phases were combined, dried, and concentrated to obtain 2-(3-methylisoxazol-5-yl)acetic acid methyl ester (4.8 g).

¹ H NMR (Methanol-d ₄ , 400MHz): δ = 6.25 (s, 1H), 3.90 (s, 2H), 3.75 (s, 3H), 2.28ppm (s, 3H).

Step 7: Synthesis of methyl 3-methyl-2-(3-methylisoxazol-5-yl)butanoate

2-(3-methylisoxazol-5-yl)acetic acid methyl ester (500mg, 3.22mmol) and potassium tert-butoxide (723.24mg, 6.45mmol) were dissolved in tetrahydrofuran (7mL), and iodopropane (766.96mg, 4.51mmol) was added. The reaction solution was reacted at 25°C for 12 hours, and the reaction was completed by LCMS detection. The reaction solution was quenched with saturated ammonium chloride (10mL), extracted three times with ethyl acetate (50mL), and the organic phases were combined, dried, concentrated, and purified by column chromatography (silicon dioxide, petroleum ether, ethyl acetate = 3/1) to obtain 3-methyl-2-(3-methylisoxazol-5-yl)butyric acid methyl ester (320mg).

MS m/z(ESI):198.2[M+H] ⁺ .

Step 8: Synthesis of 3-methyl-2-(3-methylisoxazol-5-yl)butyric acid

3-Methyl-2-(3-methylisoxazol-5-yl)butyric acid methyl ester (300 mg, 1.52 mmol) was dissolved in tetrahydrofuran (3 mL) and distilled water (3 mL), and lithium hydroxide monohydrate (191.49 mg, 4.56 mmol) was added. The reaction solution was reacted at 25°C for 3 hours, and the reaction was completed by LCMS. The reaction solution was adjusted to pH 5-6 with 1M HCl, dried, filtered, and concentrated to obtain 3-methyl-2-(3-methylisoxazol-5-yl)butyric acid (250 mg).

MS m/z(ESI):184.1[M+H] ⁺ .

Step 9: Synthesis of methyl (3S)-3-[(2S,4R)-4-hydroxy-1-[3-methyl-2-(3-methylisoxazol-5-yl)butyryl]pyrrolidine-2-amide]-3-[4-(4-methylthiazol-5-yl)phenyl]propanoate

Methyl ((3S)-3-[[(2S,4R)-4-hydroxypyrrole-2-carbonyl]amino]-3-[4-(4-methylthiazol-5-yl)phenyl]propanoate (425.17 mg, 1.09 mmol) and 3-methyl-2-(3-methylisoxazol-5-yl)butanoic acid (200 mg, 1.09 mmol) were dissolved in anhydrous N,N-dimethylformamide (10 mL), and N,N-diisopropylethylamine (950.75 μL, 5.46 mmol) and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluoro Phosphate (498.11 mg, 1.31 mmol). The reaction solution was stirred at 25 ° C for 3 hours. LCMS detected that the reaction was complete. The reaction solution was extracted with ethyl acetate (30 mL) and water (60 mL), concentrated to dryness under reduced pressure, and purified by column chromatography (silica, petroleum ether: tetrahydrofuran = 1: 4) to obtain (3S)-3-[(2S, 4R)-4-hydroxy-1-[3-methyl-2-(3-methylisoxazol-5-yl)butyryl]pyrrolidine-2-amide]-3-[4-(4-methylthiazol-5-yl)phenyl]propionic acid methyl ester (380 mg).

MS m/z(ESI):555.2[M+H] ⁺ ;

¹ H NMR (400 MHz, METHANOL-d ₄ )δ＝8.88(s,1H),7.47-7.38(m,4H),6.23(d,J＝9.5Hz,1H),5.37(t,J＝7.4,18.9Hz,1H),4.50-4.40(m,1H),3.73(d,J＝2.5Hz,3H),3.67-3.64(m,2H),3 .06-2.99(m,1H),2.96-2.90(m,1H),2.48(s,3H),2.26(s,2H),2.24-2.21(m,2H),1.99-1.94(m,1H),1.40(s,3H),1.05(dd,J＝2.6,6.7Hz,3H),0.9 0-0.84(m,3H).

Step 10: Synthesis of (3S)-3-[(2S,4R)-4-hydroxy-1-[3-methyl-2-(3-methylisoxazol-5-yl)butyryl]pyrrolidine-2-amide]-3-[4-(4-methylthiazol-5-yl)phenyl]propanoic acid

Methyl (3S)-3-[(2S,4R)-4-hydroxy-1-[3-methyl-2-(3-methylisoxazol-5-yl)butyryl]pyrrole-2-amide]-3-[4-(4-methylthiazol-5-yl)phenyl]propanoate (80 mg, 144.23 μmol) was dissolved in anhydrous tetrahydrofuran (0.5 mL) and water (0.5 mL), and sodium hydroxide (17.31 mg, 432.70 μmol) was added. The reaction solution was stirred at 25° C. for 3 hours. The reaction was completed by LCMS. The reaction solution was adjusted to pH = 3 with 1.5 M hydrochloric acid, extracted with ethyl acetate (2 mL) and water (2 mL), the organic phase was dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give (3S)-3-[(2S,4R)-4-hydroxy-1-[3-methyl-2-(3-methylisoxazol-5-yl)butyryl]pyrrolidine-2-amide]-3-[4-(4-methylthiazol-5-yl)phenyl]propanoic acid (60 mg).

MS m/z(ESI):541.1[M+H] ⁺ .

Step 11: Synthesis of tert-butyl 4-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]piperidine-1-carboxylate

Dissolve 2-[3-amino-1-(4-piperidinyl)pyrazolo[4,3-c]pyridazin-6-yl]phenol (500 mg, 1.61 mmol) in N,N-dimethylformamide (8 mL) and tetrahydrofuran (8 mL), add N-tert-butyloxycarbonyl-4-piperidone (358.20 mg, 1.93 mmol) and acetic acid (96.74 mg, 1.61 mmol). The reaction solution was stirred at 25°C for 1 hour. Sodium acetate borohydride (1.02 g, 4.83 mmol) was added, and the reaction solution was stirred at 25°C for 11 hours. LCMS detected that the reaction was complete. Water (15 mL) and dichloromethane:methanol (20:2 mL) were added to the reaction solution, the reaction mixture was separated, and the aqueous phase was extracted twice with dichloromethane:methanol (20 mL, volume ratio = 40:4). The organic phase was collected, dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to give a yellow solid 4-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]piperidine-1-carboxylic acid tert-butyl ester (680 mg, crude product).

MS m/z(ESI):494.3[M+H] ⁺ .

Step 12: Synthesis of 2-[3-amino-1-[1-(4-piperidinyl)-4-piperidinyl]pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazine-1-yl]-1-piperidinyl]piperidine-1-carboxylic acid tert-butyl ester (680 mg, 1.38 mmol) was dissolved in dichloromethane (6 mL), dioxane hydrochloride (2.07 mL, 4 M) was added, and the reaction solution was stirred at 25 ° C for 2 hours. The reaction solution was turbid yellow. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain a yellow solid 2-[3-amino-1-[1-(4-piperidinyl)-4-piperidinyl]pyrazolo[4,3-c]pyridazine-6-yl]phenol (680 mg, hydrochloride).

MS m/z(ESI):394.1[M+H] ⁺ .

Step 13: Synthesis of (2S,4R)-N-[(S)-3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-[1,4'-dipiperidinyl]-1'-yl]-1-[4-(4-methylthiazol-5-yl)phenyl]-3-oxopropyl]-4-hydroxy-1-[3-methyl-2-(3-methylisoxazol-5-yl)butanoyl]pyrrolidine-2-carboxamide

(3S)-3-[(2S,4R)-4-hydroxy-1-[3-methyl-2-(3-methylisoxazol-5-yl)butanoyl]pyrrolidine-2-amide]-3-[4-(4-methylthiazol-5-yl)phenyl]propanoic acid (25 mg, 46.24 μmol) and 2-[1-[(1,4'-dipiperidinyl)-4-yl]-3-amino-1H-pyrazolo[4,3-c] Pyridazine-6-yl]phenol (27.29 mg, 69.36 μmol) was dissolved in anhydrous N, N-dimethylformamide (0.5 mL), N, N-diisopropylethylamine (24.16 μL, 138.73 μmol) was added, and then O-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (21.10 mg, 55.49 μmol) was added. The reaction solution was stirred at 25 ° C for 3 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (Phenomenex Gemini C18 column: 5 μm silica, 30 mm diameter, 80 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 40%-60%) as eluent) to give (2S,4R)-N-[(S)-3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-[1,4'-bipiperidinyl]-1'-yl]-1-[4-(4-methylthiazol-5-yl)phenyl]-3-oxopropyl]-4-hydroxy-1-[3-methyl-2-(3-methylisoxazol-5-yl)butanoyl]pyrrolidine-2-carboxamide (Compound 008) (1.32 mg).

MS m/z (ESI): 938.5 [M+Na] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ = 8.84 (dd, J = 3.3, 9.3Hz, 1H), 8.26 (br s, 1H), 8.01 (br s, 1H), 7.54-7.46 (m, 3H ),7.46-7.40(m,1H),7.35(t,J＝7.8Hz,1H),7.04-6.97(m,2H),6.30-6.21(m,1H),5.45-5.28(m,1H), 4.65-4.54(m,4H),4.52-4.41(m,2H),4.08(d,J＝11.5H z,1H),3.95-3.83(m,1H),3.83-3.56(m,2H),3.24(d,J＝14.1Hz,2H),3.16-3.00(m,3H),2.99-2.84(m, 2H),2.59(d,J＝12.5Hz,1H),2.55-2.49(m,3H),2.41(d,J＝7.0Hz,1H),2.30(br s,1H),2.28-2.23(m,3H),2.15(d,J＝7.3Hz,1H),1.98(d,J＝9.0Hz,5H),1.45-1.25(m,2H),1.10-1.03 (m,3H),0.88(dd,J＝6.9,10.2Hz,3H).

Example 45: Synthesis of (2S,4R)-N-[(1S)-3-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidin-1-yl]-1-[4-(4-methylthiazol-5-yl)phenyl]-3-oxo-propyl]-4-hydroxy-1-[3-methyl-2-(3-methylisoxazol-5-yl)butanoyl]pyrrolidine-2-carboxamide (Compound 009)

2-[3-Amino-1-[1-[5-(piperidin-4-yl)pyrimidin-2-yl]-piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (19.13 mg, 30.83 μmol, 76% purity) and (3S)-3-[(2S,4R)-4-hydroxy-1-[3-methyl-2-(3-methylisoxazol-5-yl)butanoyl]pyrrolidine-2-amide]-3 -[4-(4-Methylthiazol-5-yl)phenyl]propionic acid (25 mg, 46.24 μmol) was dissolved in N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (11.95 mg, 92.43 μmol, 16.10 μL) and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (17.58 mg, 46.24 μmol) were added. The reaction solution was stirred at 25°C for 0.5 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (Phenomenex C18 column: 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 20%-60%) as eluent) to give (2S,4R)-N-[(1S)-3-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidin-1-yl]-1-[4-(4-methylthiazol-5-yl)phenyl]-3-oxo-propyl]-4-hydroxy-1-[3-methyl-2-(3-methylisoxazol-5-yl)butanoyl]pyrrolidine-2-carboxamide (Compound 009) (6.20 mg).

MS m/z(ESI):498.0[M+2H] ²⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.03-8.95 (m, 2H), 8.54 (s, 2H), 8.47 (d, J = 8.8Hz, 1H), 8.29 (br s, 2H), 8.17 (s,1H),7.98(br d,J＝1.5Hz,1H),7.42(br s,3H),7.06-7.02(m,3H),6.22(s,1H),5.30-5.21(m,1H ),4.99-4.87(m,2H),4.76(br s,2H),4.54-4.50(m,1H),4.44-4.34(m,1H),4.29-4.25(m,1H),3.76-3.72(m,1H),3.07-3.01(m,4H), 2.44(br s,3H),2.39(s,3H),2.20(s,3H),2.17(s,3H),1.94(br s,4H),1.23(s,1H),0.97(br d,J ＝6.3Hz,6H),0.80(br d,J＝6.5Hz,3H),0.76(br d,J＝6.5Hz,3H).

Example 46: Synthesis of Compound 010

Step 1: Synthesis of (2S,4R)-1-[2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methylbutanoic acid (909 mg, 3.02 mmol) and (2S,4R)-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (1.20 g, 3.62 mmol) were dissolved in N,N dimethylformamide (30 mL), and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (1.38 g, 3.62 mmol) and N,N-diisopropylethylamine (1.95 g, 15.1 mmol) were added. The reaction was stirred at 25° C. for 2 hours. LCMS showed that the reaction was complete. Ethyl acetate (60 mL) and water (60 mL) were added to dilute, the aqueous phase was extracted twice with ethyl acetate (60 mL), and the organic phase was washed twice with water (60 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and then filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography (silica, petroleum ether/tetrahydrofuran=100/55) to obtain (2S,4R)-1-[2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (1.84 g).

MS m/z (ESI): 615.4 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.03-8.96 (m, 1H), 8.48-8.27 (m, 1H), 7.52-7.27 (m, 5H), 6.17-6.08 (m, 1H), 5.17 -5.08(m,1H),4.96-4.84(m,1H),4.83-4.72(m,1H),4.46-4.33(m,1H),4.27(d,J＝3.5Hz,1H),4.12-4.07 (m,2H),3.70-3.61(m,3H ),3.58-3.52(m,2H),3.17(d,J＝5.3Hz,1H),2.46-2.44(m,3H),2.31-2.21(m,1H),2.03(dt,J＝4.1,7.8 Hz,1H),1.83-1.70(m,1H),1.37-1.34(m,3H),1.14-1.09(m,6H),0.96(dd,J＝3.3,6.5Hz,3H),0.85-0.79( m,3H).

Step 2: Synthesis of (2S,4R)-1-[(2S)-2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide and (2S,4R)-1-[(2R)-2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

(2S, 4R)-1-[2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (800 mg) was subjected to chiral separation (DAICEL CHIRALPAK IG column, 10 μm silica, 30 mm diameter, 250 mm length); (using 40% ethanol (containing 0.1% ammonia water) as eluent) to obtain the title product P1 (230 mg, named intermediate 9) and the title product P2 (320 mg, named intermediate 10). The two title products were then further analyzed by the following chiral HPLC analysis method.

Chiral HPLC analysis method:

Intermediate 9:

The chiral HPLC peak time was 1.686 minutes;

Intermediate 10:

The chiral HPLC peak time is 2.057 minutes.

Step 3: Synthesis of intermediate 11

Intermediate 10 (150 mg, 244 μmol) was dissolved in tetrahydrofuran (4 mL) and diluted sulfuric acid (1 M, 4.88 mL) was added. The reaction was stirred at 60 ° C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure to remove tetrahydrofuran, and saturated sodium bicarbonate was added dropwise until the pH was 7-8. The aqueous phase was extracted three times with ethyl acetate (5 mL), the collected organic layer was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated under reduced pressure to obtain the title compound intermediate 11 (130 mg).

MS m/z(ESI):541.4[M+H] ⁺ .

Step 4: Synthesis of Compound 010

2-[3-amino-1-[1-[5-(piperidin-4-yl)pyrimidin-2-yl]-piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (39.25 mg, 83.24 μmol) and intermediate 11 (15 mg, 27.75 μmol) were dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), and 1 drop of glacial acetic acid solution and sodium triacetoxyborohydride (17.64 mg, 83.24 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (Phenomenex C18 column: 5 μm silica, 30 mm diameter, 80 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 53%-73%) as eluent) to give the title compound, compound 010 (1.20 mg).

MS m/z(ESI):498.8[M+2H] ²⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.95-13.87 (m, 1H), 9.00-8.95 (m, 1H), 8.61-8.48 (m, 1H), 8.45-8.38 (m, 1H), 8.35 -8.28(m,2H),8.17-8.07(m,1H),7.52-7.40(m,2H),7.39-7.32(m,3H),7.04-6.98 (m,2H),6.62-6.35(m,2H),6.21-6.05(m,1H),5.15-5.07(m,1H),4.98-4.87(m,2H),4.83-4.74(m,2H) ,4.44-4.33(m,1H),4.32-4.22(m,3H),3.77-3.59(m,2H),3.45(br d,J＝11.0Hz,2H),3.10-2.97(m,5H),2.71(br s,2H),2.45 (s,3H),2.10(br t,J＝10.8Hz,2H),2.02-1.89 (m,5H),1.74-1.62(m,4H),1.43-1.33(m,3H),1.28-1.16(m,1H),1.00-0.93(m,3H),0.85-0.77(m,3H) .

Example 47: Synthesis of (2S,4R)-1-[2-[3-[4-[[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 011)

Step 1: Synthesis of methyl 2-[3-[4-(dimethoxymethyl)piperidin-1-yl]isoxazol-5-yl]-3-methylbutanoate

Methyl 3-methyl-2-[3-(1,1,2,2,3,3,4,4,4-nonafluorobutylsulfonyloxy)isoxazol-5-yl]butanoate (1.1 g, 2.29 mmol) and 4-(dimethoxymethyl)piperidine (400.31 mg, 2.51 mmol) were dissolved in N,N-dimethylformamide (20 mL), and N,N-diisopropylethylamine (886.15 mg, 6.86 mmol) was added. The reaction solution was reacted at 80° C. for 16 hours, and LCMS showed that the reaction was completed. The reaction solution was extracted with ethyl acetate (10 mL×3) and water (20 mL), the organic phase was washed with saturated brine (30 mL) and concentrated to dryness under reduced pressure, and the concentrate was purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 5/1) to give the product 2-[3-[4-(dimethoxymethyl)piperidin-1-yl]isoxazol-5-yl]-3-methylbutyric acid methyl ester (420 mg).

MS m/z(ESI):341.1[M+H] ⁺ ;

¹ H NMR (400MHz, CDCl ₃ ) δ5.90 (s, 1H), 4.05 (d, J = 6.7Hz, 1H), 3.77-3.66 (m, 5H), 3.47 (d, J = 8.9Hz, 1H) ,3.36(s,6H),2.88-2.73(m,2H),2.40-2.24(m,1H),1.87-1.71(m,3H),1.47-1.30(m,2H),0.99(d,J＝ 6.6Hz, 3H), 0.91 (d, J = 6.7Hz, 3H).

Step 2: Synthesis of 2-[3-[4-(dimethoxymethyl)piperidin-1-yl]isoxazol-5-yl]-3-methylbutanoic acid

2-[3-[4-(dimethoxymethyl)-1-piperidinyl]isothiazol-5-yl]-3-methylbutyric acid methyl ester (500 mg, 1.47 mmol) was dissolved in tetrahydrofuran (4 mL) and water (4 mL), sodium hydroxide (176.24 mg, 4.41 mmol) was added, and the reaction solution was reacted at 25°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was adjusted to pH 5-6 with saturated citric acid solution, and then concentrated to dryness under reduced pressure to obtain the product 2-[3-[4-(dimethoxymethyl)piperidin-1-yl]isothiazol-5-yl]-3-methylbutyric acid (460 mg).

MS m/z(ESI):294.9[M+H-MeOH] ⁺ .

Step 3: Synthesis of (2S,4R)-1-[2-[3-[4-(dimethoxymethyl)piperidin-1-yl]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrole-2-carboxamide

2-[3-[4-(Dimethoxymethyl)piperidin-1-yl]isoxazol-5-yl]-3-methylbutanoic acid (460 mg, 1.41 mmol) and (2S,4R)-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrole-2-carboxamide (544.42 mg, 1.48 mmol) were dissolved in N,N-dimethylformamide (10 mL), and triethylamine (713.07 mg, 7.05 mmol) and O-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (643.06 mg, 1.69 mmol) were added, and the reaction solution was reacted at 25°C for 1 hour. LCMS showed the reaction was complete, and the reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether/ethyl acetate = 1/0 to 3/1) to give the product (2S,4R)-1-[2-[3-[4-(dimethoxymethyl)piperidin-1-yl]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (520 mg).

MS m/z(ESI):662.1[M+Na] ⁺ .

Step 4: Synthesis of (2S,4R)-1-[2-[3-(4-formylpiperidin-1-yl)isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

(2S,4R)-1-[2-[3-[4-(dimethoxymethyl)piperidin-1-yl]isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrole-2-carboxamide (20 mg, 31.26 μmol) was dissolved in tetrahydrofuran (0.5 mL), sulfuric acid solution (1 M, 625.19 μL) was added, and the reaction solution was stirred at 65° C. for 3 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the concentrate was extracted with dichloromethane (2 mL×2) and aqueous sodium bicarbonate solution (2 mL). The organic phase was concentrated to dryness under reduced pressure to give the product (2S,4R)-1-[2-[3-(4-formylpiperidin-1-yl)isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (12 mg).

MS m/z(ESI):594.3[M+H] ⁺ .

Step 5: Synthesis of (2S,4R)-1-[2-[3-[4-[[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (14.64 mg, 47.16 μmol) and (2S,4R)-1-[2-[3-(4-formylpiperidin-1-yl)isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (28 mg, 47.16 μmol) were dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), and acetic acid (2.83 mg, 47.16 μmol) and sodium acetate borohydride (29.98 mg, 141.48 μmol) were added. The reaction solution was reacted at 25° C. for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. The product (2S,4R)-1-[2-[3-[4-[[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 011) (7 mg) was obtained by HPLC purification (Boston Green ODS column, 5 μm silica, 30 mm diameter, 150 mm length); (using water (containing 0.225% formic acid) and acetonitrile with decreasing polarity mixture (acetonitrile content: 25% to 45%) as eluent).

MS m/z (ESI): 888.3 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.10 (br s, 1H), 9.01-8.96 (m, 1H), 8.44-8.36 (m, 2H), 8.01 (br d, J＝8.2Hz, 1H ),7.48-7.34(m,5H),7.08-6.96(m,2H),6.57(br s,2H),6.18(s,1H),5.31-4.99(m,1H),4.95-4.78(m, 2H),4.35(t,J＝7.9Hz,1H),4.29(br s,1H),3.79-3.63(m,6H),3.58(br d,J＝9.9Hz,1H),3.14-3.02(m,4H),2.87-2.76(m,2H),2.46(s,5H),2.26-2.20(m,1H),2.13(br d,J ＝12.3Hz,2H),2.08-1.99(m,2H),1.88-1.76(m,3H),1.42-1.35(m,3H),1.32-1.23(m,2H),1.01-0.92(m,3H ),0.83-0.75(m,3H).

Example 48: Synthesis of Compound 012

Step 1: Synthesis of 5-bromo-4-methylthiazole

4-Methylthiazole (10 g, 100.85 mmol) was dissolved in glacial acetic acid (100 mL) at 0°C, and liquid bromine (32.23 g, 201.71 mmol) was added. The reaction solution was reacted at 50°C for 16 hours. The reaction solution was concentrated under reduced pressure, and a saturated sodium carbonate solution was added to the reaction solution to adjust the pH to 7 to quench the reaction. Water (20 mL) was added and extracted three times with dichloromethane (30 mL), and the organic phase was washed three times with water (30 mL), dried over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain 5-bromo-4-methylthiazole (31.5 g).

¹ H NMR (400MHz, Methanol-d ₄ ) δ8.92 (s, 1H), 2.41 (s, 3H).

Step 2: Synthesis of 4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiazole

Dissolve 5-bromo-4-methylthiazole (15 g, 20.14 μmol) in 1,4-dioxane (300 mL), add bis-pinacol borate (42.79 g, 18.33 μmol), potassium acetate (24.80 g, 252.74 mmol) and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (6.16 g, 8.42 mmol). After nitrogen replacement, the reaction solution was reacted at 100 ° C for 16 hours. Thin layer chromatography (petroleum ether: tetrahydrofuran = 3: 1) detected that the reaction was complete. Add water (700 mL) to the reaction solution, extract three times with dichloromethane (1500 mL), wash the organic phase with water (1500 mL) three times, dry with anhydrous sodium sulfate and concentrate to dryness under reduced pressure. Purification by column chromatography (silica, petroleum ether/ethyl acetate = 1/0 to 1/1) gave 4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiazole (8.6 g).

¹ H NMR (400MHz, Methanol-d ₄ ) δ9.06 (s, 1H), 2.63 (s, 3H), 1.20 (s, 12H).

Step 3: Synthesis of 1-[5-(4-methylthiazol-5-yl)pyrazin-2-yl]ethanone

1-(5-chloropyrazine-2-yl)ethanone (3g, 19.16mmol) was dissolved in 1,4-dioxane (80mL) and water (20mL), and 4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiazole (6.47g, 28.74mmol), potassium phosphate (12.20g, 57.48mmol) and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (1.40g, 1.92mmol) were added. After nitrogen replacement, the reaction solution was reacted at 100℃ for 16 hours. The reaction was completed by thin layer chromatography (petroleum ether: tetrahydrofuran = 5:1). Water (50mL) was added to the reaction solution and extracted three times with dichloromethane (90mL). The organic phase was washed three times with water (90mL), dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. Purification by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 5/1) gave 1-[5-(4-methylthiazol-5-yl)pyrazin-2-yl]ethanone (2.66 g).

¹ H NMR (400MHz, Methanol-d ₄ ) δ9.14 (d, J = 1.6 Hz, 1H), 9.07-9.04 (m, 2H), 2.81 (s, 3H), 2.70 (s, 3H).

Step 4: Synthesis of 1-[5-(4-methylthiazol-5-yl)pyrazin-2-yl]ethylamine

1-[5-(4-methylthiazol-5-yl)pyrazin-2-yl]ethanone (1g, 4.56mmol) was dissolved in methanol (20mL), and ammonium acetate (3.52g, 45.61mmol), sodium acetate (374.13mg, 4.56mmol), sodium cyanoborohydride (429.91mg, 6.84mmol) and acetic acid (130.42μL, 2.28mmol) were added. The reaction solution was reacted at 25°C for 16 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. Purification by column chromatography (silicon dioxide, petroleum ether/tetrahydrofuran = 1/0 to 0/1) gave 1-[5-(4-methylthiazol-5-yl)pyrazin-2-yl]ethanamine (890mg).

MS m/z(ESI):221.2[M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ9.03 (s, 1H), 9.00 (d, J = 1.5Hz, 1H), 8.77 (d, J = 1.5Hz, 1H), 4.73 (q, J = 6.8Hz,1H),2.77(s,3H),1.68(d,J=7.0Hz,3H).

Step 5: Synthesis of (2S,4R)-4-hydroxy-2-[[(R)-1-[5-(4-methylthiazol-5-yl)pyrazin-2-yl]ethyl]carbamoyl]pyrrolidine-1-carboxylic acid tert-butyl ester and (2S,4R)-4-hydroxy-2-[[(S)-1-[5-(4-methylthiazol-5-yl)pyrazin-2-yl]ethyl]carbamoyl]pyrrolidine-1-carboxylic acid tert-butyl ester

1-[5-(4-methylthiazol-5-yl)pyrazine-2-yl]ethylamine (690 mg, 3.13 mmol) was dissolved in N,N-dimethylformamide (20 mL), and (2S,4R)-1-(tert-butyloxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (869.16 mg, 3.76 mmol), 0-(7-azabenzotriazole-1-YL)-N,N,N,N-tetramethyluronium hexafluorophosphate (1.79 g, 4.70 mmol) and N,N-diisopropylethylamine (2.73 mL, 15.66 mmol) were added. The reaction solution was reacted at 25°C for 2 hours. LCMS detected that the reaction was complete. Water (50 mL) was added to the reaction solution and extracted three times with dichloromethane (90 mL). The organic phase was washed three times with water (90 mL), dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. Purification by column chromatography (silica, petroleum ether/ethyl acetate = 1/0 to 0/1) and thin layer chromatography (silica, petroleum ether/ethyl acetate = 1/2) twice, and then chiral separation (DAICEL CHIRALPAK IG column, 10 μm silica, 30 mm diameter, 250 mm length, using 40% ethanol (containing 0.1% ammonia water) as eluent) to give the title product P1 (174.2 mg, elution time of 1.731 minutes) and the title product P2 (223.9 mg, named as intermediate 3, elution time of 1.920 minutes) as a yellow solid.

Intermediate 3:

MS m/z(ESI):434.1[M+H] ⁺ .

Step 6: Synthesis of intermediate 4

Intermediate 3 (223.9 mg, 516.47 μmol) was dissolved in dioxane (2 mL), and dioxane hydrochloride (4 M, 1.29 ml) was added. The reaction solution was reacted at 25° C. for 16 hours. After LCMS detection, the reaction solution was concentrated to dryness under reduced pressure to obtain Intermediate 4 (195 mg).

MS m/z (ESI): 334.2 [M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ9.50-9.41(m,1H),8.97(d,J＝1.5Hz,1H),8.76-8.67(m,1H),5.23(q,J＝6.9 Hz,1H),4.57(s,1H),3.60(s,1H),3.38-3.33(m,2H),2.80(s,3H),2.59-2.47(m,1H),2.06-1.95(m, 1H),1.62-1.58(m,3H).

Step 7: Synthesis of Intermediate 5

Intermediate 4 (346 mg, 498.13 μmol) and N-Boc-L-tert-leucine (138.25 mg, 597.75 μmol) were dissolved in N,N-dimethylformamide (6 mL), and N,N-diisopropylethylamine ((257.52 mg, 1.99 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (284.11 mg, 747.19 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete. Water (6 mL) was added to the reaction solution and extracted three times with ethyl acetate (6 mL), and the organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure and then purified by column chromatography (petroleum ether/tetrahydrofuran = 1/2) to obtain intermediate 5 (311 mg).

MS m/z(ESI):569.2[M+Na] ⁺ .

Step 8: Synthesis of Intermediate 6

Intermediate 5 (311 mg, 500.62 μmol) was dissolved in dioxane (3 mL), and dioxane hydrochloride solution (4 M, 1.25 mL) was added. The reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain Intermediate 6 (332 mg).

MS m/z(ESI):447.0[M+H] ⁺ .

Step 9: Synthesis of compound 012

Intermediate 6 (35.20 mg, 72.88 μmol) and (4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]cyclohexane-1-carboxylic acid (75 mg, 72.88 μmol, 50% purity) were dissolved in N,N-dimethylformamide (1 mL), and N,N-dimethylformamide (37.67 mg, 291.50 μmol, 50.77 μL) and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (41.56 mg, 109.31 μmol) were added. The reaction solution was stirred at 25°C for 0.5 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (Phenomenex C18 column: 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 21%-61%) as eluent) to give the title compound 012 (4.20 mg).

MS m/z (ESI): 943.8 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.10 (d, J = 6.5Hz, 1H), 8.93-8.89 (m, 2H), 8.81-8.64 (m, 2H), 8.55-8.50 (m, 2H) ),8.35-8.32(m,1H),8.28-8.25(m,1H),8.07-7.89(m,1H),7.08-6.95(m,3H),5.03-4.86 (m,2H),4.84-4.74(m,2H),4.59-4.41(m,2H),4.41-4.35(m,1H),3.72-3.62(m,2H),3.09-3.05(m,2H) ,2.71-2.68(m,3H),2.08-1.83(m,13H),1.67-1.59(m,2H),1.46(br t,J＝6.5Hz,4H),1.28-1.21(m,1H), 0.95-0.90(m,9H).

Example 49: Synthesis of (2S,4R)-1-[(2S)-2-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]cyclohexane-1-amide]-3,3-dimethylbutyryl]-4-hydroxy-N-[1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethyl]pyrrolidine-2-carboxamide (Compound 013)

Step 1: Synthesis of 1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethanone

2-Chloro-5-acetyl-pyrimidine (2.5 g, 15.97 mmol) and 4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiazole (7.19 g, 31.93 mmol) were dissolved in anhydrous dioxane (100 mL) and water (25 mL), and cesium carbonate (15.61 g, 47.90 mmol) and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (584.17 mg, 798.37 μmol) were added. The reaction solution was stirred at 100 ° C for 16 hours under a nitrogen atmosphere. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silicon dioxide, petroleum ether: ethyl acetate = 1: 1) to obtain 1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethanone (2 g).

MS m/z (ESI): 219.9 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.30 (s, 2H), 9.18 (s, 1H), 2.88 (s, 3H), 2.65 (s, 3H).

Step 2: Synthesis of 1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethylamine

1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethanone (1.7 g, 7.75 mmol) was dissolved in anhydrous methanol (60 mL), and ammonium acetate (5.98 g, 77.53 mmol), sodium acetate (636.03 mg, 7.75 mmol), sodium cyanoborohydride (730.85 mg, 11.63 mmol) and acetic acid (221.71 μL, 3.88 mmol) were added. The reaction solution was stirred at 25 ° C for 16 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silicon dioxide, petroleum ether: tetrahydrofuran = 2: 1 to 0: 1) to obtain 1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethanamine (1 g).

MS m/z(ESI):221.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.07 (s, 1H), 8.91-8.86 (m, 2H), 4.28 (q, J = 6.8Hz, 1H), 2.84 (s, 3H), 1.34 ( d,J＝6.4Hz,3H).

Step 3: Synthesis of tert-butyl (2S,4R)-4-hydroxy-2-[1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethylcarbamoyl]pyrrolidine-1-carboxylate

1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethylamine (1 g, 4.54 mmol) and (2S, 4R)-1-tert-butyloxycarbonyl-4-hydroxy-pyrrolidine-2-carboxylic acid (1.15 g, 4.99 mmol) were dissolved in anhydrous N, N-dimethylformamide (25 mL), and N, N-diisopropylethylamine (2.37 mL, 13.62 mmol) and O-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (2.59 g, 6.81 mmol) were added. The reaction solution was stirred at 25 ° C for 16 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether:tetrahydrofuran = 5:1 to 0:1) to give (2S,4R)-4-hydroxy-2-[1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethylcarbamoyl]pyrrolidine-1-carboxylic acid tert-butyl ester (840 mg).

MS m/z(ESI):434.1[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.08-9.03 (m, 1H), 8.84-8.75 (m, 2H), 5.05-4.86 (m, 2H), 4.32-4.08 (m, 2H), 3.46 -3.36(m,1H),3.30-3.22(m,1H),2.84-2.80(m,3H),2.15-1.99(m,1H),1.49-1.42(m,3H),1.41-1.30(m, 9H).

Step 4: Synthesis of (2S,4R)-4-hydroxy-N-[1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethyl]pyrrolidine-2-carboxamide

Dissolve (2S, 4R)-4-hydroxy-2-[1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethylcarbamoyl]pyrrolidine-1-carboxylic acid tert-butyl ester (120 mg, 276.80 μmol) in anhydrous dioxane (1 mL), and add dioxane hydrochloride (4M, 692.00 μL). The reaction solution was stirred at 25 ° C for 3 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain (2S, 4R)-4-hydroxy-N-[1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethyl]pyrrolidine-2-carboxamide (120 g).

MS m/z(ESI):334.2[M+H] ⁺ .

Step 5: Synthesis of tert-butyl (2S)-1-[(2S,4R)-4-hydroxy-2-[1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethylcarbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamate

(2S,4R)-4-hydroxy-N-[1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethyl]pyrrolidine-2-carboxamide (100 mg, 299.93 μmol) and (S)-2-(tert-butyloxycarbonylamino)-3,3-dimethylbutyric acid (83.25 mg, 359.92 μmol) were dissolved in anhydrous dimethylformamide (4 mL), and N,N-diisopropylethylamine (156.73 μL, 899.80 μmol) and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (171.07 mg, 449.90 μmol) were added. The reaction solution was stirred at 25°C for 3 hours. The reaction was completed by LCMS. The mixture was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether:tetrahydrofuran = 1:2) to give tert-butyl (2S)-1-[(2S,4R)-4-hydroxy-2-[1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethylcarbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamate (40 mg).

MS m/z(ESI):569.1[M+Na] ⁺ .

Step 6: Synthesis of (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butyryl]-4-hydroxy-N-[1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethyl]pyrrolidine-2-carboxamide

Tert-butyl (2S)-1-[(2S,4R)-4-hydroxy-2-[1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethylcarbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamate (40 mg, 73.17 μmol) was dissolved in anhydrous dioxane (1 mL), and dioxane hydrochloride (4M, 182.92 μL) was added. The reaction solution was stirred at 25°C for 3 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butyryl]-4-hydroxy-N-[1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethyl]pyrrolidine-2-carboxamide (30 mg).

MS m/z(ESI):447.4[M+H] ⁺ .

Step 7: Synthesis of (2S,4R)-1-[(2S)-2-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]cyclohexanecarboxamide]-3,3-dimethyl-butyryl]-4-hydroxy-N-[1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethyl]pyrrolidine-2-carboxamide

(2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butyryl]-4-hydroxy-N-[1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethyl]pyrrolidine-2-carboxamide (25 mg, 55.98 μmol) and 4-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl] [7-azabenzotriazole]-N,N,N',N'-tetramethyluronium hexafluorophosphate (25.54 mg, 67.18 μmol) was added. The reaction solution was stirred at 25 ° C for 3 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (Phenomenex Gemini C18 column: 5 μm silica, 30 mm diameter, 80 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 40%-60%) as eluent) to give (2S,4R)-1-[(2S)-2-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]cyclohexaneamide]-3,3-dimethyl-butyryl]-4-hydroxy-N-[1-[2-(4-methylthiazol-5-yl)pyrimidin-5-yl]ethyl]pyrrolidine-2-carboxamide (Compound 013) (4.0 mg).

MS m/z (ESI): 943.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.05 (s, 1H), 8.88-8.68 (m, 2H), 8.56-8.47 (m, 2H), 8.34-8.24 (m, 2H), 8.16-8.03 (m,1H),7.82-7.62(m,1H),7.37(t,J＝7.0Hz,1H),7.09-6.98(m,2H),6.49(br s,2H),5.13(br s,1H ),4.96-4.87(m,2H),4.79(d,J＝13.1Hz,2H),4.58-4.47(m,1H),4.39(t,J＝8.2Hz,1H),4.29(br s,1H),3.62(brs,2H),3.07(d,J＝8.0Hz,2H),2.85-2.75(m,3H),2.64(br s,1H),2.56(br s,1H),2.09 -1.90(m,6H),1.89-1.70(m,5H),1.69-1.48(m,4H),1.45(d,J=7.0Hz,3H),0.94(s,9H).

Example 50: Synthesis of Compound 014

Step 1: Synthesis of trans-tert-butyl 4-[3-[[4-[4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-2-yl]oxy]cyclobutyloxy]piperidine-1-carboxylate

Trans-tert-butyl 4-[3-[(4-bromopyridin-2-yl)oxy]cyclobutoxy]piperidine-1-carboxylate (308.19 mg, 721.19 μmol) and tert-butyl N-tert-butyloxycarbonyl-N-[6-[2-(methoxymethoxy)phenyl]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-yl]carbamate (200 mg, 360.60 μmol) were dissolved in tert-amyl alcohol (20 mL), and (2-dicyclohexylphosphino-2,6-diisopropoxy-1,1-biphenyl)[2-(2-amino-1,1-biphenyl)]palladium(II) (30.16 mg, 36.06 μmol) and sodium tert-butoxide (69.31 mg, 721.19 μmol) were added. The reaction solution was subjected to microwave reaction at 140°C for 2 hours, and the reaction was complete after LCMS detection. The reaction solution was filtered, concentrated, and purified by thin layer chromatography (silica, petroleum ether/ethyl acetate = 1/1) to give trans-4-[3-[[4-[4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-2-yl]oxy]cyclobutyloxy]piperidine-1-carboxylic acid tert-butyl ester (36 mg).

MS m/z(ESI):723.3[M+Na] ⁺ .

Step 2: Synthesis of trans-2-[3-amino-1-[1-[2-[3-(piperidin-4-yloxy)cyclobutyloxy]pyridin-4-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

Trans-4-[3-[[4-[4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-2-yl]oxy]cyclobutyloxy]piperidine-1-carboxylic acid tert-butyl ester (36 mg, 51.37 μmol) was dissolved in dichloromethane (1 mL), and hydrochloric acid/dioxane (128.42 μL, 513.68 μmol) was added. The reaction solution was reacted at 25° C. for 2 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated to obtain trans-2-[3-amino-1-[1-[2-[3-(piperidin-4-yloxy)cyclobutyloxy]pyridin-4-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (30 mg).

MS m/z(ESI):557.3[M+H] ⁺ .

Step 3: Synthesis of compound 014

Trans-2-[3-amino-1-[1-[2-[3-(piperidin-4-yloxy)cyclobutyloxy]pyridin-4-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (20 mg, 33.72 μmol) and intermediate 11 (18.30 mg, 33.72 μmol) were dissolved in methanol (1 mL), and sodium acetate (11.06 mg, 134.88 μmol), acetic acid (2.02 mg, 33.72 μmol) and sodium cyanoborohydride (10.60 mg, 168.60 μmol) were added. The reaction solution was reacted at 25° C. for 5 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated and purified by preparative liquid chromatography (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia water) and acetonitrile (acetonitrile content: 36%-76%) as eluent) to give the title compound, Compound 014 (1.5 mg).

MS m/z(ESI):1084.2[M+H] ⁺ ;

¹ H NMR (DMSO-d ₆ , 400MHz): δ = 13.93 (br s, 1H), 9.12 (s, 1H), 8.93 (s, 1H), 8.61 (s, 1H), 8.52-8.56 (m, 2H ),8.11(br d,J＝7.8Hz,1H),7.96(d,J＝5.7Hz,1H),7.37(br t,J＝7.6Hz,1H),6.99-7.04(m,2H),6.45 (s,2H),6.24(s,1H),6.20(br d,J＝5.3Hz,1H),6.10(s,1H),5.11(d,J＝3.2Hz,1H),4.99(dd,J ＝13.9,6.8Hz,1H),4.91(br s,2H),4.48(br d,J＝12.8Hz,3H),4.39(br t,J＝7.9Hz,2H),4.30(br s,3H),4.21(br s,2H),3.71(br d,J＝10.5Hz,3H),3.67-3.62(m,3H),3.03-2.95(m,2H),2.70(s,3H),2.16-2.08(m,4H),2.05-1.98(m, 4H),1.93-1.85(m,4H),1.82-1.75(m,4H),1.46-1.42(m,3H),0.94(d,J＝6.6Hz,3H),0.79(d,J＝6.6Hz ,3H).

Example 51: Synthesis of 1-[[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]methyl]-N-[(1S)-1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]piperidine-4-carboxamide (Compound 015)

Step 1: Synthesis of 2-[3-amino-1-[1-[5-(hydroxymethyl)pyrimidin-2-yl]piperidin-4-yl]pyrazolo[4,3-c]pyridazin-6-yl]phenol

2-Chloro-5-hydroxymethylpyrimidine (83.84 mg, 579.98 μmol) was dissolved in N, N-dimethylformamide (4 mL), and 2-[3-amino-1-[1-[5-(hydroxymethyl)pyrimidin-2-yl]piperidin-4-yl]pyrazolo[4,3-c]pyridazin-6-yl]phenol (150 mg, 483.32 μmol) and N, N-diisopropylethylamine (336.74 μL, 1.93 mmol) were added. The reaction solution was stirred at 100°C for 2 hours. The reaction was completed by LCMS. The reaction solution was cooled to room temperature, water (8 mL) was added, and the mixture was extracted twice with ethyl acetate (15 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The mixture was purified by thin layer chromatography (petroleum ether:tetrahydrofuran = 3:1, containing 0.5% ammonia methanol solution (7N)) to give 2-[3-amino-1-[1-[5-(hydroxymethyl)pyrimidin-2-yl]piperidin-4-yl]pyrazolo[4,3-c]pyridazin-6-yl]phenol (60 mg).

MS m/z(ESI):419.2[M+H] ⁺ .

Step 2: Synthesis of 2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidine-5-carbaldehyde

2-[3-amino-1-[1-[5-(hydroxymethyl)pyrimidin-2-yl]piperidin-4-yl]pyrazolo[4,3-c]pyridazin-6-yl]phenol (20 mg, 47.80 μmol) was dissolved in dichloroethane (3 mL), and active manganese dioxide (41.55 mg, 477.95 μmol) was added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was filtered through diatomaceous earth to obtain a crude liquid, which was concentrated to dryness under reduced pressure to obtain 2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidine-5-carboxaldehyde (15 mg).

MS m/z(ESI):417.2[M+H] ⁺ .

Step 3: Synthesis of 1-[[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]methyl]-N-[(1S)-1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]piperidine-4-carboxamide

2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidine-5-carboxaldehyde (15 mg, 36.02 μmol) was dissolved in N,N-dimethylformamide (4 mL) and tetrahydrofuran (4 mL), and N-[(S)-1-[(2S,4R)-4-hydroxy-2-[[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]piperidine-4-carboxamide (20.02 mg, 36.02 μmol) and acetic acid (108.15 μg, 1.80 μmol) were added. The reaction solution was stirred at 25°C for 20 minutes. Sodium acetate borohydride (22.90 mg, 108.06 μmol) was added, and the reaction solution was stirred at 25° C. for 2.5 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (Boston Green ODS column: 5 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 23%-43%) as eluent) to purify 1-[[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]methyl]-N-[(1S)-1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]piperidine-4-carboxamide (Compound 015) (2.12 mg).

MS m/z (ESI): 979.5 [M+Na] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ = 8.90 (s, 1H), 8.40 (s, 2H), 8.32 (s, 1H), 8.04 (br d, J = 8.1Hz, 1H), 7.49-7.42 (m,4H),7.41-7.34(m,1H),7.06-7.01(m,2H),5.01-4.94(m,5H),4.59-4.54(m,1H),4.45(br s,1H),3.91-3.72(m,4H),3.26-3.14(m,4H),2.62-2.47(m,6H),2.29-2.09(m,4H),2.08-1.86(m,6H), 1.53(d,J＝7.1Hz,3H),1.07(s,9H).

Example 52: Synthesis of 1-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidine-5-carbonyl]-N-[(S)-1-[(2S,4R)-4-hydroxy-2-[[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]piperidine-4-carboxamide (Compound 016)

Step 1: Synthesis of methyl 2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidine-5-carboxylate

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (150 mg, 483.32 μmol) was dissolved in N,N-dimethylformamide (5 mL), and methyl 2-chloropyrimidine-5-carboxylate (83.41 mg, 483.32 μmol) and N,N-diisopropylethylamine (249.86 mg, 1.93 mmol) were added. The reaction solution was reacted at 100° C. for 2 hours, and the reaction was completed by LCMS detection. The reaction solution was cooled to room temperature, diluted with distilled water (10 mL), extracted three times with ethyl acetate (10 mL), the organic phases were combined, dried, concentrated, and purified by column chromatography (silica, petroleum ether/ethyl acetate = 1/1) to give 2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidine-5-carboxylic acid methyl ester (150 mg).

MS m/z(ESI):447.2[M+H] ⁺ .

Step 2: Synthesis of 2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidine-5-carboxylic acid

2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidine-5-carboxylic acid methyl ester (150 mg, 335.98 μmol) was dissolved in tetrahydrofuran (4 mL) and distilled water (4 mL), and lithium hydroxide monohydrate (42.30 mg, 1.01 mmol) was added. The reaction solution was reacted at 25°C for 12 hours, and the reaction was completed by LCMS detection. The reaction solution was adjusted to pH 5-6 with 1M hydrochloric acid, dried over anhydrous sodium sulfate, filtered, and concentrated to obtain 2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidine-5-carboxylic acid (120 mg).

MS m/z(ESI):433.2[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-[[(S)-1-[(2S,4R)-4-hydroxy-2-[[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamoyl]piperidine-1-carboxylate

(2S,4R)-1-[(S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (1 g, 2.08 mmol) and 1-tert-butyloxycarbonylpiperidine-4-carboxylic acid (524.27 mg, 2.29 mmol) were dissolved in N,N-dimethylformamide (10 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.19 g, 3.12 mmol) and N,N-diisopropylethylamine (806.01 mg, 6.24 mmol) were added. The reaction solution was reacted at 25°C for 4 hours, and the reaction was completed by LCMS detection. The reaction solution was diluted with distilled water (20 mL), extracted with ethyl acetate (15 mL×3), the organic phases were combined, dried, concentrated, and purified by column chromatography (silica, petroleum ether/ethyl acetate=1/1) to give tert-butyl 4-[[(S)-1-[(2S,4R)-4-hydroxy-2-[[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamoyl]piperidine-1-carboxylate (1.4 g).

MS m/z(ESI):556.3[M+H] ⁺ .

Step 4: Synthesis of N-[(S)-1-[(2S,4R)-4-hydroxy-2-[[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]piperidine-4-carboxamide

4-[[(S)-1-[(2S,4R)-4-hydroxy-2-[[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutane-2-yl]carbamoyl]piperidine-1-carboxylic acid tert-butyl ester (1.4 g, 2.13 mmol) was dissolved in dichloromethane (15 mL), and hydrochloric acid/dioxane (5.34 mL, 21.35 mmol) was added. The reaction solution was reacted at 25° C. for 2 hours, and the reaction was completed by LCMS detection. The reaction mixture was concentrated to give N-[(S)-1-[(2S,4R)-4-hydroxy-2-[[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]piperidine-4-carboxamide (Compound 016) (1.2 g).

MS m/z(ESI):578.2[M+Na] ⁺ .

Step 5: Synthesis of 1-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidine-5-carbonyl]-N-[(S)-1-[(2S,4R)-4-hydroxy-2-[[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]piperidine-4-carboxamide

2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidine-5-carboxylic acid (30 mg, 69.37 μmol) and N-[(S)-1-[(2S,4R)-4-hydroxy-2-[[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3,3-dithiazol-5-yl]carbamoyl]pyrrolidin-1-yl] Methyl-1-oxobutane-2-yl]piperidine-4-carboxamide (45.19 mg, 76.31 μmol) was dissolved in N,N-dimethylformamide (1.5 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (39.57 mg, 104.06 μmol) and N,N-diisopropylethylamine (35.86 mg, 277.50 μmol) were added. The reaction solution was reacted at 25°C for 2 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated and purified by preparative liquid chromatography (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 19%-59%) as eluent) to give 1-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidine-5-carbonyl]-N-[(S)-1-[(2S,4R)-4-hydroxy-2-[[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]piperidine-4-carboxamide (Compound 016) (5.7 mg).

MS m/z (ESI): 970.5 [M+H] ⁺ ;

¹ H NMR (DMSO-d ₆ , 400MHz): δ = 14.16-13.46 (m, 1H), 8.99 (s, 1H), 8.55 (s, 1H), 8.49-8.48 (m, 2H), 8.40 (br d ,J＝7.8Hz,1H),8.08(br d,J＝7.6Hz,1H),7.86(br d,J＝9.1Hz,1H),7.46-7.43(m,2H),7.41-7.39(m, 1H),7.40-7.34(m,3H),7.02(d,J＝7.8Hz,2H),6.52(br s,2H),4.99-4.87(m,5H),4.52(br d,J＝9.3Hz ,1H),4.43(br t,J＝7.9Hz,1H),4.29(br s,1H),3.65-3.56(m,2H),3.16(br s,2H),2.46(s,3H),2.00(br s,7H), 1.84-1.75(m,3H),1.68(br d,J＝11.8Hz,2H),1.59-1.45(m,2H),1.38(br d,J＝6.9Hz,3H),0.95(s,9H) .

Example 53: Synthesis of (2S,4R)-1-[(2S)-2-[2-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidin-1-yl]acetamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 017)

Step 1: Synthesis of tert-butyl 2-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidin-1-yl]acetate

2-[3-amino-1-[1-[5-(4-piperidinyl)pyrimidin-2-yl]-4-piperidinyl]pyrazolo[4,3-c]pyridazin-6-yl]phenol (40 mg, 84.83 μmol) and tert-butyl 2-bromoacetate (19.85 mg, 101.79 μmol, 15.04 μL) were dissolved in dioxane (30 mL), and N,N-diisopropylethylamine (32.89 mg, 254.48 μmol, 44.33 μL) was added. The reaction solution was stirred at 80° C. for 16 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by thin layer chromatography (silica, petroleum ether:tetrahydrofuran = 1:1) to give tert-butyl 2-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidin-1-yl]acetate (20 mg).

MS m/z(ESI):586.4[M+H] ⁺ .

Step 2: Synthesis of 2-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidin-1-yl]acetic acid

Tert-butyl 2-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidin-1-yl]acetate (20 mg, 34.15 μmol) was dissolved in dichloromethane (1 mL) and trifluoroacetic acid (0.5 mL). The reaction solution was stirred at 25°C for 4 hours, and the reaction was complete as monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure to obtain 2-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidin-1-yl]acetic acid (20 mg).

MS m/z(ESI):530.2[M+H] ⁺ .

Step 3: Synthesis of (2S,4R)-1-[(2S)-2-[2-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidin-1-yl]acetamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidin-1-yl]acetic acid (23 mg, 43.43 μmol), (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazole-5- [0043] 2-[( ... The reaction solution was concentrated to dryness under reduced pressure and purified by high performance liquid chromatography (Boston Green ODS column, 5 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 30%-50%) as eluent) to give (2S,4R)-1-[(2S)-2-[2-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidin-1-yl]acetamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 017) (6.43 mg).

MS m/z (ESI): 956.7 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.78-9.54 (m, 1H), 9.09-8.90 (m, 1H), 8.78 (d, J = 9.5Hz, 1H), 8.54 (s, 1H), 8.43(d,J＝8.6Hz,1H),8.36-8.28(m,2H),7.95(br s,1H),7.48-7.41(m,4H),7.41-7.35(m,3H),7.09-7.00 (m,3H),5.02-4.89(m,2H),4.81(br d,J＝12.9Hz,2H),4.59(br d,J＝9.0Hz,1H),4.50-4.37(m,1H), 4.32(br s,1H),4.07(br d,J＝14.4Hz,2H)3.85-3.47(m,4H),3.22-2.99(m,3H),2.83(s,1H),2.78-2.63(m,2H) ,2.44(br s,3H),2.33(s,1H),2.07(br d,J＝11.9Hz,2H),1.81(br s,1H),1.55-1.46(m,1H),1.39(d, J＝7.1Hz,4H),1.24(br s,1H),1.01-0.95(m,11H).

Example 54: Synthesis of (2S,4R)-1-[2-[3-[2-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethoxy]ethoxy]isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 018)

Step 1: Synthesis of 2-(2-benzyloxyethoxy)ethyl 4-methylbenzenesulfonate

2-(2-Benzyloxyethoxy)ethanol (5g, 25.5mmol) was dissolved in N,N-dimethylformamide (100mL), and sodium hydrogen (1.53g, 38.2mmol, 60% purity) was added under nitrogen atmosphere at 0℃. The reaction solution was stirred at 0℃ for 0.5 hours. 4-Toluenesulfonyl chloride (5.83g, 30.6mmol) was added under nitrogen atmosphere at 0℃, and the reaction solution was stirred at 25℃ for 2 hours. LCMS showed that the reaction was complete. Ice water was slowly added at 0℃ to quench the reaction, and the reaction solution was diluted with water (200mL) and ethyl acetate (250mL), and the aqueous phase was extracted twice with ethyl acetate (250mL). The organic phase was washed twice with water (200mL). The organic layer was collected, dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether/ethyl acetate = 100/20) to give the product 4-methylbenzenesulfonic acid 2-(2-benzyloxyethoxy)ethyl ester (3.5 g).

MS m/z (ESI): 373.0 [M+Na] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ = 7.78 (d, J = 8.4Hz, 2H), 7.40 (d, J = 8.0Hz, 2H), 7.36-7.22 (m, 5H), 4.57-4.45 ( m,2H),4.18-4.12(m,2H),3.68-3.64(m,2H),3.55(s,4H),2.42(s,3H).

Step 2: Synthesis of methyl 2-[3-[2-(2-benzyloxyethoxy)ethoxy]isoxazol-5-yl]-3-methylbutanoate

4-Methylbenzenesulfonic acid-2-(2-benzyloxyethoxy)ethyl ester (968mg, 2.76mmol) and 2-(3-hydroxyisoxazol-5-yl)-3-methylbutyric acid methyl ester (500mg, 2.51mmol) were dissolved in N,N dimethylformamide (20mL), and potassium carbonate (1.04g, 7.53mmol) was added. The reaction solution was stirred at 80°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was diluted with water (40mL) and ethyl acetate (40mL), and the aqueous phase was extracted twice with ethyl acetate (40mL). The organic phase was washed twice with water (40mL). The collected organic layer was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (silicon dioxide, petroleum ether/ethyl acetate = 100/15) to obtain the product 2-[3-[2-(2-benzyloxyethoxy)ethoxy]isoxazol-5-yl]-3-methylbutyric acid methyl ester (530mg).

MS m/z(ESI):378.1[M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ = 7.41-7.20 (m, 5H), 6.12-5.94 (m, 1H), 4.55 (s, 2H), 4.39-4.28 (m, 2H), 3.87-3.78 (m,2H),3.74-3.67(m,5H),3.67-3.62(m,2H),3.57(d,J＝8.8Hz,1H),2.43-2.27(m,1H),0.99(d,J ＝6.8Hz,3H),0.90(d,J＝6.8Hz,3H).

Step 3: Synthesis of methyl 2-[3-[2-(2-hydroxyethoxy)ethoxy]isoxazol-5-yl]-3-methylbutanoate

2-[3-[2-(2-benzyloxyethoxy)ethoxy]isoxazol-5-yl]-3-methylbutyric acid methyl ester (300 mg, 795 μmol) was dissolved in dichloromethane (8 mL), and titanium tetrachloride (196 mg, 1.03 mmol) was added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was diluted with water (10 mL), and the aqueous phase was extracted twice with dichloromethane (10 mL). The organic phase was washed twice with saturated sodium bicarbonate (10 mL). The collected organic layer was dried over anhydrous sodium sulfate and then filtered. The filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (silicon dioxide, petroleum ether/tetrahydrofuran=100/30) to give the product 2-[3-[2-(2-hydroxyethoxy)ethoxy]isoxazol-5-yl]-3-methylbutyric acid methyl ester (210 mg).

MS m/z(ESI):288.1[M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ = 5.62 (s, 1H), 4.01-3.85 (m, 2H), 3.46-3.37 (m, 2H), 3.35-3.29 (m, 3H), 3.29-3.24 (m,2H),3.22-3.14(m,3H),2.06-1.83(m,1H),0.59(d,J＝6.6Hz,3H),0.50(d,J＝6.8Hz,3H).

Step 4: Synthesis of methyl 3-methyl-2-[3-[2-(2-methylsulfonyloxyethoxy)ethoxy]isoxazol-5-yl]butanoate

2-[3-[2-(2-hydroxyethoxy)ethoxy]isoxazol-5-yl]-3-methylbutyric acid methyl ester (210 mg, 731 μmol) was dissolved in tetrahydrofuran (5 mL), and pyridine (173 mg, 2.19 mmol) was added. Then methylsulfonic anhydride (255 mg, 1.46 mmol) was added at 0°C, and the reaction solution was stirred at 25°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was diluted with water (10 mL) and ethyl acetate (10 mL), the organic phase was washed twice with water (10 mL), and the aqueous phase was extracted twice with ethyl acetate (10 mL). The collected organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain the product 3-methyl-2-[3-[2-(2-methylsulfonyloxyethoxy)ethoxy]isoxazol-5-yl]butyric acid methyl ester (250 mg), which was directly used in the next step.

MS m/z(ESI):366.1[M+H] ⁺ .

Step 5: Synthesis of methyl 2-[3-[2-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]ethoxy]ethoxy]isoxazol-5-yl]-3-methylbutanoate

3-Methyl-2-[3-[2-(2-methylsulfonyloxyethoxy)ethoxy]isoxazol-5-yl]butyric acid methyl ester (70.6 mg, 193 μmol) and 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (50.0 mg, 161 μmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (62.5 mg, 483 μmol) was added. The reaction solution was stirred at 80 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was diluted with water (10 mL) and ethyl acetate (10 mL), and the aqueous phase was extracted twice with ethyl acetate (10 mL). The organic phase was washed twice with water (10 mL). The organic layer was collected, dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated to dryness under reduced pressure and purified by thin layer chromatography (silica, petroleum ether/tetrahydrofuran = 1/3, 0.1% ammonia methanol) to give the product 2-[3-[2-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]ethoxy]ethoxy]isoxazol-5-yl]-3-methylbutanoic acid methyl ester (58 mg).

MS m/z(ESI):580.4[M+H] ⁺ .

Step 6: Synthesis of 2-[3-[2-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethoxy]ethoxy]isoxazol-5-yl]-3-methylbutanoic acid

2-[3-[2-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]ethoxy]ethoxy]isoxazol-5-yl]-3-methylbutanoic acid methyl ester (30 mg, 51.8 μmol) was dissolved in tetrahydrofuran (0.5 mL) and water (0.5 mL), and lithium hydroxide monohydrate (6.52 mg, 155 μmol) was added. The reaction solution was stirred at 25° C. for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, diluted with water, and then diluted hydrochloric acid (1.5 M) was added dropwise to adjust the pH to 5 to 6, followed by concentration under reduced pressure to give the product 2-[3-[2-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethoxy]ethoxy]isoxazol-5-yl]-3-methylbutanoic acid (29 mg).

MS m/z(ESI):566.5[M+H] ⁺ .

Step 7: Synthesis of (2S,4R)-1-[2-[3-[2-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethoxy]ethoxy]isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-[2-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethoxy]ethoxy]isoxazol-5-yl]-3-methylbutanoic acid (29.0 mg, 51.3 μmol) and (2S,4R)-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (18.7 mg, 56.4 μmol) were dissolved in N,N-dimethylformamide (0.5 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (29.2 mg, 76.9 μmol) and N,N-diisopropylethylamine (19.9 mg, 154 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure and purified by preparative liquid chromatography (Phenomenex C18, 5μm silica, 30mm diameter, 80mm length; using a mixture of water (containing 0.225% formic acid) and acetonitrile (30%-50%) as eluent) to obtain the product (2S,4R)-1-[2-[3-[2-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethoxy]ethoxy]isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 018) (5.64 mg).

MS m/z(ESI):440.5[M+2H] ²⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.65 (s, 1H), 9.43 (s, 1H), 9.04-8.93 (m, 1H), 8.48-8.35 (m, 2H), 8.14 (s, 1H) ),7.48-7.34(m,4H),7.30(d,J＝8.1Hz,1H),7.09-6.96(m,2H),6.54(s,2H),6.18-6.06(m,1H),5.12( d,J＝3.4Hz,1H), 4.96-4.72(m,2H),4.46-4.21(m,4H),3.94-3.48(m,9H),2.47-2.41(m,6H),2.19-1.98(m,4H),1.81-1.69(m ,1H),1.43(d,J＝5.9Hz,1H),1.39-1.27(m,3H),0.95(d,J＝6.5Hz,3H),0.84-0.70(m,4H).

Example 55: Synthesis of (2S,4R)-1-[(S)-2-[8-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]octanamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 019)

Step 1: Synthesis of 8-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]octanoic acid

2-(3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (50 mg, 161.11 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 8-bromooctanoic acid (39.54 mg, 177.22 μmol) and N,N-diisopropylethylamine (62.47 mg, 483.32 μmol) were added. The reaction solution was reacted at 80°C for 4 hours, and the reaction was completed by LCMS detection. The reaction solution was cooled to room temperature and concentrated to obtain 8-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]octanoic acid (50 mg).

MS m/z(ESI):453.3[M+H] ⁺ .

Step 2: Synthesis of (2S,4R)-1-[(S)-2-[8-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]octanamido]-3,3-dimethylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

8-[4-[3-Amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]octanoic acid (40 mg, 88.39 μmol) and (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (42.52 mg, 88.39 μmol) were dissolved in N,N-dimethylformamide (1 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (50.75 mg, 133.47 μmol) and N,N-diisopropylethylamine (34.27 mg, 265.17 μmol) were added. The reaction solution was reacted at 25°C for 5 hours, and the reaction was completed by LCMS. The reaction solution was concentrated and purified by preparative liquid chromatography (Phenomenex C18, 5 μm silica, 25 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 33%-73%) as an eluent) to obtain (2S,4R)-1-[(S)-2-[8-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]octanamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 019) (2.3 mg).

MS m/z (ESI): 879.5 [M+H] ⁺ ;

¹ H NMR (DMSO-d ₆ , 400MHz): δ = 13.99 (s, 1H), 8.99 (s, 1H), 8.52 (s, 1H), 8.39 (d, J = 7.8Hz, 1H), 8.16-8.11 (m,1H),7.84-7.78(m,1H),7.42-7.46(m,2H),7.40-7.34(m,3H),6.99(d,J＝8.1Hz,2H),6.46(s,2H ),5.12(d,J＝3.4Hz,1H),4.92(t,J＝7.4Hz,1H),4.53(br d,J＝9.3Hz,2H),4.43(t,J＝8.0Hz,1H) ,4.28(br s,1H),3.66-3.57(m,2H),2.99(br s,2H),2.46(s,3H),2.34-2.23(m,3H),2.08(br s,6H),1.86-1.77( m,3H),1.51-1.41(m,4H),1.38(d,J=7.0Hz,3H),1.28(br s,6H),0.94(s,9H).

Example 56: Synthesis of (2S,4R)-1-[(S)-2-[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]butanamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 020)

Step 1: Synthesis of ethyl 4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]butanoate

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (50 mg, 161.11 μmol) and ethyl 4-bromobutyrate (37.71 mg, 193.33 μmol, 27.73 μL) were dissolved in anhydrous N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (62.46 mg, 483.32 μmol, 84.18 μL) was added. The reaction solution was stirred at 80°C for 12 hours. The reaction was detected to be complete by thin layer chromatography (petroleum ether: tetrahydrofuran = 1:1). The reaction solution was extracted with ethyl acetate (20 mL) and water (10 mL), concentrated to dryness under reduced pressure, and purified by thin layer chromatography (silica, petroleum ether:tetrahydrofuran = 1:2) to give 4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]butanoic acid ethyl ester (57 mg).

Step 2: Synthesis of 4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]butanoic acid

Ethyl 4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]butyrate (40 mg, 94.23 μmol) and sodium hydroxide (15.08 mg, 376.92 μmol) were dissolved in methanol (1 mL) and water (1 mL). The reaction solution was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness, added with water (2 mL) to dissolve, adjusted to pH 1 with 1M HCl, and extracted with ethyl acetate (10 mL). The organic phase was concentrated to dryness under reduced pressure to obtain 4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]butyric acid (30 mg).

MS m/z(ESI):397.1[M+H] ⁺ .

Step 3: Synthesis of (2S,4R)-1-[(S)-2-[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]butyramido]-3,3-dimethylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]butanoic acid (30 mg, 75.67 μmol) and (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2 -Formamide (33.64 mg, 75.67 μmol) was dissolved in anhydrous N, N-dimethylformamide (0.25 mL), and O-(7-azabenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (43.16 mg, 113.51 μmol) and N, N-diisopropylethylamine (29.34 mg, 227.02 μmol, 39.54 μL) were added. The reaction solution was stirred at 25 ° C for 5 hours, and the reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Purification by high performance liquid chromatography (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 9%-49%) as eluent) gave (2S,4R)-1-[(S)-2-[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]butanamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 020) (1.5 mg).

MS m/z(ESI):823.4[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.80 (s, 1H), 8.99 (s, 1H), 8.46 (s, 1H), 8.39 (d, J = 7.8Hz, 1H), 8.08 (d, J＝7.3Hz,1H),7.96(s,1H),7.48-7.41(m,2H),7.41-7.32(m,3H),7.06-6.97(m,2H),6.51(s,2H),5.13 (s,1H),4.92(t,J＝6.9Hz,1H),4.70(s ,1H),4.55(d,J＝9.4Hz,1H),4.43(t,J＝8.1Hz,1H),4.30(s,1H),3.70-3.53(m,2H),3.32-3.32(m, 2H),2.46(s,5H),2.33(s,2H),2.25(s,4H),2.10-1.93(m,4H),1.87-1.76(m,2H),1.38(d,J＝7.0Hz ,3H),0.96(s,9H).

Example 57: Synthesis of (2S,4R)-1-[(S)-2-[10-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]decanoyl]-3,3-dimethylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 021)

Step 1: Synthesis of 10-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]decanoic acid

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (50 mg, 161.11 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 10-bromodecanoic acid (44.51 mg, 177.22 μmol) and N,N-diisopropylethylamine (62.47 mg, 483.32 μmol) were added. The reaction solution was reacted at 80°C for 4 hours, and the reaction was completed by LCMS detection. The reaction solution was cooled to room temperature and concentrated to obtain 10-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]decanoic acid (50 mg).

MS m/z(ESI):481.3[M+H] ⁺ .

Step 2: Synthesis of (2S,4R)-1-[(S)-2-[10-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]decanoyl]-3,3-dimethylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

10-[4-[3-Amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]decanoic acid (50 mg, 104.04 μmol) and (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (50.05 mg, 104.04 μmol) were dissolved in N,N-dimethylformamide (1 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (59.34 mg, 156.05 μmol) and N,N-diisopropylethylamine (40.34 mg, 312.11 μmol) were added. The reaction solution was reacted at 25°C for 5 hours, and the reaction was completed by LCMS. The reaction solution was concentrated and purified by preparative liquid chromatography (Phenomenex C18, 5 μm silica, 25 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 33%-73%) as an eluent) to obtain (2S,4R)-1-[(S)-2-[10-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]decanoyl]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 021) (7.7 mg).

MS m/z (ESI): 907.6 [M+H] ⁺ ;

¹ H NMR (DMSO-d ₆ , 400MHz): δ = 13.96 (br s, 1H), 8.98 (s, 1H), 8.50 (s, 1H), 8.38 (br d, J = 7.7Hz, 1H), 8.12 (br d,J＝7.7Hz,1H),7.79(br d,J＝9.0Hz,1H),7.43(s,2H),7.39(br s,3H),6.99(br d,J＝8.1Hz, 2H),6.45(br s,2H),5.11(br s,1H),4.92(br s,1H),4.52(br d,J＝9.3Hz,2H),4.43(s,1H),4.25-4.32 (m,1H),3.61(br s,2H),3.02(br s,2H),2.46(s,3H),2.37-2.23(m,3H),2.15-2.00(m,6H),1.88-1.78(m,3H),1.52-1.44 (m,4H),1.38(br d,J＝6.8Hz,3H),1.27(br s,10H),0.94(s,9H).

Example 58: Synthesis of (2S,4R)-1-[(2S)-2-[12-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]dodecanoyl]-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 022)

Step 1: Synthesis of 12-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]dodecanoic acid

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (50 mg, 161.11 μmol) and 12-bromododecanoic acid (49.48 mg, 177.22 μmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (62.47 mg, 483.32 μmol) was added. The reaction solution was reacted at 100° C. for 2 hours, and LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the product 12-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]dodecanoic acid (80 mg, crude product).

MS m/z(ESI):509.2[M+H] ⁺ .

Step 2: Synthesis of (2S,4R)-1-[(2S)-2-[12-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]dodecanoyl]-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

12-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]dodecanoic acid (80 mg, 157.28 μmol) and (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (83.22 mg, 173.01 μmol) was dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (40.65 mg, 314.56 μmol) and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (65.78 mg, 173.01 μmol) were added. The reaction solution was reacted at 25°C for 1 hour. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. The product (2S,4R)-1-[(2S)-2-[12-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]dodecanoyl]-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 022) (14.2 mg) was purified by high performance liquid chromatography (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length, using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 54%-94%) as eluent).

MS m/z(ESI):936.6[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ 14.48-13.39 (m, 1H), 8.98 (s, 1H), 8.52 (s, 1H), 8.37 (br d, J = 7.7Hz, 1H), 8.12 (br d,J＝7.4Hz,1H),7.79(br d,J＝9.2Hz,1H),7.46-7.41(m,2H),7.40-7.31(m,3H),7.02-6.94(m,2H ),6.44(s,2H),5.12(br s,1H),4.91(m,1H),4.52(br d,J＝9.2Hz,2H),4.42(br t,J＝8.0Hz,1H), 4.28(br s,1H),3.67-3.53(m,2H),3.04-2.93(m,2H),2.45(s,3H),2.35-2.23(m,3H),2.12-1.98(m,6H),1.88- 1.75(m,3H),1.50-1.40(m,4H),1.37(br d,J＝6.9Hz,3H),1.25(br s,14H),0.93(s,9H).

Example 59: Synthesis of (2S,4R)-1-[(S)-2-[9-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]nonanamide]-3,3-dimethylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 023)

Step 1: Synthesis of 9-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]nonanoic acid

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (50 mg, 161.11 μmol) was dissolved in N,N-dimethylformamide (1 mL), and 9-bromononanoic acid (42.02 mg, 177.22 μmol) and N,N-diisopropylethylamine (62.47 mg, 483.32 μmol) were added. The reaction solution was reacted at 80°C for 4 hours, and the reaction was completed by LCMS detection. The reaction solution was cooled to room temperature and concentrated to obtain 9-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]nonanoic acid (50 mg).

MS m/z(ESI):467.3[M+H] ⁺ .

Step 2: Synthesis of (2S,4R)-1-[(S)-2-[9-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]nonanamido]-3,3-dimethylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

9-[4-[3-Amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]nonanoic acid (40 mg, 88.39 μmol) and (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (41.24 mg, 88.39 μmol) were dissolved in N,N-dimethylformamide (1 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (50.75 mg, 133.47 μmol) and N,N-diisopropylethylamine (34.27 mg, 265.17 μmol) were added. The reaction solution was reacted at 25°C for 5 hours, and the reaction was completed by LCMS. The reaction solution was concentrated and purified by preparative liquid chromatography (Phenomenex C18, 5 μm silica, 25 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 33%-73%) as an eluent) to obtain (2S,4R)-1-[(S)-2-[9-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]nonanamide]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 023) (2.5 mg).

MS m/z (ESI): 893.5 [M+H] ⁺ ;

¹ H NMR (DMSO-d ₆ , 400MHz): δ = 13.99 (s, 1H), 8.99 (s, 1H), 8.52 (s, 1H), 8.38 (d, J = 7.9Hz, 1H), 8.13 (d ,J＝7.0Hz,1H),7.80(br d,J＝9.3Hz,1H),7.45-7.42(m,2H),7.40-7.34(m,3H),6.99(d,J＝8.3Hz,2H ),6.46(s,2H),5.11(d,J＝3.4Hz,1H),4.95-4.89(m,1H),4.53(br d,J＝9.4Hz,2H),4.42(t,J＝8.0 Hz,1H),4.29(br s,1H),3.65-3.57(m,2H),3.00(br d,J＝5.0Hz,2H),2.46(s,3H),2.35-2.24(m,3H),2.08(br d,J＝ 7.9Hz,6H),1.86-1.75(m,3H),1.52-1.43(m,4H),1.38(d,J＝7.0Hz,3H),1.28(br s,8H),0.94(s,9H) .

Example 60: Synthesis of (2S,4R)-1-[2-[3-[3-[4-[1-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethyl]phenyl]propoxy]isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 024)

Step 1: Synthesis of (E)-1-[4-[3-[(tetrahydro-2H-pyran-2-yl)oxy]prop-1-en-1-yl]phenyl]ethanone

4-iodoacetophenone (4g, 16.3mmol) and 2-propyleneoxytetrahydro-2H-pyran (3.47g, 24.4mmol) were dissolved in tert-butanol (100mL), and 1,3-bis(diphenylphosphine)propane (1.34g, 3.25mmol), triethylamine (4.94g, 48.8mmol) and palladium acetate (365mg, 1.63mmol) were added under nitrogen atmosphere. The reaction solution was stirred at 100°C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and column chromatography (silica, petroleum ether/ethyl acetate = 100/8) was used to obtain the product (E)-1-[4-[3-[(tetrahydro-2H-pyran-2-yl)oxy]prop-1-en-1-yl]phenyl]ethanone (650mg).

MS m/z(ESI):261.1[M+H] ⁺ ;

¹ H NMR (400MHz, CDCl ₃ ) δ = 7.91 (d, J = 8.3Hz, 2H), 7.46 (d, J = 8.3Hz, 2H), 6.68 (d, J = 16.1Hz, 1H), 6.54-6.32 (m,1H),4.71(t,J＝3.6Hz,1H),4.44(m,J＝1.5,5.3,13.6Hz,1H),4.18(m,J＝1.3,6.1,13.4Hz,1H), 4.00-3.82(m,1H),3.62-3.44(m,1H),2.62-2.54(m,3H),1.93-1.81(m,1H),1.81-1.72(m,1H),1.65-1.53(m ,4H).

Step 2: Synthesis of 1-[4-[3-[(tetrahydro-2H-pyran-2-yl)oxy]propyl]phenyl]ethanone

(E)-1-[4-[3-[(tetrahydro-2H-pyran-2-yl)oxy]prop-1-en-1-yl]phenyl]ethanone (650 mg, 2.50 mmol) was dissolved in tetrahydrofuran (15 mL), palladium carbon (350 mg, 1.25 mmol, 10% purity) was added, and the reaction was stirred at 25° C. for 2 hours under a hydrogen (15 psi) atmosphere. LCMS showed that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to give the product 1-[4-[3-[(tetrahydro-2H-pyran-2-yl)oxy]propyl]phenyl]ethanone (620 mg), which was directly used in the next step.

MS m/z(ESI):263.2[M+H] ⁺ .

Step 3: Synthesis of 1-[4-(3-hydroxypropyl)phenyl]ethanone

1-[4-[3-[(tetrahydro-2H-pyran-2-yl)oxy]propyl]phenyl]ethanone (620 mg, 2.36 mmol) was dissolved in methanol (15 mL), and p-toluenesulfonic acid (2.03 g, 11.8 mmol) was added. The reaction solution was stirred at 50 ° C for 1 hour. Thin layer chromatography (petroleum ether: ethyl acetate = 1: 1) showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, diluted with water (10 mL) and dichloromethane (15 mL), and the aqueous phase was extracted twice with dichloromethane (15 mL). The organic phase was washed twice with saturated sodium bicarbonate (15 mL). The collected organic layer was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated to dryness under reduced pressure and column chromatography (silica, petroleum ether/ethyl acetate = 100/40) was used to obtain the product 1-[4-(3-hydroxypropyl)phenyl]ethanone (310 mg).

Step 4: Synthesis of methyl 2-[3-[3-(4-acetylphenyl)propoxy]isoxazol-5-yl]-3-methylbutanoate

1-[4-(3-hydroxypropyl)phenyl]ethanone (310mg, 1.74mmol), 2-(3-hydroxyisoxazol-5-yl)-3-methyl-butyric acid methyl ester (381mg, 1.91mmol) and triphenylphosphine (684mg, 2.61mmol) were dissolved in toluene (8mL) under 0℃ ice-water bath. Under nitrogen protection, a toluene (2mL) solution of diisopropyl azodicarboxylate (528mg, 2.61mmol) was added dropwise to the above solution, and the reaction solution was stirred at 100℃ for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, diluted with water (10mL) and dichloromethane (10mL), and the aqueous phase was extracted twice with dichloromethane (10mL). The organic phase was washed twice with water (10mL) and twice with dilute hydrochloric acid (1M). The collected organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrate was purified by column chromatography (silica, petroleum ether/ethyl acetate = 100/20) to give the product methyl 2-[3-[3-(4-acetylphenyl)propoxy]isoxazol-5-yl]-3-methylbutanoate (300 mg).

MS m/z (ESI): 360.1 [M+H] ⁺ ;

¹ H NMR (400MHz, CDCl ₃ ) δ = 7.89 (d, J = 8.3Hz, 2H), 7.29 (d, J = 8.3Hz, 2H), 5.94-5.84 (m, 1H), 4.23 (t, J = 6.3Hz,2H),3.81-3.64(m,3H),3.49(d,J＝8.6Hz,1H),2.90-2.77(m,2H),2.66-2.49(m,3H),2.42-2.28(m ,1H),2.18-2.05(m,2H),1.00(d,J＝6.6Hz,3H),0.93(d,J＝6.8Hz,3H).

Step 5: Synthesis of methyl 2-[3-[3-[4-(1-hydroxyethyl)phenyl]propoxy]isoxazol-5-yl]-3-methylbutanoate

2-[3-[3-(4-acetylphenyl) propoxy] isoxazol-5-yl]-3-methylbutyric acid methyl ester (300mg, 835μmol) was dissolved in methanol (5mL), sodium borohydride (0.32g, 8.46mmol) was added under a nitrogen atmosphere at 0°C, and the reaction solution was stirred at 30°C for 48 hours. LCMS showed that the reaction was complete. The reaction solution was quenched by adding saturated ammonium chloride (5mL) and extracted twice with ethyl acetate (10mL). The collected organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure and purified by thin layer chromatography (silicon dioxide, petroleum ether/ethyl acetate=2/1) to give the product 2-[3-[3-[4-(1-hydroxyethyl)phenyl] propoxy] isoxazol-5-yl]-3-methylbutyric acid methyl ester (226mg).

MS m/z (ESI): 384.2 [M+Na] ⁺ ;

¹ H NMR (400MHz, CDCl ₃ ) δ = 7.30 (d, J = 8.0Hz, 2H), 7.19 (d, J = 8.0Hz, 2H), 5.88 (s, 1H), 4.88 (q, J = 6.4Hz ,1H),4.22(t,J＝6.3Hz,2H),3.80-3.64(m,3H),3.49(d,J＝8.8Hz,1H),2.76(t,J＝7.7Hz,2H),2.35 (qd,J＝6.7,15.3Hz,1H),2.14-2.03(m,2H),1.49(d,J＝6.4Hz,3H),1.00(d,J＝6.8Hz,3H),0.92(d, J＝6.8Hz,3H).

Step 6: Synthesis of methyl 2-[3-[3-[4-(1-chloroethyl)phenyl]propoxy]isoxazol-5-yl]-3-methylbutanoate

2-[3-[3-[4-(1-hydroxyethyl)phenyl]propoxy]isoxazol-5-yl]-3-methylbutyric acid methyl ester (170 mg, 470 μmol) was dissolved in dichloromethane (2 mL), and dichlorothionyl (83.9 mg, 706 μmol) was added under an ice bath (0° C.). The reaction was stirred at 25° C. for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the product 2-[3-[3-[4-(1-chloroethyl)phenyl]propoxy]isoxazol-5-yl]-3-methylbutyric acid methyl ester (170 mg, crude product), which was directly used in the next step.

MS m/z(ESI):344.1[M+H-HCl] ⁺ .

Step 7: Synthesis of methyl 2-[3-[3-[4-[1-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethyl]phenyl]propoxy]isoxazol-5-yl]-3-methylbutanoate

2-[3-[3-[4-(1-chloroethyl)phenyl]propoxy]isoxazol-5-yl]-3-methylbutyric acid methyl ester (170 mg, 448 μmol) and 2-[3-amino-1-(4-piperidinyl)pyrazolo[4,3-c]pyridazin-6-yl]phenol (153 mg, 492 μmol) were dissolved in N,N-dimethylformamide (4 mL), and N,N-diisopropylethylamine (174 mg, 1.34 mmol) was added. The reaction solution was stirred at 80 ° C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was diluted with water (10 mL) and ethyl acetate (10 mL), and the aqueous phase was extracted twice with ethyl acetate (10 mL). The organic phase was washed twice with water (10 mL). The collected organic layer was dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated to dryness under reduced pressure and purified by thin layer chromatography (silica, petroleum ether/tetrahydrofuran = 1/2) to give the product 2-[3-[3-[4-[1-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethyl]phenyl]propoxy]isoxazol-5-yl]-3-methylbutanoic acid methyl ester (74 mg).

MS m/z(ESI):654.5[M+H] ⁺ .

Step 8: Synthesis of 2-[3-[3-[4-[1-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethyl]phenyl]propoxy]isoxazol-5-yl]-3-methylbutanoic acid

2-[3-[3-[4-[1-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethyl]phenyl]propoxy]isoxazol-5-yl]-3-methylbutanoic acid methyl ester (60 mg, 91.8 μmol) was dissolved in tetrahydrofuran (0.5 mL) and water (0.5 mL), and lithium hydroxide monohydrate (11.6 mg, 275 μmol) was added. The reaction solution was stirred at 25° C. for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, diluted with water, and then diluted hydrochloric acid (1.5 M) was added dropwise to adjust the pH to 5. The solution was then concentrated under reduced pressure to give the product 2-[3-[3-[4-[1-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethyl]phenyl]propoxy]isoxazol-5-yl]-3-methylbutanoic acid (55 mg).

MS m/z(ESI):640.9[M+H] ⁺ .

Step 9: Synthesis of (2S,4R)-1-[2-[3-[3-[4-[1-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethyl]phenyl]propoxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-[3-[4-[1-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethyl]phenyl]propoxy]isoxazol-5-yl]-3-methylbutanoic acid (50.0 mg, 78.2 μmol) and (2S,4R)-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (31.1 mg, 93.8 μmol) were dissolved in N,N-dimethylformamide (1 mL), and HATU (35.7 mg, 93.8 μmol) and N,N-diisopropylethylamine (30.3 mg, 234 μmol) were added. The reaction was stirred at 25° C. for 2 hours. LCMS showed that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure and purified by preparative liquid chromatography (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia water) and acetonitrile (acetonitrile content: 44%-84%) as eluent) to obtain the product (2S,4R)-1-[2-[3-[3-[4-[1-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethyl]phenyl]propoxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 024) (12.4 mg).

MS m/z (ESI): 953.7 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.02 (s, 1H), 9.01-8.89 (m, 1H), 8.49 (s, 1H), 8.45-8.26 (m, 1H), 8.09 (d, J ＝8.3Hz,1H),7.48-7.29(m,5H),7.28-7.15(m,4H),7.02-6.90(m,2H),6.45(s,2H),6.10(s,1H),5.16- 5.00(m,1H),4.99-4.75(m,1H),4.55-4.32(m,2H),4.27(s, 1H),4.20-4.02(m,2H),3.79-3.62(m,1H),3.61-3.47(m,2H),3.03(s,1H),2.94(s,1H),2.74-2.63(m, 2H),2.47-2.42(m,3H),2.31-2.20(m,1H),2.18-1.90(m,8H),1.86-1.71(m,3H),1.41-1.27(m,6H),1.01- 0.90(m,3H),0.86-0.77(m,3H).

Example 61: Synthesis of (2S,4R)-1-[2-[3-[4-[[4-[[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-2-yl]oxy]piperidin-1-yl]methyl]piperidin-1-yl]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-((S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 025)

Step 1: Synthesis of tert-butyl 4-[(4-bromopyridin-2-yl)oxy]piperidine-1-carboxylate

At 0°C, sodium hydride (476.67 mg, 11.92 mmol, 60% purity) was added to a solution of 4-hydroxy-N-Boc-piperidine (2 g, 9.94 mmol) in N,N-dimethylformamide (20 mL). The reaction solution was stirred at room temperature for 30 minutes, then cooled to 0°C, and 4-bromo-2-fluoropyridine (1.75 g, 9.94 mmol) in N,N-dimethylformamide (10 mL) was added dropwise to the above reaction system. The reaction solution was reacted at 25°C for 12 hours. LCMS monitored the completion of the reaction. The reaction solution was cooled to 0°C and quenched with saturated aqueous ammonium chloride solution (15 mL). The aqueous phase was extracted three times with ethyl acetate (50 mL). The organic phases were combined, washed twice with brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by column chromatography (petroleum ether:ethyl acetate=4:1) to give tert-butyl 4-[(4-bromopyridin-2-yl)oxy]piperidine-1-carboxylate (3.2 g).

MS m/z(ESI):300.9,302.9[M+H-tBu] ⁺ ;

¹ H NMR (400MHz, MeOD) δ = 8.00 (d, J = 5.6Hz, 1H), 7.13 (dd, J = 5.2Hz, 1.6Hz, 1H), 7.02 (s, 1H), 5.26-5.20 (m, 1H),3.79-3.74(m,2H),3.35-3.33(m,1H),3.31-3.29(m,1H),2.00-1.97(m,2H),1.72-1.67(m,2H),1.48( s,9H).

Step 2: Synthesis of tert-butyl 4-[[4-[4-[3-[bis(tert-butyloxycarbonyl)amino]-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-2-yl]oxy]piperidine-1-carboxylate

Tert-butyl 4-[(4-bromopyridin-2-yl)oxy]piperidine-1-carboxylate (149.34 g, 418.02 μmol), tert-butyl N-tert-butoxycarbonyl-N-[6-[2-(methoxymethoxy)phenyl]-1-(4-piperidinyl)pyrazolo[4,3-c]pyridazin-3-yl]carbamate (231.85 mg, 209.01 μmol, 50% purity), cesium carbonate (136.20 mg, 418.02 μmol) and methanesulfonic acid (2-dicyclohexylphosphine-2',6'-diisopropoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium (II) (17.48 mg, 20.90 μmol) were dissolved in tetrahydrofuran (8 mL), and then replaced with nitrogen three times, and the yellow reaction solution was stirred at 80°C for 16 hours. The reaction was completed by LCMS monitoring. The reaction solution was cooled to room temperature, diluted with water (10 mL), extracted three times with ethyl acetate (30 mL), and the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by thin layer chromatography (silica, petroleum ether: ethyl acetate = 2: 1) to obtain 4-[[4-[4-[3-[bis(tert-butyloxycarbonyl)amino]-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]pyridin-2-yl]oxy]piperidine-1-carboxylic acid tert-butyl ester (75 mg).

MS m/z(ESI):753.3[M+Na] ⁺ .

Step 3: Synthesis of 2-[3-amino-1-[1-[2-(piperidin-4-yloxy)pyridin-4-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[[4-[4-[3-(tert-butyloxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]-2-pyridinyl]oxy]piperidine-1-carboxylic acid tert-butyl ester (75 mg, 102.62 μmol) was dissolved in 1,4-dioxane (2 mL), and then a solution of hydrochloric acid in dioxane (2 mL, 4 M) was added. The reaction solution was stirred at 25°C for 2 hours. LCMS monitored the completion of the reaction. The reaction solution was concentrated to dryness to obtain 2-[3-amino-1-[1-[2-(piperidin-4-yloxy)pyridin-4-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (70 mg, hydrochloride), which was used directly in the next step without purification.

MS m/z(ESI):487.3[M+H] ⁺ .

Step 4: Synthesis of (2S,4R)-1-[2-[3-[4-[[4-[[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-2-yl]oxy]piperidin-1-yl]methyl]piperidin-1-yl]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-((S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

(2S,4R)-1-[2-[3-(4-Formylpiperidin-1-yl)isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (30.51 mg, 51.38 μmol) and 2-[3-amino-1-[1-[2-(piperidin-4-yloxy)pyridin-4-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (50.00 mg, 51.38 μmol, 50% purity) were dissolved in tetrahydrofuran (2 mL) and N,N-dimethylformamide (2 mL). Then, sodium triacetyl borohydride (32.67 mg, 154.14 μmol) and acetic acid (308.55 μg, 5.14 μmol) were added to the reaction system. The reaction solution was stirred at 50°C for 4 hours. After the reaction was complete as monitored by LCMS, the reaction solution was concentrated to dryness and then purified by preparative liquid chromatography (Phenomenex C18, 5μm silica, 30mm diameter, 80mm length; using a mixture of water (containing 0.05% ammonia water) and acetonitrile (acetonitrile content: 50%-70%) as eluent) to obtain (2S,4R)-1-[2-[3-[4-[[4-[[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-2-yl]oxy]piperidin-1-yl]methyl]piperidin-1-yl]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-((S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 025) (3.9mg).

MS m/z(ESI):1065.1[M+H] ⁺ ;

¹ H NMR (400MHz, MeOD) δ = 8.90 (s, 1H), 8.30 (s, 1H), 8.04-8.02 (m, 1H), 7.48-7.44 (m, 1H), 7.40-7.36 (m, 3H) ,7.31-7.29(m,2H),7.04-7.01(m,2H),6.63-6.61(m,1H),6.25(s,1H),6.09-6.06(m,1H),5.06-5.02(m, 2H),4.53-4.51(m,1H),4.16-4.13(m,2H),3.76 -3.65(m,5H),3.22-3.18(m,2H),2.92-2.88(m,4H),2.51(s,3H),2.50-2.49(m,3H),2.23-2.21(m,3H) ,2.05-1.99(m,5H),1.86-1.84(m,3H),1.67-1.65(m,1H),1.55-1.53(m,3H),1.35-1.33(m,3H),1.29-1.25( m,4H),1.08-1.06(m,3H),0.93-0.89(m,3H).

Example 62: Synthesis of Compound 026

Step 1: Synthesis of (2S,4R)-1-[(S)-2-[3-[4-(dimethoxymethyl)piperidin-1-yl]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide and (2S,4R)-1-[(R)-2-[3-[4-(dimethoxymethyl)piperidin-1-yl]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

(2S, 4R)-1-[2-[3-[4-(dimethoxymethyl)-1-piperidinyl]isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (300.00 mg, 468.89 μmol) was purified by chiral preparation (DAICEL CHIRALPAK AD column, 10 μm silica, 30 mm diameter, 250 mm length; using isopropanol (containing 0.1% ammonia water) as eluent) to obtain the title product P1 (75 mg, named intermediate 1) and another title product P2 (170 mg, named intermediate 1-1). The two title products were then further analyzed by the following chiral HPLC analysis conditions.

Chiral HPLC analysis method:

Intermediate 1:

The chiral HPLC peak time was 1.744 minutes;

MS m/z(ESI):640.4[M+H] ⁺ .

Intermediate 1-1:

The chiral HPLC peak time was 2.036 minutes;

MS m/z(ESI):640.6[M+H] ⁺ .

Step 2: Synthesis of Intermediate 2

Intermediate 1 (75.00 mg, 117.22 μmol) was dissolved in tetrahydrofuran (1 mL), hydrochloric acid solution (2 M, 586.12 μL) was added, and the reaction solution was stirred at 65 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the concentrate was extracted with ethyl acetate (15 mL×2) and sodium bicarbonate aqueous solution (2 mL). The organic phase was concentrated under reduced pressure to dryness to obtain the product intermediate 2 (65 mg).

MS m/z(ESI):594.5[M+H] ⁺ .

Step 3: Synthesis of Compound 026

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (37.37 mg, 120.42 μmol) and intermediate 2 (65.00 mg, 109.48 μmol) were dissolved in N,N-dimethylformamide (2 mL) and tetrahydrofuran (2 mL), and acetic acid (6.57 mg, 109.48 μmol) and sodium acetate borohydride (69.61 mg, 328.43 μmol) were added. The reaction solution was reacted at 25°C for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Boston Green ODS column, 5 μm silica, 30 mm diameter, 150 mm length); (using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 20%-40%) as eluent) gave the product Compound 026 (47.5 mg).

MS m/z (ESI): 888.3 [M+H] ⁺ ;

¹ H NMR(400MHz, DMSO-d ₆ )δ13.66(br s,1H),9.02-8.96(m,1H),8.53-8.39(m,1H),8.30(br s,1H),8.14-7.96 (m,1H),7.51-7.30(m,5H),7.06-6.96(m,2H),6.58-6.45(m,2H),6.18(br s,1H),5.14(d,J＝3.5Hz, 1H),5.05-4.58(m,2H),4.56-4.37(m,1H),4.27(br s,1H),3.81-3.50(m,5H),3.05(br s,2H),2.77(br s,2H),2.45(s,5H),2.32-1.87(m,8H),1.87-1.58(m,4H),1.49-1.31(m,3H),1.23(br s,2H),0.96(d ,J＝6.8Hz,3H),0.86-0.79(m,3H).

Example 63: Synthesis of Compound 027

Step 1: Synthesis of intermediate 2-1

The intermediate 1-1 (140 mg, 218.82 μmol) prepared by the method of Example 62 was dissolved in tetrahydrofuran (2 mL), and hydrochloric acid solution (2M, 1.09 mL) was added, and the reaction solution was stirred at 65° C. for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the concentrate was extracted with ethyl acetate (20 mL×2) and sodium bicarbonate aqueous solution (2 mL), and the organic phase was concentrated under reduced pressure to dryness to obtain the title product intermediate 2-1 (120 mg).

MS m/z(ESI):594.5[M+H] ⁺ .

Step 2: Synthesis of Compound 027

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (69.00 mg, 222.32 μmol) and intermediate 2-1 (120 mg, 202.11 μmol) were dissolved in N,N-dimethylformamide (3 mL) and tetrahydrofuran (3 mL), and acetic acid (36.41 mg, 606.33 μmol) and sodium acetate borohydride (128.51 mg, 606.33 μmol) were added. The reaction solution was reacted at 25°C for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Boston Green ODS column, 5 μm silica, 30 mm diameter, 150 mm length); (using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 20%-40%) as eluent) gave the title product Compound 027 (84.7 mg).

MS m/z (ESI): 888.4 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.94 (br s, 1H), 8.98 (s, 1H), 8.50 (br s, 1H), 8.42 (d, J = 7.5Hz, 1H), 8.15- 8.04(m,1H),7.48-7.41(m,2H),7.40-7.33(m,3H),7.04-6.96(m,2H),6.49(s,2H),6.14(s,1H),5.13( d,J＝3.8Hz,1H),4.91(t,J＝7.2Hz,1H),4.72-4.52(m,1H),4.36(t,J＝7.9Hz,1H),4.28(br s,1H),3.76-3.61(m,3H),3.57(d,J＝9.8Hz,1H),3.43(d,J＝10.8Hz,2H),3.21-2.88(m,2H),2.77(t ,J＝11.9Hz,2H),2.46(s,3H),2.24-1.98(m,8H),1.95-1.83(m,2H),1.82-1.71(m,3H),1.38(d,J＝7.0 Hz,3H),1.18(br s,2H),0.95(d,J＝6.4Hz,3H),0.79(d,J＝6.4Hz,3H).

Example 64: Synthesis of (2S,4R)-1-[2-[3-[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carbonyl]piperidin-1-yl]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 028)

Step 1: Synthesis of tert-butyl 1-[5-(1-methoxy-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl]piperidine-4-carboxylate

4-Boc-piperidine (100 mg, 539.77 μmol) and 3-methyl-2-[3-(1,1,2,2,3,3,4,4,4-nonafluorobutylsulfonyloxy)isoxazol-5-yl]butyric acid methyl ester (311.74 mg, 647.73 μmol) were dissolved in N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (209.29 mg, 1.62 mmol, 282.06 μL) was added. The reaction solution was stirred at 80 ° C for 2 hours. The reaction was complete as monitored by LCMS. Water (5 mL) was added to the reaction solution and extracted three times with dichloromethane (15 mL). The organic phase was washed three times with water (5 mL) and dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure and then purified by column chromatography (petroleum ether/ethyl acetate=3/1) to give tert-butyl 1-[5-(1-methoxy-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl]piperidine-4-carboxylate (96 mg).

MS m/z(ESI):367.2[M+H] ⁺ .

Step 2: Synthesis of 2-[3-(4-tert-butoxycarbonyl-1-piperidin-1-yl)isoxazol-5-yl]-3-methylbutanoic acid

1-[5-(1-methoxy-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl]piperidine-4-carboxylic acid tert-butyl ester (76 mg, 207.39 μmol) was dissolved in tetrahydrofuran (0.5 mL) and water (0.5 mL). Lithium hydroxide monohydrate (14.90 mg, 622.18 μmol) was added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS monitored the reaction to be complete. Water (3 mL) was added to the reaction solution and extracted three times with ethyl acetate (15 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure to obtain 2-[3-(4-tert-butyloxycarbonyl-1-piperidin-1-yl)isoxazol-5-yl]-3-methylbutanoic acid (58 mg).

MS m/z(ESI):353.2[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 1-[5-[1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]isoxazol-3-yl]piperidine-4-carboxylate

2-[3-(4-tert-Butyloxycarbonyl-1-piperidin-1-yl)isoxazol-5-yl]-3-methylbutanoic acid (58 mg, 164.57 μmol) and (2S,4R)-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (60.55 mg, 164.57 μmol) were dissolved in dichloromethane (1 mL), and N,N-diisopropylethylamine (63.81 mg, 493.72 μmol, 86.00 μL) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (93.86 mg, 246.86 μmol) were added. The reaction solution was stirred at 25°C for 2 hours, and the reaction was completed after LCMS monitoring. Water (3 mL) was added to the reaction solution and extracted three times with dichloromethane (15 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. The product was purified by thin layer chromatography (silicon dioxide, petroleum ether/tetrahydrofuran = 1/2) to give 1-[5-[1-[(2S, 4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]isoxazol-3-yl]piperidine-4-carboxylic acid tert-butyl ester (48 mg).

MS m/z (ESI): 666.3 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.01-8.95 (m, 1H), 8.43-8.19 (m, 1H), 7.49-7.30 (m, 4H), 6.16-6.11 (m, 1H), 5.13 -4.99(m,1H),4.97-4.85(m,1H),4.46-4.33(m,1H),4.32-4.23(m,1H),3.74-3.66(m,1H),3.58-3.48(m, 3H), 3.49-3.38(m,1H),2.92-2.53(m,4H),2.45(d,J＝3.3Hz,3H),1.86-1.72(m,3H),1.56-1.42(m,3H),1.41- 1.39(m,9H),1.38-1.33(m,3H),0.95(dd,J＝3.3,6.5Hz,3H),0.80(dd,J＝6.7,11.4Hz,3H).

Step 4: Synthesis of 1-[5-[1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]isoxazol-3-yl]piperidine-4-carboxylic acid

1-[5-[1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]isoxazol-3-yl]piperidine-4-carboxylic acid tert-butyl ester (48 mg, 72.09 μmol) was dissolved in dichloromethane (0.5 mL), and trifluoroacetic acid (82.20 mg, 720.89 μmol, 53.38 μL) was added. The reaction solution was stirred at 25° C. for 2 hours. The reaction was completed by LCMS monitoring. The reaction solution was concentrated to dryness under reduced pressure to give 1-[5-[1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]isoxazol-3-yl]piperidine-4-carboxylic acid (40 mg).

MS m/z(ESI):632.2[M+H] ⁺ .

Step 5: Synthesis of (2S,4R)-1-[2-[3-[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carbonyl]piperidin-1-yl]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

1-[5-[1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3-methyl-1-oxobutan-2-yl]isoxazol-3-yl]piperidine-4-carboxylic acid (40 mg, 65.60 μmol) and 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4, 3-c]pyridazine-6-yl]phenol (20.36 mg, 65.60 μmol) was dissolved in N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (25.44 mg, 196.81 μmol, 34.28 μL) and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (27.44 mg, 72.16 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was complete when monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by high performance liquid chromatography (Phenomenex C18 column, 5 μm silica, 30 mm diameter, 80 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 40%-60%) as eluent) to give (2S,4R)-1-[2-[3-[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidine-1-carbonyl]piperidin-1-yl]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 028) (11.5 mg).

MS m/z (ESI): 902.4 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.88 (br s, 1H), 9.02-8.91 (m, 1H), 8.51 (s, 1H), 8.32 (d, J = 12.4Hz, 1H), 8.09 (br d,J＝7.8Hz,1H),7.47-7.31(m,5H),7.05-6.96(m,2H),6.50(br s,2H),6.20-6.12(m,1H),5.12(br d,J＝3.1Hz,1H),4.96-4.79(m,2H),4.66-4.52(m,1H),4.46-4.35(m,1H),4.32-4.13(m,2H),3.71-3.50( m,4H),3.04-2.78(m,4H),2.75-2.63(m,1H),2.47-2.44(m,3H),2.36-2.10(m,2H),2.03-1.89(m,4H), 1.87-1.74(m,1H),1.71-1.58(m,4H),1.46(br d,J＝6.9Hz,1H),1.42-1.20(m,3H),0.96(br dd,J＝2.4,6.2Hz,3H),0.81(br dd,J＝6.7,10.7Hz,3H).

Example 65: Synthesis of (2S,4R)-1-[(2S)-2-[11-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]undecanoylamino]-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 029)

Step 1: Synthesis of 11-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]undecanoic acid

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (50.0 mg, 161 μmol) and 11-bromondecanoic acid (47.0 mg, 177 μmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (62.5 mg, 483 μmol) was added. The reaction solution was stirred at 80 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to obtain a brown liquid 11-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]undecanoic acid (63 mg), which was used directly in the next step without purification.

MS m/z(ESI):495.4[M+H] ⁺ .

Step 2: Synthesis of (2S,4R)-1-[(2S)-2-[11-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]undecanoylamino]-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

11-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]undecanoic acid (63.0 mg, 127 μmol) and (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (73.5 mg, 153 μmol) were dissolved in N,N dimethylformamide (0.5 mL), and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (58.1 mg, 153 μmol) and N,N-diisopropylethylamine (49.4 mg, 382 μmol) were added. The reaction was stirred at 25°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure and purified by preparative liquid chromatography (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 50%-90%) as eluent) to obtain the product (2S, 4R)-1-[(2S)-2-[11-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]undecanoylamino]-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 029) (13.31 mg).

MS m/z (ESI): 921.7 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.99 (s, 1H), 8.98 (s, 1H), 8.51 (s, 1H), 8.38 (d, J = 7.8Hz, 1H), 8.12 (dd, J＝1.4,8.4Hz,1H),7.80(d,J＝9.3Hz,1H),7.55-7.23(m,5H),7.07-6.86(m,2H),6.46(s,2H),5.11(d ,J＝3.5Hz,1H),4.99-4.85(m,1H),4.52(d,J＝9.3Hz,2H),4 .41(t,J＝8.0Hz,1H),4.27(s,1H),3.65-3.53(m,2H),2.98(d,J＝6.0Hz,2H),2.45(s,3H),2.35- 2.28(m,2H),2.28-2.18(m,1H),2.15-1.97(m,6H),1.87-1.75(m,3H),1.51-1.39(m,4H),1.37(d,J=7.0 Hz,3H),1.25(s,12H),0.93(s,9H).

Example 66: Synthesis of Compound 030

Intermediate 11 (40 mg, 76.25 μmol) and 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (26.03 mg, 83.88 μmol) were dissolved in tetrahydrofuran (0.5 mL) and N,N-dimethylformamide (0.5 mL), and a drop of acetic acid and sodium triacetoxyborohydride (48.48 mg, 228.76 μmol) were added. The mixture was stirred at 25°C for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (YMC-Pack CN column: 5 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 20%-40%) as eluent) to give the title compound, compound 030 (14.8 mg).

MS m/z(ESI):835.5[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.99 (br s, 1H), 8.98 (s, 1H), 8.51 (s, 1H), 8.43 (d, J = 7.6Hz, 1H), 8.12 (d ,J＝7.1Hz,1H),7.45-7.40(m,2H),7.36(br d,J＝8.1Hz,3H),7.02-6.94(m,2H),6.46(s,2H),6.12(s ,1H),4.97-4.88(m,1H),4.62-4.51(m,1H),4.38(t,J＝7.9Hz,1H),4.30-4.26(m,3H),3.71-3.63(m,2H ),3.46(br d,J＝11.3Hz,3H),3.06(br d,J＝10.9Hz,3H),2.78(br t,J＝5.3Hz,2H),2.45(s,3H),2.30-2.22(m,3H ),2.08(br dd,J＝12.1Hz,2.5,2H),1.84(br d,J＝9.1Hz,2H),1.38(d,J＝7.0Hz,3H),0.96(br d,J＝6.4 Hz,3H),0.81(d,J＝6.6Hz,3H).

Example 67: Synthesis of (2S,4R)-1-[2-[3-[7-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]heptyloxy]isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 031)

Step 1: Synthesis of methyl 2-[3-(7-bromoheptyloxy)isoxazol-5-yl]-3-methylbutanoate

2-(3-hydroxyisoxazol-5-yl)-3-methyl-butyric acid methyl ester (300 mg, 1.51 mmol), 7-bromo-1-heptanol (323 mg, 1.66 mmol) and triphenylphosphine (593 mg, 2.26 mmol) were dissolved in toluene (10 mL) under 0 ° C ice water bath. Under nitrogen protection, a toluene (1 mL) solution of diisopropyl azodicarboxylate (457 mg, 2.26 mmol) was added dropwise to the above solution, and the reaction solution was stirred at 100 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, diluted with water (10 mL) and dichloromethane (10 mL), and the aqueous phase was extracted twice with dichloromethane (10 mL). The organic phase was washed once with brine (10 mL) and twice with dilute hydrochloric acid (0.5 M). The collected organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The concentrate was purified by column chromatography (silica, petroleum ether/ethyl acetate = 100/10) to give the product methyl 2-[3-(7-bromoheptyloxy)isoxazol-5-yl]-3-methylbutanoate (430 mg).

MS m/z (ESI): 377.9 [M+H] ⁺ ;

¹ H NMR (400MHz, CDCl ₃ ) δ = 5.99-5.76 (s, 1H), 4.20 (t, J = 6.6Hz, 2H), 3.81-3.63 (s, 3H), 3.48 (d, J = 8.8Hz, 1H),3.41(t,J＝6.8Hz,2H),2.44-2.27(m,1H),1.92-1.71(m,4H),1.49-1.35(m,6H),1.00(d,J＝6.6Hz ,3H),0.92(d,J＝6.8Hz,3H).

Step 2: Synthesis of methyl 2-[3-[7-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]heptyloxy]isoxazol-5-yl]-3-methylbutanoate

2-[3-(7-bromoheptyloxy)isoxazol-5-yl]-3-methylbutyric acid methyl ester (100 mg, 266 μmol) and 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (82.5 mg, 266 μmol) were dissolved in N,N-dimethylformamide (3 mL), and N,N-diisopropylethylamine (103 mg, 797 μmol) was added. The reaction solution was stirred at 80 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was diluted with water (10 mL) and ethyl acetate (10 mL), and the aqueous phase was extracted twice with ethyl acetate (10 mL). The organic phase was washed twice with water (10 mL). The organic layer was collected, dried over anhydrous sodium sulfate and filtered, and the filtrate was concentrated to dryness under reduced pressure and purified by thin layer chromatography (silica, petroleum ether/tetrahydrofuran = 1/1, 0.1% ammonia methanol) to give the product 2-[3-[7-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]heptyloxy]isoxazol-5-yl]-3-methylbutanoic acid methyl ester (76 mg).

MS m/z(ESI):606.3[M+H] ⁺ .

Step 3: Synthesis of 2-[3-[7-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]heptyloxy]isoxazol-5-yl]-3-methylbutanoic acid

2-[3-[7-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]heptyloxy]isoxazol-5-yl]-3-methylbutanoic acid methyl ester (76 mg, 125 μmol) was dissolved in tetrahydrofuran (1 mL) and water (1 mL), and lithium hydroxide monohydrate (15.8 mg, 376 μmol) was added. The reaction solution was stirred at 25° C. for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, diluted with water, and then diluted hydrochloric acid (1.5 M) was added dropwise to adjust the pH to 5 to 6, followed by concentration under reduced pressure to give the product 2-[3-[7-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]heptyloxy]isoxazol-5-yl]-3-methylbutanoic acid (74 mg, crude) which was directly used in the next step.

MS m/z(ESI):592.2[M+H] ⁺ .

Step 4: Synthesis of (2S,4R)-1-[2-[3-[7-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]heptyloxy]isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-[7-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]heptyloxy]isoxazol-5-yl]-3-methylbutanoic acid (52.0 mg, 87.9 μmol) and (2S,4R)-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (32.0 mg, 96.7 μmol) were dissolved in N,N-dimethylformamide (1.5 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (40.1 mg, 105 μmol) and N,N-diisopropylethylamine (34.1 mg, 264 μmol) were added. The reaction was stirred at 25°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure and purified by preparative liquid chromatography (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 43%-83%) as eluent) to obtain the product (2S, 4R)-1-[2-[3-[7-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]heptyloxy]isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 031) (30.8 mg).

MS m/z (ESI): 905.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.00 (s, 1H), 9.01-8.91 (m, 1H), 8.51 (s, 1H), 8.47-8.27 (m, 1H), 8.19-8.07 (m ,1H),7.52-7.27(m,5H),7.05-6.93(m,2H),6.46(s,2H),6.17-6.02(m,1H),5.18-5.00(m,1H),4.94-4.73 (m,1H),4.61-4.47(m,1H),4.46-4.34(m,1H),4. 28(s,1H),4.20-4.05(m,2H),3.79-3.60(m,1H),3.60-3.50(m,1H),3.47-3.41(m,1H),2.99(s,2H), 2.48-2.42(m,3H),2.37-2.21(m,3H),2.08(d,J＝6.8Hz,5H),1.88-1.62(m,5H),1.48-1.24(m,11H),1.02- 0.90(m,3H),0.87-0.78(m,3H).

Example 68: (2S,4R)-1-[(2R)-2-[3-[9-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]nonyloxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxylate Synthesis of amine and (2S,4R)-1-[(2S)-2-[3-[9-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]nonyloxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

Step 1: Synthesis of 9-bromononenal

9-bromo-1-nonanol (1 g, 4.48 mmol) was dissolved in dichloromethane (10 mL), pyridinium chlorochromate (1.45 g, 6.72 mmol) and silica (1.35 g, 22.41 mmol) were added, and the reaction solution was stirred at 25 ° C for 12 hours. Thin layer chromatography (silica, petroleum ether: ethyl acetate = 10: 1) showed that the reaction was complete. The reaction solution was filtered and concentrated to dryness under reduced pressure to obtain the product 9-bromononanal (1 g).

¹ H NMR (400MHz, CDCl ₃ ) δ9.76 (s, 1H), 3.40 (t, J = 6.8Hz, 2H), 2.42 (dt, J = 1.4, 7.3Hz, 2H), 1.85 (m, 2H) ,1.63(br t,J＝6.7Hz,2H),1.48-1.38(m,2H),1.32(s,6H).

Step 2: Synthesis of 9-bromo-1,1-dimethoxynonane

9-Bromononanal (1 g, 4.52 mmol) and trimethyl orthoformate (575.59 mg, 5.42 mmol) were dissolved in methanol (10 mL), and dioxane hydrochloride (4 M, 33.90 μL) was added. The reaction solution was stirred at 65 ° C for 2 hours, and thin layer chromatography (silica, petroleum ether: ethyl acetate = 10: 1) showed that the reaction was complete. The reaction solution was quenched with sodium bicarbonate (20 mg), concentrated, and the concentrate was extracted with ethyl acetate and water. The organic phase was concentrated to dryness under reduced pressure to obtain the product 9-bromo-1,1-dimethoxynonane (1 g).

¹ H NMR (400MHz, CDCl ₃ ) δ4.35 (t, J = 5.7Hz, 1H), 3.40 (t, J = 6.8Hz, 2H), 3.31 (s, 6H), 1.85 (m, 2H), 1.63 -1.55(m,2H),1.41(d,J＝6.6Hz,2H),1.31(s,8H).

Step 3: Synthesis of methyl 2-[3-(9,9-dimethoxynonyloxy)isoxazol-5-yl]-3-methylbutanoate

9-Bromo-1,1-dimethoxynonane (73.77 mg, 276.10 μmol) and 2-(3-hydroxyisoxazol-5-yl)-3-methylbutyric acid methyl ester (50 mg, 251.00 μmol) were dissolved in acetonitrile (2 mL), potassium carbonate (69.38 mg, 502.00 μmol) was added, and the reaction solution was reacted at 60° C. for 12 hours. LCMS showed that the reaction was complete. The reaction solution was filtered and concentrated, and the concentrate was purified by thin layer chromatography (silicon dioxide, petroleum ether/ethyl acetate=3:1) to obtain the product 2-[3-(9,9-dimethoxynonyloxy)isoxazol-5-yl]-3-methylbutyric acid methyl ester (50 mg).

MS m/z(ESI):354.2[M-MeOH] ⁺ ;

¹ H NMR (400MHz, CDCl ₃ ) δ5.87 (s, 1H), 4.35 (t, J = 5.7Hz, 1H), 4.19 (t, J = 6.5Hz, 2H), 3.72 (s, 3H), 3.50 -3.45(m,1H),3.31(s,6H),2.39-2.30(m,1H),1.80-1.70(m,2H),1.61-1.56(m,2H),1.45-1.38(m,2H) ,1.31(br s,8H),1.00(d,J＝6.6Hz,3H),0.92(d,J＝6.7Hz,3H).

Step 4: Synthesis of 2-[3-(9,9-dimethoxynonyloxy)isoxazol-5-yl]-3-methylbutanoic acid

2-[3-(9,9-dimethoxynonyloxy)isoxazol-5-yl]-3-methylbutyric acid methyl ester (50 mg, 129.70 μmol) was dissolved in tetrahydrofuran (1 mL) and water (1 mL), and lithium hydroxide monohydrate (5.44 mg, 129.70 μmol) was added. The reaction solution was reacted at 25°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was adjusted to pH 5-6 with 1M hydrochloric acid solution, and then extracted with ethyl acetate (25 mL×3). The organic phase was concentrated to dryness under reduced pressure to obtain the product 2-[3-(9,9-dimethoxynonyloxy)isoxazol-5-yl]-3-methylbutyric acid (45 mg).

MS m/z(ESI):340.1[M-MeOH] ⁺ .

Step 5: Synthesis of (2S,4R)-1-[2-[3-(9,9-dimethoxynonyloxy)isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-(9,9-Dimethoxynonyloxy)isoxazol-5-yl]-3-methylbutyric acid (45 mg, 121.14 μmol) and (2S,4R)-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (49.02 mg, 133.26 μmol) were dissolved in N,N-dimethylformamide (2 mL), and triethylamine (36.77 mg, 363.42 μmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (50.67 mg, 133.26 μmol) were added, and the reaction solution was reacted at 25°C for 1 hour. LCMS showed that the reaction was completed. The reaction solution was extracted with ethyl acetate (15 mL×3) and water (4 mL). The organic phase was concentrated to dryness under reduced pressure. The concentrate was purified by thin layer chromatography (silica, petroleum ether/tetrahydrofuran=1/2) to give (2S,4R)-1-[2-[3-(9,9-dimethoxynonyloxy)isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (29 mg).

MS m/z(ESI):653.2[M-MeOH] ⁺ .

Step 6: Synthesis of (2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(9-oxononyloxy)isoxazol-5-yl]butyryl]-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

((2S,4R)-1-[2-[3-(9,9-dimethoxynonyloxy)isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (29 mg, 42.34 μmol) was dissolved in tetrahydrofuran (1 mL), hydrochloric acid solution (2 M, 211.71 μL) was added, and the reaction solution was stirred at 25 °C for 1 h. LC MS showed that the reaction was complete. The reaction solution was adjusted to pH 8-9 with saturated sodium bicarbonate solution, then extracted with ethyl acetate (15 mL × 3), and the organic phase was concentrated to dryness under reduced pressure to obtain the product (2S, 4R)-4-hydroxy-1-[3-methyl-2-[3-(9-oxononyloxy)isoxazol-5-yl]butyryl]-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (29 mg).

MS m/z(ESI):639.2[M+H] ⁺ .

Step 7: (2S,4R)-1-[(2R)-2-[3-[9-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]nonyloxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide and (2S,4R)-1-[(2S)-2-[3-[9-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]nonyloxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-Amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (15.50 mg, 49.94 μmol) and ((2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(9-oxononyloxy)isoxazol-5-yl]butanoyl]-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (29 mg, 45.40 μmol) was dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), and acetic acid (2.73 mg, 45.40 μmol) and sodium acetate borohydride (28.86 mg, 136.19 μmol) were added. The reaction solution was reacted at 25 ° C for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Purified by high performance liquid chromatography (Boston Green ODS column, 5 μm silica, 30 mm diameter, 150 mm length; using water (containing 0.225% formic acid) and acetonitrile with decreasing polarity (acetonitrile content: 26%-46%) as eluent) to give the title product P1 (1.9 mg, first peak, HPLC retention time of 4.472 minutes, compound 032) and the title product P2 (11.6 mg, second peak, HPLC retention time of 4.496 minutes, compound 032-1).

Compound 032:

MS m/z (ESI): 934.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.69(s,1H),9.26(br s,1H),9.04-8.92(m,1H),8.47-8.28(m,2H),8.00(br d ,J＝7.9Hz,1H),7.45-7.28(m,5H),7.09-6.95(m,2H),6.54(br s,2H),6.15-5.97(m,1H),5.21-4.76(m, 3H),4.41(t,J＝7.8Hz,1H),4.26(br s,1H),4.17-4.06(m,2H),3.79-3.63(m,3H),3.05(br d,J＝11.3Hz,3H),2.44(s,3H),2.35-2.27(m,3H),2.13(br d,J＝12.5Hz,3H),2.04(br d,J＝11.8Hz,1H ),1.81-1.61(m,6H),1.37-1.28(m,13H),0.95(br d,J＝6.4Hz,3H),0.82(br d,J＝6.6Hz,3H).

Compound 032-1:

MS m/z(ESI):933.9[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ14.11-12.66(m,1H),9.30(br s,1H),8.98(s,1H),8.48-8.34(m,2H),8.00(d, J＝8.3Hz,1H),7.48-7.40(m,2H),7.38-7.32(m,3H),7.07-6.96(m,2H),6.54(br s,2H),6.06(s,1H), 5.21-4.75(m,3H),4.35(t,J＝7.9Hz,1H),4.28(br s,1H),4.13(m,2H),3.78-3.64(m,3H),3.11-3.03(m,3H),2.45(s,3H),2.38-2.28(m,3H),2.19-2.08( m,3H),2.07-1.97(m,1H),1.82-1.60(m,6H),1.37(d,J＝7.0Hz,4H),1.32(br s,9H),0.95(d,J＝6.4 Hz,3H),0.83-0.77(m,3H).

Example 69: trans-(2S,4R)-1-[(S)-2-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]cyclohexane-1-amido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxylate Synthesis of amine and cis-(2S,4R)-1-[(S)-2-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]cyclohexane-1-amido]-3,3-dimethylbutyryl]-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide

Step 1: Synthesis of tert-butyl (S)-[1-(6-bromopyridin-3-yl)ethyl]carbamate

(S)-1-(6-bromopyridin-3-yl)ethylamine (900 mg, 4.48 mmol) was dissolved in dichloromethane (45 mL), triethylamine (1.36 g, 13.43 mmol) was added and stirred at 0°C for 0.5 hours, then di-tert-butyl dicarbonate (1.47 g, 6.71 mmol) was added to the reaction solution and stirred at 0°C for 0.5 hours. The reaction solution was stirred at 30°C for 2 hours. Water (30 mL) was added to the reaction solution, and the solution was extracted three times with dichloromethane (45 mL). The organic phase was washed three times with water (45 mL) and dried over anhydrous sodium sulfate. The organic phase was filtered and concentrated to dryness under reduced pressure, and then purified by column chromatography (silicon dioxide, petroleum ether/tetrahydrofuran = 1/0 to 6/1) to obtain (S)-tert-butyl (1-(6-bromopyridin-3-yl)ethyl)carbamate (1.11 g).

MS m/z (ESI): 300.7 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.31 (d, J = 2.3Hz, 1H), 7.70-7.58 (m, 2H), 7.50 (d, J = 7.5Hz, 1H), 4.68-4.56 ( m,1H),1.42-1.27(m,12H).

Step 2: Synthesis of (S)-tert-butyl [1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]carbamate

Dissolve (S)-tert-butyl [1-(6-bromopyridin-3-yl)ethyl]carbamate (1.1 g, 3.65 mmol) in 1,4-dioxane (20 mL) and water (5 mL), add 4-methyl-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)thiazole (1.64 g, 7.30 mmol), cesium carbonate (3.57 g, 10.95 mmol) and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (267.24 mg, 365.00 μmol). The reaction solution was stirred at 100 ° C for 3 hours. LCMS monitored the reaction to be complete. Add water (10 mL) to the reaction solution, extract three times with dichloromethane (30 mL), wash the organic phase with water (30 mL) three times, and dry over anhydrous sodium sulfate. The organic phase was filtered and concentrated to dryness under reduced pressure, and then purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 2/1) to give (S)-tert-butyl [1- [6- (4-methylthiazol-5-yl) pyridin-3-yl] ethyl] carbamate (700 mg).

MS m/z (ESI): 320.2 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.00 (s, 1H), 8.53 (d, J = 1.6Hz, 1H), 7.81 (dd, J = 2.3, 8.3Hz, 1H), 7.71 (d, J＝8.1Hz,1H),7.53(d,J＝7.6Hz,1H),4.74-4.63(m,1H),2.65(s,3H),1.37(s,12H).

Step 3: Synthesis of (S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethylamine

Dissolve (S)-tert-butyl [1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]carbamate (700 mg, 2.19 mmol) in dichloromethane (5 mL), add dioxane hydrochloride (4M, 5.48 mL). The reaction solution was stirred at 25°C for 2 hours. LCMS monitored the reaction to be complete. The reaction solution was concentrated to dryness under reduced pressure to obtain (S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethylamine (690 mg).

MS m/z (ESI): 220.2 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.14 (s, 1H), 8.87 (s, 1H), 8.77 (d, J = 1.9 Hz, 2H), 8.14 (dd, J = 2.3, 8.3 Hz, 1H),7.83(d,J＝8.3Hz,1H),3.16(s,1H),2.68(s,3H),1.58(d,J＝6.9Hz,3H).

Step 4: Synthesis of tert-butyl (2S,4R)-4-hydroxy-2-[[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]aminocarbonyl)pyrrolidine-1-carboxylate

(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethylamine (620 mg, 2.42 mmol) was dissolved in N,N-dimethylformamide (13 mL), O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.11 g, 2.91 mmol) and N,N-diisopropylethylamine (939.89 mg, 7.27 mmol) were added, and the reaction solution was stirred at 25°C for 0.5 hours, and (2S,4R)-1-(tert-butyloxycarbonyl)-4-hydroxypyrrolidine-2-carboxylic acid (560.56 mg, 2.42 mmol) was added to the reaction solution. The reaction solution was stirred at 25°C for 2 hours. The reaction was complete when monitored by LCMS. Water (15 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (30 mL). The organic phase was washed three times with water (30 mL) and dried over anhydrous sodium sulfate. The organic phase was filtered and concentrated to dryness under reduced pressure, and then purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 1/2) to obtain tert-butyl (2S, 4R)-4-hydroxy-2-[[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]aminocarbonyl)pyrrolidine-1-carboxylate (630 mg).

MS m/z(ESI):433.2[M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ8.93 (s, 1H), 8.58 (d, J = 2.0Hz, 1H), 7.85 (dd, J = 2.4, 8.2Hz, 1H), 7.70 (d, J＝8.0Hz,1H),5.10(q,J＝7.2Hz,1H),4.35(d,J＝7.5Hz,2H),3.94-3.87(m,1H),3.57-3.46(m,2H), 2.67(s,3H),2.29-2.15(m,1H),1.55(d,J＝7.3Hz,3H),1.42(s,9H).

Step 5: Synthesis of (2S,4R)-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide

Tert-butyl (2S,4R)-4-hydroxy-2-[[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]aminocarbonyl)pyrrolidine-1-carboxylate (100 mg, 231.20 μmol) was dissolved in 1,4-dioxane (2 mL), and a dioxane hydrochloride solution (4M, 577.99 μL) was added. The reaction solution was stirred at 25°C for 2 hours. LCMS monitored the reaction to be complete. The reaction solution was concentrated to dryness under reduced pressure to obtain (2S,4R)-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide (110 mg).

MS m/z(ESI):333.2[M+H] ⁺ .

Step 6: Synthesis of tert-butyl ((S)-1-[(2S,4R)-4-hydroxy-2-[[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]aminocarbonyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamate

(2S,4R)-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide (110 mg, 298.20 μmol) and (S)-2-[(tert-butyloxycarbonyl)amino]-3,3-dimethylbutyric acid (68.97 mg, 298.20 μmol) were dissolved in N,N-dimethylformamide (3 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (136.06 mg, 357.84 μmol) and N,N-diisopropylethylamine (115.62 mg, 894.60 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was completed by LCMS monitoring. Water (10 mL) was added to the reaction solution, and the mixture was extracted three times with dichloromethane (15 mL). The organic phase was washed three times with water (15 mL) and dried over anhydrous sodium sulfate. The organic phase was filtered and concentrated to dryness under reduced pressure, and then purified by column chromatography (silicon dioxide, petroleum ether/tetrahydrofuran = 1/0 to 2/3) to obtain tert-butyl (((S)-1-[(2S,4R)-4-hydroxy-2-[[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]aminocarbonyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamate (100 mg).

MS m/z(ESI):546.4[M+H] ⁺ .

Step 7: Synthesis of (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide

Tert-butyl ((S)-1-((2S,4R)-4-hydroxy-2-(((S)-1-(6-(4-methylthiazol-5-yl)pyridin-3-yl)ethyl)aminocarbonyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (100 mg, 183.25 μmol) was dissolved in dichloromethane (1 mL), and dioxane hydrochloride (4M, 458.13 μL) was added. The reaction solution was stirred at 25° C. for 2 hours. LCMS monitored the reaction to be complete. The reaction solution was concentrated to dryness under reduced pressure to obtain (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide (140 mg).

MS m/z(ESI):446.3[M+H] ⁺ ;

Step 8: trans-(2S,4R)-1-[(S)-2-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]cyclohexane-1-amido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide and the synthesis of cis-(2S,4R)-1-[(S)-2-[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]cyclohexane-1-amido]-3,3-dimethylbutyryl]-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide

(2S,4R)-1-[(S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide (40 mg, 49.79 μmol) and 4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1 -yl]pyrimidin-5-yl]cyclohexane-1-carboxylic acid (51.24 mg, 49.79 μmol, 50% purity) was dissolved in N,N-dimethylformamide (1 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (22.72 mg, 59.75 μmol) and N,N-diisopropylethylamine (32.17 mg, 248.94 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was complete when monitored by LCMS. Water (0.5 mL) was added to the reaction solution to quench the reaction, and then purified by high performance liquid chromatography (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (0.225% formic acid) and acetonitrile with decreasing polarity (31%-71%) as eluent) to obtain the title product P1 (2.6 mg, first peak, LCMS retention time of 1.205 minutes, compound 033) and another title product P2 (8.4 mg, second peak, LCMS retention time of 1.210 minutes, compound 033-1).

Compound 033:

MS m/z(ESI):942.4[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.91 (s, 1H), 9.00 (s, 1H), 8.54 (s, 2H), 8.46 (d, J = 7.1Hz, 1H), 8.34-8.28 ( m,2H),8.11(d,J＝7.9Hz,1H),7.83-7.67(m,3H),7.42-7.33(m,1H),7.05-6.97(m,2H),6.45(s,2H) ,5.12(s,1H),4.93(dd, J＝7.3,14.3Hz,2H),4.80(d,J＝12.0Hz,2H),4.54-4.47(m,1H),4.41(t,J＝7.9Hz,1H),4.28(s,1H), 3.60(s,2H),3.12-3.02(m,2H),2.65(s,3H),2.04-1.44(m,16H),1.41(d,J＝7.0Hz,3H),0.95(s,9H) .

Compound 033-1:

MS m/z(ESI):942.4[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.91 (s, 1H), 9.00 (s, 1H), 8.59-8.49 (m, 2H), 8.46 (d, J = 7.5Hz, 1H), 8.27 ( s,2H),8.11(d,J＝7.5Hz,1H),7.79(dd,J＝2.1,8.4Hz,1H),7.69(d,J＝8.2Hz,1H),7.63(d,J＝9.4 Hz,1H),7.40-7.33(m,1H),7.05-6.97(m,2H),6.45(s ,2H),5.12(d,J＝2.6Hz,1H),5.01-4.86(m,2H),4.80(d,J＝12.6Hz,2H),4.58-4.49(m,1H),4.41(t, J＝8.1Hz,1H),4.28(s,1H),3.62(s,2H),3.14-2.96(m,2H),2.65(s,3H),2.09-1.45(m,16H),1.41(d ,J＝7.0Hz,3H),0.95(s,9H).

Example 70: Synthesis of (2S,4R)-1-[(2S)-2-[5-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pentanamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 034)

Step 1: Synthesis of methyl 5-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pentanoate

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (50 mg, 161.11 μmol) was dissolved in N,N-dimethylformamide (2 mL), and methyl 5-bromovalerate (34.57 mg, 177.22 μmol) and N,N-diisopropylethylamine (84.18 μL, 483.32 μmol) were added. The reaction solution was stirred at 80°C for 2 hours. The reaction was completed by LCMS. The reaction solution was cooled to room temperature, water (8 mL) was added, and the mixture was extracted twice with ethyl acetate (10 mL). The organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to dryness under reduced pressure and purified by thin layer chromatography (petroleum ether:tetrahydrofuran = 1:1) to give 5-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pentanoic acid methyl ester (30 mg).

MS m/z(ESI):425.2[M+H] ⁺ .

Step 2: Synthesis of 5-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pentanoic acid

5-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pentanoic acid methyl ester (30 mg, 70.67 μmol) was dissolved in methanol (1 mL) and water (1 mL), and sodium hydroxide (5.65 mg, 141.34 μmol) was added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, hydrochloric acid (1.5M) was added to adjust the pH to 3, water (2 mL) and ethyl acetate (2 mL) were added, and the aqueous phase was extracted twice with ethyl acetate (4 mL), the organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 5-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pentanoic acid (20 mg).

MS m/z(ESI):411.2[M+H] ⁺ .

Step 3: Synthesis of (2S,4R)-1-[(2S)-2-[5-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pentanamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

5-[4-[3-Amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pentanoic acid (15 mg, 36.54 μmol) was dissolved in N,N-dimethylformamide (1 mL), and (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (16.25 mg, 36.54 μmol), O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (16.67 mg, 43.85 μmol) and N,N-diisopropylethylamine (19.10 μL, 109.63 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (Phenomenex C18 column: 3 μm silica, 30 mm diameter, 75 mm length; using water (containing 0.05% ammonia) and acetonitrile with decreasing polarity mixture (acetonitrile content: 31%-71%) as eluent) to obtain (2S,4R)-1-[(2S)-2-[5-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pentanamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 034) (3.2 mg).

MS m/z(ESI):837.6[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.99 (s, 1H), 8.99 (s, 1H), 8.52 (s, 1H), 8.39 (br d, J = 7.5Hz, 1H), 8.13 (br d,J＝7.4Hz,1H),7.83(br d,J＝9.1Hz,1H),7.47-7.32(m,5H),6.99(br d,J＝8.0Hz,2H),6.47(br s, 2H),5.12(br d,J＝3.1Hz,1H),4.98-4.88(m,1H),4.54(br d,J＝9.3Hz,2H),4.43(br t,J＝8.0Hz,1H) ,4.29(br s,1H),3.62(br s,2H),2.98(br s,2H),2.46(s,3H),2.29(br d,J＝6.8Hz,1H),2.21-1.98(m,6H),1.82(br d,J＝16.1 Hz,3H), 1.59-1.41(m,5H),1.38(br d,J＝6.9Hz,3H),1.24(s,1H),0.95(s,9H).

Example 71: Synthesis of (2S,4R)-1-[(2S)-2-[6-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexanamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 035)

Step 1: Synthesis of 6-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexanoic acid

2-[3-amino-1-(4-piperidinyl)pyrazolo[4,3-c]pyridazin-6-yl]phenol (40.0 mg, 128.89 μmol) and 6-bromohexanoic acid (27.65 mg, 141.77 μmol) were dissolved in N,N-dimethylformamide (0.8 mL), and N,N-diisopropylethylamine (49.97 mg, 386.66 μmol) was added. The reaction solution was stirred at 80°C for 4 hours. LCMS showed that the reaction was complete, and the reaction solution was used directly in the next step without treatment.

MS m/z(ESI):425.1[M+H] ⁺ .

Step 2: Synthesis of (2S,4R)-1-[(2S)-2-[6-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexanamido]-3,3-dimethylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

6-[4-[3-Amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexanoic acid (40 mg, 94.23 μmol) and (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (41.89 mg, 94.23 μmol) were dissolved in N,N-dimethylformamide (0.8 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (42.99 mg, 113.08 μmol) and N,N-diisopropylethylamine (36.54 mg, 282.69 μmol) were added. The reaction was stirred at 25°C for 4 hours. LCMS showed that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure and purified by preparative liquid chromatography (Phenomenex C18, 5μm silica, 30mm diameter, 150mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 35%-65%) as eluent) to obtain the product (2S,4R)-1-[(2S)-2-[6-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexanamide]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 035) (6.0 mg).

MS m/z (ESI): 851.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.99 (br s, 1H), 9.01-8.94 (m, 1H), 8.51 (s, 1H), 8.39 (d, J = 7.9Hz, 1H), 8.12 (d,J＝7.2Hz,1H),7.82(d,J＝9.3Hz,1H),7.46-7.41(m,2H),7.40-7.32(m,3H),7.02-6.96(m,2H), 6.46(s,2H),5.12(br s,1H),4.91(t,J＝7.2Hz,1H),4.53(d,J＝9.3Hz,2H),4.42(t,J＝8.0Hz,1H) ,4.28(br s,1H),3.69-3.55(m,2H),2.99(br s,2H),2.45(s,3H),2.36-2.22(m,4H),2.10-2.00(m,5H),1.89-1.74 (m,3H),1.48(td,J＝6.8,13.4Hz,4H),1.37(d,J＝7.0Hz,3H),1.31-1.24(m,2H),0.94(s,9H).

Example 72: Synthesis of (2S,4R)-1-[2-[3-[8-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]octyloxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 036)

Step 1: Synthesis of methyl 2-[3-(8-bromooctyloxy)isoxazol-5-yl]-3-methylbutanoate

Methyl 2-(3-hydroxyisoxazol-5-yl)-3-methylbutyrate (300 mg, 1.51 mmol), 8-bromooctan-1-ol (377.93 mg, 1.81 mmol, 309.78 μL) and triphenylphosphine (592.50 mg, 2.26 mmol) were dissolved in anhydrous tetrahydrofuran (4 mL), and diisopropyl azodicarboxylate (456.79 mg, 2.26 mmol, 439.22 μL) was added at 0°C under a nitrogen atmosphere. The reaction solution was stirred at 65°C for 4 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silicon dioxide, petroleum ether: tetrahydrofuran = 3:1) to obtain methyl 2-[3-(8-bromooctyloxy)isoxazol-5-yl]-3-methylbutyrate (280 mg).

MS m/z(ESI):390.1,392.1[M+H] ⁺ .

Step 2: Synthesis of methyl 2-[3-[8-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]octyloxy]isoxazol-5-yl]-3-methylbutanoate

2-[3-(8-bromooctyloxy)isoxazol-5-yl]-3-methylbutyric acid methyl ester (100 mg, 256.21 μmol) and 2-[3-amino-1-(4-piperidinyl)pyrazolo[4,3-c]pyridazin-6-yl]phenol (87.47 mg, 281.83 μmol) were dissolved in N,N-dimethylformamide (1.5 mL), and N,N-diisopropylethylamine (99.34 mg, 768.62 μmol, 133.88 μL) was added. The reaction solution was stirred at 80° C. for 2 hours. The reaction was completed by LCMS. The reaction solution was extracted with ethyl acetate (20 mL) and water (10 mL), concentrated to dryness under reduced pressure, and purified by thin layer chromatography (silica, petroleum ether:tetrahydrofuran = 1:1) to give methyl 2-[3-[8-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]octyloxy]isoxazol-5-yl]-3-methylbutanoate (128 mg).

MS m/z(ESI):620.4[M+H] ⁺ .

Step 3: Synthesis of 2-[3-[8-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]octyloxy]isoxazol-5-yl]-3-methylbutanoic acid

2-[3-[8-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]octyloxy]isoxazol-5-yl]-3-methylbutyric acid methyl ester (128 mg, 206.53 μmol) was dissolved in methanol (2 mL), and then sodium hydroxide aqueous solution (4M, 206.53 μL) was added. The reaction solution was stirred at 25°C for 12 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness, added with water (2 mL) to dissolve, adjusted to pH 1 with 1M HCl, and then concentrated to dryness under reduced pressure to obtain 2-[3-[8-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]octyloxy]isoxazol-5-yl]-3-methylbutyric acid (100 mg).

MS m/z(ESI):606.3[M+H] ⁺ .

Step 4: Synthesis of (2S,4R)-1-[2-[3-[8-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]octyloxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-[8-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]octyloxy]isoxazol-5-yl]-3-methylbutanoic acid (50 mg, 82.55 μmol), (2S,4R)-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (27.36 mg, 82.55 μmol) were added. g, 82.55 μmol), O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (37.66 mg, 99.05 μmol) and N,N-diisopropylethylamine (32.01 mg, 247.64 μmol, 43.13 μL) were dissolved in anhydrous N,N-dimethylformamide (4 mL), and the reaction solution was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. The product was purified by high performance liquid chromatography (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 49%-89%) as eluent) to give (2S,4R)-1-[2-[3-[8-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]octyloxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 036) (3.2 mg).

MS m/z (ESI): 919.5 [M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ8.95-8.82 (m, 1H), 8.28 (d, J = 3.5Hz, 1H), 8.07-7.94 (m, 1H), 7.50-7.37 (m, 5H ),7.07-6.96(m,2H),6.02(d,J＝7.8Hz,1H),5.03(d,J＝7.0,11.3Hz,1H),4.63-4.50(m,2H),4.24-4.10( m,2H),3.89-3.60(m,3H),3.24(d,J＝7.5Hz,2H ),2.56(s,2H),2.52-2.48(m,3H),2.39(d,J＝6.8Hz,4H),2.27-2.16(m,1H),2.06-1.91(m,3H),1.85- 1.72(m,2H),1.61(d,J＝7.0Hz,2H),1.52(d,J＝7.0,10.5Hz,4H),1.41(d,J＝7.0Hz,9H),1.07(d,J ＝6.5Hz,3H),0.96-0.89(m,3H).

Example 73: Synthesis of (2S,4R)-1-[2-[3-[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]butoxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 037)

Step 1: Synthesis of methyl 2-[3-(4,4-dimethoxybutoxy)isoxazol-5-yl]-3-methylbutanoate

2-(3-hydroxyisoxazol-5-yl)-3-methylbutyric acid methyl ester (300 mg, 1.51 mmol), 4-chlorobutyraldehyde dimethyl acetal (275.81 mg, 1.81 mmol) and cesium carbonate (1.47 g, 4.52 mmol) were dissolved in anhydrous N,N-dimethylformamide (20 mL). The reaction solution was stirred at 80 ° C for 6 hours. LCMS detected that the reaction was complete. The reaction solution was extracted with ethyl acetate (40 mL) and water (10 mL). The organic phases were combined and washed three times with saturated brine (10 mL), then dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. Purification by thin layer chromatography (silicon dioxide, petroleum ether: tetrahydrofuran = 1:2) gave 2-[3-(4,4-dimethoxybutoxy)isoxazol-5-yl]-3-methylbutyric acid methyl ester (188 mg).

MS m/z(ESI):338.1[M+Na] ⁺ .

Step 2: Synthesis of 2-[3-(4,4-dimethoxybutoxy)isoxazol-5-yl]-3-methylbutanoic acid

2-[3-(4,4-dimethoxybutoxy)isoxazol-5-yl]-3-methylbutyric acid methyl ester (188 mg, 596.14 μmol) and sodium hydroxide (47.69 mg, 1.19 mmol) were dissolved in methanol (1 mL) and water (1 mL). The reaction solution was stirred at 25 ° C for 4 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness, added with water (2 mL) to dissolve, adjusted to pH 1 with 1M HCl, and then extracted three times with ethyl acetate (10 mL), and the organic phase was concentrated to dryness under reduced pressure to obtain 2-[3-(4,4-dimethoxybutoxy)isoxazol-5-yl]-3-methylbutyric acid (188 mg).

MS m/z(ESI):324.1[M+Na] ⁺ .

Step 3: Synthesis of (2S,4R)-1-[2-[3-(4,4-dimethoxybutoxy)isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-(4,4-dimethoxybutoxy)isoxazol-5-yl]-3-methylbutanoic acid (188 mg, 623.89 μmol), (2S,4R)-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (248.13 mg, 748.67 μmol), O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (355.83 mg, 935.84 μmol) and N,N-diisopropylethylamine (241.90 mg, 1.87 mmol, 326.01 μL) were dissolved in N,N-dimethylformamide (2 mL). The reaction solution was stirred at 25° C. for 4 hours. The reaction was completed by LCMS. The reaction solution was diluted with water (5 mL), then extracted with ethyl acetate (40 mL), and the organic phase was concentrated to dryness under reduced pressure and purified by thin layer chromatography (silica, petroleum ether:tetrahydrofuran = 1:2) to give (2S,4R)-1-[2-[3-(4,4-dimethoxybutoxy)isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (100 mg).

MS m/z(ESI):637.3[M+Na] ⁺ .

Step 4: Synthesis of (2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(4-oxobutoxy)isoxazol-5-yl]butanoyl]-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

(2S,4R)-1-[2-[3-(4,4-dimethoxybutoxy)isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (100 mg, 162.67 μmol) was dissolved in anhydrous tetrahydrofuran (2 mL) and dilute hydrochloric acid (2M, 0.1 mL). The reaction solution was stirred at 25°C for 4 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness to obtain (2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(4-oxobutoxy)isoxazol-5-yl]butyryl]-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (80 mg).

MS m/z(ESI):569.2[M+H] ⁺ .

Step 5: Synthesis of (2S,4R)-1-[2-[3-[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]butoxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

(2S,4R)-4-Hydroxy-1-[3-methyl-2-[3-(4-oxobutoxy)isoxazol-5-yl]butanoyl]-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (80 mg, 140.68 μmol), 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (43.66 mg, 140.68 μmol) and acetic acid (16.90 mg, 281.35 μmol, 16.09 μL) were dissolved in anhydrous tetrahydrofuran (0.5 mL), the reaction solution was stirred at 25°C for 1 hour, and sodium acetate borohydride (89.44 mg, 422.03 μmol) was added. The reaction solution was stirred at 25°C for 15 hours, and the reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by high performance liquid chromatography (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using water (containing 0.225% formic acid) and acetonitrile with decreasing polarity mixtures (acetonitrile content: 12%-52%) as eluent) to obtain (2S, 4R)-1-[2-[3-[4-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]butoxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 037) (13.2 mg).

MS m/z (ESI): 863.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.99(s,1H),9.05-8.96(m,1H),8.52(s,1H),8.46-8.26(m,1H),8.13(d,J ＝8.4Hz,1H),7.55-7.22(m,5H),7.06-6.91(m,2H),6.48(s,2H),6.09(d,J＝2.0Hz,1H),4.96-4.84(m, 1H),4.64-4.49(m,1H),4.47-4.35(m,1H),4. 27(s,1H),4.22-4.07(m,2H),3.80-3.53(m,4H),3.09-2.94(m,2H),2.48-2.43(m,3H),2.43-2.35(m,2H ),2.31-2.21(m,1H),2.17-1.91(m,5H),1.90-1.60(m,5H),1.58(d,J＝13.4Hz,2H),1.47-1.30(m,3H), 1.03-0.65(m,6H).

Example 74: Synthesis of (2S,4R)-1-[(2S)-2-[7-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]heptylamido]-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 038)

Step 1: Synthesis of 7-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]heptanoic acid

2-[3-amino-1-(4-piperidinyl)pyrazolo[4,3-c]pyridazin-6-yl]phenol (50 mg, 161.11 μmol) and 7-bromoheptanoic acid (37.05 mg, 177.22 μmol) were dissolved in N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (62.47 mg, 483.32 μmol) was added. The reaction solution was reacted at 80° C. for 4 hours, and LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the product 7-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]heptanoic acid (50 mg, crude product).

Step 2: Synthesis of (2S,4R)-1-[(2S)-2-[7-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]heptylamido]-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

7-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]heptanoic acid (50 mg, 114.02 μmol) and (2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (50 mg, 114.02 μmol) were added. 5.76 mg, 125.42 μmol) was dissolved in N,N-dimethylformamide (1 mL), N,N-diisopropylethylamine (44.21 mg, 342.06 μmol) and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (47.69 mg, 125.42 μmol) were added, and the reaction solution was reacted at 25°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. Purification by high performance liquid chromatography (Phenomenex C18, 5 μm silica, 30 mm diameter, 80 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 45%-65%) as eluent) gave the product (2S,4R)-1-[(2S)-2-[7-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]heptylamide]-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 038) (17.4 mg).

MS m/z (ESI): 865.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.99 (s, 1H), 8.98 (s, 1H), 8.51 (s, 1H), 8.38 (br d, J = 7.8Hz, 1H), 8.12 (br d,J＝7.0Hz,1H),7.81(br d,J＝9.3Hz,1H),7.45-7.41(m,2H),7.39-7.32(m,3H),7.03-6.95(m,2H), 6.46(s,2H),5.11(d,J＝3.5Hz,1H),4.99-4.85(m,1H),4.52(br d,J＝9.3Hz,2H),4.42(t,J＝8.0Hz,1H),4.32-4.23(m,1H),3.64-3.56(m,2H),3.32(br s,2H),2.98( br d,J＝5.0Hz,2H),2.45(s,3H),2.34-2.26(m,3H),2.07(br s,4H),1.86-1.75(m,3H),1.51-1.40(m, 4H),1.37(d,J=7.0Hz,3H),1.27(br s,4H),0.94(s,9H).

Example 75: Synthesis of (2S,4R)-1-[(2S)-2-[13-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]tridecanoyl]-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 039)

Step 1: Synthesis of 13-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]tridecanoic acid

2-[3-amino-1-(4-piperidinyl)pyrazolo[4,3-c]pyridazin-6-yl]phenol (50 mg, 161.11 μmol) and 12-bromotridecanoic acid (51.97 mg, 177.22 μmol) were dissolved in N,N-dimethylformamide (1.5 mL), and N,N-diisopropylethylamine (62.47 mg, 483.32 μmol) was added. The reaction solution was reacted at 80° C. for 2 hours, and LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the product 13-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]tridecanoic acid (59 mg, crude product).

MS m/z(ESI):523.3[M+H] ⁺ ;

Step 2: Synthesis of (2S,4R)-1-[(2S)-2-[13-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]tridecanoyl]-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

13-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]tridecanoic acid (59 mg, 112.88 μmol) and (2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (55.20 mg, 124.17 μmol) was dissolved in N,N-dimethylformamide (1.5 mL), and N,N-diisopropylethylamine (43.77 mg, 338.64 μmol) and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (47.21 mg, 124.17 μmol) were added. The reaction solution was reacted at 25°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. Purification by high performance liquid chromatography (Phenomenex C18, 5 μm silica, 30 mm diameter, 80 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 65%-85%) as eluent) gave the product (2S,4R)-1-[(2S)-2-[13-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]tridecanoyl]-3,3-dimethyl-butanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 039) (7.6 mg).

MS m/z (ESI): 949.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.06-13.86 (m, 1H), 9.05-8.92 (m, 1H), 8.50 (s, 1H), 8.37 (br d, J = 7.8Hz, 1H) ,8.20-8.04(m,1H),7.78(br d,J＝9.3Hz,1H),7.48-7.26(m,5H),7.08-6.89(m,2H),6.45(s,2H),5.10( d,J＝3.1Hz,1H),4.92-4.90(m,1H),4.51(br d,J＝9.3Hz,2H),4.41(t,J＝7.9Hz,1H),4.33-4.23(m,1H),3.66-3.54(m,2H),3.04-2.92(m,2H),2.45 (s,3H),2.36-2.19(m,4H),2.13-1.98(m,6H),1.86-1.74(m,3H),1.57-1.39(m,5H),1.37(br d,J＝7.0 Hz,3H),1.24(br s,14H),0.93(s,9H).

Example 76: Synthesis of (2S,4R)-1-[2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]propoxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 040)

Step 1: Synthesis of methyl 2-[3-(3-bromopropoxy)isoxazol-5-yl]-3-methylbutanoate

Methyl 2-(3-hydroxyisoxazol-5-yl)-3-methylbutyrate (100 mg, 502.00 μmol) and 3-bromopropanol (76.75 mg, 552.20 μmol) were dissolved in toluene (2 mL), and triphenylphosphine (197.50 mg, 753.00 μmol) was added at 0°C. Diisopropyl azodicarboxylate (152.26 mg, 753.00 μmol) was dissolved in toluene (0.5 mL) and added to the reaction solution under nitrogen protection at 0°C. The reaction solution was stirred at 100°C for 2 hours under nitrogen protection. LCMS monitored the reaction to be complete. The reaction solution was concentrated to dryness under reduced pressure, and then purified by column chromatography (silicon dioxide, petroleum ether/ethyl acetate = 3/1) to obtain methyl 2-[3-(3-bromopropoxy)isoxazol-5-yl]-3-methylbutyrate (160 mg).

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 6.21 (s, 1H), 4.26 (t, J = 6.1Hz, 2H), 3.713.65 (m, 4H), 3.63 (t, J = 6.6Hz, 2H),2.33-2.19(m,3H),0.93(d,J＝6.8Hz,3H),0.85(d,J＝6.8Hz,3H).

Step 2: Synthesis of methyl 2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]propoxy]isoxazol-5-yl]-3-methylbutanoate

2-[3-(3-bromopropoxy)isoxazol-5-yl]-3-methylbutyric acid methyl ester (60 mg, 187.40 μmol) was dissolved in N,N-dimethylformamide (2 mL), and 2-[3-amino-1-(4-piperidinyl)pyrazolo[4,3-c]pyridazine-6-yl]phenol (58.16 mg, 187.40 μmol) and N,N-diisopropylethylamine (72.66 mg, 562.19 μmol) were added. The reaction solution was stirred at 80 ° C for 4 hours. LCMS monitored the reaction to be complete. Saturated brine (6 mL) was added to the reaction solution and extracted three times with ethyl acetate (2 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. The residue was then purified by thin layer chromatography (petroleum ether/tetrahydrofuran = 1/1) to give methyl 2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]propoxy]isoxazol-5-yl]-3-methylbutanoate (32 mg).

MS m/z(ESI):550.5[M+H] ⁺ .

Step 3: Synthesis of 2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]propoxy]isoxazol-5-yl]-3-methylbutanoic acid

2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]propoxy]isoxazol-5-yl]-3-methylbutyric acid methyl ester (32 mg, 58.22 μmol) was dissolved in tetrahydrofuran (200 μL) and water (200 μL). Lithium hydroxide monohydrate (7.33 mg, 174.67 μmol) was added. The reaction solution was stirred at 25°C for 2 hours. LCMS monitored the reaction to be complete. The pH was adjusted to 3 to 4 with 1.5M hydrochloric acid, and the reaction solution was concentrated to dryness under reduced pressure to obtain 2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]propoxy]isoxazol-5-yl]-3-methylbutyric acid (30 mg).

MS m/z(ESI):536.5[M+H] ⁺ .

Step 4: Synthesis of (2S,4R)-1-[2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]propoxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]propoxy]isoxazol-5-yl]-3-methylbutanoic acid (30 mg, 56.01 μmol) and (2S,4R)-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (18.56 mg, 56.01 μmol) were dissolved in N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (21.72 mg, 168.04 μmol) and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (25.56 mg, 67.22 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. LCMS monitored the reaction to be complete. The reaction solution was concentrated to dryness under reduced pressure and purified by HPLC (Phenomenex C18 column, 5 μm silica, 30 mm diameter, 80 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 30%-50%) as eluent) to obtain (2S,4R)-1-[2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]propoxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 040) (1.2 mg).

MS m/z (ESI): 850.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.88-13.59 (m, 1H), 9.07-8.93 (m, 1H), 8.51 (s, 2H), 8.08 (br d, J = 6.9Hz, 1H) ,7.47-7.31(m,5H),7.05-6.96(m,2H),6.54(br s,2H),6.14-6.10(m,1H),5.19(s,1H),4.95-4.73(m,2H ),4.48-4.33(m,1H),4.26(br d,J＝6.0Hz,3H),3.81-3.54(m,3H),3.50-3.38(m,3H),2.45(d,J＝4.1Hz,3H),2.40-2.34(m,2H),2.30 -1.89(m,9H),1.83-1.69(m,1H),1.37(t,J＝7.1Hz,3H),0.97(dd,J＝2.8,6.5Hz,3H),0.85-0.78(m,3H ).

Example 77: Synthesis of Compound 041

Step 1: Synthesis of Intermediate 11-1

Intermediate 9 (60 mg, 97.60 μmol) was dissolved in tetrahydrofuran (1.3 mL), and 1.5 M hydrochloric acid (1.3 mL) was added. The mixture was stirred at 65 ° C for 2 hours. LCMS detected that the reaction was complete. Saturated sodium carbonate was added to the reaction solution to adjust the pH to 8 to 9, and then saturated brine (2 mL) was added, and extracted three times with ethyl acetate (2 mL). The organic phase was washed three times with water (2 mL), and the organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure to obtain the title compound intermediate 11-1.

MS m/z(ESI):541.3[M+H] ⁺ .

Step 2: Synthesis of Compound 041

Intermediate 11-1 (50 mg, 92.48 μmol) and 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (31.57 mg, 101.73 μmol) were dissolved in tetrahydrofuran (0.5 mL) and N,N-dimethylformamide (0.5 mL), and a drop of acetic acid and sodium triacetoxyborohydride (58.80 mg, 277.45 μmol) were added. The mixture was stirred at 25°C for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (YMC-Pack CN column: 5 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 40%-60%) as eluent) to give the title compound, compound 041 (12.4 mg).

MS m/z(ESI):835.5[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.09-13.83 (m, 1H), 9.03-8.83 (m, 1H), 8.58-8.43 (m, 1H), 8.33 (br d, J = 7.9Hz, 1H),8.20-8.05(m,1H),7.51-7.26(m,5H),7.05-6.93(m,2H),6.51-6.36(m,2H),6.19-6.05(m,1H),5.17- 5.01(m,1H),5.00-4.84(m,1H),4.63-4.48(m,1H),4.46-4.37(m,1H),4.32-4.19(m,3H),3 .76(d,J＝8.6Hz,1H),3.60-3.42(m,2H),3.11-2.98(m,2H),2.79-2.65(m,2H),2.44(s,3H),2.32-2.20 (m,3H),2.14-1.99(m,3H),1.93-1.72(m,3H),1.54-1.40(m,1H),1.35(d,J＝6.9Hz,2H),1.00-0.74(m ,6H).

Example 78: Synthesis of (2S,4R)-1-[2-[3-[5-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pentyloxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 042)

Step 1: Synthesis of methyl 2-[3-[4-(1,3-dioxolan-2-yl)butoxy]isoxazol-5-yl]-3-methylbutanoate

Dissolve 2-(3-hydroxyisoxazol-5-yl)-3-methylbutyric acid methyl ester (300 mg, 1.51 mmol) and 2-(4-chlorobutyl)-1,3-dioxolane (297.52 mg, 1.81 mmol) in N,N-dimethylformamide (8 mL), add potassium carbonate (624.41 mg, 4.52 mmol). The reaction solution was stirred at 80 ° C for 2 hours. The reaction was completed by thin layer chromatography (petroleum ether: ethyl acetate = 2: 1). Water (6 mL) was added to the reaction solution and extracted with ethyl acetate (6 mL) three times. The organic phase was washed three times with saturated brine (6 mL) and dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure and then purified by thin layer chromatography (silica, petroleum ether/ethyl acetate = 2/1) to give methyl 2-[3-[4-(1,3-dioxolan-2-yl)butoxy]isoxazol-5-yl]-3-methylbutanoate (273 mg).

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 6.19 (s, 1H), 4.77 (t, J = 4.7Hz, 1H), 4.14 (t, J = 6.4Hz, 2H), 3.89-3.81 (m, 2H),3.80-3.70(m,2H),3.67-3.65(m,3H),3.31(s,1H),2.34-2.23(m,1H),1.78-1.68(m,2H),1.64-1.55( m,2H),1.50-1.40(m,2H),0.92(d,J＝6.6Hz,3H),0.84(d,J＝6.6Hz,3H).

Step 2: Synthesis of 2-[3-[4-(1,3-dioxolan-2-yl)butoxy]isoxazol-5-yl]-3-methylbutanoic acid

Dissolve methyl 2-[3-[4-(1,3-dioxolan-2-yl)butoxy]isoxazol-5-yl]-3-methylbutanoate (173 mg, 528.45 μmol) in tetrahydrofuran (1 mL) and water (1 mL). Add lithium hydroxide monohydrate (66.53 mg, 1.59 mmol). The reaction solution was stirred at 25°C for 1 hour. LCMS monitored the reaction to be complete. Adjust the pH to 5-6 with 1.5M hydrochloric acid, add water (2 mL) to the reaction solution and extract three times with ethyl acetate (2 mL), wash the organic phase with saturated brine (2 mL) three times, dry the organic phase with anhydrous sodium sulfate and concentrate to dryness under reduced pressure to obtain 2-[3-[4-(1,3-dioxolan-2-yl)butoxy]isoxazol-5-yl]-3-methylbutanoic acid (148 mg).

MS m/z(ESI):314.2[M+H] ⁺ .

Step 3: Synthesis of (2S,4R)-1-[2-[3-[4-(1,3-dioxolan-2-yl)butoxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-[4-(1,3-dioxolan-2-yl)butoxy]isoxazol-5-yl]-3-methylbutanoic acid (148 mg, 472.32 μmol) and (2S,4R)-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (156.54 mg, 472.32 μmol) were dissolved in dichloromethane (4 mL), and N,N-diisopropylethylamine (183.13 mg, 1.42 mmol) and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (215.51 mg, 566.7 μmol) were added. The reaction solution was stirred at 25°C for 1 hour. The reaction was completed by LCMS monitoring. Water (4 mL) was added to the reaction solution and extracted three times with ethyl acetate (4 mL). The organic phase was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure. Purification by column chromatography (petroleum ether/tetrahydrofuran = 1/2) gave (2S,4R)-1-[2-[3-[4-(1,3-dioxolan-2-yl)butoxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (150 mg).

MS m/z (ESI): 627.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.04-8.88 (m, 1H), 8.46-8.20 (m, 1H), 7.44-7.32 (m, 3H), 6.89-6.59 (m, 1H), 6.17 -6.03(m,1H),5.10(t,J＝3.8Hz,1H),4.98-4.86(m,1H),4.76(td,J＝4.8,9.9Hz,1H),4.55-4.32(m,1H ),4.31-4.21(m,1H),4.16-4.04(m,2H),3.90-3. 81(m,2H),3.77-3.61(m,3H),3.63-3.36(m,2H),2.46-2.43(m,3H),2.30-2.20(m,1H),2.18(s,1H), 2.08-1.98(m,1H),1.82-1.65(m,3H),1.64-1.53(m,2H),1.48-1.37(m,4H),0.96(dd,J＝3.4,6.4Hz,3H), 0.81(dd,J＝13.3,6.8Hz,3H).

Step 4: Synthesis of (2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(5-oxopentyloxy)isoxazol-5-yl]butyryl]-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

(2S,4R)-1-[2-[3-[4-(1,3-dioxolan-2-yl)butoxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (30 mg, 47.87 μmol) was dissolved in tetrahydrofuran (638.20 μL), and 1.5 M hydrochloric acid (638.20 μL) was added. The reaction solution was stirred at 65°C for 2 hours. The reaction was completed by LCMS monitoring. The reaction solution was concentrated to dryness under reduced pressure to obtain (2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(5-oxopentyloxy)isoxazol-5-yl]butyryl]-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (24 mg).

MS m/z(ESI):583.3[M+H] ⁺ .

Step 5: Synthesis of (2S,4R)-1-[2-[3-[5-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pentyloxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

(2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(5-oxopentyloxy)isoxazol-5-yl]butyryl]-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (24 mg, 41.19 μmol) and 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (12.78 mg, 41.19 μmol) were dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), and acetic acid (2.47 mg, 41.19 μmol) and sodium triacetoxyborohydride (26.19 mg, 123.56 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was completed by LCMS monitoring. The reaction solution was concentrated to dryness under reduced pressure and purified by high performance liquid chromatography (Phenomenex C18 column, 5 μm silica, 30 mm diameter, 80 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 55%-75%) as eluent) to give (2S,4R)-1-[2-[3-[5-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pentyloxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 042) (4.7 mg).

MS m/z(ESI):878.3[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.07-13.87 (m, 1H), 9.01-8.78 (m, 1H), 8.51 (s, 1H), 8.43-8.26 (m, 1H), 8.20-8.06 (m,1H),7.52-7.26(m,5H),7.05-6.92(m,2H),6.45(s,2H),6.13-5.88(m,1H),5.14-4.97(m,1H),4.95 -4.84(m,1H),4.62-4.47(m,1H),4.39(td,J＝7.8,18.3Hz,1H),4.27(br d,J＝1.5Hz,1H),4.20-4.08(m,2H),3.79-3.59(m,1H),3.59-3.39(m,2H),2.99(br s,2H),2.47-2.44(m ,3H),2.39-2.26(m,3H),2.15-1.99(m,5H),1.87-1.67(m,5H),1.53-1.30(m,7H),0.96(br dd,J＝3.3,6.3 Hz,3H),0.86-0.73(m,3H).

Example 79: Synthesis of (2S,4R)-1-[2-[3-[6-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexyloxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 043)

Step 1: Synthesis of methyl 2-[3-(6-bromohexyloxy)isoxazol-5-yl]-3-methylbutanoate

Methyl 2-(3-hydroxyisoxazol-5-yl)-3-methylbutyrate (300 mg, 1.51 mmol), 6-bromo-1-hexanol (299.96 mg, 1.66 mmol) and triphenylphosphine (592.50 mg, 2.26 mmol) were dissolved in anhydrous toluene (4 mL), and diisopropyl azodicarboxylate (456.79 mg, 2.26 mmol) was added at 0°C under a nitrogen atmosphere. The reaction solution was stirred at 100°C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether: ethyl acetate = 5:1) to obtain methyl 2-[3-(6-bromohexyloxy)isoxazol-5-yl]-3-methylbutyrate (240 mg).

MS m/z(ESI):362.0,363.9[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 6.18 (s, 1H), 4.14 (t, J = 6.5Hz, 2H), 3.68 (s, 1H), 3.66 (s, 3H), 3.53 (t, J＝6.7Hz,2H),2.39-2.21(m,1H),1.83-1.82(m,2H),1.73-1.70(m,2H),1.49-1.33(m,4H),0.93(d,J＝ 6.6Hz, 3H), 0.84 (d, J = 6.7Hz, 3H).

Step 2: Synthesis of 2-[3-[6-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexyloxy]isoxazol-5-yl]-3-methyl-butyric acid methyl ester

2-[3-(6-bromohexyloxy)isoxazol-5-yl]-3-methylbutyric acid methyl ester (100 mg, 276.05 μmol) and 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (85.67 mg, 276.05 μmol) were dissolved in N,N-dimethylformamide (0.5 mL), and N,N-diisopropylethylamine (107.03 mg, 828.14 μmol, 144.25 μL) was added. The reaction solution was stirred at 80° C. for 4 hours. The reaction was completed by LCMS. The reaction solution was extracted with ethyl acetate (20 mL) and water (10 mL), and the organic phase was concentrated to dryness under reduced pressure and purified by thin layer chromatography (silica, petroleum ether:tetrahydrofuran = 1:1) to give 2-[3-[6-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexyloxy]isoxazol-5-yl]-3-methyl-butyric acid methyl ester (110 mg).

MS m/z(ESI):592.2[M+H] ⁺ .

Step 3: Synthesis of 2-[3-[6-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexyloxy]isoxazol-5-yl]-3-methylbutanoic acid

2-[3-[6-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexyloxy]isoxazol-5-yl]-3-methyl-butyric acid methyl ester (110 mg, 185.91 μmol) was dissolved in anhydrous tetrahydrofuran (1 mL) and water (1 mL), and then lithium hydroxide monohydrate (23.40 mg, 557.72 μmol) was added. The reaction solution was stirred at 25° C. for 12 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness, and water (2 mL) was added to dissolve it. The pH value was adjusted to 3-4 with 1 M HCl, and then concentrated to dryness under reduced pressure to obtain 2-[3-[6-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexyloxy]isoxazol-5-yl]-3-methylbutanoic acid (180 mg).

MS m/z(ESI):578.2[M+H] ⁺ .

Step 4: Synthesis of (2S,4R)-1-[2-[3-[6-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexyloxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-[6-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexyloxy]isoxazol-5-yl]-3-methylbutanoic acid (160 mg, 138.49 μmol, 50%), (2S,4R)-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (45.9 0mg, 138.49μmol), O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (63.19mg, 166.18μmol) and N,N-diisopropylethylamine (53.70mg, 415.46μmol, 72.37μL) were dissolved in anhydrous N,N-dimethylformamide (1.5mL), and the reaction solution was stirred at 25°C for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. The product was purified by high performance liquid chromatography (Kromasil Eternity XT column, 10 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 32%-82%) as eluent) to give (2S,4R)-1-[2-[3-[6-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexyloxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 043) (14.8 mg).

MS m/z(ESI):891.9[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.03-8.94 (m, 1H), 8.51 (s, 1H), 8.48-8.30 (m, 1H), 8.12 (d, J = 8.0Hz, 1H), 7.54-7.27(m,5H),7.07-6.90(m,2H),6.47(s,2H),6.08(s,1H),4.95-4.83(m,1H),4.53(d,J＝6.0Hz, 1H),4.46-4.33(m,1H),4.27(br s,1H),4.20-4.06(m,2H),3.80-3.61(m,2H),3.59-3.53(m,1H),2.98(br s,2H),2.47-2.43(m,3H),2.35-2.26(m,3H),2.06(br s,5H),1.88-1.61(m,5H),1.52-1.23(m,10H),1.01 -0.90(m,3H),0.81(dd,J＝6.7,13.4Hz,3H).

Example 80: (2S,4R)-1-[(R)-2-[3-[10-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]decyloxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxylate Synthesis of amine and (2S,4R)-1-[(S)-2-[3-[10-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]decyloxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

Step 1: Synthesis of methyl 2-[3-(10-bromodecyloxy)isoxazol-5-yl]-3-methylbutanoate

2-(3-hydroxyisoxazol-5-yl)-3-methylbutyric acid methyl ester (300 mg, 1.51 mmol) and 10-bromodecan-1-ol (392.91 mg, 1.66 mmol) were dissolved in toluene (10 mL), and diisopropyl azodicarboxylate (456.79 mg, 2.26 mmol) and triphenylphosphine (592.50 mg, 2.26 mmol) were added. After nitrogen replacement of the reaction liquid three times, the reaction liquid was stirred at 100 ° C for 2 hours. The reaction was complete as monitored by LCMS. The reaction liquid was concentrated to dryness under reduced pressure. Purification by column chromatography (silicon dioxide, petroleum ether/tetrahydrofuran = 1/0 to 20/1) gave 2-[3-(10-bromodecanyloxy)isoxazol-5-yl]-3-methylbutyric acid methyl ester (260 mg).

MS m/z(ESI):418.2,420.2[M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ6.00 (s, 1H), 4.18 (t, J = 6.5Hz, 2H), 3.72 (s, 3H), 3.57 (d, J = 8.8Hz, 1H) ,3.44(t,J＝6.8Hz,2H),2.35(qd,J＝6.8,15.5Hz,1H),1.89-1.71(m,4H),1.49-1.32(m,12H),1.00(d,J ＝6.8Hz,3H),0.91(d,J＝6.8Hz,3H).

Step 2: Synthesis of methyl 2-[3-[10-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]decyloxy]isoxazol-5-yl]-3-methylbutanoate

2-[3-(10-bromodecyloxy)isoxazol-5-yl]-3-methylbutyric acid methyl ester (100 mg, 239.03 μmol) and 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (74.18 mg, 239.03 μmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (92.68 mg, 717.08 μmol) was added. The reaction solution was stirred at 80° C. for 16 hours. The reaction was completed by LCMS monitoring. The reaction solution was concentrated to dryness under reduced pressure and then purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 0/1) to give methyl 2-[3-[10-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]decyloxy]isoxazol-5-yl]-3-methylbutanoate (110 mg).

MS m/z(ESI):648.9[M+H] ⁺ .

Step 3: Synthesis of 2-[3-[10-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]decyloxy]isoxazol-5-yl]-3-methylbutanoic acid

Dissolve 2-[3-[10-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]decyloxy]isoxazol-5-yl]-3-methylbutanoic acid methyl ester (110 mg, 169.80 μmol) in tetrahydrofuran (1 mL) and water (1 mL), and add sodium hydroxide (20.38 mg, 509.41 μmol). The reaction solution was stirred at 25°C for 2 hours. The reaction was complete by LCMS monitoring. Dilute hydrochloric acid was added to the reaction solution to adjust the pH to 3-4. The reaction solution was concentrated to dryness under reduced pressure to obtain crude 2-[3-[10-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]decyloxy]isoxazol-5-yl]-3-methylbutanoic acid (124 mg).

MS m/z(ESI):634.8[M+H] ⁺ .

Step 4: (2S,4R)-1-[(R)-2-[3-[10-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]decyloxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide and (2S,4R)-1-[(S)-2-[3-[10-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]decyloxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-[10-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]decyloxy]isoxazol-5-yl]-3-methylbutanoic acid (104 mg, 164.10 μmol) and (2S,4R)-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl ] Pyrrolidine-2-carboxamide (38.07 mg, 114.87 μmol) was dissolved in N,N-dimethylformamide (1.5 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (74.87 mg, 196.91 μmol) and N,N-diisopropylethylamine (63.62 mg, 492.29 μmol) were added. The reaction solution was stirred at 25 ° C for 1 hour. LCMS monitored the reaction to be complete. Water (0.1 mL) was added to the reaction solution to quench the reaction. High performance liquid chromatography (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; (using decreasingly polar mixtures of water (0.225% formic acid) and acetonitrile (acetonitrile content: 28%-68%) as eluent) gave the title product P1 (8.3 mg, compound 044, LCMS retention time of 3.928 minutes) and the title product P2 (7.96 mg, compound 044-1, LCMS retention time of 3.961 minutes).

Compound 044:

MS m/z (ESI): 947.9 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.77(s,1H),9.01-8.97(m,1H),8.44(s,1H),8.43-8.29(m,1H),8.06(d,J ＝8.1Hz,1H),7.50-7.28(m,6H),7.00(d,J＝8.2Hz,2H),6.51(s,2H),6.06(s,1H),5.11(s,1H),4.94 -4.84(m, 1H),4.71(s,1H),4.46-4.33(m,1H),4.27(s,1H),4.15-4.08(m,2H),2.45(s,3H),2.30-1.92(m,10H) ,1.82-1.51(m,7H),1.37-1.25(m,17H),0.96(d,J＝6.6Hz,3H),0.82(d,J＝6.7Hz,3H).

Compound 044-1:

MS m/z (ESI): 947.9 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.15(s,1H),8.99(s,1H),8.49-8.38(m,2H),7.91(s,1H),7.56-7.33(m,6H ),7.27-7.18(m,1H),7.08-7.03(m,2H),6.99-6.93(m,1H),6.07(s,1H),4.96-4.82(m,2H),4.36(t,J ＝7.9Hz,1H),4 .28(s,1H),4.13(t,J＝6.3Hz,2H),3.73-3.62(m,3H),3.18-2.95(m,4H),2.45(s,3H),2.31-1.96(m ,6H),1.70(d,J＝6.2Hz,4H),1.43-1.18(m,18H),0.95(d,J＝6.5Hz,3H),0.79(d,J＝6.6Hz,3H).

Example 81: (2S,4R)-1-[(R)-2-[3-[11-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]undecyloxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxylate Synthesis of amine and (2S,4R)-1-[(S)-2-[3-[11-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]undecyloxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

Step 1: Synthesis of methyl 2-[3-(11-bromodencyloxy)isoxazol-5-yl]-3-methylbutanoate

Dissolve 2-(3-hydroxyisoxazol-5-yl)-3-methylbutyric acid methyl ester (300 mg, 1.51 mmol) and 11-bromondecane-1-ol (416.14 mg, 1.66 mmol) in toluene (10 mL), add diisopropyl azodicarboxylate (456.79 mg, 2.26 mmol) and triphenylphosphine (592.50 mg, 2.26 mmol). The reaction solution was stirred at 100 ° C for 16 hours. The reaction was complete as monitored by LCMS. Water (15 mL) was added to the reaction solution, extracted three times with ethyl acetate (30 mL), and the organic phase was washed three times with water (30 mL) and dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure. Purification by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 3/1) gave methyl 2-[3-(11-bromodencyloxy)isoxazol-5-yl]-3-methylbutanoate (500 mg).

MS m/z(ESI):432.3,434.3[M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ6.00 (s, 1H), 4.17 (t, J = 6.5Hz, 2H), 3.72 (s, 3H), 3.57 (d, J = 8.8Hz, 1H) ,3.44(t,J＝6.8Hz,2H),2.41-2.28(m,1H),1.88-1.72(m,4H),1.47-1.30(m,14H),1.00(d,J＝6.8Hz,3H ),0.91(d,J＝6.8Hz,3H).

Step 2: Synthesis of methyl 2-[3-[11-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]undecyloxy]isoxazol-5-yl]-3-methylbutanoate

2-[3-(11-bromondecyloxy)isoxazol-5-yl]-3-methylbutyric acid methyl ester (100 mg, 231.27 μmol) and 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (71.78 mg, 231.27 μmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (89.67 mg, 693.82 μmol) was added. The reaction solution was stirred at 80° C. for 4 hours. The reaction was completed by LCMS monitoring. Water (1 mL) was added to the reaction solution to quench the reaction. The reaction solution was concentrated to dryness under reduced pressure and then purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 1/2) to give methyl 2-[3-[11-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]undecyloxy]isoxazol-5-yl]-3-methylbutanoate (200 mg).

MS m/z (ESI): 662.6 [M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ8.27 (s, 1H), 8.01 (dd, J = 1.5, 8.3Hz, 1H), 7.38-7.31 (m, 1H), 7.03-6.97 (m, 2H ),6.00(s,1H),4.53(s,1H),4.18(t,J＝6.5Hz,2H),3.71(s,3H),3.56(d,J＝8.8Hz ,1H),3.19(s,2H),2.55-2.47(m,2H),2.42-2.29(m,5H),1.99(s,2H),1.82-1.71(m,2H),1.60(s,2H ),1.35(s,14H),0.99(d,J＝6.7Hz,3H),0.90(d,J＝6.7Hz,3H).

Step 3: Synthesis of 2-[3-[11-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]undecyloxy]isoxazol-5-yl]-3-methylbutanoic acid

Dissolve 2-[3-[11-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]undecyloxy]isoxazol-5-yl]-3-methylbutanoic acid methyl ester (100 mg, 151.10 μmol) in tetrahydrofuran (1 mL) and water (1 mL), and add sodium hydroxide (18.13 mg, 453.29 μmol). The reaction solution was stirred at 25°C for 2 hours. LCMS monitored the reaction to be complete. Dilute hydrochloric acid was added to the reaction solution to adjust the pH to 3-4. The reaction solution was concentrated to dryness under reduced pressure to obtain crude 2-[3-[11-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]undecyloxy]isoxazol-5-yl]-3-methylbutanoic acid (126 mg).

MS m/z(ESI):648.6[M+H] ⁺ .

Step 4: (2S,4R)-1-[(R)-2-[3-[11-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]undecyloxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide and (2S,4R)-1-[(S)-2-[3-[11-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]undecyloxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-[11-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]undecyloxy]isoxazol-5-yl]-3-methylbutanoic acid (120 mg, 185.24 μmol) and (2S,4R)-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl [7-Azabenzotriazole] pyrrolidine-2-carboxamide (61.39 mg, 185.24 μmol) was dissolved in N,N-dimethylformamide (1.5 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (84.52 mg, 222.29 μmol) and N,N-diisopropylethylamine (71.82 mg, 555.72 μmol) were added. The reaction solution was stirred at 25 ° C for 16 hours. LCMS monitored the reaction to be complete. Water (0.5 mL) was added to the reaction solution to quench the reaction. Purification by high performance liquid chromatography (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (0.225% formic acid) and acetonitrile (acetonitrile content: 30%-70%) as eluent) gave the title product P1 (5.8 mg, compound 045, LCMS retention time of 4.740 minutes) and the title product P2 (4.4 mg, compound 045-1, LCMS retention time of 4.754 minutes).

Compound 045:

MS m/z(ESI):962.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.77(s,1H),9.01-8.94(m,1H),8.44(s,1H),8.42-8.25(m,1H),8.06(d,J ＝8.1Hz,1H),7.50-7.26(m,6H),7.03-6.98(m,2H),6.49(s,2H),6.06(s,1H),5.11(s,1H),4.93-4.82( m,1H),4.69(s,1H),4.39(td, J＝7.7,19.9Hz,1H),4.27(s,1H),4.15-4.09(m,2H),3.77-3.60(m,2H),2.45(d,J＝1.8Hz,3H),2.31-2.13 (m,4H),2.10-1.89(m,4H),1.85-1.51(m,6H),1.38-1.24(m,20H),1.03-0.92(m,3H),0.91-0.80(m,3H) .

Compound 045-1:

MS m/z(ESI):962.1[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.17 (s, 1H), 8.99 (s, 1H), 8.40 (s, 1H), 7.91 (d, J = 5.5Hz, 1H), 7.50-7.33 ( m,6H),7.20(s,1H),7.07(s,1H),7.05(d,J＝6.6Hz,2H),6.94(s,1H),6.06(s,1H),4.94-4.83(m ,2H),4.36(t,J＝7.6Hz,1H),4.28(s,1H), 4.13(t,J＝6.4Hz,2H),3.77-3.66(m,4H),3.64(d,J＝9.8Hz,2H),3.05(d,J＝10.0Hz,2H),2.45(s,3H ),2.14(d,J＝11.5Hz,4H),1.69(d,J＝6.3Hz,3H),1.39-1.25(m,22H),0.95(d,J＝6.4Hz,3H),0.79(d ,J＝6.6Hz,3H).

Example 82: Synthesis of (2S,4R)-1-[2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-2-oxoethoxy]isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 046)

Step 1: Synthesis of methyl 2-[3-(2-tert-butoxy-2-oxoethoxy)isoxazol-5-yl]-3-methylbutanoate

Methyl 2-(3-hydroxyisoxazol-5-yl)-3-methylbutyrate (200 mg, 1.00 mmol), tert-butyl bromoacetate (235.00 mg, 1.20 mmol, 178.03 μL) and potassium carbonate (416.28 mg, 3.01 mmol) were dissolved in anhydrous N,N-dimethylformamide (6 mL). The black reaction solution was stirred at 80 ° C for 4 hours. The reaction was completed by thin layer chromatography (petroleum ether: ethyl acetate = 3: 1). The reaction solution was extracted with ethyl acetate (40 mL) and water (10 mL), and the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. Purification by thin layer chromatography (silicon dioxide, petroleum ether: ethyl acetate = 3: 1) gave methyl 2-[3-(2-tert-butoxy-2-oxoethoxy)isoxazol-5-yl]-3-methylbutyrate (150 mg).

¹ H NMR (400MHz, DMSO-d ₆ ) δ6.28(s,1H),4.73(s,2H),3.77-3.61(m,4H),2.39-2.21(m,1H),1.41(s,9H ),0.93(d,J＝6.7Hz,3H),0.85(d,J＝6.7Hz,3H).

Step 2: Synthesis of 2-[5-(1-methoxy-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl]oxyacetic acid

2-[3-(2-tert-butoxy-2-oxoethoxy)isoxazol-5-yl]-3-methylbutyric acid methyl ester (140 mg, 446.79 μmol) was dissolved in dichloromethane (4 mL) and trifluoroacetic acid (2 mL). The reaction solution was stirred at 25 ° C for 4 hours. Thin layer chromatography (silica, petroleum ether: ethyl acetate = 3: 1) monitored the completion of the reaction. The reaction solution was concentrated to dryness to obtain 2-[5-(1-methoxy-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl]oxyacetic acid (160 mg).

Step 3: Synthesis of methyl 2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-2-oxoethoxy]isoxazol-5-yl]-3-methylbutanoate

2-[5-(1-methoxy-3-methyl-1-oxobutan-2-yl)isoxazol-3-yl]oxyacetic acid (160 mg, 621.99 μmol), 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (193.04 mg, 621.99 μmol), O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (260.15 mg, 684.19 μmol) and N,N-diisopropylethylamine (241.16 mg, 1.87 mmol, 325.02 μL) were dissolved in N,N-dimethylformamide (4 mL). The reaction solution was stirred at 25° C. for 4 hours. The reaction was completed by LCMS. The reaction solution was extracted with water (5 mL) and ethyl acetate (40 mL). The organic phase was concentrated under reduced pressure and purified by thin layer chromatography (silica, petroleum ether:tetrahydrofuran = 1:2) to give methyl 2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-2-oxoethoxy]isoxazol-5-yl]-3-methylbutanoate (200 mg).

MS m/z(ESI):550.1[M+H] ⁺ .

Step 4: Synthesis of 2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-2-oxoethoxy]isoxazol-5-yl]-3-methylbutanoic acid

2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]-2-oxoethoxy]isoxazol-5-yl]-3-methylbutanoic acid methyl ester (100 mg, 181.96 μmol) was dissolved in anhydrous methanol (2 mL), and sodium hydroxide solution (4M, 181.96 μL) was added. The reaction solution was stirred at 25 ° C for 4 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. The product was purified by HPLC (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 16%-56%) as eluent) to give 2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-2-oxoethoxy]isoxazol-5-yl]-3-methylbutanoic acid (20 mg).

MS m/z(ESI):536.1[M+H] ⁺ .

Step 5: Synthesis of (2S,4R)-1-[2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-2-oxoethoxy]isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-2-oxoethoxy]isoxazol-5-yl]-3-methylbutanoic acid (20 mg, 37.34 μmol), (2S,4R)-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxylate Amine (14.85 mg, 44.81 μmol), O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (17.04 mg, 44.81 μmol) and diisopropylethylamine (14.48 mg, 112.03 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and the reaction solution was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by high performance liquid chromatography (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 18%-58%) as eluent) to give (2S,4R)-1-[2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-2-oxoethoxy]isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 046) (13.75 mg).

MS m/z (ESI): 849.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.05-8.93(m,1H),8.56-8.36(m,2H),8.00(s,1H),7.56-7.27(m,5H),7.13-6.93 (m,2H),6.68(s,1H),6.18(d,J＝1.9Hz,1H),5.18-5.00(m,2H),4.96-4.78(m,2H),4.52(d,J＝11.9 Hz,1H),4.47-4 .36(m,1H),4.29(s,1H),3.90(s,1H),3.81-3.64(m,2H),3.62-3.41(m,4H),2.88-2.76(m,1H),2.46 (t,J＝4.1Hz,4H),2.25(d,J＝5.9Hz,1H),2.10-1.85(m,5H),1.51-1.31(m,3H),1.03-0.74(m,6H).

Example 83: Synthesis of Compound 047

2-[3-(Methylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (30 mg, 55.49 μmol) and intermediate 11 (30 mg, 55.49 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL) and anhydrous tetrahydrofuran (0.5 mL), and acetic acid (3.17 μL, 55.49 μmol) and sodium triacetoxyborohydride (35.28 mg, 166.47 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (Boston Prime C18 column: 5 μm silica, 30 mm diameter, 80 mm length; using decreasingly polar mixtures of water (containing 0.025% ammonia) and acetonitrile (acetonitrile content: 45%-65%) as eluent) to give the title compound, compound 047 (6.8 mg).

MS m/z(ESI):849.3[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.98 (br s, 1H), 8.98 (s, 1H), 8.49 (s, 1H), 8.43 (d, J = 7.5Hz, 1H), 8.12 (d ,J＝7.8Hz,1H),7.49-7.41(m,2H),7.40-7.32(m,3H),7.08(d,J＝4.3Hz,1H),7.04-6.95(m,2H),6.13( s,1H),5.12(d,J＝3.5Hz,1H),4.97-4.85(m,1H),4.58(br s,1H),4.37(t,J＝7.9Hz,1H),4.29(br s,3H),3.78-3.62(m,2H),3.46(d,J＝10.8Hz,1H),3.08(br s,2H),2.96(d,J＝4.8Hz,3H),2.78(br s ,2H),2.45(s,3H),2.31-2.08(m,5H),2.08-1.99(m,1H),1.90-1.73(m,3H),1.50-1.34(m,3H),1.01-0.93 (m,3H),0.87-0.77(m,3H).

Example 84: Synthesis of Compound 048

Intermediate 11 (49.76 mg, 92.04 μmol) and 2-[3-amino-1-(pyrrolidin-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (50 mg, 101.24 μmol) were dissolved in tetrahydrofuran (200 μL) and N,N-dimethylformamide (200 μL), and acetic acid (5.53 mg, 92.04 μmol) and sodium triacetoxyborohydride (58.52 mg, 276.11 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was complete when monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by high performance liquid chromatography (Phenomenex C18 column, 5 μm silica, 30 mm diameter, 80 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 30%-50%) as eluent) to give compound 048 (7.5 mg).

MS m/z(ESI):821.9[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.95-13.75 (m, 1H), 8.99 (s, 1H), 8.53 (s, 1H), 8.43 (d, J = 7.6Hz, 1H), 8.06 ( dd,J＝1.4,8.3Hz,1H),7.46-7.42(m,2H),7.38-7.34(m,3H),7.03-6.96(m,2H),6.56(br s,2H),6.09(d ,J＝2.4Hz,1H),5.34(br d,J＝4.5Hz,1H),5.12(d,J＝2.6Hz,1H),4.94-4.86(m,1H),4.39-4.26(m,4H),3.73-3.61(m,2H),3.45 (br d,J＝9.5Hz,2H),3.01-2.76(m,4H),2.45(s,3H),2.31-2.09(m,4H),2.06-1.99(m,1H),1.77(ddd, J＝4.6,8.1,12.7Hz,1H),1.37(dd,J＝2.0,7.0Hz,3H),0.95(d,J＝6.5Hz,3H),0.78(d,J＝6.6Hz,3H).

Example 85: Synthesis of (2S,4R)-1-[2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethoxy]isoxazol-5-yl]-3-methylbutyryl]-N-[(1S)-1-[2-fluoro-4-(4-methylthiazol-5-yl)phenyl]ethyl]-4-hydroxypyrrolidine-2-carboxamide (Compound 049)

Step 1: Synthesis of (R,E)-N-(4-bromo-2-fluorobenzylidene)-2-methylpropane-2-sulfenamide

2-Fluoro-4-bromobenzaldehyde (10 g, 49.26 mmol) and (R)-(+)-2-methyl-2-propanesulfinamide (5.43 g, 44.78 mmol) were dissolved in dichloromethane (200 mL), and pyridine p-toluenesulfonate (562.68 mg, 2.24 mmol) and magnesium sulfate (53.90 g, 447.81 mmol) were added. The reaction solution was reacted at 25°C for 16 hours, and magnesium sulfate (26.95 g, 223.90 mmol) was added. The reaction solution was reacted at 25°C for 24 hours, and the reaction was completed by thin layer chromatography (silicon dioxide, petroleum ether/ethyl acetate = 5/1). The reaction solution was filtered, the filtrate was concentrated, and purified by column chromatography (silicon dioxide, petroleum ether/ethyl acetate = 5/1) to obtain (R, E)-N-(4-bromo-2-fluorobenzylidene)-2-methylpropane-2-sulfinamide (4.9 g).

¹ H NMR (400 MHz, Methanol-d ₄ ) δ＝8.79(s,1H),7.96(t,J＝8.0Hz,1H),7.60-7.53(m,2H),1.29(s,9H). Step 2: Synthesis of (R)-N-[(S)-1-(4-bromo-2-fluorophenyl)ethyl]-2-methylpropane-2-sulfenamide

(R,E)-N-(4-bromo-2-fluorobenzylidene)-2-methylpropane-2-sulfinamide (4.9 g, 16.00 mmol) was dissolved in dichloromethane (60 mL), cooled to -45 ° C, and methylmagnesium chloride (8 mL, 3 M) was added under nitrogen protection. The reaction solution was reacted at -45 ° C for 1 hour, and methylmagnesium chloride (2.67 mL, 3 M) was added. The reaction solution was reacted at -45 ° C for 5 hours. LCMS detected that the reaction was complete. The reaction was heated to 0 ° C, quenched with saturated ammonium chloride solution (10 mL), diluted with water (50 mL), extracted three times with dichloromethane (30 mL), and the organic phases were combined, dried, and concentrated to obtain (R)-N-[(S)-1-(4-bromo-2-fluorophenyl)ethyl]-2-methylpropane-2-sulfinamide (4.8 g).

MS m/z(ESI):322.0,324.0[M+H] ⁺ .

Step 3: Synthesis of (S)-1-(4-bromo-2-fluorophenyl)ethylamine

(R)-N-[(S)-1-(4-bromo-2-fluorophenyl)ethyl]-2-methylpropane-2-sulfenamide (4.8 g, 14.90 mmol) was dissolved in methanol (50 mL), and dioxane hydrochloride (7.45 mL, 4 M) was added. The reaction solution was reacted at 25° C. for 4 hours, and the reaction was completed by LCMS. The reaction solution was concentrated, tetrahydrofuran (10 mL) was added, and stirred at 25° C. for 30 minutes, filtered, and the filter cake was freed from the solvent in vacuo to obtain (S)-1-(4-bromo-2-fluorophenyl)ethylamine (3.3 g, HCl salt).

MS m/z (ESI): 219.9 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 8.84 (br s, 3H), 7.71 (t, J = 8.3Hz, 1H), 7.64 (dd, J = 1.9, 10.0Hz, 1H), 7.54 (dd ,J＝1.8,8.4Hz,1H),4.56(br d,J＝6.3Hz,1H),1.52(d,J＝6.9Hz,3H).

Step 4: Synthesis of tert-butyl (2S,4R)-2-[[(S)-1-(4-bromo-2-fluorophenyl)ethyl]carbamoyl]-4-hydroxypyrrolidine-1-carboxylate

(S)-1-(4-bromo-2-fluorophenyl)ethylamine (3.1 g, 12.18 mmol) and Boc-L-hydroxyproline (3.10 g, 13.40 mmol) were dissolved in N,N-dimethylformamide (30 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (5.09 g, 13.40 mmol) and N,N-diisopropylethylamine (4.72 g, 36.54 mmol) were added. The reaction solution was reacted at 25° C. for 4 hours, and the reaction was completed by LCMS detection. The reaction solution was diluted with water (50 mL), extracted three times with ethyl acetate (30 mL), the organic phases were combined, dried, concentrated, and purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/1) to give (2S,4R)-2-[[(S)-1-(4-bromo-2-fluorophenyl)ethyl]carbamoyl]-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester (4 g).

MS m/z(ESI):432.8[M+H] ⁺ .

Step 5: Synthesis of tert-butyl (2S,4R)-2-[[(S)-1-[2-fluoro-4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]-4-hydroxypyrrolidine-1-carboxylate

Tert-butyl (2S,4R)-2-[[(S)-1-(4-bromo-2-fluorophenyl)ethyl]carbamoyl]-4-hydroxypyrrolidine-1-carboxylate (210 mg, 486.91 μmol) and 4-methylthiazole-5-boronic acid pinacol ester (164.41 mg, 730.36 μmol) were dissolved in dioxane (4 mL) and water (1 mL), and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (35.63 mg, 48.69 μmol) and potassium phosphate (206.71 mg, 973.81 μmol) were added. The reaction solution was reacted at 80°C for 12 hours under nitrogen protection, and the reaction was completed by LCMS detection. The reaction solution was cooled to room temperature and filtered. The filtrate was diluted with water (5 mL) and extracted three times with ethyl acetate (30 mL). The organic phases were combined, dried, concentrated, and purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/2) to give (2S,4R)-2-[[(S)-1-[2-fluoro-4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester (150 mg).

MS m/z(ESI):450.3[M+H] ⁺ .

Step 6: Synthesis of (2S,4R)-N-[(S)-1-[2-fluoro-4-(4-methylthiazol-5-yl)phenyl]ethyl]-4-hydroxypyrrolidine-2-carboxamide

(2S,4R)-2-[[(S)-1-[2-fluoro-4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]-4-hydroxypyrrolidine-1-carboxylic acid tert-butyl ester (150 mg, 333.68 μmol) was dissolved in dichloromethane (2 mL), and dioxane hydrochloride (834.19 μL, 4 M) was added. The reaction solution was reacted at 25° C. for 2 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated to obtain (2S,4R)-N-[(S)-1-[2-fluoro-4-(4-methylthiazol-5-yl)phenyl]ethyl]-4-hydroxypyrrolidine-2-carboxamide (150 mg).

MS m/z(ESI):350.0[M+H] ⁺ .

Step 7: Synthesis of (2S,4R)-1-[2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methyl-butyryl]-N-[(1S)-1-[2-fluoro-4-(4-methylthiazol-5-yl)phenyl]ethyl]-4-hydroxypyrrolidine-2-carboxamide

(2S,4R)-N-[(S)-1-[2-fluoro-4-(4-methylthiazol-5-yl)phenyl]ethyl]-4-hydroxypyrrolidine-2-carboxamide (150 mg, 388.72 μmol) and 2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methyl-butyric acid (117.13 mg, 388.72 μmol) were dissolved in N,N-dimethylformamide (3 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (162.58 mg, 427.59 μmol) and N,N-diisopropylethylamine (150.72 mg, 1.17 mmol) were added. The reaction solution was reacted at 25° C. for 2 hours, and the reaction was completed when LCMS was detected. The reaction mixture was diluted with water (5 mL), extracted three times with ethyl acetate (30 mL), the organic phases were combined, dried, concentrated, and purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/1) to give (2S, 4R)-1-[2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methyl-butyryl]-N-[(1S)-1-[2-fluoro-4-(4-methylthiazol-5-yl)phenyl]ethyl]-4-hydroxypyrrolidine-2-carboxamide (160 mg).

MS m/z(ESI):633.4[M+H] ⁺ .

Step 8: Synthesis of (2S,4R)-N-[(1S)-1-[2-fluoro-4-(4-methylthiazol-5-yl)phenyl]ethyl]-4-hydroxy-1-[3-methyl-2-[3-(2-oxoethoxy)isoxazol-5-yl]butyryl]pyrrolidine-2-carboxamide

(2S,4R)-1-[2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methyl-butyryl]-N-[(1S)-1-[2-fluoro-4-(4-methylthiazol-5-yl)phenyl]ethyl]-4-hydroxypyrrolidine-2-carboxamide (140 mg, 221.26 μmol) was dissolved in tetrahydrofuran (3 mL), and 1 M sulfuric acid (0.2 mL) was added. The reaction solution was reacted at 70° C. for 2 hours, and the reaction was completed by LCMS. The reaction solution was adjusted to pH 7-8 with saturated sodium carbonate, diluted with water (5 mL), extracted three times with ethyl acetate (15 mL), and the organic phases were combined, dried, and concentrated to give (2S,4R)-N-[(1S)-1-[2-fluoro-4-(4-methylthiazol-5-yl)phenyl]ethyl]-4-hydroxy-1-[3-methyl-2-[3-(2-oxoethoxy)isoxazol-5-yl]butyryl]pyrrolidine-2-carboxamide (130 mg).

MS m/z(ESI):559.1[M+H] ⁺ .

Step 9: Synthesis of (2S,4R)-1-[2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethoxy]isoxazol-5-yl]-3-methylbutyryl]-N-[(1S)-1-[2-fluoro-4-(4-methylthiazol-5-yl)phenyl]ethyl]-4-hydroxypyrrolidine-2-carboxamide

(2S,4R)-N-[(1S)-1-[2-fluoro-4-(4-methylthiazol-5-yl)phenyl]ethyl]-4-hydroxy-1-[3-methyl-2-[3-(2-oxoethoxy)isoxazol-5-yl]butyryl]pyrrolidine-2-carboxamide (40 mg, 71.60 μmol) and 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (22.22 mg, 71.60 μmol) were dissolved in methanol (1 mL), and sodium cyanoborohydride (22.50 mg, 358.02 μmol) and acetic acid (4.30 mg, 71.60 μmol) were added. The reaction solution was reacted at 25°C for 5 hours, and the reaction was completed by LCMS. The reaction solution was concentrated and purified by preparative liquid chromatography (column: Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 22%-62%) as eluent) to give (2S,4R)-1-[2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethoxy]isoxazol-5-yl]-3-methylbutanoyl]-N-[(1S)-1-[2-fluoro-4-(4-methylthiazol-5-yl)phenyl]ethyl]-4-hydroxypyrrolidine-2-carboxamide (Compound 049) (13.20 mg).

MS m/z (ESI): 853.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.98 (br s, 1H), 9.02 (d, J = 5.5Hz, 1H), 8.58-8.41 (m, 2H), 8.13 (br d, J = 8.4 Hz,1H),7.45-7.26(m,4H),6.99(br d,J＝7.9Hz,2H),6.45(br s,2H),6.12(d,J＝4.6Hz,1H),5.18-4.97 (m,2H),4.61-4.51(m,1H),4.48-4.36(m,1H),4.29(br d,J＝5.6Hz,3H),3.79-3.64(m,1H),3.61-3.44(m,2H),3.07(br d,J＝11.1Hz,2H),2.83-2.74(m,2H), 2.48-2.45(m,3H),2.32-2.21(m,3H),2.14-2.01(m,3H),1.89-1.72(m,3H),1.49-1.35(m,3H),1.01-0.80(m ,6H).

Example 86: Synthesis of (2S,4R)-1-[2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethoxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]pyrrolidine-2-carboxamide (Compound 050)

Step 1: Synthesis of tert-butyl (S)-[1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]carbamate

(S)-[1-(5-bromopyridin-2-yl)ethyl]carbamic acid tert-butyl ester (450mg, 1.49mmol) and 4-methylthiazole (296.30mg, 2.99mmol) were dissolved in N,N-dimethylformamide (10mL), and palladium acetate (16.77mg, 74.71μmol) and potassium acetate (293.27mg, 2.99mmol) were added. The reaction solution was reacted at 90°C for 16 hours under nitrogen protection, and the reaction was completed by LCMS detection. The reaction solution was cooled to room temperature, filtered, and the filtrate was concentrated and purified by column chromatography (silicon dioxide, petroleum ether/ethyl acetate = 1/1) to obtain (S)-[1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]carbamic acid tert-butyl ester (430mg).

MS m/z(ESI):320.1[M+H] ⁺ .

Step 2: Synthesis of (S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethylamine

(S)-[1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]aminomethyl tert-butyl ester (430 mg, 1.35 mmol) was dissolved in dichloromethane (6 mL), and dioxane hydrochloride (3.37 mL, 13.46 mmol) was added. The reaction solution was reacted at 25° C. for 2 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated to give (S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethylamine (390 mg).

MS m/z(ESI):220.0[M+H] ⁺ .

Step 3: Synthesis of tert-butyl (2S,4R)-4-hydroxy-2-[[(S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]carbamoyl]pyrrolidine-1-carboxylate

(S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethylamine (390 mg, 1.78 mmol) was dissolved in N,N-dimethylformamide (10 mL), and (2S,4R)-1-tert-butyloxycarbonyl-4-hydroxypyrrolidine-2-carboxylic acid (493.48 mg, 2.13 mmol), O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphate (1.01 g, 2.67 mmol) and N,N-diisopropylethylamine (1.15 g, 8.89 mmol) were added. The reaction solution was reacted at 25° C. for 16 hours, and the reaction was completed after LCMS detection. The reaction solution was diluted with distilled water (15 mL), extracted three times with ethyl acetate (50 mL), and the organic phases were combined, dried, concentrated, and purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/2) to give tert-butyl (2S, 4R)-4-hydroxy-2-[[(S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]carbamoyl]pyrrolidine-1-carboxylate (510 mg).

Step 4: Synthesis of (2S,4R)-4-hydroxy-N-[(S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]pyrrolidine-2-carboxamide

Tert-butyl (2S, 4R)-4-hydroxy-2-[[(S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]carbamoyl]pyrrolidine-1-carboxylate (510 mg, 1.18 mmol) was dissolved in dichloromethane (10 mL), and dioxane hydrochloride (2.95 mL, 11.79 mmol, 4 M) was added. The reaction solution was reacted at 25° C. for 2 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated to obtain (2S, 4R)-4-hydroxy-N-[(S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]pyrrolidine-2-carboxamide (580 mg).

MS m/z(ESI):333.2[M+H] ⁺ .

Step 5: Synthesis of (2S,4R)-1-[2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]pyrrolidine-2-carboxamide

2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methylbutyric acid (81.69 mg, 271.09 μmol) and (2S,4R)-4-hydroxy-N-[(S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]pyrrolidine-2-carboxamide (100 mg, 271.09 μmol) were dissolved in N,N-dimethylformamide (3 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (113.38 mg, 298.20 μmol) and N,N-diisopropylethylamine (105.11 mg, 813.27 μmol) were added. The reaction solution was reacted at 25° C. for 2 hours, and the reaction was completed by LCMS detection. The reaction solution was diluted with water (10 mL), extracted three times with ethyl acetate (10 mL), the organic phases were combined, dried, concentrated, and purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/2) to give (2S, 4R)-1-[2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]pyrrolidine-2-carboxamide (90 mg).

MS m/z(ESI):616.5[M+H] ⁺ .

Step 6: Synthesis of (2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(2-oxoethoxy)isoxazol-5-yl]butyryl]-N-[(1S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]pyrrolidine-2-carboxamide

(2S,4R)-1-[2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]pyrrolidine-2-carboxamide (90 mg, 146.17 μmol) was dissolved in tetrahydrofuran (2 mL), and 1 M sulfuric acid (176.33 mg, 1.80 mmol) was added. The reaction solution was reacted at 70° C. for 5 hours, and the reaction was completed by LCMS. The reaction solution was adjusted to pH 7-8 with sodium carbonate, dried, filtered, and the filtrate was concentrated to give (2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(2-oxoethoxy)isoxazol-5-yl]butyryl]-N-[(1S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]pyrrolidine-2-carboxamide (75 mg).

MS m/z(ESI):560.1[M+NH ₄ ] ⁺ .

Step 7: Synthesis of (2S,4R)-1-[2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethoxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]pyrrolidine-2-carboxamide

(2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(2-oxoethoxy)isoxazol-5-yl]butyryl]-N-[(1S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]pyrrolidine-2-carboxamide (40 mg, 73.85 μmol) and 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (22.92 mg, 73.85 μmol) were dissolved in methanol (1 mL), and acetic acid (443.50 μg, 7.39 μmol) and sodium cyanoborohydride (23.21 mg, 369.26 μmol) were added. The reaction solution was reacted at 25°C for 4 hours, and the reaction was completed when LCMS was detected. The reaction solution was concentrated and purified by preparative liquid chromatography (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 6%-46%) as eluent) to give (2S,4R)-1-[2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethoxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[5-(4-methylthiazol-5-yl)pyridin-2-yl]ethyl]pyrrolidine-2-carboxamide (Compound 050) (4.23 mg).

MS m/z(ESI):836.4[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.05 (br s, 1H), 9.15-9.10 (m, 1H), 8.74-8.63 (m, 1H), 8.58-8.38 (m, 2H), 8.17 ( br d,J＝8.3Hz,1H),8.01-7.88(m,1H),7.47-7.36(m,2H),7.07-7.01(m,2H),6.51(br s,2H),6.18(d,J＝1.9Hz,1H),5.23-4.92(m,2H),4.66-4.57(m,1H),4.54-4.43(m,1H),4.37-4.28(m, 3H),3.84-3.71(m,1H),3.65-3.49(m,2H),3.11(br d,J＝11.5Hz,2H),2.87-2.76(m,2H),2.51(d,J＝4.8 Hz,3H),2.31(br d,J＝13.4Hz,3H),2.18-2.05(m,3H),1.89(br d, J＝8.4Hz, 3H), 1.46 (dd, J＝7.1, 9.4Hz, 3H), 1.01 (t, J＝6.8Hz, 3H), 0.87 (dd, J＝6.7, 13.7Hz, 3H).

Example 87: Synthesis of (2S,4R)-1-[2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethoxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide (Compound 051)

Step 1: Synthesis of (2S,4R)-1-[2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide

2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methylbutyric acid (100 mg, 331.86 μmol) and (2S,4R)-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide (110.32 mg, 331.86 μmol) were dissolved in N,N-dimethylformamide (3 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (151.42 mg, 398.23 μmol) and N,N-diisopropylethylamine (128.67 mg, 995.57 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was completed by LCMS monitoring. The reaction solution was filtered and concentrated to dryness under reduced pressure, and then purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 0/1) to give (2S,4R)-1-[2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide (110 mg).

MS m/z(ESI):616.5[M+H] ⁺ .

Step 2: Synthesis of (2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(2-oxoethoxy)isoxazol-5-yl]butyryl]-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide

(2S,4R)-1-[2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide (50 mg, 81.20 μmol) was dissolved in tetrahydrofuran (1 mL), and 1 M sulfuric acid (1 M, 974.44 μL) was added. The reaction solution was stirred at 70° C. for 1 hour. The reaction was completed by LCMS monitoring. Sodium bicarbonate was added to the reaction solution to adjust the pH to 7-8, and then the reaction solution was concentrated to dryness under reduced pressure to obtain a crude product of (2S, 4R)-4-hydroxy-1-[3-methyl-2-[3-(2-oxoethoxy)isoxazol-5-yl]butyryl]-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide (62 mg).

MS m/z(ESI):542.3[M+H] ⁺ .

Step 3: Synthesis of (2S,4R)-1-[2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethoxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide

(2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(2-oxoethoxy)isoxazol-5-yl]butanoyl]-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide (57 mg, 105.24 μmol) and 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (26.13 mg, 84.19 μmol) were dissolved in N,N-dimethylformamide (1 mL) and tetrahydrofuran (1 mL), and sodium acetate borohydride (66.91 mg, 315.72 μmol) and acetic acid (631.99 μg, 10.52 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was completed by LCMS monitoring. The reaction solution was concentrated to dryness under reduced pressure and purified by high performance liquid chromatography (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (0.05% ammonia) and acetonitrile (acetonitrile content: 21%-61%) as eluent) to give (2S,4R)-1-[2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethoxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[6-(4-methylthiazol-5-yl)pyridin-3-yl]ethyl]pyrrolidine-2-carboxamide (Compound 051) (11.83 mg).

MS m/z(ESI):836.4[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.98 (s, 1H), 9.04-8.97 (m, 1H), 8.62-8.36 (m, 3H), 8.13 (d, J = 8.4Hz, 1H), 7.82-7.65(m,2H),7.37(t,J＝6.9Hz,1H),7.04-6.95(m,2H),6.45(s,2H),6.12(d,J＝5.1Hz,1H),5.12 (d,J＝3.4Hz,1H),5.00-4.87(m,1H),4.64-4.49 (m,1H),4.33-4.19(m,3H),3.30(s,3H),3.13-2.99(m,2H),2.85-2.73(m,2H),2.70-2.60(m,4H),2.36 -2.22(m,3H),2.16-2.00(m,3H),1.89-1.71(m,3H),1.46-1.35(m,3H),0.97(t,J＝5.8Hz,3H),0.83(dd ,J＝6.7,13.3Hz,3H).

Example 88: Synthesis of Compound 052

Step 1: Synthesis of intermediate 7

The intermediate 4 (300.32 mg, 630.53 μmol) prepared by the method of Example 48 was dissolved in N,N-dimethylformamide (8 mL), and N,N-diisopropylethylamine (244.47 mg, 1.89 mmol) and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (287.69 mg, 756.63 μmol) were added. The reaction solution was stirred at 25°C for 3 hours. LCMS monitored the reaction to be complete. The organic phase was concentrated to dryness under reduced pressure and then purified by column chromatography (silicon dioxide, petroleum ether/tetrahydrofuran = 1/0 to 3/2) to obtain the title intermediate 7 compound (280 mg).

MS m/z(ESI):617.3[M+H] ⁺ .

Step 2: Synthesis of intermediate 8

Dissolve intermediate 7 (50 mg, 81.07 μmol) in tetrahydrofuran (1 mL) and add dilute sulfuric acid (1 M, 972.88 μL). Stir the reaction solution at 70°C for 2 hours. LCMS monitors the reaction to be complete. Add sodium bicarbonate to the reaction solution to adjust the pH to 7-8, then extract with ethyl acetate (3 mL) three times, and dry the organic phase with anhydrous sodium sulfate. Filter the organic phase and concentrate under reduced pressure to dryness to obtain the title intermediate 8 compound (43 mg).

MS m/z(ESI):543.5[M+H] ⁺ .

Step 3: Synthesis of Compound 052

Intermediate 8 (38 mg, 70.03 μmol) and 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (21.73 mg, 70.03 μmol) were dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), and sodium acetate borohydride (44.53 mg, 210.10 μmol) and acetic acid (420.54 μg, 7.00 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was complete when monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure and then purified by high performance liquid chromatography (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (0.05% ammonia water) and acetonitrile (acetonitrile content: 23%-63%) as eluent) to give the title compound 052 (2.72 mg).

MS m/z (ESI): 837.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ14.01 (d, J = 2.3 Hz, 1H), 9.15-9.09 (m, 1H), 8.92 (d, J = 11.0 Hz, 1H), 8.63-8.55 ( m,1H),8.54-8.46(m,1H),8.13(d,J＝8.3Hz,1H),7.42-7.31(m,1H),6.99(d,J＝8.0Hz,2H),6.47(s ,2H),6.16-6.08(m,1H),5.20-5.09(m,1H),5.06-4.89(m,1H),4. 62-4.34(m,2H),4.33-4.21(m,3H),3.32(s,3H),3.07(d,J＝10.5Hz,2H),2.76(d,J＝18.3Hz,2H),2.72 -2.66(m,4H),2.32-2.21(m,3H),2.15-2.00(m,3H),1.83(t,J＝11.8Hz,3H),1.49-1.40(m,3H),1.01-0.92 (m,3H),0.83(dd,J＝6.7,13.9Hz,3H).

Example 89: Synthesis of Compound 053

2-[3-amino-1-(piperidin-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (10 mg, 32.22 μmol) and intermediate 11 (17.42 mg, 32.22 μmol) were dissolved in N,N-dimethylformamide (1 mL) and tetrahydrofuran (1 mL), and acetic acid (5.80 mg, 96.66 μmol), sodium acetate (5.29 mg, 64.44 μmol) and sodium acetate borohydride (20.49 mg, 96.66 μmol) were added. The reaction solution was reacted at 25 ° C for 1 hour. The reaction was completed by LCMS. Water (0.2 mL) was added to the reaction solution to quench the reaction. The reaction solution was concentrated to dryness under reduced pressure and purified by HPLC (Boston Green ODS column, 5 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 23%-43%) as eluent) to give the title compound, compound 053 (3.7 mg).

MS m/z (ESI): 835.7 [M+H] ⁺ ;

¹ H NMR(400MHz, DMSO-d ₆ )δ14.03(br s,1H),8.98(s,1H),8.65-8.55(m,1H),8.47-8.37(m,1H),8.19-8.14( m,1H),7.47-7.41(m,2H),7.39-7.31(m,3H),7.07-6.88(m,2H),6.48(s,2H),6.10(d,J＝5.0Hz,1H) ,5.10(br s,1H),4.94-4.81(m,1H),4.75-4.62(m,1H),4.35(t,J＝7.9Hz,1H),4.27(br d,J＝5.0Hz,3H),3.73-3.60(m,2H),3.08(br d,J＝7.5Hz,2H),2.97(br s,2H),2.77(br s,2H),2.45( s,3H),2.23(m,2H),2.10-1.97(m,2H),1.92-1.63(m,4H),1.43-1.31(m,3H),1.01-0.88(m,3H),0.77( m,3H).

Example 90: Synthesis of Compound 054

2-[3-amino-1-(8-azabicyclo[3.2.1]octan-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (15 mg, 40.23 μmol) and intermediate 11 (21.75 mg, 40.23 μmol) were dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), and acetic acid (7.25 mg, 120.69 μmol), sodium acetate (9.90 mg, 120.69 μmol) and sodium acetate borohydride (25.58 mg, 120.69 μmol) were added. The reaction solution was reacted at 25°C for 2 hours. The reaction was completed by LCMS. Water (0.2 mL) was added to the reaction solution to quench the reaction. The reaction solution was concentrated to dryness under reduced pressure and purified by HPLC (Boston Green ODS column, 5 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 23%-43%) as eluent) to give the title compound, compound 054 (5.13 mg).

MS m/z (ESI): 861.6 [M+H] ⁺ ;

¹ H NMR(400MHz, DMSO-d ₆ )δ14.06(br s,1H),8.98(s,1H),8.55(s,1H),8.47-8.39(m,1H),8.17-8.14(m, 1H),7.47-7.41(m,2H),7.40-7.32(m,3H),7.05-6.94(m,2H),6.47(br d,J＝7.4Hz,2H),6.13(s,1H), 5.12(br s,1H),4.97-4.77(m,2H),4.37(m,1H),4.31-4.21(m,3H),3.74-3.63(m,3H),2.86(br t,J＝5.8Hz,2H),2.45(s,3H),2.33-2.14(m,3H),2.09-1.89(m,5H),1.88-1.58(m,5H),1.38(br d,J ＝7.0Hz,3H),0.96(br d,J＝6.4Hz,3H),0.85-0.77(m,3H).

Example 91: Synthesis of Compound 055

2-[3-amino-1-(piperidin-4-ylmethyl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (20 mg, 55.43 μmol) and intermediate 11 (23.97 mg, 44.34 μmol) were dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), and acetic acid (3.33 mg, 55.43 μmol), sodium acetate (9.09 mg, 110.85 μmol) and sodium acetate borohydride (35.24 mg, 166.28 μmol) were added. The reaction solution was reacted at 25°C for 1 hour. The reaction was completed by LCMS. Water (0.5 mL) was added to the reaction solution to quench the reaction. The reaction solution was concentrated to dryness under reduced pressure and purified by HPLC (Boston Green ODS column, 5 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 18%-38%) as eluent) to give the title compound, compound 055 (16.4 mg).

MS m/z (ESI): 849.4 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.96 (br s, 1H), 8.98 (s, 1H), 8.49 (s, 1H), 8.41 (d, J = 7.8Hz, 1H), 8.12 (dd ,J＝1.6,8.4Hz,1H),7.47-7.42(m,2H),7.39-7.33(m,3H),7.02-6.96(m,2H),6.52-6.40(m,2H),6.07(s ,1H),5.09(br s,1H),4.94-4.85(m,1H),4.36(t,J＝7.9Hz,1H),4.27(br s,1H),4.19(br t,J＝5.5Hz ,2H),4.10(br d,J＝7.0Hz,2H),3.69(br dd,J＝4.4,10.6Hz,1H),3.63(d,J＝9.8Hz,1H),3.55-3.40(m,1H),2.87(d, J＝10.9Hz,2H),2.63(t,J＝5.5Hz,2H),2.45(s,3H),2.30-2.14(m,2H),2.02(m,1H),1.97-1.90(m,2H ),1.77(m,1H),1.43(br d,J＝7.3Hz,2H),1.37(d,J＝7.0Hz,3H),1.31(m,2H),0.98-0.91(m,3H), 0.82-0.74(m,3H).

Example 92: Synthesis of (2S,4R)-1-[2-[3-[[1-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]propan-2-yl]oxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 056)

Step 1: Synthesis of methyl 2-[3-(2-bromo-1-methyl-ethoxy)isoxazol-5-yl]-3-methylbutanoate

2-(3-hydroxyisoxazol-5-yl)-3-methylbutyric acid methyl ester (50 mg, 251.00 μmol) and 1-bromo-2-propanol (55.82 mg, 301.20 μmol) were dissolved in toluene (5 mL), and triphenylphosphine (98.75 mg, 376.50 μmol) and diisopropyl azodicarboxylate (76.13 mg, 376.50 μmol) were added under nitrogen. The reaction solution was reacted at 100 ° C for 2 hours. The reaction was complete as detected by LCMS. The reaction solution was concentrated, and the concentrate was extracted with dichloromethane (3 mL × 3) and water (5 mL), and the organic phase was concentrated to dryness under reduced pressure. Purification by thin layer chromatography (silica, petroleum ether/ethyl acetate = 3:1) gave the product 2-[3-(2-bromo-1-methyl-ethoxy)isoxazol-5-yl]-3-methylbutyric acid methyl ester (40 mg).

MS m/z(ESI):319.7,321.7[M+H] ⁺ .

Step 2: Synthesis of methyl 2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-1-methyl-ethoxy]isoxazol-5-yl]-3-methylbutanoate

2-[3-(2-bromo-1-methyl-ethoxy)isoxazol-5-yl]-3-methylbutyric acid methyl ester (40 mg, 124.93 μmol) and 2-[3-amino-1-(4-piperidinyl)pyrazolo[4,3-c]pyridazin-6-yl]phenol (38.77 mg, 124.93 μmol) were dissolved in N,N-dimethylformamide (2 mL) and N,N-diisopropylethylamine (48.44 mg, 374.79 μmol), and the reaction solution was reacted at 80° C. for 12 hours. The reaction was completed by LCMS. The reaction solution was extracted with dichloromethane (15 mL×3) and water (3 mL), and the organic phase was concentrated to dryness under reduced pressure and purified by thin layer chromatography (silica, petroleum ether/tetrahydrofuran=2/3) to give 2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-1-methyl-ethoxy]isoxazol-5-yl]-3-methylbutanoic acid methyl ester (10 mg).

MS m/z(ESI):550.1[M+H] ⁺ .

Step 3: Synthesis of 2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-1-methyl-ethoxy]isoxazol-5-yl]-3-methylbutanoic acid

2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-1-methyl-ethoxy]isoxazol-5-yl]-3-methylbutyric acid methyl ester (10 mg, 18.19 μmol) was dissolved in tetrahydrofuran (0.5 mL) and water (0.5 mL), and lithium hydroxide monohydrate (2.29 mg, 54.58 μmol) was added. The reaction solution was reacted at 25° C. for 1 hour, and LCMS showed that the reaction was completed. The reaction solution was adjusted to pH 5-6 with 1 M hydrochloric acid solution and then concentrated to dryness under reduced pressure to give the product 2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-1-methyl-ethoxy]isoxazol-5-yl]-3-methylbutanoic acid (10 mg).

MS m/z(ESI):536.4[M+H] ⁺ .

Step 4: Synthesis of (2S,4R)-1-[2-[3-[[1-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]propan-2-yl]oxy]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-1-methyl-ethoxy]isoxazol-5-yl]-3-methylbutanoic acid (10 mg, 18.67 μmol) and ((2S,4R)-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (8.24 mg, 22.41 μmol) were added. l) was dissolved in N,N-dimethylformamide (1 mL), triethylamine (7.56 mg, 74.68 μmol) and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (7.10 mg, 18.67 μmol) were added, and the reaction solution was reacted at 25°C for 1 hour. LCMS showed that the reaction was complete. Water (0.5 mL) was added to the reaction solution to quench the reaction. The reaction solution was concentrated to dryness under reduced pressure. Purified by high performance liquid chromatography (Boston Green ODS column, 5μm silica, 30mm diameter, 150mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content: 20%-40%) as eluent) to obtain (2S,4R)-1-[2-[3-[[1-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]propan-2-yl]oxy]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 056) (3.01mg).

MS m/z (ESI): 849.6 [M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ8.87 (s, 1H), 8.24 (s, 1H), 7.99 (br d, J = 7.5Hz, 1H), 7.43 (d, J = 4.2Hz, 3H ),7.36-7.31(m,2H),7.00(br d,J＝8.1Hz,2H),6.07(s,1H),5.08-5.00(m,3H),4.58(br s,2H),4.52( br s,1H),4.44(br s,1H),3.71(br d,J＝9.9Hz,1H),3.64(br d,J＝10.5Hz,1H),3.53-3.41(m,2H),3.00 (br d,J＝9.0Hz,2H),2.86(br s,1H),2.48(s,3H),2.39(br d,J＝13.3Hz,3H),2.25-2.15(m,1H),2.06(br d,J＝10.1Hz,2H),2.00-1.93(m,1H),1.52(dd,J＝1.8,7.0Hz,3H),1.43(br dd,J＝3.5,6.0Hz,3H),1.07( d,J＝6.5Hz,3H),0.91(br d,J＝6.7Hz,3H).

Example 93: Synthesis of (2S,4R)-1-[(2S)-2-[2-[2-[3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octan-8-yl]ethoxy]acetamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 057)

Step 1: Synthesis of (2S,4R)-1-[(2S)-2-[2-(2-bromoethoxy)acetamido]-3,3-dimethylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-(2-Bromoethoxy)acetic acid (50.0 mg, 273 μmol) and (2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (131 mg, 273 μmol) were dissolved in N,N-dimethylformamide (3 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (114 mg, 301 μmol) and N,N-diisopropylethylamine (106 mg, 820 μmol) were added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was diluted with water (10 mL) and ethyl acetate (10 mL), and the aqueous phase was extracted twice with ethyl acetate (10 mL). The organic phase was washed twice with water (10 mL). The collected organic layer was dried over anhydrous sodium sulfate and then filtered, and the filtrate was concentrated under reduced pressure and purified by thin layer chromatography (silica, dichloromethane/methanol=10/1) to obtain the product (2S,4R)-1-[(2S)-2-[2-(2-bromoethoxy)acetamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (57 mg).

MS m/z(ESI):608.9,610.9[M+H] ⁺ .

Step 2: Synthesis of (2S,4R)-1-[(2S)-2-[2-[2-[3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octan-8-yl]ethoxy]acetamido]-3,3-dimethylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

(2S,4R)-1-[(2S)-2-[2-(2-bromoethoxy)acetamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (27.2 mg, 44.6 μmol) and 2-[3-amino-1-(8-azabicyclo[3.2.1]octan-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (15.0 mg, 44.6 μmol) were dissolved in N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (17.3 mg, 134 μmol) was added. The reaction solution was stirred at 80° C. for 16 hours. LCMS showed that the reaction was complete. The reaction solution was filtered and the filtrate was purified by preparative liquid chromatography (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia water) and acetonitrile (acetonitrile content: 39%-79%) as eluent) to obtain the product (2S,4R)-1-[(2S)-2-[2-[2-[3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octan-8-yl]ethoxy]acetamido]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 057) (1.92 mg).

MS m/z (ESI): 865.4 [M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ = 8.90-8.83 (m, 1H), 8.39 (s, 1H), 8.08 (d, J = 6.8Hz, 1H), 7.42-7.38 (m, 2H), 7.37-7.31(m,3H),7.02-6.97(m,2H),4.69(s,1H),4.59-4.50(m,1H),4.43(s,1H),4.19-4.00(m,2H), 3.89-3.77(m,3H), 3.73(dd,J＝3.6,11.4Hz,1H),3.66-3.55(m,2H),2.95(s,2H),2.54(s,2H),2.48-2.45(m,3H),2.17(s, 3H),2.08-1.90(m,4H),1.81(s,2H),1.42(d,J＝7.0Hz,3H),1.33-1.22(m,1H),1.05-1.01(m,9H).

Example 94: Synthesis of (2S,4R)-1-[2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]azetidin-1-yl]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 058)

Step 1: Synthesis of tert-butyl 3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]azetidine-1-carboxylate

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (150 mg, 387 μmol) and 1-Boc-3-azetidinone (99.3 mg, 580 μmol) were dissolved in tetrahydrofuran (3 mL) and N,N-dimethylformamide (3 mL), acetic acid (70.0 mg, 1.16 mmol) was added, and stirred at 25 ° C for 30 minutes. Finally, sodium acetate borohydride (246 mg, 1.16 mmol) was added. The reaction was stirred at 25 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, diluted with water (10 mL) and ethyl acetate (10 mL), the aqueous phase was extracted twice with ethyl acetate (10 mL), and the organic phase was washed twice with water (10 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography (silica, petroleum ether/ethyl acetate=100/35) to give the product 3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]azetidine-1-carboxylic acid tert-butyl ester (130 mg).

MS m/z(ESI):466.1[M+H] ⁺ .

Step 2: Synthesis of 2-[3-amino-1-[1-(azetidin-3-yl)piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]azetidine-1-carboxylic acid tert-butyl ester (130 mg, 279 μmol) was dissolved in dichloromethane (2 mL), and trifluoroacetic acid (318 mg, 2.79 mmol) was added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure to give the product 2-[3-amino-1-[1-(azetidine-3-yl)piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (100 mg, crude product) which was directly used in the next step.

MS m/z(ESI):366.1[M+H] ⁺ .

Step 3: Synthesis of methyl 2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]azetidin-1-yl]isoxazol-5-yl]-3-methylbutanoate

2-[3-amino-1-[1-(azetidin-3-yl)piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (100 mg, 274 μmol) and 3-methyl-2-[3-(1,1,2,2,3,3,4,4,4-nonafluorobutylsulfonyloxy)isoxazol-5-yl]butyric acid methyl ester (145 mg, 301 μmol) were dissolved in N,N-dimethylformamide (2 mL), N,N-diisopropylethylamine (212 mg, 1.64 mmol) was added, and the reaction solution was stirred at 80 ° C for 16 hours. LCMS showed that the reaction was complete. Ethyl acetate (10 mL) and water (10 mL) were added for dilution, the aqueous phase was extracted twice with ethyl acetate (10 mL), and the organic phase was washed twice with water (10 mL). The collected organic layer was dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure and purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 100/40) to give the product 2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]azetidin-1-yl]isoxazol-5-yl]-3-methylbutanoic acid methyl ester (50 mg, 50% purity).

MS m/z(ESI):547.4[M+H] ⁺ .

Step 4: Synthesis of 2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]azetidin-1-yl]isoxazol-5-yl]-3-methylbutanoic acid

2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]azetidin-1-yl]isoxazol-5-yl]-3-methylbutanoic acid methyl ester (50 mg, 45.7 μmol, 50% purity) was dissolved in tetrahydrofuran (0.5 mL) and water (0.5 mL), and lithium hydroxide monohydrate (5.76 mg, 137 μmol) was added. The reaction solution was stirred at 25° C. for 2 hours. LCMS showed that the reaction was complete. The reaction solution was extracted twice with ethyl acetate (3 mL), the organic phase (3 mL) was washed twice with water, citric acid (1.5 M) was added dropwise to the aqueous phase to adjust the pH to 5 to 6, and then concentrated under reduced pressure to give the product 2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]azetidin-1-yl]isoxazol-5-yl]-3-methylbutanoic acid (25 mg, crude product), which was directly used in the next step.

MS m/z(ESI):533.4[M+H] ⁺ .

Step 5: Synthesis of (2S,4R)-1-[2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]azetidin-1-yl]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]azetidin-1-yl]isoxazol-5-yl]-3-methylbutanoic acid (25.0 mg, 47.0 μmol) and (2S,4R)-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (31.1 mg, 93.9 μmol) were dissolved in N,N-dimethylformamide (1 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (21.4 mg, 56.3 μmol) and N,N-diisopropylethylamine (36.4 mg, 282 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was filtered and the filtrate was purified by preparative liquid chromatography (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 27%-67%) as eluent) to obtain the product (2S, 4R)-1-[2-[3-[3-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]azetidin-1-yl]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 058) (5.32 mg).

MS m/z (ESI): 846.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.96 (s, 1H), 9.03-8.91 (m, 1H), 8.50 (s, 1H), 8.46-8.20 (m, 1H), 8.12 (d, J ＝8.1Hz,1H),7.52-7.29(m,5H),6.99(d,J＝7.9Hz,2H),6.46(s,2H),6.00-5.75(m,1H),5.16-4.99(m, 1H),4.97-4.82(m,1H),4.65-4.50(m,1H),4.47-4.32 (m,1H),4.28(s,1H),4.02-3.87(m,2H),3.77-3.64(m,3H),3.63-3.39(m,3H),2.99-2.86(m,2H),2.45 (d,J＝3.0Hz,3H),2.29-2.18(m,1H),2.12-1.99(m,5H),1.96-1.70(m,3H),1.51-1.30(m,3H),1.01-0.85 (m,3H),0.85-0.74(m,3H).

Example 95: Synthesis of (2S,4R)-4-hydroxy-1-[(2S)-2-[6-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexanamido]-3,3-dimethylbutyryl]-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 059)

Step 1: Synthesis of 6-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexanoic acid

2-[3-(Methylamino)-1-(4-piperidinyl)pyrazolo[4,3-c]pyridazin-6-yl]phenol (30.0 mg, 92.5 μmol) and 6-bromohexanoic acid (36.1 mg, 185 μmol) were dissolved in N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (59.8 mg, 462 μmol) was added. The reaction solution was stirred at 80 ° C for 3 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to obtain a brown liquid 6-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexanoic acid (40 mg), which was used directly in the next step without purification.

MS m/z(ESI):439.3[M+H] ⁺ .

Step 2: Synthesis of (2S,4R)-4-hydroxy-1-[(2S)-2-[6-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexanamido]-3,3-dimethylbutyryl]-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

6-[4-[6-(2-Hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexanoic acid (40.0 mg, 91.2 μmol) and (2S,4R)-1-[(2S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (60.8 mg, 137 μmol) were dissolved in N,N-dimethylformamide (1 mL), and O-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (41.6 mg, 109 μmol) and N,N-diisopropylethylamine (58.9 mg, 456 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was filtered and the filtrate was purified by preparative liquid chromatography (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content: 34%-74%) as eluent) to obtain the product (2S, 4R)-4-hydroxy-1-[(2S)-2-[6-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]hexanamide]-3,3-dimethylbutanoyl]-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 059) (6.78 mg).

MS m/z(ESI):865.7[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.99 (s, 1H), 8.98 (s, 1H), 8.49 (s, 1H), 8.38 (d, J = 7.8Hz, 1H), 8.13 (d, J＝7.0Hz,1H),7.82(d,J＝9.3Hz,1H),7.46-7.42(m,2H),7.40-7.33(m,3H),7.07(d,J＝5.0Hz,1H), 7.02-6.95(m,2H),5.12(d,J＝3.3Hz,1H),4.97-4.84 (m,1H),4.60-4.48(m,2H),4.42(t,J＝8.0Hz,1H),4.28(s,1H),3.60(s,2H),3.03-2.94(m,5H), 2.36-2.24(m,4H),2.19-1.97(m,6H),1.88-1.75(m,3H),1.58-1.41(m,5H),1.37(d,J＝7.0Hz,3H),1.35- 1.21(m,3H),0.95(s,9H).

Example 96: Synthesis of Compound 301

2-[3-[(methyl-d ₃ )amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (18a) (15 mg, 41.22 μmol) and intermediate 2-1 (24.48 mg, 41.22 μmol) were dissolved in methanol (1.5 mL), and sodium acetate (13.53 mg, 164.90 μmol), acetic acid (247.56 μg, 4.12 μmol) and sodium cyanoborohydride (12.95 mg, 206.12 μmol) were added. The reaction was carried out at 25° C. for 2 hours, and the reaction was completed by LCMS. The reaction solution was concentrated and purified by preparative liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 35%-75%) as eluent) to give compound 301 (12.83 mg).

MS m/z (ESI): 905.4 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.99 (s, 1H), 8.99 (s, 1H), 8.50 (s, 1H), 8.42 (d, J = 7.6Hz, 1H), 8.13 (br d ,J＝7.1Hz,1H),7.49-7.32(m,6H),7.05-6.98(m,3H),6.13(s,1H),5.13(d,J＝3.6Hz,1H),4.95-4.88( m,1H),4.61-4.53(m,1H),4.37(t,J＝7.8Hz,1H),4.29(br s,1H),3.72(br dd,J＝4.2,10.3Hz,1H),3.64 (br d,J＝11.8Hz,2H),3.57(d,J＝9.9Hz,1H),3.43(br d,J＝11.1Hz,1H),3.00(br d,J＝8.9Hz,2H),2.76( br t,J＝11.2Hz,2H),2.46(s,3H),2.25-2.17(m,4H),2.13(br t,J＝11.2Hz,3H),2.07-2.01(m,1H),1.86 -1.76(m,5H),1.39(d,J＝6.9Hz,3H),1.16(br d,J＝11.0Hz,2H),0.99-0.94(m,3H),0.85-0.79(m,3H) .

Example 97: Synthesis of Compound 303

2-[1-(piperidin-4-yl)-3-(propylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (20a) (35.37 mg, 90.95 μmol), intermediate 2-1 (45 mg, 75.79 μmol), acetic acid (13.65 mg, 227.37 μmol, 13.00 μL) and sodium acetate (18.65 mg, 227.37 μmol) were dissolved in anhydrous tetrahydrofuran (1.5 mL) and anhydrous N,N-dimethylformamide (1.5 mL), and sodium acetate borohydride (89.44 mg, 422.03 μmol) was added. The reaction solution was stirred at 25°C for 4 hours. After the reaction was completed by LCMS, the reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 48%-88%) as eluent) gave compound 303 (28.73 mg).

MS m/z (ESI): 930.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ14.02(s,1H),8.99(s,1H),8.56-8.39(m,2H),8.18-8.09(m,1H),7.51-7.30(m ,5H),7.13(br t,J＝5.7Hz,1H),7.05-6.94(m,2H),6.13(s,1H),5.12(d,J＝3.6Hz,1H),4.92(br t, J＝6.9Hz,1H),4.57(br s,1H),4.42-4.23(m,2H),3.79-3.53(m,4H),3.49-3.38(m,2H),3.00(br d,J＝8.3Hz,2H),2.76(br t,J＝11.6Hz,2H),2.46(s,5H),2.22(br d,J＝7.0Hz,3H),2.14-2.08(m,3H ),2.07-1.97(m,1H), 1.88-1.65(m,8H),1.39(d,J＝7.0Hz,2H),1.28-1.03(m,3H),1.01-0.92(m,6H), 0.86-0.77(m,3H).

Example 98: Synthesis of Compound 305

N-[6-(2-hydroxyphenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-yl]cyclopropanamide (22a) (34.42 mg, 90.95 μmol), intermediate 2-1 (45 mg, 75.79 μmol), acetic acid (9.10 mg, 151.58 μmol) and sodium acetate (12.43 mg, 151.58 mol) were dissolved in anhydrous tetrahydrofuran (1.5 mL) and anhydrous N,N-dimethylformamide (1.5 mL). The reaction solution was stirred at 25°C for 1 hour, and sodium acetate borohydride (48.19 mg, 227.37 μmol) was added. The reaction solution was stirred at 25°C for 3 hours, and the reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 35%-75%) as eluent) gave compound 305 (38.63 mg).

MS m/z(ESI):957.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.25 (s, 1H), 11.08 (s, 1H), 8.99 (s, 1H), 8.81 (s, 1H), 8.43 (d, J = 7.5Hz, 1H),8.15(d,J＝6.9Hz,1H),7.52-7.30(m,5H),7.09-6.82(m,2H),6.13(s,1H),5.17-4.99(m,1H),4.92 (t,J＝7.3Hz,1H),4.81(s,1H),4.37(t,J＝7.9Hz,1H),4.29(s,1H),3.78-3.68(m,1H),3.68-3.50( m ,3H),3.48-3.40(m,1H),3.01(s,2H),2.83-2.69(m,2H),2.46(s,3H),2.24(d,J＝5.9Hz,3H),2.18- 2.08(m,4H),2.02(d,J＝12.3Hz,2H),1.98-1.86(m,2H),1.84-1.64(m,4H),1.39(d,J＝7.0Hz,3H),1.16 (d,J＝9.9Hz,2H),1.00-0.93(m,3H),0.92-0.86(m,4H),0.86-0.77(m,3H).

Example 99: Synthesis of Compound 310

2-[3-(Cyclopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (20 mg, 51.70 μmol) and intermediate 2-1 (30.69 mg, 51.70 μmol) were dissolved in methanol (2 mL), and sodium acetate (16.96 mg, 206.78 μmol), acetic acid (310.44 μg, 5.17 μmol) and sodium cyanoborohydride (16.24 mg, 258.48 μmol) were added. The reaction solution was reacted at 25° C. for 2 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated and purified by preparative liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 39%-79%) as eluent) to give compound 310 (11.89 mg).

MS m/z (ESI): 928.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.98 (s, 1H), 8.99 (s, 1H), 8.51 (s, 1H), 8.42 (br d, J = 7.6Hz, 1H), 8.13 (br d,J＝7.4Hz,1H),7.47-7.35(m,7H),7.02-6.98(m,2H),6.13(s,1H),5.13(d,J＝3.6Hz,1H),4.92(br t,J＝7.2Hz,1H),4.59(br s,1H),4.37(br t,J＝7.8Hz,1H),4.29(br s,1H),3.72(br dd,J＝4.3,10.8Hz ,2H),3.64(br d,J＝11.9Hz,2H),3.57(br d,J＝9.8Hz,2H),3.01(br d,J＝9.5Hz,3H),2.81(dt,J＝3.3,6.8Hz,2H), 2.75(br d,J＝10.9Hz,2H),2.46(s,3H),2.23(br d,J＝2.9Hz,2H),2.17-2.10(m,3H),1.86(br d,J＝9.8 Hz,2H),1.78(br d,J＝8.8Hz,3H),1.39(br d,J＝7.0Hz,3H),1.16(br d,J＝9.9Hz,2H),0.98-0.94(m, 3H),0.80(br d,J＝6.6Hz,3H),0.77-0.72(m,2H),0.62-0.57(m,2H).

Example 100: Synthesis of Compound 311

2-[1-(piperidin-4-yl)-3-[(2,2,2-trifluoroethyl)amino]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (26a) (15 mg, 34.98 μmol) and intermediate 2-1 (20.77 mg, 34.98 μmol) were dissolved in methanol (1 mL), and sodium acetate (11.48 mg, 139.91 μmol), acetic acid (210.05 ug, 3.50 μmol) and sodium cyanoborohydride (10.99 mg, 174.89 μmol) were added. The reaction solution was reacted at 25° C. for 2 hours, and the reaction was completed after LCMS detection. The reaction solution was concentrated and purified by preparative liquid chromatography (Phenomenex Luna C18 column, 5 μm silica, 30 mm diameter, 80 mm length; using a mixture of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content 35%-75%) as eluent) to give compound 311 (8.48 mg).

MS m/z (ESI): 970.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.50-13.01 (m, 1H), 9.01-8.98 (m, 1H), 8.47 (s, 1H), 8.42 (d, J = 7.5Hz, 1H), 8.06-7.96(m,2H),7.49-7.43(m,2H),7.42-7.36(m,3H),7.07-7.01(m,2H),6.19(s,1H),4.96-4.85(m,2H ),4.36(t,J＝7.9Hz,1H),4.33-4.19(m,3H),3.77-3.67(m,5H),3.59(br d,J＝9.8Hz,1H),3.51-3.46(m,2H),3.32-3.20(m,2H),3.14-3.02(m,4H),2.82(br t,J＝11.3Hz,2H), 2.46(s,3H),2.29-2.18(m,1H),2.14(br d,J＝12.8Hz,2H),2.09-1.98(m,2H),1.87-1.76(m,3H),1.38(d ,J＝7.0Hz,3H),1.33-1.22(m,2H),0.96(br d,J＝6.3Hz,3H),0.83-0.77(m,3H).

Example 101: Synthesis of Compound 313

2-[3-[(methyl-d ₃ )amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (18a) (15 mg, 41.22 μmol) and intermediate 11 (22.29 mg, 41.22 μmol) were dissolved in methanol (1.5 mL), and sodium acetate (13.53 mg, 164.90 μmol), acetic acid (247.56 μg, 4.12 μmol) and sodium cyanoborohydride (12.95 mg, 206.12 μmol) were added. The reaction solution was reacted at 25° C. for 2 hours, and the reaction was completed by LCMS. The reaction solution was concentrated and purified by preparative liquid chromatography (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 34%-74%) as eluent) to give compound 313 (9.34 mg).

MS m/z (ESI): 852.4 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.01-13.96 (m, 1H), 8.99 (s, 1H), 8.49 (s, 1H), 8.43 (br d, J = 7.5Hz, 1H), 8.16 -8.10(m,1H),7.49-7.33(m,6H),7.05-6.98(m,3H),6.13(s,1H),5.12(d,J＝3.6Hz,1H),4.97-4.88(m ,1H),4.58(br t,J＝11.0Hz,1H),4.38(br t,J＝7.9Hz,1H),4.32-4.27(m,3H),3.75-3.64(m,2H),3.46( br d,J＝10.0Hz,2H),3.08(br d,J＝10.1Hz,2H),2.78(br t,J＝4.8Hz,2H),2.46(s,3H),2.30-2.23(m,2H ),2.22-2.11(m,2H),2.04(br t,J＝8.9Hz,1H),1.88-1.75(m,2H),1.38(d,J＝7.0Hz,3H),1.00-0.95(m ,3H),0.86-0.80(m,3H).

Example 102: Synthesis of (2S,4R)-1-[(S)-2-[2-[4-[[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetyl]amino]-3,3-dimethylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 223)

Step 1: Synthesis of tert-butyl 4-[[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]methyl]piperidine-1-carboxylate

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (240.11 mg, 773.66 μmol) and 1-BOC-4-piperidinecarboxaldehyde (150 mg, 703.32 μmol) were dissolved in N,N-dimethylformamide (2 mL) and tetrahydrofuran (2 mL), and acetic acid (42.24 mg, 703.32 μmol) and sodium triacetoxyborohydride (447.19 mg, 2.11 mmol) were added. The reaction solution was stirred at 25°C for 1 hour. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure, 5 mL of ethyl acetate was added and stirred at 25°C for 2 hours, and the filter cake was concentrated to dryness under reduced pressure to obtain tert-butyl 4-[[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]methyl]piperidine-1-carboxylate (205 mg).

MS m/z(ESI):508.3[M+H] ⁺ .

Step 2: Synthesis of 2-[3-amino-1-[1-(piperidin-4-ylmethyl)piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]-1-piperidinyl]methyl]piperidine-1-carboxylic acid tert-butyl ester (205 mg, 403.84 μmol) was dissolved in dichloromethane (2 mL), and dioxane hydrochloride (4M, 504.80 μL) was added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain 2-[3-amino-1-[1-(piperidin-4-ylmethyl)piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (228 mg).

MS m/z(ESI):408.1[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 2-[4-[[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetate

2-[3-amino-1-[1-(piperidin-4-ylmethyl)piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (80 mg, 196.31 μmol) was dissolved in N,N-dimethylformamide (1.5 mL), and N,N-diisopropylethylamine (76.12 mg, 588.94 μmol) was added. Tert-butyl bromoacetate (38.29 mg, 196.31 μmol) was added at 0°C, and the reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete. Water (2 mL) was added to the reaction solution and extracted three times with ethyl acetate (15 mL), and the organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure and then purified by thin layer chromatography (silica: dichloromethane/methanol = 10/1) to give tert-butyl 2-[4-[[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetate (58 mg).

MS m/z(ESI):522.3[M+H] ⁺ .

Step 4: Synthesis of 2-[4-[[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetic acid

Tert-butyl 2-[4-[[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetate (30 mg, 57.51 μmol) was dissolved in dichloromethane (0.5 mL), and dioxane hydrochloride (4M, 71.89 μL) was added. The reaction solution was stirred at 25° C. for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain 2-[4-[[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetic acid (20 mg).

MS m/z(ESI):466.2[M+H] ⁺ .

Step 5: Synthesis of (2S,4R)-1-[(S)-2-[2-[4-[[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetyl]amino]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[4-[[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetic acid (8 mg, 17.18 μmol) and (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2 -formamide (8.40 mg, 18.90 μmol) was dissolved in anhydrous N,N-dimethylformamide (0.5 mL), and N,N-diisopropylethylamine (8.88 mg, 68.74 μmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (7.19 mg, 18.90 μmol) were added, and the reaction solution was stirred at 25° C. for 2 hours, and the reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. The product was purified by HPLC (Phenomenex C18 column, 5 μm silica, 30 mm diameter, 80 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 50%-70%) as eluent) to give (2S,4R)-1-[(S)-2-[2-[4-[[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetyl]amino]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 223) (2.68 mg).

MS m/z(ESI):892.8[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.03-13.95 (m, 1H), 8.98 (s, 1H), 8.56-8.45 (m, 2H), 8.13 (br d, J = 7.3Hz, 1H) ,7.76(br d,J＝9.8Hz,1H),7.46-7.41(m,2H),7.38-7.34(m,3H),7.02-6.96(m,2H),6.46(br s,2H),5.14(d,J＝3.4Hz,1H),4.89(quin,J＝6.9Hz,1H),4.57-4.37(m,3H),4.31-4.24(m,1H),3.64-3.53 (m,2H),3.03-2.93(m,3H),2.91-2.80(m,3H),2.45(s,3H),2.22(br d,J＝6.9Hz,2H),2.08(br s,5H ),1.85-1.73(m,5H),1.60-1.44(m,2H),1.38(d,J=7.0Hz,3H),1.25-1.11(m,3H),0.94(s,9H).

Example 103: Synthesis of (2S,4R)-4-hydroxy-1-[2-[3-[2-[3-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octan-8-yl]ethoxy]isoxazol-5-yl]-3-methylbutyryl]-N-[(S)-1-[4-(4-methylthiazol-5)-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 228)

Step 1: Synthesis of (2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(2-oxoethoxy)isoxazol-5-yl]butyryl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

(2S,4R)-1-[2-[3-(2,2-diethoxyethoxy)isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (300 mg, 488.00 μmol) was dissolved in anhydrous tetrahydrofuran (6.5 mL) and 1.5 M hydrochloric acid (6.51 mL) was added. The reaction solution was stirred at 60° C. for 2 hours. LCMS detected that the reaction was complete. Saturated sodium bicarbonate was added to the reaction solution to adjust the pH to 7-8, and the reaction solution was concentrated to dryness under reduced pressure. Water (7 mL) was added to the reaction solution and extracted three times with ethyl acetate (15 mL). The organic phase was washed three times with water (7 mL) and dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure to give (2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(2-oxoethoxy)isoxazol-5-yl]butyryl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (260 mg).

MS m/z(ESI):541.6[M+H] ⁺ .

Step 2: Synthesis of (2S,4R)-4-hydroxy-1-[2-[3-[2-[3-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octan-8-yl]ethoxy]isoxazol-5-yl]-3-methylbutyryl]-N-[(S)-1-[4-(4-methylthiazol-5)-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[1-(8-Azabicyclo[3.2.1]octan-3-yl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (7.16 mg, 18.50 μmol) and (2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(2-oxoethoxy)isoxazol-5-yl]butanoyl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine -2-Formamide (10 mg, 18.5 μmol) was dissolved in anhydrous N,N-dimethylformamide (0.5 mL) and anhydrous tetrahydrofuran (0.5 mL), and acetic acid (1.11 mg, 18.50 μmol), sodium acetate (4.55 mg, 55.49 μmol) and sodium triacetoxyborohydride (11.76 mg, 55.49 μmol) were added, and the reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. The product was purified by HPLC (Phenomenex C18-1 column, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia and ammonium bicarbonate) and acetonitrile (acetonitrile content 50%-70%) as eluent) to give (2S,4R)-4-hydroxy-1-[2-[3-[2-[3-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octan-8-yl]ethoxy]isoxazol-5-yl]-3-methylbutanoyl]-N-[(S)-1-[4-(4-methylthiazol-5)-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 228) (1.1 mg).

MS m/z (ESI): 875.4 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.01-8.95 (m, 1H), 8.54-8.49 (m, 1H), 8.46-8.28 (m, 1H), 8.21-8.13 (m, 1H), 7.45 -7.29(m,5H),7.10-7.04(m,1H),7.02-6.98(m,1H),6.16-6.04(m,2H),4.99-4.75(m,2H),4.47-4. 33(m,1H),4.30-4.20(m,3H),3.00-2.91(m,4H),2.84-2.77(m,2H),2.45(s,3H),2.34-2.25(m,4H), 2.05-1.86(m,6H),1.80-1.72(m,1H),1.68-1.60(m,2H),1.49-1.42(m,1H),1.36(br dd,J＝7.0,11.8Hz,3H) ,1.23(s,2H),0.96(br dd,J＝3.4,6.4Hz,3H),0.84-0.78(m,3H).

Example 104: Synthesis of Compound 304

2-[3-(Isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (28.82 mg, 74.11 μmol) and intermediate 2-1 (40 mg, 67.37 μmol) were dissolved in tetrahydrofuran (1 mL) and N,N-dimethylformamide (1 mL), and acetic acid (12.14 mg, 202.11 μmol), sodium triacetoxyborohydride (42.84 mg, 202.11 μmol) and anhydrous sodium acetate (42.84 mg, 202.11 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was complete when monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by HPLC (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 49%-89%) as eluent) to give compound 304 (25.01 mg).

MS m/z (ESI): 930.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.03 (br s, 1H), 9.02-8.95 (m, 1H), 8.49 (s, 1H), 8.42 (d, J = 7.8Hz, 1H),8.16-8.10(m,1H),7.49-7.40(m,2H),7.39-7.31(m,3H),7.03-6.94(m,2H),6.92(d,J＝7.6Hz,1H) ,6.13(s,1H),5.12(d,J＝3.6Hz,1H),4.91(q,J＝6.9Hz,1H),4.61-4.52(m,1H),4.36(t,J＝7.8Hz, 1H),4.31-4.23(m,1H),4.08-3.98(m,1H),3.71(br dd,J＝4.5,10.5Hz,1H),3.63(br d,J＝11.6Hz,2H),3.56(d,J＝9.9Hz,1H),3.46-3.40(m,1H),3.04-2.95(m,2H),2.82-2.68(m,2H),2.45 (s,3H),2.25-2.00(m,8H),1.88-1.61(m,6H),1.38(d,J＝7.0Hz,3H),1.28(d,J＝6.4Hz,6H),1.21- 1.09(m,2H),0.99-0.92(m,3H),0.85-0.75(m,3H).

Example 105: Synthesis of Compound 306

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]-6-chlorophenol (31.79 mg, 83.37 μmol) and intermediate 2-1 (45 mg, 75.79 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL) and anhydrous tetrahydrofuran (0.5 mL), and sodium acetate (8.65 mg, 227.37 μmol), acetic acid (13.00 μL, 227.37 μmol) and sodium triacetoxyborohydride (48.19 mg, 227.37 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (BostonLuna C18 column: 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content 15%-55%) as eluent) to give compound 306 (6.8 mg).

MS m/z (ESI): 923.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ15.51 (br s, 1H), 8.98 (s, 1H), 8.63 (s, 1H), 8.43 (d, J = 7.5Hz, 1H), 8.19-8.13 (m,1H),7.54(d,J＝6.8Hz,1H),7.49-7.41(m,2H),7.39-7.34(m,2H),7.01(t,J＝7.9Hz,1H),6.57( br s,2H),6.16-5.97(m,1H),5.13(br s,1H),4.91(quin,J＝6.8Hz,1H),4.74-4.51(m,1H),4.36(t,J＝ 7.8Hz,1H),4.29(br s,1H),3.76-3.53(m,3H),2.99(br d,J＝4.8Hz,2H),2.75(br t,J＝11.4Hz,2H),2.52(d,J＝1.8Hz,3H ),2.45(s,3H),2.26-2.17(m,3H),2.13-1.98(m,4H),1.91-1.62(m,6H),1.49-1.33(m,3H),1.14(br d, J＝11.3Hz,2H),0.99-0.92(m,3H),0.85-0.76(m,3H).

Example 106: Synthesis of Compound 308

N-[6-(3-chloro-2-hydroxyphenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-yl]cyclopropanecarboxamide (13.91 mg, 33.69 μmol), intermediate 2-1 (20 mg, 33.69 μmol), acetic acid (6.07 mg, 101.06 μmol, 5.78 μL) and sodium acetate (8.29 mg, 101.06 μmol) were dissolved in anhydrous tetrahydrofuran (0.5 mL) and N,N-dimethylformamide (0.5 mL). The reaction solution was stirred at 25°C for 1 hour, and sodium acetate borohydride (21.42 mg, 101.06 μmol) was added. The reaction solution was stirred at 25°C for 3 hours, and the reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.225% formic acid) and acetonitrile (acetonitrile content 36%-76%) as eluent) gave compound 308 (13.2 mg).

MS m/z(ESI):496.1[M+2H] ²⁺ ;

¹ H NMR (400MHz, DMSO-d6) δ14.85 (s, 1H), 11.16 (s, 1H), 8.97 (d, J = 12.4Hz, 2H), 8.42 (d, J = 7.6Hz, 1H), 8.19(d,J＝7.3Hz,1H),7.58(d,J＝7.9Hz,1H),7.49-7.29(m,4H),7.06(t,J＝7.9Hz,1H),6.13(s,1H ),5.12(d,J＝3.6Hz,1H),4.97-4.76(m,2H),4.36(t,J ＝7.8Hz,1H),4.28(s,1H),3.77-3.53(m,4H),3.48-3.40(m,1H),3.02(d,J＝4.8Hz,2H),2.76(t,J＝ 11.5Hz,2H),2.46(s,3H),2.23(d,J＝5.5Hz,3H),2.16-1.91(m,8H),1.86-1.64(m,4H),1.38(d,J＝6.9 Hz, 3H), 1.16 (d, J = 10.6Hz, 2H), 1.01-0.74 (m, 10H).

Example 107: Synthesis of Compound 318

2-[3-amino-1-[1-[5-(piperidin-4-yl)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (9.53 mg, 20.21 μmol), intermediate 2-1 (10 mg, 16.84 μmol), acetic acid (3.03 mg, 50.53 μmol, 2.89 μL) and sodium acetate (4.14 mg, 50.53 μmol) were dissolved in anhydrous tetrahydrofuran (1.5 mL) and anhydrous N,N-dimethylformamide (1.5 mL), and sodium acetate borohydride (10.71 mg, 50.53 μmol) was added. The reaction solution was stirred at 25°C for 4 hours, and the reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.5% ammonia) and acetonitrile (acetonitrile content 43%-83%) as eluent) gave compound 318 (7.8 mg).

MS m/z (ESI): 1049.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.93 (s, 1H), 9.12-8.94 (m, 1H), 8.55 (s, 1H), 8.43 (d, J = 7.6Hz, 1H), 8.33 ( s,2H),8.12(d,J＝8.0Hz,1H),7.49-7.33(m,5H),7.06-6.97(m,2H),6.48(s,2H),6.13(s,1H),5.13 (d,J＝3.3Hz,1H),4.92(t,J＝7.3Hz,2H),4.80(d,J＝13.0Hz,2H),4.37(t,J＝7.8Hz,1H),4.28(s ,1H),3.75-3.68(m, 1H),3.63(d,J＝12.5Hz,2H),3.57(d,J＝10.0Hz,1H),3.43(d,J＝10.4Hz,1H),3.14-3.01(m,2H),2.95( d,J＝9.9Hz,2H),2.75(t,J＝11.5Hz,2H),2.46(s,3H),2.29-2.12(m,4H),2.06-1.90(m,7H),1.84-1.59 (m,8H),1.38(d,J=7.0Hz,3H),1.25-1.05(m,2H),1.02-0.91(m,3H),0.84-0.73(m,3H).

Example 108: Synthesis of Compound 318-1

2-[3-amino-1-[8-[5-(piperidin-4-yl)pyrimidin-2-yl]-8-azabicyclo[3.2.1]octan-3-yl]pyrazolo[4,3-c]pyridazin-6-yl]phenol (synthesized according to Example 116) (10 mg, 18.72 μmol) and intermediate 2-1 (11.11 mg, 18.72 μmol) were dissolved in N,N-dimethylformamide (1 mL) and tetrahydrofuran (1 mL), and acetic acid (3.37 mg, 56.16 μmol), sodium acetate (4.61 mg, 56.16 μmol) and sodium acetate borohydride (11.90 mg, 56.16 μmol) were added. The reaction solution was reacted at 25° C. for 1 hour. The reaction was completed by LCMS. Water (0.2 mL) was added to the reaction solution to quench the reaction. The reaction solution was concentrated to dryness under reduced pressure and purified by HPLC (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 55%-95%) as eluent) to give compound 318-1 (2.2 mg).

MS m/z(ESI):538.8[M+2H] ²⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ14.02 (s, 1H), 9.02-8.96 (m, 1H), 8.60 (s, 1H), 8.34 (s, 2H), 8.17 (d, J = 7.5 Hz,1H),7.49-7.34(m,5H),7.24-6.93(m,2H),6.5(s,1H),5.32(t,J＝4.4Hz,1H),5.25-5.08(m,2H) ,4.91(br t,J＝7.0Hz,1H),4.74(br s,2H),4.44-4.25(m,2H),3.72-3.53(m,5H),2.96(br s,1H),2.75( br s,2H),2.45(br s,3H),2.27-1.94(m,13H),1.89-1.59(m,9H),1.52-1.28(m,6H),1.18-1.06(m,3H),0.95(br d,J＝6.5Hz ,3H),0.79(br d,J＝6.8Hz,3H).

Example 109: Synthesis of Compound 319

Step 1: Synthesis of tert-butyl 4-[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]-3,6-dihydropyridine-1(2H)-carboxylate

2-[1-[1-(5-bromopyrimidin-2-yl)piperidin-4-yl]-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (prepared with reference to Example 115) (180 mg, 373.95 μmol) and N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester (138.75 mg, 448.74 μmol) were dissolved in 1,4-dioxane (4 mL) and water (1 mL), and cesium carbonate (365.52 mg, 1.12 mmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (30.54 mg, 37.40 μmol) were added. The reaction solution was stirred at 100° C. for 16 hours. The reaction was monitored to be complete by LCMS. Water (10 mL) was added to the reaction solution and extracted three times with ethyl acetate (15 mL). The organic phases were combined and washed three times with water (15 mL), dried over anhydrous sodium sulfate, filtered and concentrated to dryness under reduced pressure. Purification by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 2/1) gave 4-[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]-3,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (97 mg).

MS m/z(ESI):584.2[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidine-1-carboxylate

4-[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]-3,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (87 mg, 149.05 μmol) was dissolved in tetrahydrofuran (2 mL), and palladium carbon (10% purity, 50 mg) was added. The reaction solution was stirred at 25° C. for 16 hours. LCMS monitored the reaction to be complete. The reaction solution was filtered and concentrated to dryness under reduced pressure to obtain 4-[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidine-1-carboxylic acid tert-butyl ester (190 mg).

MS m/z(ESI):586.1[M+H] ⁺ .

Step 3: Synthesis of 2-[3-(methylamino)-1-[1-[5-(4-piperidinyl)pyrimidin-2-yl]-4-piperidinyl]pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)pyrazolo[4,3-c]pyridazin-1-yl]-1-piperidinyl]pyrimidin-5-yl]piperidine-1-carboxylic acid tert-butyl ester (70 mg, 119.52 μmol) was dissolved in 1,4-dioxane (2 mL), and dioxane hydrochloride (4M, 298.79 μL) was added. The reaction solution was stirred at 25° C. for 16 hours. LCMS monitored the reaction to be complete. The reaction solution was concentrated to dryness under reduced pressure to obtain 4-[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidine-1-carboxylic acid tert-butyl ester (84 mg).

MS m/z(ESI):486.2[M+H] ⁺ .

Step 4: Synthesis of compound 319

4-[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidine-1-carboxylic acid tert-butyl ester (79.13 mg, 75.79 μmol, 50% pure hydrochloride) and intermediate 2-1 (30 mg, 50.53 μmol) were dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), and sodium acetic acid borohydride (32.13 mg, 151.58 μmol), sodium acetate (8.29 mg, 101.06 μmol) and acetic acid (303.43 μg, 5.05 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was complete when monitored by LCMS. The reaction solution was concentrated under reduced pressure to remove tetrahydrofuran and then purified by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (0.225% formic acid) and acetonitrile (acetonitrile content 15%-55%) as eluent) to obtain compound 319 (17.52 mg).

MS m/z(ESI):1064.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.92 (s, 1H), 9.00-8.95 (m, 1H), 8.52 (s, 1H), 8.42 (d, J = 7.5Hz, 1H), 8.32 ( s,2H),8.16-8.09(m,1H),7.46-7.41(m,2H),7.40-7.33(m,3H),7.08(d,J＝5.0Hz,1H),7.04-6.97(m, 2H),6.12(s,1H),5.13(d,J＝3.5Hz,1H),4.93-4.80(m,4H),4.36(t,J＝7.9Hz,1H),4.28(s, 1H),3.75-3.52(m,4H),3.07(t,J＝11.0Hz,3H),3.01-2.93(m,2H),2.91(d,J＝5.0Hz,3H),2.74(t,J ＝11.5Hz,2H),2.45(s,3H),2.21(s,3H),2.08-1.86(m,8H),1.79-1.61(m,8H),1.37(d,J＝7.0Hz,3H) ,1.13(d,J＝10.5Hz,2H),0.94(d,J＝6.8Hz,3H),0.79(d,J＝6.5Hz,3H).

Example 110: Synthesis of Compound 322

Step 1: Synthesis of 2-[1-[1-(5-bromopyridin-2-yl)piperidin-4-yl]-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

2-[3-(Methylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (300 mg, 831.39 μmol, hydrochloride) was dissolved in dimethyl sulfoxide (10 mL), and 5-bromo-2-fluoropyridine (175.58 mg, 997.67 μmol) and N,N-diisopropylethylamine (217.22 μL, 1.25 mmol) were added. The reaction solution was stirred at 100° C. for 12 hours. The reaction was completed by LCMS. Water (15 mL) was added, and the mixture was extracted twice with ethyl acetate (20 mL). The organic layer was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The mixture was purified by column chromatography (petroleum ether: ethyl acetate = 2: 1) to give 2-[1-[1-(5-bromopyridin-2-yl)piperidin-4-yl]-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (300 mg).

MS m/z(ESI):471.9,481.9[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-[6-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-3',6'-dihydro-[3,4'-bipyridine]-1'(2'H)-carboxylate

2-[1-[1-(5-bromo-2-pyridyl)-4-piperidinyl]-3-(methylamino)pyrazolo[4,3-c]pyridazin-6-yl]phenol (300 mg, 624.53 μmol) was dissolved in anhydrous dioxane (12 mL) and water (3 mL), and N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester (386.22 mg, 1.25 mmol) and anhydrous cesium carbonate (610.46 mg, 1.87 mmol) were added. Under the protection of nitrogen, [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (51 mg, 62.45 μmol) was added, and the reaction solution was heated to 90°C and stirred for 16 hours. LCMS detected that the reaction was complete. The reaction solution was cooled to 25°C, concentrated to dryness under reduced pressure, and purified by thin layer chromatography (petroleum ether: ethyl acetate = 2: 1) to give 4-[6-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-3',6'-dihydro-[3,4'-bipyridine]-1'(2'H)-carboxylic acid tert-butyl ester (310 mg).

MS m/z(ESI):583.2[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-[6-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-3-yl]piperidine-1-carboxylate

4-[6-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-3',6'-dihydro-[3,4'-bipyridine]-1'(2'H)-carboxylic acid tert-butyl ester (260 mg, 446.20 μmol) was dissolved in tetrahydrofuran (10 mL) and methanol (10 mL), wet palladium carbon (400 mg, 446.20 μmol) was added, and replaced with hydrogen (15 Psi), and the reaction solution was stirred at 25° C. for 2 hours. LCMS detected that the reaction was complete. The reaction solution was filtered through celite, and the filtrate was collected and concentrated to dryness under reduced pressure to give tert-butyl 4-[6-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-3-yl]piperidine-1-carboxylate (310 mg, crude product).

MS m/z(ESI):585.3[M+H] ⁺ .

Step 4: Synthesis of 2-[3-(methylamino)-1-[1-[5-(piperidin-4-yl)pyridin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[6-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-3-yl]piperidine-1-carboxylic acid tert-butyl ester (100 mg, 171.02 μmol) was dissolved in methanol (2 mL) and dichloromethane (4 mL), and dioxane hydrochloride (427.56 μL, 4 M) was added, and the mixture was stirred at 25° C. for 2 hours. After LCMS detection, the reaction was completed, and the mixture was concentrated to dryness under reduced pressure to obtain 2-[3-(methylamino)-1-[1-[5-(piperidin-4-yl)pyridin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (100 mg, hydrochloride).

MS m/z(ESI):485.2[M+H] ⁺ .

Step 5: Synthesis of compound 322

2-[3-(Methylamino)-1-[1-[5-(piperidin-4-yl)pyridin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (10 mg, 20.64 μmol, hydrochloride) was dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), and intermediate 2-1 (12.65 mg, 20.64 μmol), acetic acid (1.24 mg, 20.64 μmol) and sodium acetate (6.77 mg, 82.54 μmol) were added. The reaction was stirred at 25°C for 0.5 hours. Sodium acetate borohydride (13.12 mg, 61.91 μmol) was added, and the reaction was stirred at 25°C for 2.5 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Purification by preparative liquid chromatography (Phenomenex Luna C18 column: 3 μm silica, 30 mm diameter, 75 mm length; using water (containing a mixture of aqueous ammonia (0.5%) and acetonitrile (acetonitrile content 52%-92%) as eluent) gave compound 322 (4.24 mg).

MS m/z(ESI):532.1[M+2H] ²⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.94 (s, 1H), 9.02-8.94 (m, 1H), 8.52 (s, 1H), 8.42 (d, J = 7.8Hz, 1H), 8.12 ( d,J＝7.6Hz,1H),8.02(d,J＝2.1Hz,1H),7.51-7.41(m,3H),7.36(br d,J＝8.3Hz,3H),7.09(br d,J ＝4.9Hz,1H),7.03-6.95(m,2H),6.88(br d,J＝8.8Hz,1H),6.16-5.97(m,1H),5.16-5.01(m,1H),4.98-4.79 (m,2H),4.47(br d,J＝12.8Hz,2H),4.36(t,J＝7.9Hz,1H),4.28(br s,1H),3.71(br d,J＝6.3Hz,1H),3.66-3.52(m,3H ),3.46-3.38(m,3H),3.03-2.87(m,7H),2.74(br t,J＝11.6Hz,2H),2.45(s,3H),2.23-2.12(m,3H),2.08 -1.88(m,6H),1.80-1.59(m,8H),1.37(br d,J＝7.0Hz,3H),1.19-1.09(m,2H),1.00-0.89(m,3H),0.84- 0.74(m,3H).

Example 111: Synthesis of Compound 323-1

Step 1: Synthesis of 2-[1-[1-(5-bromopyrimidin-2-yl)piperidin-4-yl]-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

2-[3-(Isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (106 mg, 272.57 μmol) and 2-chloro-5-bromopyrimidine (57.99 mg, 299.8 μmol) were dissolved in N,N-dimethylformamide (3 mL), and N,N-diisopropylethylamine (140.91 mg, 1.09 mmol) was added. The reaction solution was stirred at 80°C for 2 hours. The reaction was complete as monitored by LCMS. Water (3 mL) was added to the reaction solution and extracted three times with ethyl acetate (15 mL), and the organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure and then purified by thin layer chromatography (silica: petroleum ether/tetrahydrofuran = 3/1) to give 2-[1-[1-(5-bromopyrimidin-2-yl)piperidin-4-yl]-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (121 mg).

MS m/z(ESI):509.0,511.0[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-[2-[4-[6-(2-hydroxyphenyl)-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]-3,6-dihydropyridine-1(2H)-1-carboxylate

2-[1-[1-(5-bromopyrimidin-2-yl)piperidin-4-yl]-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (121 mg, 237.53 μmol) and N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester (88.14 mg, 285.04 μmol) were dissolved in dioxane (4 mL) and water (1 mL), and potassium carbonate (98.49 mg, 712.60 μmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (19.40 mg, 23.75 μmol) were added. The reaction solution was stirred at 80°C under nitrogen atmosphere for 2 hours. The reaction was complete when monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (petroleum ether/tetrahydrofuran=3/1) to give tert-butyl 4-[2-[4-[6-(2-hydroxyphenyl)-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]-3,6-dihydropyridine-1(2H)-1-carboxylate (115 mg).

MS m/z(ESI):612.3[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-[2-[4-[6-(2-hydroxyphenyl)-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidine-1-carboxylate

4-[2-[4-[6-(2-hydroxyphenyl)-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]-3,6-dihydropyridine-1(2H)-1-carboxylic acid tert-butyl ester (25 mg, 40.87 μmol) was dissolved in tetrahydrofuran (3 mL), and wet palladium carbon (40.87 μmol, 10% purity) was added. The reaction solution was stirred at 25° C. for 12 hours under hydrogen protection. LCMS monitored the reaction to be complete. The reaction solution was concentrated to dryness under reduced pressure to obtain 4-[2-[4-[6-(2-hydroxyphenyl)-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidine-1-carboxylic acid tert-butyl ester (22 mg).

MS m/z(ESI):614.3[M+H] ⁺ .

Step 4: Synthesis of 2-[3-(isopropylamino)-1-[1-[5-(piperidin-4-yl)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[2-[4-[6-(2-hydroxyphenyl)-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidine-1-carboxylic acid tert-butyl ester (22 mg, 35.85 μmol) was dissolved in dichloromethane (0.4 mL), and dioxane hydrochloride (4M, 179.23 μL) was added. The reaction solution was stirred at 25° C. for 2 hours. LCMS monitored the reaction to be complete, and the reaction solution was concentrated to dryness under reduced pressure to obtain 2-[3-(isopropylamino)-1-[1-[5-(piperidin-4-yl)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (16 mg).

MS m/z(ESI):514.3[M+H] ⁺ .

Step 5: Synthesis of compound 323-1

2-[3-(Isopropylamino)-1-[1-[5-(piperidin-4-yl)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (16 mg, 31.15 μmol) and intermediate 11 (16.84 mg, 31.15 μmol) were dissolved in tetrahydrofuran (0.5 mL) and N,N-dimethylformamide (0.5 mL), and acetic acid (1.87 mg, 31.15 μmol), sodium triacetoxyborohydride (19.81 mg, 93.45 μmol) and sodium acetate (7.67 mg, 93.45 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was completed by LCMS monitoring. The reaction solution was concentrated to dryness under reduced pressure and purified by high performance liquid chromatography (Phenomenex C18 column, 5 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia water) and acetonitrile (acetonitrile content 51%-91%) as eluent) to obtain compound 323-1 (11.25 mg).

MS m/z(ESI):1038.9[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.94 (s, 1H), 8.98 (s, 1H), 8.51 (s, 1H), 8.43 (br d, J = 7.8Hz, 1H), 8.32 (s ,2H),8.11(br d,J＝7.8Hz,1H),7.49-7.41(m,2H),7.39-7.33(m,3H),7.04-6.96(m,2H),6.91(br d,J＝7.5Hz,1H),6.12(s,1H),5.12(d,J＝3.3Hz,1H),4.93-4.80(m,4H),4.42-4.21(m,4H),4.05-3.93 (m,1H),3.79-3.60(m,3H),3.14-2.96(m,4H),2.71(br s,2H),2.45(s,3H),2.16-1.85(m,8H),1.82- 1.62(m,5H),1.37(brd,J＝7.0Hz,3H),1.23(br d,J＝6.5Hz,7H),0.96(br d,J＝6.5Hz,3H),0.80(br d, J＝6.5Hz,3H).

Example 112: Synthesis of Compound 323-2

2-[3-(Isopropylamino)-1-[1-[5-(piperidin-4-yl)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (20.38 mg, 37.05 μmol) and intermediate 2-1 (20 mg, 33.69 μmol) were dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), and acetic acid (6.07 mg, 101.06 μmol), sodium acetate (8.29 mg, 101.06 μmol) and sodium acetate borohydride (21.42 mg, 101.06 μmol) were added. The reaction solution was reacted at 25°C for 1 hour. The reaction was completed by LCMS. Water (0.2 mL) was added to the reaction solution to quench the reaction. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 59%-99%) as eluent) gave compound 323-2 (10.11 mg).

MS m/z(ESI):1092.0[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.94 (br s, 1H), 8.98 (s, 1H), 8.51 (s, 1H), 8.42 (br d, J = 7.5Hz, 1H), 8.31 ( s,2H),8.11(d,J＝8.0Hz,1H),7.47-7.41(m,2H),7.40-7.33(m,3H),7.05-6.96(m,2H),6.90(d,J＝ 7.8Hz,1H),6.19-5.94(m,1H),5.13(br s,1H),4.97-4.76(m,4H),4.36(t,J＝7.8Hz,1H),4.28(br s,1H),3.99(qd,J＝6.5,13.5Hz,1H),3.76-3.68(m,1H),3.66-3.53(m,4H),3.07(br t,J＝10.4Hz,2H), 2.95(br d,J＝10.0Hz,2H),2.74(br t,J＝11.4Hz,2H),2.45(s,3H),2.39(br s,1H),2.29-2.12(m,3H), 2.06-1.88(m,7H),1.85-1.55(m,8H),1.49-1.33(m,3H),1.23(d,J＝6.5Hz,6H),1.13(br d,J＝11.8Hz,2H),1.01-0.90(m,3H),0.85-0.74(m,3H).

Example 113: Synthesis of Compound 227

2-[3-(Methylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (15 mg, 46.24 μmol) and intermediate 2-1 (27.46 mg, 46.24 μmol) were dissolved in N,N-dimethylformamide (0.6 mL) and tetrahydrofuran (0.6 mL), and sodium acetate borohydride (29.40 mg, 138.73 μmol) and acetic acid (2.78 mg, 46.2 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was complete when monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure and then purified by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (0.05% ammonia water) and acetonitrile (acetonitrile content 29%-69%) as eluent) to give compound 227 (7.63 mg).

MS m/z (ESI): 902.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.10-13.90 (m, 1H), 8.99 (s, 1H), 8.50 (s, 1H), 8.42 (br d, J = 7.6Hz, 1H), 8.13 (br d,J＝7.5Hz,1H),7.49-7.41(m,2H),7.39-7.31(m,3H),7.08(br d,J＝4.9Hz,1H),7.04-6.95(m,2H),6.13(s,1H),5.12(d,J＝3.5Hz,1H),5.01-4.86(m,1H),4.62-4.53 (m,1H),4.40-4.23(m,2H),3.75-3.54(m,4H),3.48-3.38( m,2H),3.06-2.93(m,5H),2.84-2.70(m,2H),2.25-1.95(m,9H),1.87-1.69(m,6H),1.48-1.35(m,3H), 1.28-1.07(m,3H),0.98-0.91(m,3H),0.83-0.73(m,3H).

Example 114: Synthesis of (2S,4R)-4-hydroxy-1-[2-[3-[2-[3-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]pyrrolidin-1-yl]ethoxy]isoxazol-5-yl]-3-methylbutyryl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 229)

2-[3-(Methylamino)-1-(pyrrolidin-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (6.89 mg, 22.20 μmol), (2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(2-oxoethoxy)isoxazol-5-yl]butanoyl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-yl] Formamide (10.00 mg, 18.50 μmol), acetic acid (2.22 mg, 37.00 μmol, 2.12 μL) and sodium acetate (4.55 mg, 55.50 μmol) were dissolved in anhydrous tetrahydrofuran (0.5 mL) and N,N-dimethylformamide (0.5 mL). The reaction solution was stirred at 25 ° C for 1 hour, and sodium acetate borohydride (11.76 mg, 55.50 μmol) was added. The reaction solution was stirred at 25 ° C for 3 hours, and the reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 34%-74%) as eluent) gave (2S,4R)-4-hydroxy-1-[2-[3-[2-[3-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]pyrrolidin-1-yl]ethoxy]isoxazol-5-yl]-3-methylbutanoyl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 229) (2.28 mg).

MS m/z (ESI): 835.4 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.87 (br s, 1H), 9.10-8.93 (m, 1H), 8.52 (br d, J = 3.1Hz, 1H), 8.46-8.24 (m, 1H) ),8.06(br d,J＝7.9Hz,1H),7.58-7.26(m,6H),7.11(br d,J＝4.5Hz,1H),7.04-6.90(m,2H),6.14-6.01( m,1H),5.29(s,1H),5.11(s,1H),4.98-4.79(m,1H),4.40-4.21(m,4H),3.83-3.59(m,3H),3.02-2.80( m,7H), 2.47-2.44(m,3H),2.29-2.12(m,3H),2.10-1.97(m,1H),1.78(s,1H),1.49-1.30(m,4H),0.96(d,J＝3.4 Hz,3H),0.80(d,J＝13.6Hz,3H).

Example 115: Synthesis of Compound 278

Step 1: Synthesis of 2-[1-[1-(5-bromopyrimidin-2-yl)piperidin-4-yl]-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

2-[3-(Methylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (340 mg, 800.91 μmol, 85% pure hydrochloride) and 5-bromo-2-chloro-pyrimidine (185.90 mg, 961.09 μmol) were dissolved in N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (310.54 mg, 2.40 mmol) was added. The reaction solution was stirred at 100°C for 2 hours. LCMS monitored the reaction to be complete. Water (20 mL) was added to the reaction solution and extracted twice with dichloromethane (100 mL). The organic phases were combined and dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure. Purification by column chromatography (silica, tetrahydrofuran/dichloromethane = 1/0 to 1/50) gave 2-[1-[1-(5-bromopyrimidin-2-yl)piperidin-4-yl]-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (400 mg).

MS m/z(ESI):481.0,482.9[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]-3,6-dihydropyridine-1(2H)-carboxylate

2-[1-[1-(5-bromopyrimidin-2-yl)piperidin-4-yl]-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (180 mg, 373.95 μmol) and N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester (138.75 mg, 448.74 μmol) were dissolved in 1,4-dioxane (4 mL) and water (1 mL), and cesium carbonate (365.52 mg, 650.34 μmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (30.54 mg, 37.40 μmol) were added. The reaction solution was replaced with nitrogen three times and stirred at 100°C for 16 hours under nitrogen atmosphere. The reaction was completed by LCMS monitoring. Water (10 mL) was added to the reaction solution and extracted three times with ethyl acetate (15 mL). The organic phases were combined and washed three times with water (15 mL), filtered and concentrated to dryness under reduced pressure. Purification by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 2/1) gave 4-[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]-3,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (97 mg).

MS m/z(ESI):584.2[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidine-1-carboxylate

4-[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]-3,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (87 mg, 149.05 μmol) was dissolved in tetrahydrofuran (2 mL), and palladium carbon (10% purity, 50 mg) was added. The reaction solution was stirred at 25°C for 16 hours. LCMS monitored the reaction to be complete. The reaction solution was filtered and concentrated to dryness under reduced pressure to obtain a crude product of 4-[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidine-1-carboxylic acid tert-butyl ester (190 mg).

MS m/z(ESI):586.1[M+H] ⁺ .

Step 4: Synthesis of 2-[3-(methylamino)-1-[1-[5-(piperidin-4-yl)pyrimidin-2-yl]piperidin-4-yl]pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidine-1-carboxylic acid tert-butyl ester (90 mg, 153.66 μmol) was dissolved in 1,4-dioxane (2 mL), and a dioxane hydrochloride solution (4M, 384.16 μL, 1.54 mmol) was added. The reaction solution was stirred at 25° C. for 16 hours. LCMS monitored the reaction to be complete. The reaction solution was concentrated to dryness under reduced pressure to obtain a crude hydrochloride solid 2-[3-(methylamino)-1-[1-[5-(piperidin-4-yl)pyrimidin-2-yl]piperidin-4-yl]pyrazolo[4,3-c]pyridazin-6-yl]phenol (110 mg).

MS m/z(ESI):486.2[M+H] ⁺ .

Step 5: Synthesis of compound 278

2-[3-(Methylamino)-1-[1-[5-(piperidin-4-yl)pyrimidin-2-yl]piperidin-4-yl]pyrazolo[4,3-c]pyridazin-6-yl]phenol (57.94 mg, 55.49 μmol, 50% pure hydrochloride) and intermediate 11 (25 mg, 46.24 μmol) were dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), and sodium acetic acid borohydride (29.40 mg, 138.73 μmol), sodium acetate (7.59 mg, 92.48 μmol) and acetic acid (277.69 μg, 4.62 μmol) were added. The reaction solution was stirred at 25°C for 16 hours. The reaction was completed by LCMS monitoring. The reaction solution was concentrated under reduced pressure to remove tetrahydrofuran and then purified by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (0.225% formic acid) and acetonitrile (acetonitrile content 15%-55%) as eluent) to give compound 278 (11.94 mg).

MS m/z (ESI): 1011.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.92 (s, 1H), 8.98 (s, 1H), 8.52 (s, 1H), 8.44 (d, J = 7.5Hz, 1H), 8.32 (s, 2H),8.12(d,J＝7.3Hz,1H),7.49-7.33(m,6H),7.09(d,J＝5.0Hz,1H),7.04-6.97(m,2H),6.12(s,1H ),5.12(s,1H),4.96-4.79(m,4H),4.37(t,J＝7.7Hz,1H),4.27 (d,J＝5.3Hz,3H),3.75-3.62(m,2H),3.45(d,J＝11.0Hz,2H),3.11-2.98(m,4H),2.91(d,J＝4.8Hz, 3H),2.70(s,2H),2.45(s,3H),2.14-1.91(m,7H),1.84-1.58(m,5H),1.37(d,J＝7.3Hz,3H),0.96(d ,J＝6.3Hz,3H),0.80(d,J＝6.8Hz,3H).

Example 116: Synthesis of Compound 281

Step 1: Synthesis of 2-[3-amino-1-[8-(5-bromopyrimidin-2-yl)-8-azabicyclo[3.2.1]octan-3-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

2-[3-amino-1-(8-azabicyclo[3.2.1]octan-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (83 mg, 246.74 μmol) and 2-chloro-5-bromopyrimidine (52.50 mg, 271.41 μmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (127.56 mg, 986.95 μmol) was added. The reaction solution was stirred at 80° C. for 2 hours. The reaction was complete as monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure and then purified by thin layer chromatography (petroleum ether/ethyl acetate = 1/1) to give 2-[3-amino-1-[8-(5-bromopyrimidin-2-yl)-8-azabicyclo[3.2.1]octan-3-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (68 mg).

MS m/z(ESI):493.0,494.9[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-[2-[3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octan-8-yl]pyrimidin-5-yl]-3,6-dihydropyridine-1(2H)-carboxylate

2-[3-amino-1-[8-(5-bromopyrimidin-2-yl)-8-azabicyclo[3.2.1]octan-3-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (75 mg, 152.02 μmol) and N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid pinacol ester (47.01 mg, 152.02 μmol) were dissolved in dioxane (2 mL) and water (0.5 mL), and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (12.41 mg, 15.20 μmol) and potassium carbonate (63.03 mg, 456.06 μmol) were added. The reaction solution was stirred at 100°C for 2 hours under nitrogen protection. The reaction was complete when monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (petroleum ether/tetrahydrofuran=1/1) to give tert-butyl 4-[2-[3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octan-8-yl]pyrimidin-5-yl]-3,6-dihydropyridine-1(2H)-carboxylate (32 mg).

MS m/z(ESI):596.4[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-[2-[3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octan-8-yl]pyrimidin-5-yl]piperidine-1-carboxylate

4-[2-[3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]-8-azabicyclo[3.2.1]octan-8-yl]pyrimidin-5-yl]-3,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (32 mg, 53.72 μmol) was dissolved in anhydrous methanol (3 mL), and wet palladium carbon (537.19 μmol, 10% purity) was added. The reaction solution was stirred at 25° C. for 12 hours. LCMS monitored the reaction to be complete. The reaction solution was filtered and the filtrate was concentrated to dryness under reduced pressure to obtain tert-butyl 4-[2-[3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octan-8-yl]pyrimidin-5-yl]piperidine-1-carboxylate (15 mg).

MS m/z(ESI):598.5[M+H] ⁺ .

Step 4: Synthesis of 2-[3-amino-1-[8-[5-(piperidin-4-yl)pyrimidin-2-yl]-8-azabicyclo[3.2.1]octan-3-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[2-[3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]-8-azabicyclo[3.2.1]octan-8-yl]pyrimidin-5-yl]piperidine-1-carboxylic acid tert-butyl ester (15 mg, 25.10 μmol) was dissolved in anhydrous dichloromethane (2 mL), and then dioxane hydrochloride (4M, 31.37 μL) was added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain 2-[3-amino-1-[8-[5-(piperidin-4-yl)pyrimidin-2-yl]-8-azabicyclo[3.2.1]octan-3-yl]-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (10 mg).

MS m/z(ESI):498.4[M+H] ⁺ .

Step 5: Synthesis of compound 281

2-[3-amino-1-[8-[5-(piperidin-4-yl)pyrimidin-2-yl]-8-azabicyclo[3.2.1]octan-3-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (10 mg, 18.72 μmol) and intermediate 11 (10.12 mg, 18.72 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL) and anhydrous tetrahydrofuran (0.5 mL), acetic acid (1.12 mg, 18.72 μmol) and sodium triacetoxyborohydride (11.91 mg, 56.17 μmol) were added, and the reaction solution was stirred at 25° C. for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex C18 column, 5 μm silica, 30 mm diameter, 80 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 50%-70%) as eluent) gave Compound 281 (1.1 mg).

MS m/z(ESI):512.1[M+2H] ²⁺ ;

¹ H NMR (400 MHz, DMSO-d ₆ )δ＝14.04-13.84(m,1H),9.02-8.90(m,1H),8.62-8.41(m,1H),8.38-8.26(m,3H),8.20-8.03(m,1H),7.50-7.30(m,5H),7.05-6.90(m,2H),6.59-6 .44(m,2H),6.19-6.09(m,1H),5.22-5.09(m,1H),4.97-4.85(m,1H),4.80-4.64(m,3H),4.47-4.34(m,1H),4.31-4.20(m,3H),3.06-2.98(m,4H), 2.45(br s,3H),2.16-1.99(m,11H),1.97-1.89(m,2H),1.87-1.78(m,2H),1.76-1.61(m,5H),1.46(br d,J＝6.9Hz,1H),1.39-1.35(m,2H),1.26-1.13(m,1H) ,1.03-0.91(m,3H),0.82(br dd,J=6.8,13.4Hz,3H).

Example 117: Synthesis of (2S,4R)-4-hydroxy-1-[2-[3-[[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethyl]-methylamino]isoxazol-5-yl]-3-methylbutyryl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 284)

Step 1: Synthesis of methyl 2-[3-[(2,2-dimethoxyethyl)(methyl)amino]isoxazol-5-yl]-3-methylbutanoate

3-Methyl-2-[3-(1,1,2,2,3,3,4,4,4-nonafluorobutylsulfonyloxy)isoxazol-5-yl]butanoic acid methyl ester (300 mg, 623.33 μmol) and 2,2-dimethoxy-N-methyl-ethylamine (81.70 mg, 685.66 μmol) were dissolved in N,N-dimethylacetamide (5 mL), triethylamine (189.22 mg, 1.87 mmol) was added, and the reaction solution was reacted at 80° C. for 12 hours. LCMS showed that the reaction was completed. The reaction solution was extracted with ethyl acetate (8 mL×3) and water (10 mL). The organic phase was washed with saturated brine (20 mL) and concentrated to dryness under reduced pressure. The concentrate was purified by thin layer chromatography (silica, petroleum ether/tetrahydrofuran=3/1) to give methyl 2-[3-[(2,2-dimethoxyethyl)(methyl)amino]isoxazol-5-yl]-3-methylbutanoate (57 mg).

MS m/z (ESI): 301.1 [M+H] ⁺ ;

¹ H NMR (400MHz, CDCl ₃ ) δ5.86 (s, 1H), 4.53 (t, J = 5.3Hz, 1H), 3.72 (s, 3H), 3.46 (d, J = 8.8Hz, 1H), 3.40 (s,6H),3.33(t,J＝5.1Hz,2H),2.34(qd,J＝6.8,15.5Hz,1H),1.00(d,J＝6.7Hz,3H),0.92(d,J＝ 6.7Hz,3H).

Step 2: Synthesis of 2-[3-[(2,2-dimethoxyethyl)(methyl)amino]isoxazol-5-yl]-3-methylbutanoic acid

2-[3-[(2,2-dimethoxyethyl)(methyl)amino]isoxazol-5-yl]-3-methylbutyric acid methyl ester (55 mg, 183.12 mmol) was dissolved in tetrahydrofuran (1 mL), and a monohydrated lithium hydroxide aqueous solution (2M, 274.68 μL) was added. The reaction solution was reacted at 25° C. for 1 hour, and LCMS showed that the reaction was complete. The reaction solution was adjusted to a pH of 5 to 6 with a 2M hydrochloric acid solution, and then extracted three times with ethyl acetate (10 mL). The organic phase was concentrated to dryness under reduced pressure to obtain 2-[3-[(2,2-dimethoxyethyl)(methyl)amino]isoxazol-5-yl]-3-methylbutyric acid (50 mg).

MS m/z(ESI):287.1[M+H] ⁺ .

Step 3: Synthesis of (2S,4R)-1-[2-[3-[(2,2-dimethoxyethyl)(methyl)amino]isoxazol-5-yl]-3-methylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[3-[(2,2-Dimethoxyethyl)(methyl)amino]isoxazol-5-yl]-3-methylbutanoic acid (50 mg, 174.63 mmol) and (2S,4R)-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (70.67 mg, 192.09 mmol) were dissolved in N,N-dimethylformamide (2 mL), and triethylamine (88.35 mg, 873.14 μmol) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (73.04 mg, 192.09 mmol) were added, and the reaction solution was reacted at 25°C for 1 hour. LCMS showed that the reaction was completed. The reaction solution was quenched with water (3 mL), then extracted three times with ethyl acetate (15 mL). The organic phase was concentrated to dryness under reduced pressure and purified by thin layer chromatography (silica, petroleum ether/tetrahydrofuran = 1/2) to give (2S,4R)-1-[2-[3-[(2,2-dimethoxyethyl)(methyl)amino]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (52 mg).

MS m/z(ESI):600.2[M+H] ⁺ .

Step 4: Synthesis of (2S,4R)-4-hydroxy-1-[3-methyl-2-[3-[methyl(2-oxoethyl)amino]isoxazol-5-yl]butyryl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

(2S,4R)-1-[2-[3-[(2,2-dimethoxyethyl)(methyl)amino]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (50 mg, 83.37 μmol) was dissolved in tetrahydrofuran (1 mL), hydrochloric acid solution (2M, 416.85 μL) was added, and the reaction solution was stirred at 25° C. for 12 hours. LCMS showed that the reaction was complete. The reaction solution was adjusted to pH 8-9 with aqueous sodium bicarbonate solution, extracted three times with ethyl acetate (10 mL), and the organic phase was concentrated to dryness under reduced pressure to give (2S, 4R)-4-hydroxy-1-[3-methyl-2-[3-[methyl(2-oxoethyl)amino]isoxazol-5-yl]butyryl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (34 mg).

MS m/z(ESI):576.2[M+Na] ⁺ .

Step 5: Synthesis of (2S,4R)-4-hydroxy-1-[2-[3-[[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethyl]-methylamino]isoxazol-5-yl]-3-methylbutyryl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

((2S,4R)-4-hydroxy-1-[3-methyl-2-[3-[methyl(2-oxoethyl)amino]isoxazol-5-yl]butanoyl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (34 mg, 61.41 μmol) and 2-[3-(methylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (24.37 mg, 67.55 μmol) were added to the mixture. l) was dissolved in N,N-dimethylformamide (1 mL) and tetrahydrofuran (1 mL), and acetic acid (11.06 mg, 184.22 μmol), sodium acetate (15.11 mg, 184.22 μmol) and sodium acetate borohydride (39.04 mg, 184.22 μmol) were added. The reaction solution was reacted at 25 °C for 1 hour. The reaction was completed by LCMS. The reaction solution was quenched with water (0.2 mL) and concentrated to dryness under reduced pressure. The reaction solution was analyzed by high performance liquid chromatography (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 36%-76%) as eluent) to give (2S,4R)-4-hydroxy-1-[2-[3-[[2-[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]ethyl]-methylamino]isoxazol-5-yl]-3-methylbutanoyl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 284) (9.66 mg).

MS m/z(ESI):862.3[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.98(s,1H),9.01-8.93(m,1H),8.52-8.46(m,1H),8.45-8.23(m,1H),8.12(br d,J＝8.3Hz,1H),7.50-7.39(m,2H),7.39-7.28(m,3H),7.08(br d,J＝4.8Hz,1H),7.02-6.95(m,2H), 6.12-5.91(m,1H),5.12(d,J＝3.5Hz,1H),4.96-4.83(m,1H),4.56(br s,1H),4.47-4.34(m,1H),4.28(br s,1H),3.77-3.64(m,1H),3.55(br d,J＝10.0Hz,1H),3.49-3.37(m,2H),3.32-3.21(m,2H),3.05(br s, 2H),2.98-2.92(m,3H),2.91-2.83(m,3H),2.45(s,3H),2.32-1.93(m,7H),1.81(br dd,J＝9.0,13.3Hz,3H ),1.48-1.31(m,3H),1.03-0.90(m,3H),0.85-0.76(m,3H).

Example 118: Synthesis of Compound 302

Intermediate 2-1 (38.5 mg, 64.84 μmol) and 2-(3-(ethylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (26.33 mg, 77.81 μmol) were dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), sodium acetate (15.96 mg, 194.53 μmol) and acetic acid (11.68 mg, 194.53 μmol, 11.13 μL) were added at 25°C, and the reaction solution was stirred at 25°C for 1.5 hours. Then sodium acetate borohydride (41.23 mg, 194.53 μmol) was added, and the reaction solution was stirred at 25°C for 0.5 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure and the concentrate was purified by preparative liquid chromatography (Boston Prime C18 column: 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 55%-75%) as eluent) to obtain compound 302 (22.16 mg).

MS m/z (ESI): 916.7 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.00 (s, 1H), 8.99 (s, 1H), 8.50 (s, 1H), 8.41 (d, J = 7.9Hz, 1H), 8.13 (d, J＝7.5Hz,1H),7.47-7.42(m,2H),7.38(d,J＝8.3Hz,3H),7.08(t,J＝5.6Hz,1H),7.03-6.97(m,2H), 6.13(s,1H),5.11(d,J＝3.6Hz,1H),4.96-4.88(m,1H),4.57(br s,1H),4.38(t,J＝8.0Hz,1H),4.29( br s,1H),3.72(br dd,J＝4.4,10.8Hz,1H),3.64(br d,J＝11.9Hz,2H),3.57(d,J＝9.9Hz,1H),3.45-3.41(m,2H),3.00(br d ,J＝7.9Hz,2H),2.83-2.72(m,2H),2.46(s,3H),2.22(br d,J＝6.4Hz,4H),2.18-2.08(m,5H),1.87-1.74 (m,6H),1.39(d,J＝7.0Hz,3H),1.27(t,J＝7.1Hz,3H),1.17(br d,J＝9.8Hz,2H),0.96(br d,J＝6.5Hz,3H),0.80(d,J＝6.8Hz,3H).

Example 119: Synthesis of (2S,4R)-4-hydroxy-1-[(S)-2-[2-[4-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetamido]-3,3-dimethylbutyryl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 309)

Step 1: Synthesis of tert-butyl 4-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidine-1-carboxylate

2-[3-(Methylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (270 mg, 832.36 μmol) and tert-butyl 4-formylpiperidine-1-carboxylate (213.02 mg, 998.83 μmol) were dissolved in dichloromethane (1.5 mL) and N,N-dimethylformamide (1.5 mL). Acetic acid (249.92 mg, 4.16 mmol, 238.02 μL) was added at 25°C, and the reaction solution was stirred at 25°C for 1 hour. Sodium triacetoxyborohydride (529.23 mg, 2.50 mmol) was added at 25°C, and the reaction solution was stirred at 25°C for 15 minutes. LCMS showed that the reaction was complete. The reaction solution was adjusted to pH = 8 with aqueous sodium carbonate solution, extracted three times with ethyl acetate (20 mL), the organic phase was concentrated under reduced pressure, and the concentrate was purified by column chromatography (silica, tetrahydrofuran/petroleum ether (tetrahydrofuran ratio 0-38%) as eluent) to give 4-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidine-1-carboxylic acid tert-butyl ester (412 mg).

MS m/z(ESI):522.5[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d6) δ = 13.99 (s, 1H), 8.50 (s, 1H), 8.52-8.47 (m, 1H), 8.13 (dd, J = 1.5, 8.4Hz, 1H), 7.40 -7.33(m,1H),7.07(q,J＝4.8Hz,1H),7.03-6.97(m,2H),4.62-4.51(m,1H),3.94(br d,J＝10.1Hz,4H) ,3.24(t,J＝5.8Hz,1H),2.97(d,J＝4.9Hz,3H),2.80-2.59(m,4H),2.18(br d,J＝5.0Hz,2H),2.14-2.07(m,2H),1.86-1.79(m,2H),1.74-1.69(m,2H),1.62(br d,J＝13.6Hz,2H), 1.39(s,9H).

Step 2: Synthesis of 2-[3-(methylamino)-1-[1-(piperidin-4-ylmethyl)piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

Tert-butyl 4-((4-(6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)methyl)piperidine-1-carboxylate (412 mg, 789.80 μmol) was dissolved in methanol (5 mL), and then dioxane hydrochloride (4M, 1.97 mL) was added, and the reaction solution was stirred at 25° C. for 1 hour. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure to give 2-[3-(methylamino)-1-[1-(piperidin-4-ylmethyl)piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (332 mg).

MS m/z(ESI):422.4[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d6) δ = 10.70 (br s, 1H), 9.21 (br s, 1H), 9.10 (br d, J = 7.5Hz, 1H), 7.91 (br s, 1H), 7.44 (br t,J＝7.1Hz,1H),7.15(br d,J＝8.1Hz,1H),7.02(t,J＝7.3Hz,1H),4.96(br t,J＝11.4Hz,1H), 3.72(br d,J＝10.9Hz,4H),3.27-3.23(m,3H),3.07(br d,J＝5.1Hz,2H),2.98(s,2H),2.87-2.77(m,4H),2.17-2.01(m,4H),1.76(br d,J＝11.5Hz,2H),1.50( br d,J＝11.8Hz,2H).

Step 3: Synthesis of tert-butyl 2-[4-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetate

2-[3-(Methylamino)-1-[1-(piperidin-4-ylmethyl)piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (210 mg, 498.18 μmol) and N,N-diisopropylethylamine (193.16 mg, 1.49 mmol, 260.32 μL) were dissolved in N,N-dimethylformamide (4 mL), tert-butyl bromoacetate (97.17 mg, 498.18 μmol, 73.61 μL) was added at 0°C under nitrogen protection, and the reaction solution was stirred at 25°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was diluted with water (10 mL), extracted with ethyl acetate three times (20 mL), and the organic phase was concentrated under reduced pressure. The concentrate was purified by column chromatography (silica, tetrahydrofuran/petroleum ether (tetrahydrofuran ratio 0-46%) as eluent) to give tert-butyl 2-[4-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetate (137 mg).

MS m/z(ESI):536.3[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d6) δ = 14.00 (s, 1H), 8.50 (s, 1H), 8.13 (d, J = 7.3Hz, 1H), 7.40-7.32 (m, 1H), 7.09 (br d,J＝5.0Hz,1H),6.99(d,J＝8.0Hz,2H),4.62-4.49(m,1H),3.22(t,J＝5.8Hz,1H),3.06(s,2H), 2.96(br d,J＝5.0Hz,4H),2.81(br d,J＝10.5Hz,2H),2.19-2.06(m,8H),1.82(br d,J＝10.3Hz,2H),1.68(brd,J＝12.3Hz,2H),1.41(s,9H),1.36(s,3H).

Step 4: Synthesis of 2-[4-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetic acid

Tert-butyl 2-[4-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetate (137 mg, 255.75 μmol) was dissolved in dichloromethane (1.5 mL), trifluoroacetic acid (291.61 mg, 2.56 mmol, 189.35 μL) was added at 25°C, and the reaction solution was stirred at 25°C for 16 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure to give 2-[4-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetic acid (122 mg).

MS m/z(ESI):480.5[M+H] ⁺ .

Step 5: Synthesis of (2S,4R)-4-hydroxy-1-[(S)-2-[2-[4-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetamido]-3,3-dimethylbutyryl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[4-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetic acid (40 mg, 83.41 μmol) and (2S,4R)-1-[(S)-2-amino-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine -2-Formamide (44.50 mg, 100.09 μmol) was dissolved in N,N-dimethylformamide (1 mL), N,N-diisopropylethylamine (53.90 mg, 417.04 μmol, 72.64 μL) and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (38.06 mg, 100.09 μmol) were added at 25°C, and the reaction solution was stirred at 25°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure, and the concentrate was purified by preparative liquid chromatography (Boston Prime C18 column: 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile content 50%-70%) as eluent) to give (2S,4R)-4-hydroxy-1-[(S)-2-[2-[4-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]piperidin-1-yl]acetamido]-3,3-dimethylbutanoyl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 309) (18.57 mg).

MS m/z (ESI): 906.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d6) δ = 13.99 (br s, 1H), 8.99 (s, 1H), 8.52-8.42 (m, 2H), 8.13 (br d, J = 7.3Hz, 1H), 7.77 (br d,J＝9.6Hz,1H),7.48-7.41(m,2H),7.40-7.31(m,3H),7.07(br d,J＝5.0Hz,1H),7.03-6.95(m,2H ),5.14(d,J＝3.4Hz,1H),4.90(q,J＝7.1Hz,1H),4.62-4.41(m,3H),4.29(br s,1H),3.65-3.53(m,2H ), 3.06-2.93(m,6H),2.92-2.77(m,3H),2.46(s,3H),2.26-2.02(m,9H),1.88-1.69(m,5H),1.59-1.46(m,1H ),1.43-1.31(m,3H),1.25-1.09(m,2H),0.95(s,9H).

Example 120: Synthesis of (2S,4R)-1-[(S)-2-[[2-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]oxy]acetylamino]-3,3-dimethylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 211)

Step 1: Synthesis of tert-butyl 2-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]oxyacetate

Tert-butyl 2-(2-chloropyrimidin-5-yl)oxyacetate (100 mg, 408.71 μmol) and 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (126.84 mg, 408.71 μmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (158.47 mg, 1.23 mmol) was added. The reaction solution was stirred at 120°C for 16 hours, and the reaction was complete after LCMS monitoring. The reaction solution was concentrated to dryness under reduced pressure, and then purified by thin layer chromatography (silica, petroleum ether/ethyl acetate = 1/1) to obtain the title compound (45 mg).

MS m/z(ESI):519.4[M+H] ⁺ .

Step 2: Synthesis of 2-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]oxyacetic acid

Tert-butyl 2-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]pyrimidin-5-yl]oxyacetate (45 mg, 86.78 μmol) was dissolved in dichloromethane (1 mL), and trifluoroacetic acid (462.00 mg, 4.05 mmol) was added. The reaction solution was stirred at 25°C for 16 hours, and the reaction was complete after LCMS monitoring. The reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (74 mg).

MS m/z(ESI):463.1[M+H] ⁺ .

Step 4: Synthesis of (2S,4R)-1-[(S)-2-[[2-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]oxy]acetylamino]-3,3-dimethylbutyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (Compound 211)

2-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]oxyacetic acid (64 mg, 111.02 μmol) and (2S,4R)-1-[(S)-2-amino-3,3-dimethyl-butanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)benzene] [7-Azabenzotriazol-1-yl]-N,N,N,N-tetramethyluronium hexafluorophosphonate (46.43 mg, 122.12 μmol) and N,N-diisopropylethylamine (71.74 mg, 555.09 μmol) were added to the mixture. The reaction mixture was stirred at 25°C for 2 hours and the reaction was complete after monitoring by LCMS. Water (0.1 mL) was added to the reaction solution to quench the reaction, and then the product was purified by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile ratio 16%-56%) as eluent) to give (2S,4R)-1-[(S)-2-[[2-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]oxy]acetylamino]-3,3-dimethylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (25.93 mg).

MS m/z (ESI): 889.7 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.98 (s, 1H), 8.99 (s, 1H), 8.54 (s, 1H), 8.46 (d, J = 7.8Hz, 1H), 8.25 (s, 2H),8.08(d,J＝8.3Hz,1H),7.92(d,J＝9.3Hz,1H),7.46-7.35(m,6H),7.01(d,J＝7.8Hz,2H),6.52( s,2H),5.15(s,1H),4.92(t,J＝7.2Hz,2H),4.71(d,J＝13.8Hz,3H),4.66(s,2H),4.58(d,J＝9 . 3Hz,1H),4.45(t,J＝8.2Hz,1H),4.29(s,1H),3.63-3.55(m,2H),3.06(t,J＝10.9Hz,2H),2.45(s,3H ),1.95-1.88(m,3H),1.78(ddd,J＝4.8,8.5,12.8Hz,1H),1.38(d,J＝7.0Hz,3H),0.95(s,9H).

Example 121: Synthesis of Compound 331

Step 1: Synthesis of trans-tert-butyl 4-(3-((4-(4-hydroxypiperidin-1-yl)pyridin-2-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate

Piperidin-4-ol (0.94 g, 9.36 mmol), trans-4-(3-((4-bromopyridin-2-yl)oxy)cyclobutyloxy)piperidine-1-carboxylic acid tert-butyl ester (2 g, 4.68 mmol), potassium tert-butoxide (1.05 g, 9.36 mmol) and Ruphos-Pd-G ₃ (391.27 mg, 468.02 μmol) were added to tetrahydrofuran (20 mL). The reaction solution was stirred at 70°C for 18 h under a nitrogen atmosphere. LCMS showed that the reaction was complete. The reaction solution was cooled and poured into water (20 mL), extracted with ethyl acetate (30 mL*3), the organic layers were combined and washed with water (40 mL*2), washed with saturated brine (40 mL), and dried over anhydrous sodium sulfate. The desiccant was filtered off, the filtrate was concentrated under reduced pressure, and purified by column chromatography (silica, dichloromethane/methanol=10/1) to obtain the title compound (1.1 g).

MS m/z(ESI):448.3[M+H] ⁺ .

Step 2: Synthesis of trans-tert-butyl 4-(3-((4-(4-((methylsulfonyl)oxy)piperidin-1-yl)pyridin-2-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate

Trans-4-(3-((4-(4-hydroxypiperidin-1-yl)pyridin-2-yl)oxy)cyclobutyloxy)piperidine-1-carboxylic acid tert-butyl ester (1.1 g, 2.46 mmol) and triethylamine (541 mg, 4.92 mmol) were added to dichloromethane (5 mL), and methylsulfonyl chloride (561 mg, 4.92 mmol) was added under an ice-water bath, and the reaction solution was stirred at 25 ° C for 2 h. LCMS showed that the reaction was complete. 30 mL of water was added to the reaction solution for dilution, and it was extracted with ethyl acetate (50 mL*3), and the organic layers were combined and washed with water (40 mL*2), washed with saturated brine (40 mL), and dried over anhydrous sodium sulfate. The desiccant was filtered off, and the filtrate was concentrated under reduced pressure. The title compound (600 mg) was purified by column chromatography (silicon dioxide, ethyl acetate/petroleum ether=5/1).

MS m/z(ESI):526.3[M+H] ⁺ .

Step 3: Synthesis of trans-tert-butyl 4-(3-((4-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1))-yl)piperidin-1-yl)pyridin-2-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate

6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-3-amine (650.24 mg, 2.40 mmol), trans-4-(3-((4-((methylsulfonyl)oxy)piperidin-1-yl)pyridin-2-yl)oxy)cyclobutyloxy)piperidine-1-carboxylic acid tert-butyl ester (700 mg, 1.33 mmol) and potassium carbonate (865.58 mg, 2.66 mmol) were added to N,N-dimethylformamide (10 mL), and the reaction solution was reacted at 80°C for 18 h. LCMS showed that the reaction was complete. 20 mL of water was added to the reaction solution for dilution, and it was extracted with ethyl acetate (20 mL*3). The organic layers were combined and washed with water (30 mL*2), washed with saturated brine (50 mL), and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure and purified by column chromatography (silica, ethyl acetate/petroleum ether=9/1) to give the title compound (400 mg).

MS m/z(ESI):701.4[M+H] ⁺ .

Step 4: Synthesis of trans-2-(3-amino-1-(1-(2-(3-(piperidin-4-yloxy)cyclobutyloxy)pyridin-4-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

Trans-4-(3-((4-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazine-1))-yl)piperidin-1-yl)pyridin-2-yl)oxy)cyclobutyloxy)piperidine-1-carboxylic acid tert-butyl ester (350 mg, 499.41 μmol) was added to methanol (2 mL) and dichloromethane (2 mL). 4M hydrochloric acid dioxane solution (6 mL) was added to the reaction solution at room temperature. The reaction solution was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated, 30 mL of saturated sodium bicarbonate aqueous solution was added under ice-water bath to adjust pH=8, and extracted with dichloromethane:methanol=10:1 (20 mL*3), the organic layers were combined and washed with water (40 mL*2), washed with saturated brine (40 mL), and dried over anhydrous sodium sulfate. The desiccant was removed by filtration and the filtrate was concentrated under reduced pressure to give the title compound (270 mg).

MS m/z(ESI):557.3[M+H] ⁺ .

Step 5: Synthesis of compound 331

Intermediate 2-1 (20 mg, 33.69 μmol), trans-2-(3-amino-1-(1-(2-(3-(piperidin-4-yloxy)cyclobutyloxy)pyridin-4-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (18.75 mg, 33.69 μmol) and acetic acid (202.11 μg, 3.37 μmol) were added to N,N-dimethylformamide (1 mL), and sodium triacetoxyborohydride (28.43 mg, 134.74 μmol) was added under ice-water bath. The reaction solution was stirred at 50° C. for 2 h. LCMS showed that the reaction was complete. The reaction solution was concentrated and then purified by preparative liquid chromatography (Phenomenex C18, 5 μm silica, 30 mm diameter, 80 mm length; using a mixture of water (containing 0.05% ammonium bicarbonate) and acetonitrile (acetonitrile content: 60%-70%) as the eluent) to give compound 331 (3 mg).

MS m/z (ESI): 1134.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.90(s,1H),8.99(s,1H),8.52(s,1H),8.45–8.38(m,1H),8.13–8.06(m,1H ),7.82–7.76(m,1H),7.50–7.40(m,2H),7.42–7.32(m,3H),7.06–6.97(m,2H),6.65– 6.58(m,1H),6.47(s,2H),6.22–6.16(m,1H),6.11(s,1H),5.23–5.18(m,1H),5.15–5.10(m,1H),4.95– 4.87(m,2H),4.41–4.26(m,4H),4.14–4.06(m,2H),3.73–3.6 8(m,1H),3.64–3.52(m,3H),3.46–3.41(m,3H),3.11–3.00(m,2H),2.78–2.70(m,2H),2.46(s,3H), 2.33–2.28(m,4H),2.14–2.07(m,2H),2.06–1.93(m,4H),1.93–1 .85(m,2H),1.82–1.74(m,3H),1.75–1.67(m,2H),1.66–1.61(m,1H),1.48–1.40(m,2H),1.41–1.34(m, 3H),1.08–1.02(m,4H),1.00–0.91(m,3H),0.88–0.75(m,3H).

Example 122: Synthesis of Compound 332

Step 1: Synthesis of trans-4-[3-[2-[3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octan-8-yl]pyrimidin-5-yl]oxycyclobutyloxy]piperidine-1-carboxylic acid tert-butyl ester

2-[3-amino-1-(8-azabicyclo[3.2.1]octan-3-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (200 mg, 536.41 μmol) and trans-4-[3-(2-chloropyrimidin-5-yl)oxycyclobutyloxy]piperidine-1-carboxylic acid tert-butyl ester (205.91 mg, 536.41 μmol) were dissolved in dimethyl sulfoxide (5 mL), and N,N-diisopropylethylamine (5 mL) was added. The reaction solution was reacted at 140°C for 48 hours, and the reaction was completed by LCMS detection. The reaction solution was cooled to room temperature, diluted with water (15 mL), extracted with ethyl acetate (10 mL*3), the organic phases were combined, dried with sodium sulfate, concentrated, and purified by column chromatography (silicon dioxide, petroleum ether/tetrahydrofuran = 1/1) to obtain the title compound (42 mg).

MS m/z(ESI):684.3[M+H] ⁺ .

Step 2: Synthesis of trans-2-[3-amino-1-[8-[5-[3-(piperidin-4-yloxy)cyclobutyloxy]pyrimidin-2-yl]-8-azabicyclo[3.2.1]octan-3-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

Trans-4-[3-[2-[3-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]-8-azabicyclo[3.2.1]octan-8-yl]pyrimidin-5-yl]oxycyclobutyloxy]piperidine-1-carboxylic acid tert-butyl ester (42 mg, 61.42 μmol) was dissolved in dichloromethane (3 mL), and dioxane hydrochloride (153.55 μL, 614.22 μmol) was added. The reaction solution was reacted at 25° C. for 2 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated to obtain the title compound (40 mg).

MS m/z(ESI):584.3[M+H] ⁺ .

Step 3: Synthesis of compound 332

Trans-2-[3-amino-1-[8-[5-[3-(piperidin-4-yloxy)cyclobutyloxy]pyrimidin-2-yl]-8-azabicyclo[3.2.1]octan-3-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (30 mg, 48.38 μmol) and intermediate 2-1 (25.85 mg, 43.54 μmol) were dissolved in N,N-dimethylformamide (1 mL) and tetrahydrofuran (1 mL), and sodium acetate borohydride (30.76 mg, 145.13 μmol), sodium acetate (15.87 mg, 193.50 μmol) and acetic acid (2.91 mg, 48.38 μmol) were added. The reaction solution was reacted at 25°C for 2 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated and purified by preparative liquid chromatography (Phenomenex C18 column: 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile of decreasing polarity (acetonitrile ratio: 57%-97%) as the eluent) to give compound 332 (7.4 mg).

MS m/z (ESI): 1161.4 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.01 (s, 1H), 8.99 (s, 1H), 8.57 (s, 1H), 8.41 (d, J = 7.6Hz, 1H), 8.19-8.14 ( m,3H),7.48-7.43(m,2H),7.41-7.36(m,3H),7.05-6.98(m,2H),6.44(s,2H),6.11(s,1H),5.21-5.12( m,1H),5.11(d,J＝3.7Hz,1H),4.92(br t,J＝7.2Hz,1H),4.82(br t,J＝5.2Hz,1H),4.67(br s,2H),4.40-4.32(m,2H),4.28(br s,1H),3.75-3.68(m,1H),3.66-3.54(m,4H),2.78-2.68(m,4H),2.46 (s,3H),2.36(br t,J＝5.7Hz,4H),2.26-2.18(m,4H),2.14(br s,6H),2.01(br d,J＝10.1Hz,3H),1.80 (br d,J＝8.1Hz,5H),1.71(br d,J＝12.8Hz,3H),1.46(br d,J＝6.8Hz,2H),1.38(d,J＝7.0Hz,3H), 1.10(br d,J＝11.9Hz,2H),0.95(br d,J＝6.6Hz,3H),0.79(d,J＝6.6Hz,3H).

Example 123: Synthesis of Compound 333

Step 1: Synthesis of tert-butyl 3-hydroxy-8-azabicyclo[3.2.1]octane-8-carboxylate

Dissolve N-Boc-nortropinone (25 g, 110.97 mmol) in anhydrous methanol (300 mL), add sodium borohydride (8.41 g, 222.22 mmol) at 0°C. Stir the reaction solution at 0°C for 2 hours. Thin layer chromatography (petroleum ether: tetrahydrofuran = 2:1) detects that the reaction is complete. Quench the reaction solution with saturated ammonium chloride (70 mL), concentrate, add ethyl acetate (50 mL) and water (200 mL) to dilute, then extract with ethyl acetate (60 mL) three times, dry the organic phase with anhydrous sodium sulfate, filter, and concentrate to dryness under reduced pressure to obtain the title compound (20 g).

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 4.62-4.49 (m, 1H), 4.08-3.95 (m, 2H), 3.93-3.84 (m, 1H), 3.33 (s, 1H), 2.11 (br d,J＝4.0Hz,1H),1.78(br s,4H),1.60(br d,J＝12.3Hz,2H),1.41-1.37(m,9H).

Step 2: Synthesis of 3-hydroxy-8-azabicyclo[3.2.1]octane-8-carboxylic acid allyl ester

Dissolve tert-butyl 3-hydroxy-8-azabicyclo[3.2.1]octane-8-carboxylate (10g, 43.99mmol) in anhydrous methanol (50mL), add dioxane hydrochloride (4M, 65.99mL). The reaction solution was stirred at 25℃ for 4 hours. Thin layer chromatography (petroleum ether:tetrahydrofuran = 3:1, potassium permanganate) detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain a crude residue. The crude residue was dissolved in anhydrous tetrahydrofuran (60mL) and water (30mL), sodium carbonate (8.03g, 75.77mmol) was added, and a solution of allyl chloroformate (5.02g, 41.68mmol) in tetrahydrofuran (40mL) was added at 0℃. The reaction solution was stirred at 25℃ for 12 hours. Thin layer chromatography (petroleum ether:tetrahydrofuran = 3:1) detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, ethyl acetate (20 mL) and water (100 mL) were added, and then extracted three times with ethyl acetate (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to obtain the title compound (2.5 g).

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 6.04-5.82 (m, 1H), 5.27 (ddd, J = 1.6, 8.5, 17.2Hz, 1H), 5.22-5.14 (m, 1H), 4.63-4.48 (m,3H),4.11(br s,2H),3.95-3.85(m,1H),2.15(br d,J＝3.3Hz,1H),1.83(br s,4H),1.64(br d,J ＝18.1Hz,2H),1.35(br s,1H).

Step 3: Synthesis of allyl 3-((methylsulfonyl)oxy)-8-azabicyclo[3.2.1]octane-8-carboxylate

Dissolve 3-hydroxy-8-azabicyclo[3.2.1]octane-8-carboxylic acid allyl ester (2.5 g, 11.83 mmol) in anhydrous tetrahydrofuran (25 mL), add pyridine (2.87 mL, 35.50 mmol), and add a solution of methylsulfonic anhydride (3.09 g, 17.75 mmol) in tetrahydrofuran (30 mL) at 0°C. The reaction solution was stirred at 25°C for 3 hours. The reaction was completed by thin layer chromatography (petroleum ether: tetrahydrofuran = 3:1, potassium permanganate). The reaction solution was concentrated to dryness under reduced pressure, ethyl acetate (20 mL) and water (100 mL) were added, and extracted three times with ethyl acetate (50 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated to dryness under reduced pressure to obtain the title compound (2.8 g).

MS m/z(ESI):290.1[M+H] ⁺ .

Step 4: Synthesis of 3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)-8-azabicyclo[3.2.1]octane-8-carboxylic acid allyl ester

3-((Methylsulfonyl)oxy)-8-azabicyclo[3.2.1]octane-8-carboxylic acid allyl ester (10 g, 34.56 mmol) was dissolved in dimethylformamide (47 mL) and sodium iodide (5.18 g, 34.56 mmol) was added. After the reaction solution was stirred at 25 ° C for 1 hour, cesium carbonate (15.01 g, 46.08 mmol) and 6-chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (3.91 g, 23.04 mmol) were added to the reaction. The reaction solution was stirred at 80 ° C for 15 hours. LCMS detected that the reaction was complete. Dichloromethane (50 mL) and water (700 mL) were added to the reaction solution, extracted three times with dichloromethane (70 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain the title compound (2.5 g).

MS m/z(ESI):363.1[M+H] ⁺ .

Step 5: Synthesis of 3-[3-[bis(tert-butoxycarbonyl)amino]-6-chloropyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylic acid allyl ester

3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)-8-azabicyclo[3.2.1]octane-8-carboxylic acid allyl ester (2.5 g, 6.89 mmol) was dissolved in dichloromethane (25 mL), and Boc anhydride (4.51 g, 20.67 mmol), pyridine (2.18 g, 27.56 mmol) and 4-dimethylaminopyridine (168.36 mg, 1.36 mmol) were added. The reaction solution was stirred at 25 ° C for 12 hours. LCMS detected that the reaction was complete. The reaction solution was washed with water (20 mL) three times, the organic phase was concentrated under reduced pressure, and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain the title compound (3.01 g).

MS m/z(ESI):563.2[M+H] ⁺ .

Step 6: Synthesis of 3-(3-(tert-butoxycarbonylamino)-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)-8-azabicyclo[3.2.1]octane-8-carboxylic acid allyl ester

3-[3-[bis(tert-butoxycarbonyl)amino]-6-chloropyrazolo[4,3-c]pyridazine-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylic acid allyl ester (3.01 g, 5.35 mmol) was dissolved in methanol (8 mL) and water (8 mL), and sodium hydroxide (1.82 g, 45.44 mmol) was added. The reaction solution was stirred at 25 ° C for 12 hours. LCMS detected that the reaction was complete. Water (10 mL) was poured into the reaction solution, extracted three times with ethyl acetate (18 mL), and the organic phase was dried over sodium sulfate, filtered, and concentrated under reduced pressure to obtain the title compound (1.8 g, crude product).

MS m/z(ESI):463.2[M+H] ⁺ .

Step 7: Synthesis of tert-butyl (1-(8-azabicyclo[3.2.1]octan-3-yl)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-3-yl)carbamate

3-(3-(tert-Butyloxycarbonylamino)-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl)-8-azabicyclo[3.2.1]octane-8-carboxylic acid allyl ester (1.8 g, 3.89 mmol) and (2-(methoxymethoxy)phenyl)boronic acid (849.11 mg, 4.67 mmol) were dissolved in dioxane (20 mL) and water (20 mL), and potassium carbonate (1.61 g, 11.66 mmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (635.06 mg, 777.66 μmol) were added. The reaction solution was stirred at 80°C for 15 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated, dissolved in dichloromethane, filtered, and purified by column chromatography (dichloromethane: methanol = 10: 1) to obtain the title compound (1.8 g).

MS m/z(ESI):481.3[M+H] ⁺ .

Step 8: Synthesis of trans-2-(3-amino-1-(8-(6-3-(piperidin-4-oxy)cyclobutyloxy)pyridin-3-yl)-8-azabicyclo[3.2.1]octan-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

Tert-butyl (1-(8-azabicyclo[3.2.1]octan-3-yl)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-3-yl)carbamate (127.03 mg, 297.27 μmol) and tert-butyl trans-4-(3-((5-bromopyridin-2-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate (127.03 mg, 297.27 μmol) were dissolved in dioxane (1 mL), and sodium tert-butoxide (85.71 mg, 891.81 μmol) and methanesulfonic acid (2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium(II) (50.32 mg, 59.45 mol) were added. The reaction mixture was replaced with nitrogen three times and then stirred at 110°C for 15 hours under a nitrogen atmosphere. The reaction solution was filtered to obtain a crude residue. The obtained crude residue was dissolved in ethyl acetate (0.5 mL) and a solution of hydrochloric acid in ethyl acetate (4M, 256.29 μL) was added. The reaction was stirred at 25°C for 1 hour. LCMS monitored the completion of the reaction. The reaction solution was directly concentrated and purified by preparative high performance liquid chromatography (Phenomenex C18 column: 3 μm silica, 30 mm diameter, 75 mm length; a mixture of water (containing 0.05% ammonia) and acetonitrile with decreasing polarity (acetonitrile ratio: 45%-85%) was used as the eluent) to obtain the title compound (25.4 mg).

MS m/z(ESI):583.3[M+H] ⁺ .

Step 9: Synthesis of compound 333

Trans-2-(3-amino-1-(8-(6-(3-(piperidin-4-oxy)cyclobutyloxy)pyridin-3-yl)-8-azabicyclo[3.2.1]octan-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (25 mg, 42.90 μmol) and intermediate 2-1 (21.23 mg, 35.75 μmol) were dissolved in N,N-dimethylformamide (0.3 mL) and tetrahydrofuran (0.3 mL), and sodium acetate (4.40 mg, 53.63 μmol) was added. After reacting at 25°C for half an hour, acetic acid (6.44 mg, 107.26 μmol, 6.13 μL) and sodium acetate borohydride (22.73 mg, 107.26 μmol) were added. The reaction was stirred at 25°C for 2 hours. The reaction was completed by LCMS. The reaction solution was directly filtered and purified by preparative HPLC (Phenomenex C18 column: 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile of decreasing polarity (acetonitrile ratio: 57%-97%) as eluent) to give compound 333 (2.73 mg).

MS m/z(ESI):581.0[M+2H] ²⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ = 8.88 (s, 1H), 8.70 (br s, 1H), 8.43 (s, 1H), 8.26-8.01 (m, 2H), 7.54-7.15 (m, 5H),7.11-6.95(m,2H),6.80(br d,J＝9.01Hz,1H),6.10(s,1H),4.54-4.37(m,4H),3.94-3.81(m,3H), 3.73-3.44(m,7H),2.93-2.73(m,4H),2.52(br s,1H),2.49(s,3H),2.47-2.34(m,4H),2.24(br d,J＝6.88Hz,2H),2.20-2.03(m,7H),1.94-1.78(m,4H),1.66-1.57(m,3H),1.53(br d,J＝7.13Hz,3H), 1.45-1.14(m,8H),1.11-0.80(m,6H).

Example 124: Synthesis of Compound 334-1

Step 1: Synthesis of exo-8-azabicyclo[3.2.1]octan-3-ol hydrochloride

To a solution of exo-3-hydroxy-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (4 g, 17.6 mmol) in dichloromethane (20 mL) was added dioxane hydrochloride (4 M, 8.00 mL), and the mixture was stirred at 25° C. for 2 hours. The mixture was concentrated to dryness, and the residue was slurried with ether and dried in vacuo to give the title compound (2.88 g).

MS m/z(ESI):128.2[M+H] ⁺ .

Step 2: Synthesis of exo-3-hydroxy-8-azabicyclo[3.2.1]octane-8-carboxylic acid benzyl ester

Triethylamine (890 mg, 8.80 mmol, 1.23 mL) and benzyl chloroformate (564 mg, 3.30 mmol) were added to a solution of exo-8-azabicyclo[3.2.1]octan-3-ol hydrochloride in dichloromethane (5 mL), and the mixture was stirred at 25° C. for 2 hours. The mixture was diluted with dichloromethane, washed with water and brine, dried and concentrated to dryness to give a crude product, which was purified by column chromatography (silica, dichloromethane: methanol = 20: 1) to give the title compound (340 mg).

MS m/z(ESI):262.1[M+H] ⁺ .

Step 3: Synthesis of exo-3-methylsulfonyloxy-8-azabicyclo[3.2.1]octane-8-carboxylic acid benzyl ester

Methanesulfonyl chloride (223 mg, 1.95 mmol) and triethylamine (394 mg, 3.9 mmol) were added to a dichloromethane solution (10 mL) of exo-3-hydroxy-8-azabicyclo[3.2.1]octane-8-carboxylic acid benzyl ester (340 mg, 1.30 mmol). The mixture was stirred at room temperature for 3 hours. The mixture was diluted with dichloromethane, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to dryness. The residue was purified by silica gel chromatography (silicon dioxide, dichloromethane: methanol = 20: 1) to give the title compound (330 mg).

MS m/z(ESI):340.1[M+H] ⁺ .

Step 4: Synthesis of endo-3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)-8-azabicyclo[3.2.1]octane-8-carboxylic acid benzyl ester

To a solution of 6-chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (2.4 g, 11.65 mmol) in N,N-dimethylformamide (10 mL) was added exo-3-methylsulfonyloxy-8-azabicyclo[3.2.1]octane-8-carboxylic acid benzyl ester (2.77 g, 8.15 mmol) and cesium carbonate (15.14 g, 46.59 mmol). The mixture was stirred at 80 ° C for 16 hours. The mixture was diluted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, filtered and concentrated to dryness. The residue was purified by silica gel chromatography (silicon dioxide, petroleum ether: ethyl acetate = 1: 2) to give the title compound (3 g).

MS m/z(ESI):413.1[M+H] ⁺ .

Step 5: Synthesis of endo-3-[3-[bis(tert-butyloxycarbonyl)amino]-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylic acid benzyl ester

Endo-3-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)-8-azabicyclo[3.2.1]octane-8-carboxylic acid benzyl ester (3 g, 5.81 mmol) was dissolved in dichloromethane (30 mL), di-tert-butyl dicarbonate (5.07 g, 23.25 mmol), triethylamine (1.76 g, 17.44 mmol) and 4-dimethylaminopyridine (1.42 g, 11.63 mmol) were added, and the reaction solution was stirred at 25° C. for 12 hours. The reaction solution was purified by column chromatography (silica, petroleum ether:ethyl acetate=2:1) to obtain the title compound.

MS m/z(ESI):613.1[M+H] ⁺ .

Step 6: Synthesis of endo-3-[3-(tert-butyloxycarbonylamino)-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylic acid benzyl ester

Endo-3-[3-[bis(tert-butyloxycarbonyl)amino]-6-chloro-1H-pyrazolo[4,3-c]pyridazine-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylic acid benzyl ester (1.9 g, 3.10 mmol) was dissolved in methanol (20 mL) and potassium carbonate (1.28 g, 9.30 mmol) was added. The mixture was stirred at 80 ° C for 1 hour. The mixture was diluted with ethyl acetate, washed with water and brine, dried over anhydrous sodium sulfate, filtered and concentrated to dryness. The residue was purified by silica gel chromatography (silicon dioxide, petroleum ether: ethyl acetate = 1: 1) to give the title compound (1.4 g).

MS m/z(ESI):513.2[M+H] ⁺ .

Step 7: Synthesis of endo-3-[3-(tert-butyloxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylic acid benzyl ester

Endo-3-[3-(tert-butyloxycarbonylamino)-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylic acid benzyl ester (1.4 g, 2.73 mmol) and [2-(methoxymethoxy)phenyl]boronic acid (744 mg, 4.09 mmol) were dissolved in dioxane (20 mL) and water (5 mL), and potassium phosphate (1.74 g, 8.19 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (222 mg, 272 μmol) were added thereto. The mixture was replaced with nitrogen three times and stirred at 80° C. for 12 hours under a nitrogen atmosphere. The mixture was diluted with ethyl acetate, washed with water and brine, dried and concentrated to dryness to give the crude product. The residue was purified by silica gel chromatography (silica, petroleum ether:ethyl acetate=1:1) to give the title compound (1.5 g).

MS m/z(ESI):615.2[M+H] ⁺ .

Step 8: Synthesis of tert-butyl (1-(endo-8-azabicyclo[3.2.1]octan-3-yl)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-3-yl)carbamate

Endo-3-[3-(tert-butyloxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazine-1-yl]-8-azabicyclo[3.2.1]octane-8-carboxylic acid benzyl ester (820 mg, 1.33 mmol) was dissolved in methanol (10 mL), palladium carbon (70 mg, 10% wt) was added thereto, the mixture was replaced with hydrogen 3 times and stirred at room temperature under a hydrogen balloon for 12 hours. The mixture was filtered and the filtrate was concentrated to dryness to give a crude product, and the residue was purified by silica gel chromatography (silica, dichloromethane: methanol = 10: 1) to give the title compound (442 mg).

MS m/z(ESI):481.2[M+H] ⁺ .

Step 9: Synthesis of tert-butyl 4-[trans-3-[[4-[endo-3-[3-(tert-butoxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octan-8-yl]pyridin-2-yl]oxy]cyclobutyloxy]piperidine-1-carboxylate

Under nitrogen atmosphere, to a solution of tert-butyl (1-(endo-8-azabicyclo[3.2.1]octan-3-yl)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-3-yl)carbamate (200 mg, 416 μmol) and tert-butyl 4-[(1r,3r)-3-[(4-bromopyridin-2-yl)oxy]cyclobutyloxy]piperidine-1-carboxylate (177.85 mg, 416 μmol) in dioxane (10 mL) were added methanesulfonic acid (2-dicyclohexylphosphino-2',6'-diisopropoxy-1,1'-biphenyl)(2-amino-1,1'-biphenyl-2-yl)palladium(II) (51 mg, 62 μmol) and potassium tert-butoxide (139 mg, 1.25 mmol). The mixture was replaced with nitrogen three times and stirred at 100° C. for 12 hours under a nitrogen atmosphere. The mixture was diluted with ethyl acetate, washed with water and brine, dried and concentrated to dryness to give a crude product, which was purified by flash chromatography (silica, petroleum ether:ethyl acetate=1:1) to give the title compound (100 mg).

MS m/z(ESI):827.4[M+H] ⁺ .

Step 10: Synthesis of 2-[3-amino-1-[endo-8-[2-[trans-3-(piperidin-4-yloxy)cyclobutyloxy]pyridin-4-yl]-8-azabicyclo[3.2.1]octan-3-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[trans-3-[[4-[endo-3-[3-(tert-butoxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]-8-azabicyclo[3.2.1]octan-8-yl]pyridin-2-yl]oxy]cyclobutyloxy]piperidine-1-carboxylic acid tert-butyl ester (180 mg, 217 μmol) was dissolved in dichloromethane (5 mL) and methanol (1 mL), and dioxane hydrochloride (4M, 1 mL) was added thereto, and stirred at 25° C. for 2 hours. The reaction solution was spin-dried, extracted with dichloromethane and sodium bicarbonate aqueous solution, and the organic phase was dried and spin-dried to obtain the title compound (105 mg).

MS m/z(ESI):583.2[M+H] ⁺ .

Step 11: Synthesis of compound 334

2-[3-amino-1-[endo-8-[2-[trans-3-(piperidin-4-yloxy)cyclobutyloxy]pyridin-4-yl]-8-azabicyclo[3.2.1]octan-3-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (40 mg, 68.65 μmol) and (2S,4R)-1-[(2R)-2-[3-(4-formyl-1-piperidinyl)isoxazol-5-yl]- 3-Methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (40.76 mg, 68.65 μmol) was dissolved in N,N-dimethylformamide (3 mL), acetic acid (4.12 mg, 68 μmol) and sodium acetate borohydride (43 mg, 205 μmol) were added thereto, and the mixture was stirred at room temperature for 3 hours. The mixture was filtered and purified by preparative HPLC (Phenomenex C18, 5 μm silica, 30 mm diameter, 80 mm length; using a mixture of water (containing 0.05% ammonium bicarbonate) and acetonitrile (acetonitrile content: 60%-70%) as eluent) to obtain compound 334-1 (33 mg).

MS m/z (ESI): 1160.6 [M+H] ⁺ ;

¹ H NMR (600MHz, DMSO-d ₆ ) δ13.83(s,1H),9.02–8.97(m,1H),8.44–8.39(m,1H),8.26(s,1H),8.06–8.00(m ,1H),7.82–7.77(m,1H),7.47–7.43(m,2H),7.38–7.32(m,3H),7.00–6.94(m,2H),6.56–6.51 (m,1H),6.51–6.47(m,2H),6.14–6.09(m,2H),5.25–5.20(m,1H),5.13–5.10(m,1H),4.94–4.90(m,1H) ,4.70–4.64(m,1H),4.39–4.34(m,3H),4.33–4.27(m,2H),3.73–3.69(m ,1H),3.63–3.59(m,2H),3.57–3.55(m,1H),3.44–3.40(m,2H),2.76–2.65(m,4H),2.57–2.55(m,1H),2.48 –2.45(m,3H),2.37–2.27(m,4H),2.23–2.16(m,3H),2.13–2.08(m,2 H),2.06–1.87(m,8H),1.82–1.76(m,3H),1.74–1.68(m,2H),1.68–1.59(m,1H),1.47–1.40(m,2H),1.40– 1.36(m,3H),1.14–1.05(m,2H),0.98–0.92(m,3H),0.82–0.74(m,3H).

Example 125: Synthesis of Compound 334-2

Step 1: Synthesis of tert-butyl 4-(trans-3-((4-(endo-3-hydroxy-8-azabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate

Endo-8-azabicyclo[3.2.1]octan-3-ol (1.18 g, 9.36 mmol), tert-butyl 4-(trans-3-((4-bromopyridin-2-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate (2 g, 4.68 mmol), potassium tert-butoxide (1.05 g, 9.36 mmol) and Ruphos-Pd-G ₃ (391.27 mg, 468.02 μmol) were added to tetrahydrofuran (20 mL). The reaction solution was stirred at 70 ° C for 18 h under nitrogen atmosphere. LCMS showed that the reaction was complete. The reaction solution was cooled and poured into water (20 mL), extracted with ethyl acetate (30 mL*3), and the organic layers were combined and washed with water (40 mL*2), washed with saturated brine (40 mL), and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure and purified by column chromatography (silica, dichloromethane/methanol=10/1) to give the title compound (400 mg).

MS m/z(ESI):474.2[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-(trans-3-((4-(endo-3-((methylsulfonyl)oxy)-8-azabicyclo[3.2.1]oct-8-yl)pyridin-2-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate

Tert-butyl 4-(trans-3-((4-(endo-3-hydroxy-8-azabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate (380.00 mg, 802.36 μmol) and triethylamine (243.57 mg, 2.41 mmol) were added to dichloromethane (5 mL), and methanesulfonyl chloride (183.82 mg, 1.60 mmol) was added under ice-water bath, and the reaction solution was stirred at 25 ° C for 2 h. LCMS showed that the reaction was complete. 30 mL of water was added to the reaction solution for dilution, and it was extracted with ethyl acetate (50 mL*3), and the organic layers were combined and washed with water (40 mL*2), washed with saturated brine (40 mL), and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure and purified by column chromatography (silica, ethyl acetate/petroleum ether=5/1) to give the title compound (300 mg).

MS m/z(ESI):552.3[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-(trans-3-((4-(exo-3-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)-8-azabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate

6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-3-amine (221.27 mg, 815.67 μmol), tert-butyl 4-(trans-3-((4-(endo-3-((methylsulfonyl)oxy)-8-azabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate (300.00 mg, 543.78 μmol) and potassium carbonate (353.46 mg, 1.09 mmol) were added to N,N-dimethylformamide (5 mL), and the reaction solution was reacted at 80°C for 18 h. LCMS showed that the reaction was complete. 20 mL of water was added to the reaction solution for dilution, extracted with ethyl acetate (20 mL*3), the organic layers were combined and washed with water (20 mL*2), washed with saturated brine (20 mL), and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure and purified by column chromatography (silica, ethyl acetate/petroleum ether = 9/1) to give the title compound (55 mg).

MS m/z(ESI):727.4[M+H] ⁺ .

Step 4: Synthesis of 2-(3-amino-1-(exo-8-(2-(trans-3-(piperidin-4-yloxy)cyclobutyloxy)pyridin-4-yl)-8-azabicyclo[3.2.1]oct-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

Tert-butyl 4-(trans-3-((4-(exo-3-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)-8-azabicyclo[3.2.1]octan-8-yl)pyridin-2-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate (52 mg, 71.54 μmol) was dissolved in dichloromethane (1 mL) and methanol (1 mL), and a 4M HCl solution in dioxane (1 mL) was added at room temperature. The reaction solution was stirred at room temperature for 2 h. LCMS showed that the reaction was complete. The reaction solution was directly concentrated under reduced pressure to give the title compound (62 mg).

MS m/z(ESI):583.3[M+H] ⁺ .

Step 5: Synthesis of compound 334-2

Intermediate 2-1 (40 mg, 48.05 μmol), 2-(3-amino-1-(exo-8-(2-(trans-3-(piperidin-4-yloxy)cyclobutyloxy)pyridin-4-yl)-8-azabicyclo[3.2.1]oct-3-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (35.66 mg, 48.05 μmol) and acetic acid (514.85 μg, 4.81 μmol) were added to tetrahydrofuran (1 mL) and N,N-dimethylformamide (1 mL). The reaction solution was stirred at 50° C. for 2 h. LCMS showed that the reaction was complete. The reaction solution was concentrated and then purified by preparative liquid chromatography (Phenomenex C18, 5 μm silica, 30 mm diameter, 80 mm length; using a mixture of water (containing 0.05% ammonium bicarbonate) and acetonitrile (acetonitrile content: 60%-70%) as eluent) to give compound 334-2 (4 mg).

MS m/z (ESI): 1060.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ14.03(s,1H),8.98(s,1H),8.56(s,1H),8.44–8.37(m,1H),8.17–8.10(m,1H ),7.82–7.76(m,1H),7.55–7.29(m,5H),7.07–6.96(m,2H),6.54–6.46(m,3H),6.13–6.07(m,2H),5.26–5.19 (m,1H),5.14–5.08(m,2H),4.96–4.87(m,1H),4.48–4.43(m,2H),4.39–4.24(m,3H),4.15–4 .07(m,2H),3.76–3.68(m,1H),3.65–3.52(m,3H),3.20–3.14(m,6H),2.78–2.65(m,4H),2.48–2.43(m, 3H),2.39–2.25(m,4H),2.25–2.15(m,4H),2.15–2.05(m,4H),2.05–1.89(m,2H),1.84–1.67(m,5H),1.49– 1.35(m,4H),1.14–1.06(m,2H),1.00–0.92(m,3H),0.89–0.76(m,3H).

Example 126: Synthesis of Compound 340

Step 1: Synthesis of tert-butyl 4-(trans-3-((2-(methylthio)pyrimidin-5-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate

5-Bromo-2-(methylthio)pyrimidine (1.13 g, 5.53 mmol), tert-butyl 4-(trans-3-hydroxycyclobutyloxy)piperidine-1-carboxylate (500 mg, 1.84 mmol), cesium carbonate (1.20 g, 3.69 mmol) and methanesulfonic acid-2-(di-tert-butylphosphino)-3,6-dimethoxy-2',4',6'-triisopropyl-1,1'-biphenyl(2-amino-1,1'-biphenyl-2-yl)palladium(II) (155.70 mg, 184.26 μmol) were added to toluene (8 mL), and the reaction solution was stirred at 100° C. for 18 h under nitrogen atmosphere. LCMS showed that the reaction was complete. The reaction solution was cooled and poured into water (20 mL), extracted with ethyl acetate (30 mL*3), the organic layers were combined and washed with water (40 mL*2), washed with saturated brine (40 mL), and dried over anhydrous sodium sulfate. The desiccant was filtered off, the filtrate was concentrated under reduced pressure, and purified by column chromatography (silicon dioxide, petroleum ether/ethyl acetate = 3/2) to obtain the title compound (166 mg).

MS m/z(ESI):396.2[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-(trans-3-((2-(methylsulfonyl)pyrimidin-5-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate

4-(trans-3-((2-(methylthio)pyrimidin-5-yl)oxy)cyclobutyloxy)piperidine-1-carboxylic acid tert-butyl ester (100 mg, 252.83 μmol) was added to tetrahydrofuran (3 mL), potassium peroxymonosulfonate (621.97 mg, 1.01 mmol) was dissolved in H ₂ O (5 mL) and then slowly added dropwise to the reaction solution at 0°C. The reaction solution was stirred at room temperature for 3 h. LCMS showed that the reaction was complete. 5 mL of saturated sodium sulfite solution was added to the reaction solution to quench, and the solution was extracted with ethyl acetate (15 mL*3), dried, filtered, and the filtrate was concentrated to obtain the title compound (103 mg).

MS m/z(ESI):450.2[M+Na] ⁺ .

Step 3: Synthesis of tert-butyl 4-(trans-3-((2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate

Tert-butyl 4-(trans-3-((2-(methylsulfonyl)pyrimidin-5-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate (50 mg, 116.96 μmol), piperidine-4-ol (236.59 mg, 2.34 mmol) and potassium carbonate (161.40 mg, 1.17 mmol) were added to N,N-dimethylformamide (2 mL) and acetonitrile (2 mL), and the reaction solution was stirred at 100 ° C for 18 h. LCMS showed that the reaction was complete. The reaction solution was cooled to room temperature, diluted with 15 mL of water, extracted with ethyl acetate (20 mL*3), washed with saturated brine (40 mL), and dried over anhydrous sodium sulfate. The desiccant was filtered off, the filtrate was concentrated under reduced pressure, and the title compound (10 mg) was purified by column chromatography (silicon dioxide, petroleum ether/ethyl acetate=3/2).

Step 4: Synthesis of tert-butyl 4-(trans-3-((2-(4-((methylsulfonyl)oxy)piperidin-1-yl)pyrimidin-5-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate

Tert-butyl 4-(trans-3-((2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate (420 mg, 936.34 μmol) and triethylamine (284.25 mg, 2.81 mmol) were added to dichloromethane (5 mL), and methylsulfonyl chloride (214.52 mg, 1.87 mmol) was added under an ice-water bath. The reaction solution was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. 30 mL of water was added for dilution, ethyl acetate was extracted (30 mL*3), washed with saturated brine (40 mL), and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, the filtrate was concentrated under reduced pressure, and the title compound (40 mg) was purified by column chromatography (silicon dioxide, petroleum ether/ethyl acetate = 1/1).

Step 5: Synthesis of tert-butyl 4-(trans-3-((2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1)-yl)piperidin-1-yl)pyrimidin-5-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate

6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-3-amine (29.36 mg, 108.23 μmol), tert-butyl 4-(trans-3-((2-(4-((methylsulfonyl)oxy)piperidin-1-yl)pyrimidin-5-yl)oxy)cyclobutyloxy)piperidine-1-carboxylate (38 mg, 72.15 μmol) and cesium carbonate (70.35 mg, 216.46 μmol) were added to N,N-dimethylformamide (1 mL). The reaction solution was stirred at 80°C for 18 h. LCMS showed that the reaction was complete. 30 mL of water was added for dilution, ethyl acetate was extracted (30 mL*3), washed with saturated brine (40 mL), and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, the filtrate was concentrated under reduced pressure, and the title compound (23 mg) was purified by column chromatography (silicon dioxide, petroleum ether/ethyl acetate = 1/9).

MS m/z(ESI):702.4[M+H] ⁺ .

Step 6: Synthesis of 2-(3-amino-1-(1-(5-(trans-3-(piperidin-4-yloxy)cyclobutyloxy)pyrimidin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

4-(trans-3-((2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1)-yl)piperidin-1-yl)pyrimidin-5-yl)oxy)cyclobutyloxy)piperidine-1-carboxylic acid tert-butyl ester (20 mg, 28.50 μmol) was added to dichloromethane (0.3 mL) and methanol (0.1 mL), and 4M HCl dioxane solution (2 mL) was added at room temperature. The reaction solution was stirred at 25°C for 1 h. LCMS showed that the reaction was complete. The reaction solution was concentrated, 30 mL of saturated sodium bicarbonate aqueous solution was added under an ice-water bath to adjust the pH to 8, and extracted with dichloromethane:methanol=10:1 (20 mL*3), the organic layers were combined and washed with water (40 mL*2), washed with saturated brine (40 mL), and dried over anhydrous sodium sulfate. The desiccant was removed by filtration and the filtrate was concentrated under reduced pressure to give the title compound (23 mg).

MS m/z(ESI):558.3[M+H] ⁺ .

Step 7: Synthesis of compound 340

2-(3-amino-1-(1-(5-(trans-3-(piperidin-4-yloxy)cyclobutyloxy)pyrimidin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (20 mg, 35.87 μmol), intermediate 2-1 (17.04 mg, 28.69 μmol) and acetic acid (215.19 μg, 3.59 μmol) were added to N,N-dimethylformamide (1 mL), and sodium triacetoxyborohydride (30.41 mg, 143.46 μmol) was added under ice-water bath. The reaction solution was stirred at 50°C for 2 h. LCMS showed that the reaction was complete. The reaction solution was concentrated and then purified by preparative liquid chromatography (Phenomenex C18, 5 μm silica, 30 mm diameter, 80 mm length; using a mixture of water (containing 0.05% ammonium bicarbonate) and acetonitrile (acetonitrile content: 60%-70%) as the eluent) to give Compound 340 (1.5 mg).

MS m/z (ESI): 1135.6 [M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ8.87(s,1H),8.28(s,1H),8.05(s,2H),8.04–7.98(m,1H),7.48–7.31(m,5H ),7.04–6.95(m,2H),6.10(s,1H),5.08–4.98(m,2H),4.63–4.38(m,5H),3.89–3.79(m,2H),3.78–3.66(m ,3H),3.65–3.58(m,4H),3 .21–3.07(m,4H),2.94–2.83(m,3H),2.82–2.75(m,1H),2.49–2.28(m,7H),2.21–2.10(m,4H),2.06–1.90( m,5H),1.90–1.72(m,4H),1.56–1.46(m,2H),1.41–1.23(m,4H),1.08–1.02(m,3H),0.92–0.85(m,3H).

Example 127: Synthesis of Compound 346

Step 1: Synthesis of tert-butyl 4-[trans-3-[2-[4-[6-(2-hydroxyphenyl)-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]oxycyclobutyloxy]piperidine-1-carboxylate

2-[3-(Isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (300 mg, 771.42 μmol) and tert-butyl 4-[trans-3-(2-chloropyrimidin-5-yl)oxycyclobutyloxy]piperidine-1-carboxylate (325.74 mg, 848.56 μmol) were dissolved in dimethyl sulfoxide (3 mL), and N,N-diisopropylethylamine (498.50 mg, 3.86 mmol) was added. The reaction was reacted at 140°C for 16 hours, and the reaction was completed by LCMS. The reaction solution was cooled to room temperature, diluted with water (15 mL), extracted with ethyl acetate (10 mL*3), the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (silicon dioxide, petroleum ether/ethyl acetate = 1/1) to obtain the title compound (150 mg).

MS m/z(ESI):700.4[M+H] ⁺ .

Step 2: Synthesis of 2-[3-(isopropylamino)-1-[1-[5-[trans-3-(piperidin-4-yloxy)cyclobutyloxy]pyrimidin-2-yl]piperidin-4-yl]pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[trans-3-[2-[4-[6-(2-hydroxyphenyl)-3-(isopropylamino)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]pyrimidin-5-yl]oxycyclobutyloxy]piperidine-1-carboxylic acid tert-butyl ester (150 mg, 214.33 μmol) was dissolved in methanol (1 mL), and dioxane hydrochloride (535.84 μL, 2.14 mmol) was added. The reaction solution was reacted at 25° C. for 12 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated to obtain the title compound (150 mg, 235.78 μmol).

MS m/z(ESI):600.2[M+H] ⁺ .

Step 3: Synthesis of compound 346

2-[3-(Isopropylamino)-1-[1-[5-[trans-3-(piperidin-4-yloxy)cyclobutyloxy]pyrimidin-2-yl]piperidin-4-yl]pyrazolo[4,3-c]pyridazin-6-yl]phenol (30 mg, 47.16 μmol) and intermediate 2-1 (28.00 mg, 47.16 μmol) were dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), and sodium acetate borohydride (29.98 mg, 141.47 μmol), sodium acetate (15.47 mg, 188.62 μmol) and acetic acid (2.83 mg, 47.16 μmol) were added. The reaction solution was reacted at 25°C for 2 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated and purified by preparative liquid chromatography (Phenomenex C18 column: 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile ratio 28%-68%) as eluent) to give compound 346 (13.01 mg).

MS m/z (ESI): 1177.4 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.95 (br s, 1H), 9.01-8.98 (m, 1H), 8.52 (s, 1H), 8.41 (d, J = 7.5Hz, 1H), 8.14 -8.11(m,3H),7.48-7.43(m,2H),7.39-7.35(m,3H),7.03-6.99(m,2H),6.90(d,J＝7.8Hz,1H),6.11(s ,1H),4.94-4.85(m,2H),4.81(t,J＝5.3Hz,1H),4.74(br d,J＝12.3Hz,2H),4.40-4.28(m,3H),4.00(dd,J＝6.5,14.1Hz,1H),3.72(br d,J＝5.8Hz,1H),3.59(br dd ,J＝10.5,19.1Hz,2H),3.11-3.02(m,2H),2.76-2.69(m,2H),2.46(s,3H),2.34(br t,J＝5.5Hz,5H),2.28 -2.15(m,2H),2.11(br d,J＝6.8Hz,2H),2.05-1.93(m,8H),1.85-1.74(m,4H),1.74-1.58(m,4H),1.49-1.42(m,2H),1.38(br d ,J＝7.0Hz,3H),1.24(d,J＝6.5Hz,6H),1.10(br d,J＝10.3Hz,3H),0.98-0.93(m,3H),0.82-0.76(m,3H ).

Example 128: Synthesis of Compound 366

Step 1: Synthesis of 6-chloro-N-isopropyl-1H-pyrazolo[4,3-c]pyridazin-3-amine

6-Chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (100 mg, 589 μmol) and acetone (205 mg, 3.54 mmol) were dissolved in N,N-dimethylformamide (1 mL), and acetic acid (0.1 mL) and sodium triacetylborohydride (749 mg, 3.54 mmol) were added. The mixture was stirred at room temperature for 3 hours. The reaction solution was diluted with ethyl acetate, washed with water and brine, dried and concentrated to dryness to obtain a crude product, which was purified by flash column chromatography (silica, petroleum ether: ethyl acetate = 1:1) to obtain the title compound (40 mg).

MS m/z(ESI):212.1[M+H] ⁺ .

Step 2: Synthesis of N-isopropyl-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-3-amine

6-Chloro-N-isopropyl-1H-pyrazolo[4,3-c]pyridazine-3-amine (30 mg, 141.74 μmol) and [2-(methoxymethoxy)phenyl]boronic acid (38 mg, 212 μmol) were dissolved in dioxane (1 mL) and water (0.2 mL), and potassium phosphate (90 mg, 425 μmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (9.95 mg, 14.17 μmol) were added. The mixture was replaced with nitrogen three times and stirred at 80°C for 12 hours under a nitrogen atmosphere. The mixture was diluted with ethyl acetate (40 mL), washed with water (30 mL) and brine (30 mL), dried and concentrated to dryness to obtain a crude product, and the residue was purified by silica gel column chromatography (silica, petroleum ether: ethyl acetate = 1:1) to obtain the title compound (40 mg).

MS m/z(ESI):314.1[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-[trans-3-[[4-[4-[3-(isopropylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-2-yl]oxy]cyclobutyloxy]piperidine-1-carboxylate

4-[trans-3-[[4-(4-methanesulfonyloxypiperidin-1-yl)pyridin-2-yl]oxy]cyclobutyloxy]piperidine-1-carboxylic acid tert-butyl ester (550 mg, 1.05 mmol) was dissolved in N,N-dimethylformamide (10 mL), and N-isopropyl-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-3-amine (393.44 mg, 1.26 mmol) and cesium carbonate (1.02 g, 3.14 mmol) were added. The mixture was stirred at 80° C. for 16 hours. The mixture was filtered and concentrated to dryness, and the residue was purified by preparative HPLC (chromatographic column: Gemini NX C18 5 μm*10*150 mm; mobile phase: A: 0.05% TFA v/v, B: acetonitrile; B%: 40%-45%, 12 min) to give the title compound (800 mg).

MS m/z(ESI):743.4[M+H] ⁺ .

Step 4: Synthesis of 2-[3-(isopropylamino)-1-[1-[2-[trans-3-(piperidin-4-yloxy)cyclobutyloxy]pyridin-4-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

Tert-butyl 4-[trans-3-[[4-[4-[3-(isopropylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-2-yl]oxy]cyclobutyloxy]piperidine-1-carboxylate (30 mg, 40.38 μmol) was dissolved in methanol (0.5 mL) and dichloromethane (2 mL), dioxane hydrochloride (4M, 1 mL) was added thereto, and the reaction solution was stirred at 25° C. for 2 hours. The mixture was concentrated to dryness, and the residue was washed with diethyl ether and dried in vacuo to give the title compound (30 mg).

MS m/z(ESI):599.3[M+H] ⁺ .

Step 5: Synthesis of compound 366

2-[3-(isopropylamino)-1-[1-[2-[trans-3-(piperidin-4-yloxy)cyclobutyloxy]pyridin-4-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (15 mg, 25.05 μmol) and intermediate 2-1 (17.85 mg, 30.06 μmol) were dissolved in N,N-dimethylformamide (4 mL), and acetic acid (1.50 mg, 25.05 μmol) and sodium triacetyl borohydride (15.93 mg, 75.16 μmol) were added thereto. The mixture was stirred at room temperature for 16 hours. The mixture was concentrated to dryness, and the residue was purified by thin layer chromatography (silica, dichloromethane: methanol = 10: 1) to give compound 366 (9.8 mg).

MS m/z (ESI): 1176.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.72–9.57(m,1H),9.00(s,1H),8.51(s,1H),8.43(d,J＝7.6Hz,1H),8.00( d,J＝7.9Hz,1H),7.93(d,J＝7.3Hz,1H),7.49–7.31(m,6H),7.17–7.00(m,3H),6.96(d,J＝7.5Hz,1H ),6.32(s,1H),6.17(s,1H),5.31–5.26(m,1H),5 .09–4.87(m,3H),4.45–4.26(m,6H),4.01–3.93(m,2H),3.05–2.88(m,5H),2.84–2.71(m,3H),2.46(s, 3H),2.12–1.95(m,10H),1.83–1.74(m,4H),1.40–1.36(m,3H),1.27–1.20(m,14H),0.97–0.92(m,3H),0.80– 0.76(m,3H).

Example 129: Synthesis of Compound 375

2-[3-(Cyclopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (25.05 mg, 64.74 μmol) and intermediate 11 (35 mg, 64.74 μmol) were dissolved in N,N-dimethylformamide (1 mL) and tetrahydrofuran (1 mL), and sodium acetate (21.24 mg, 258.96 μmol), acetic acid (3.89 mg, 64.74 μmol) and sodium acetate borohydride (41.16 mg, 194.22 μmol) were added. The reaction solution was reacted at 25° C. for 2 hours, and the reaction was completed by LCMS detection. The reaction mixture was concentrated and purified by preparative liquid chromatography (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile ratio 14%-54%) as eluent) to give compound 375 (16.20 mg).

MS m/z (ESI): 875.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.98 (br s, 1H), 8.99 (s, 1H), 8.51 (s, 1H), 8.43 (d, J = 7.8Hz, 1H), 8.13 (d ,J＝7.0Hz,1H),7.49-7.41(m,3H),7.40-7.35(m,3H),7.07-6.96(m,2H),6.18-5.94(m,1H),4.92-4.88(m ,1H),4.70-4.55(m,1H),4.38(t,J＝7.9Hz,1H),4.30(br s,3H),3.76-3.63(m,2H),3.46(br d,J＝10.3Hz,2H),3.08(br d,J＝9.8Hz,2H),2.86-2.73(m,3H),2.46(s,3H),2.31-2.16(m,5H),2.08- 1.99(m,1H),1.91-1.75(m,3H),1.38(d,J＝7.0Hz,3H),1.00-0.94(m,3H),0.86-0.79(m,3H),0.77-0.69( m,2H),0.63-0.53(m,2H).

Example 130: Synthesis of Compound 378

2-Chloro-6-[3-(isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (25.0 mg, 59.1 μmol) and intermediate 2-1 (31.9 mg, 53.7 μmol) were dissolved in tetrahydrofuran (0.5 mL) and N,N-dimethylformamide (0.5 mL), sodium acetate (13.2 mg, 161 μmol), acetic acid (9.67 mg, 161 μmol) and sodium acetate borohydride (34.1 mg, 161 μmol) were added, and the reaction was stirred at 25° C. for 2 hours. LCMS showed that the reaction was complete. The reaction solution was filtered and the filtrate was purified by preparative liquid chromatography (Boston Prime C18, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile (acetonitrile ratio 65%-85%) as eluent) to obtain compound 378 (18.24 mg).

MS m/z (ESI): 964.5 [M+H] ⁺ ;

¹ H NMR (400 MHz, DMSO-d ₆ )δ＝15.56(s,1H),8.98(s,1H),8.60(s,1H),8.42(d,J＝7.8Hz,1H),8.15(d,J＝8.3Hz,1H),7.54(d,J＝7.0Hz,1H),7.49-7.41(m,2H),7.40-7.32(m,2H) ,7.11-6.94(m,2H),6.19-5.95(m,1H),5.18-5.00(m,1H),4.91(m,J＝6.9Hz,1H),4.65-4.51(m,1H),4.36(t,J＝7.8Hz,1H),4.28(s,1H),4.01(qd,J =6.6,13.6 Hz,1H),3.71(dd,J＝4.1,10.4Hz,1H),3.63(d,J＝11.5Hz,2H),3.56(d,J＝10.0Hz,1H),3.47-3.40(m,1H),3.00(d,J＝8.3Hz,2H),2.75(t,J＝11.2Hz,2H),2 .45(s,3H),2.26-1.98(m,8H),1.90-1.66(m,6H),1.51-1.33(m,3H),1.28(d,J＝6.3Hz,6H),1.15(d,J＝12.0Hz,2H),1.01-0.90(m,3H),0.87-0.75( m,3H).

Example 131: Synthesis of Compound 386

Step 1: Synthesis of tert-butyl 4-(4-chloropyridin-2-yl)-3,6-dihydro-2H-pyridine-1-carboxylate

2-Bromo-4-chloro-pyridine (1 g, 5.20 mmol) and tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (1.61 g, 5.20 mmol) were dissolved in dioxane (8 mL) and water (2 mL), and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (76.04 mg, 103.93 μmol) and sodium carbonate (1.65 g, 15.59 mmol) were added. The reaction solution was reacted at 90° C. for 4 hours under a nitrogen atmosphere, and the reaction was completed by LCMS detection. The reaction solution was cooled to room temperature and filtered. The filtrate was diluted with water (20 mL) and extracted with ethyl acetate (15 mL*3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated and purified by column chromatography (silica, petroleum ether/ethyl acetate = 3/1) to give the title compound (1.5 g).

MS m/z(ESI):239.0[M+H-56] ⁺ .

Step 2: Synthesis of tert-butyl 4-(4-(3-((tert-butyloxycarbonyl)amino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)-3',6'-dihydro-[2,4'-bipyridine]-1'(2'H)-carboxylate

Tert-butyl (6-(2-(methoxymethoxy)phenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-yl)carbamate (300 mg, 660.04 μmol) and tert-butyl 4-(4-chloro-2-pyridyl)-3,6-dihydro-2H-pyridine-1-carboxylate (214.02 mg, 726.04 μmol) were dissolved in dioxane (8 mL), and (2-dicyclohexylphosphino-2,6-diisopropoxy-1,1-biphenyl)[2-(2-amino-1,1-biphenyl)]palladium (II) (55.20 mg, 66.00 μmol) and potassium tert-butoxide (222.19 mg, 1.98 mmol) were added. The reaction solution was reacted at 100° C. under nitrogen for 12 hours, and the reaction was completed by LCMS detection. The reaction solution was cooled to room temperature and filtered. The filtrate was diluted with water (20 mL) and extracted with ethyl acetate (15 mL*3). The organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/2) to give the title compound (190 mg).

MS m/z(ESI):713.4[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-[4-[4-[3-(tert-butoxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-2-yl]piperidine-1-carboxylate

4-(4-(3-((tert-butyloxycarbonyl)amino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidin-1-yl)-3',6'-dihydro-[2,4'-bipyridine]-1'(2'H)-carboxylic acid tert-butyl ester (190 mg, 266.54 μmol) was dissolved in tetrahydrofuran (5 mL), and wet palladium carbon (100 mg, 26.65 μmol) was added. The reaction solution was reacted at 25°C for 6 hours under 15 psi hydrogen, and the reaction was complete when detected by LCMS. The reaction solution was filtered, and the filtrate was concentrated to obtain a crude product. The crude product was dissolved in methanol (5 mL), and dichlorodicyanobenzoquinone (90.76 mg, 399.81 μmol) was added, and the reaction was carried out at 25°C for 2 hours. The reaction was complete when detected by LCMS. The reaction solution was concentrated and purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/1) to give the title compound (150 mg).

MS m/z(ESI):717.2[M+H] ⁺ .

Step 4: Synthesis of 2-[3-amino-1-[1-[2-(piperidin-4-yl)pyridin-4-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[4-[4-[3-(tert-butyloxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]pyridin-2-yl]piperidine-1-carboxylic acid tert-butyl ester (150 mg, 209.83 μmol) was dissolved in methanol (3 mL), and dioxane hydrochloride (524.58 μL, 2.10 mmol) was added. The reaction solution was reacted at 25° C. for 12 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated to obtain the title compound (140 mg).

MS m/z(ESI):471.2[M+H] ⁺ .

Step 5: Synthesis of compound 386

2-[3-amino-1-[1-[2-(piperidin-4-yl)pyridin-4-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (30 mg, 59.17 μmol) and intermediate 2-1 (31.62 mg, 53.25 μmol) were dissolved in N,N-dimethylformamide (1 mL) and tetrahydrofuran (1 mL), and sodium acetate borohydride (37.62 mg, 177.50 μmol), sodium acetate (19.42 mg, 236.67 μmol) and acetic acid (3.55 mg, 59.17 μmol) were added. The reaction solution was reacted at 25°C for 2 hours, and the reaction was completed by LCMS detection. The reaction solution was concentrated and purified by preparative liquid chromatography (Boston Prime C18 column: 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile of decreasing polarity (acetonitrile ratio 44%-84%) as eluent) to give compound 386 (13.2 mg).

MS m/z (ESI): 1048.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.90 (br s, 1H), 8.99 (s, 1H), 8.52 (s, 1H), 8.41 (d, J = 7.7Hz, 1H), 8.13-8.08 (m,2H),7.48-7.42(m,2H),7.40-7.34(m,3H),7.04-6.99(m,2H),6.81-6.72(m,2H),6.47(s,2H),6.12 (s,1H),5.12(br d,J＝3.1Hz,1H),4.96-4.85(m,2H),4.37(t,J＝7.9Hz,1H),4.29(br s,1H),4.17( br d,J＝12.8Hz,2H),3.71(br dd,J＝4.2,10.5Hz,1H),3.63(br d,J＝11.5Hz,2H),3.56(br d,J＝9.9Hz,1H) ,3.06(brt,J＝12.0Hz,2H),2.95(br d,J＝10.1Hz,2H),2.75(br t,J＝11.3Hz,2H),2.46(s,3H),2.26-2.15( m,3H),2.09-1.89(m,8H),1.85-1.66(m,9H),1.38(d,J＝7.0Hz,3H),1.13(br d,J＝10.5Hz,2H),0.99-0.92(m,3H),0.86-0.76(m,3H).

Example 132: Synthesis of Compound 394

Step 1: Synthesis of tert-butyl 4-[[(4-bromopyridine-2-carbonyl)amino]methyl]piperidine-1-carboxylate

Dissolve 4-bromopyridine-2-carboxylic acid (2g, 9.90mmol) in N,N-dimethylformamide (80mL), add tert-butyl 4-aminomethylpiperidine-1-carboxylate (2.12g, 9.90mmol), O-(7-azabenzotriazole-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (4.52g, 11.88mol) and N,N-diisopropylethylamine (5.17mL, 29.70mmol). The reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete. Add water (200mL), extract the reaction mixture twice with ethyl acetate (200mL), combine the organic phases, add anhydrous sodium sulfate to dry, filter, and concentrate to dryness under reduced pressure. Purify by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain the title compound (3.06g).

MS m/z(ESI):341.9,343.9[M+H-56] ⁺ ;

Step 2: Synthesis of tert-butyl 4-((4-(4-(3-((tert-butoxycarbonyl)amino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)))piperidin-1-yl)pyridineamide)methyl)piperidine-1-carboxylate

Tert-butyl (6-(2-(methoxymethoxy)phenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-yl)carbamate (200 mg, 440.02 μmol) and tert-butyl 4-[[(4-bromopyridine-2-carbonyl)amino]methyl]piperidine-1-carboxylate (210.31 mg, 528.03 μmol) were dissolved in toluene (2 mL), and cesium carbonate (200.72 mg, 616.03 μmol), (R)-(+)-2,2-bis(diphenylphosphino)-1,1-binaphthyl (54.80 mg, 88.00 μmol) and palladium acetate (19.76 mg, 88.00 μmol) were added. The reaction solution was reacted at 100° C. under nitrogen for 16 hours, and the reaction was completed by LCMS detection. The reaction solution was cooled to room temperature and filtered. The filtrate was diluted with water (15 mL) and extracted with ethyl acetate (10 mL*3). The organic phases were combined, dried, filtered, and the filtrate was concentrated. The title compound (50 mg) was purified by thin layer chromatography (silica, petroleum ether/tetrahydrofuran = 1/1).

MS m/z(ESI):772.2[M+H] ⁺ .

Step 3: Synthesis of 4-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)-N-(piperidin-4-ylmethyl)picolinamide

Tert-butyl 4-((4-(4-(3-((tert-butoxycarbonyl)amino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyridineamide)methyl)piperidine-1-carboxylate (50 mg, 64.77 μmol) was dissolved in methanol (1 mL), and dioxane hydrochloride (161.94 μL, 647.75 μmol) was added. The reaction solution was reacted at 25° C. for 12 hours, and the reaction was completed by LCMS. The reaction solution was concentrated to obtain the title compound (50 mg).

MS m/z(ESI):528.5[M+H] ⁺ .

Step 4: Synthesis of compound 394

4-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)-N-(piperidin-4-ylmethyl)picolinamide (30 mg, 53.18 μmol) and intermediate 2-1 (31.58 mg, 53.18 μmol) were dissolved in N,N-dimethylformamide (1 mL) and tetrahydrofuran (1 mL), and sodium acetate borohydride (45.09 mg, 212.74 μmol), sodium acetate (21.81 mg, 265.92 μmol) and acetic acid (3.19 mg, 53.18 μmol) were added. The reaction solution was reacted at 25°C for 2 hours, and the reaction was completed by LCMS detection. The reaction mixture was concentrated and purified by preparative liquid chromatography (Phenomenex C18: 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile ratio 6%-46%) as eluent) to give compound 394 (8.66 mg).

MS m/z(ESI):1105.6[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.88 (s, 1H), 8.99 (s, 1H), 8.67 (br t, J = 6.0Hz, 1H), 8.52 (s, 1H), 8.41 (d ,J＝7.5Hz,1H),8.24(d,J＝6.0Hz,1H),8.10(d,J＝7.5Hz,1H),7.52(s,1H),7.44(d,J＝8.3Hz,2H ),7.40-7.35(m,3H),7.09(br d,J＝3.5Hz,1H),7.05-6.99(m,2H),6.47(s,2H),6.11(s,1H),4.96-4.88(m,2H),4.37(t,J＝7.7Hz ,1H),4.29(s,1H),4.22(br d,J＝13.3Hz,2H),3.72(br d,J＝6.0Hz,1H),3.63-3.55(m,2H),3.21-3.12( m,3H),2.83(br d,J＝9.5Hz,2H),2.73(br t,J＝10.8Hz,2H),2.46(s,3H),2.28-2.10(m,5H),2.07-1.97(m,5H),1.91-1.76(m,4H),1.71(br d,J ＝11.5Hz,3H),1.61(br d,J＝12.8Hz,3H),1.38(d,J＝7.3Hz,3H),1.24-1.08(m,5H),0.95(br d,J＝6.5Hz ,3H),0.82-0.77(m,3H).

Example 133: Synthesis of Compound 398

Step 1: Synthesis of ethyl 2-[4-(dimethoxymethyl)piperidin-1-yl]acetate

Dissolve 4-(dimethoxymethyl)piperidine (5 g, 31.40 mmol) in acetonitrile (100 mL) at 0°C, add ethyl bromoacetate (6.29 g, 37.68 mmol) and N,N-diisopropylethylamine (16.41 mL, 94.21 mmol). Stir the reaction solution at 25°C for 5 hours. LCMS detected that the reaction was complete. The reaction liquid was concentrated to dryness under reduced pressure and purified by column chromatography (petroleum ether: tetrahydrofuran = 1:1) to obtain the title compound (3.64 g).

MS m/z(ESI):246.2[M+1] ⁺ .

Step 2: Synthesis of 2-[4-(dimethoxymethyl)piperidin-1-yl]acetic acid

Ethyl 2-[4-(dimethoxymethyl)piperidin-1-yl]acetate (3.64 g, 14.84 mmol) was dissolved in anhydrous tetrahydrofuran (15 mL) and water (15 mL), and lithium hydroxide monohydrate (1.87 g, 44.51 mmol) was added. The reaction solution was heated to 25°C and stirred for 16 hours. The reaction was completed by LCMS. Dilute hydrochloric acid (1.5 M) was added dropwise to the reaction solution until pH = 6, and the solution was concentrated to dryness under reduced pressure to obtain the title compound (3.22 g, crude product).

MS m/z(ESI):217.9[M+H] ⁺ .

Step 3: Synthesis of (2S,4R)-1-[(2S)-2-[[2-[4-(dimethoxymethyl)piperidin-1-yl]acetyl]amino]-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

2-[4-(Dimethoxymethyl)piperidin-1-yl]acetic acid (1.45 g, 6.65 mmol) was dissolved in N,N-dimethylformamide (15 mL), and (2S,4R)-1-[(S)-2-amino-3,3-dimethyl-butyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (3.2 g, 6.65 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (1.53 g, 7.98 mmol) and pyridine (15 mL) were added. The reaction was stirred at 25°C for 2 hours. The reaction was completed by LCMS. The residue was concentrated to dryness under reduced pressure and purified by column chromatography (dichloromethane: methanol = 10: 1) to obtain the title compound (2 g).

MS m/z (ESI): 644.3 [M+H] ⁺ ;

Step 4: Synthesis of (2S,4R)-1-((S)-2-(2-(4-formylpiperidin-1-yl)acetylamino)-3,3-dimethylbutyryl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

(2S,4R)-1-[(2S)-2-[[2-[4-(dimethoxymethyl)piperidin-1-yl]acetyl]amino]-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (1 g, 1.55 mmol) was dissolved in tetrahydrofuran (5 mL), diluted hydrochloric acid (4.66 mL, 2 M) was added, and the mixture was stirred at 50°C for 2 hours. After LCMS detection, solid sodium bicarbonate was added to the reaction mixture until pH = 8, water (8 mL) was added, the aqueous phase was extracted twice with ethyl acetate (20 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (750 mg).

MS m/z(ESI):598.3[M+H] ⁺

Step 5: Synthesis of compound 398

(2S,4R)-1-((S)-2-(2-(4-formylpiperidin-1-yl)acetylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (45 mg, 75.28 μmol) and 2-(3-(ethylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)-2-carboxamide (45 mg, 75.28 μmol) were added. Phenol (31.04 mg, 82.81 μmol) was dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), acetic acid (13.56 mg, 225.84 μmol) and sodium acetate (12.35 mg, 150.56 μmol) were added, the reaction solution was reacted at 25 ° C for 1 hour, sodium acetate borohydride (47.86 mg, 225.84 μmol) was added, and then reacted at 25 ° C for 1 hour. The reaction was completed by LCMS. Water (0.2 mL) was added to the reaction solution to quench the reaction. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile ratio 45%-85%) as eluent) gave compound 398 (20.81 mg).

MS m/z (ESI): 920.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ14.00 (s, 1H), 8.98 (s, 1H), 8.49 (s, 1H), 8.45 (d, J = 7.5Hz, 1H), 8.12 (d, J＝7.0Hz,1H),7.76(d,J＝9.5Hz,1H),7.47-7.41(m,2H),7.40-7.32(m,3H),7.07(t,J＝5.5Hz,1H), 7.03-6.96(m,2H),5.13(d,J＝3.5Hz,1H),4.89(t,J＝7.2Hz,1H),4.61-4.40(m,3H),4.28(br s,1H), 3.66-3.52(m,2H),3.42(br d,J＝6.0Hz,2H),3.05-2.94(m,3H),2.91-2.77(m,3H),2.45(s,3H),2.23-2.03(m,9H),1.88-1.70(m, 5H),1.53(br s,1H),1.38(d,J＝7.0Hz,3H),1.26(t,J＝7.0Hz,3H),1.21-1.10(m,2H),0.94(s,9H) .

Example 134: Synthesis of Compound 400

(2S,4R)-1-((S)-2-(2-(4-formylpiperidin-1-yl)acetylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (45 mg, 75.28 μmol) and 2-(1-(piperidin-4-yl)-3-(propylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)-2-carboxamide (45 mg, 75.28 μmol) were added. Phenol (32.20 mg, 82.81 μmol) was dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), acetic acid (13.56 mg, 225.84 μmol) and sodium acetate (12.35 mg, 150.56 μmol) were added, the reaction solution was reacted at 25 ° C for 1 hour, sodium acetate borohydride (47.86 mg, 225.84 μmol) was added, and then reacted at 25 ° C for 1 hour. The reaction was completed by LCMS. Water (0.2 mL) was added to the reaction solution to quench the reaction. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile ratio 48%-88%) as eluent) gave Compound 400 (29.32 mg).

MS m/z (ESI): 934.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ14.00 (s, 1H), 8.98 (s, 1H), 8.51-8.42 (m, 2H), 8.12 (d, J = 7.3Hz, 1H), 7.76 ( br d,J＝9.8Hz,1H),7.48-7.40(m,2H),7.39-7.31(m,3H),7.14-7.06(m,1H),7.02-6.95(m,2H),5.13(d ,J＝3.3Hz,1H),4.89(t,J＝7.0Hz,1H),4.61-4.48(m,2H),4.45(t,J＝8.4Hz,1H),4.28(br s,1H), 3.58(br d,J＝7.5Hz,2H),3.46-3.40(m,2H),3.05-2.94(m,3H),2.91-2.77(m,3H),2.45(s,3H),2.24-2.03(m, 9H),1.87-1.67(m,7H),1.53(br s,1H),1.38(d,J＝7.0Hz,3H),1.14(br s,2H),1.00-0.91(m,12H).

Example 135: Synthesis of Compound 401

(2S,4R)-1-[(2S)-2-[[2-(4-(2S,4R)-1-((S)-2-(2-(4-formylpiperidin-1-yl)acetylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (46.35 mg, 77.54 μmol) was dissolved in N,N-dimethylformamide (1.5 mL) and tetrahydrofuran (1.5 mL), and 2-(3-(cyclopropylamino)-1 -(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (30 mg, 69.96 μmol, hydrochloride), acetic acid (4.66 mg, 77.54 μmol) and sodium acetate (25.44 mg, 310.18 μmol). Stir the reaction at 25°C for 1 hour. Add sodium acetate borohydride (49.30 mg, 232.63 μmol), stir the reaction at 25°C for 1 hour, and LCMS detects that the reaction is complete. The reaction solution is concentrated to dryness under reduced pressure. Purify by preparative liquid chromatography (Phenomenex C18 column: 3 μm silica, 30 mm diameter, 75 mm length; use water (containing ammonia (0.5%) and acetonitrile with decreasing polarity (acetonitrile ratio 45%-85%) as eluent) to obtain compound 401 (13.80 mg).

MS m/z (ESI): 932.6 [M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ = 8.77 (s, 1H), 8.12 (s, 1H), 7.89 (br d, J = 7.9Hz, 1H), 7.36-7.27 (m, 4H), 7.22 (br t,J＝7.5Hz,1H),6.92-6.85(m,2H),4.91(q,J＝6.8Hz,1H),4.54-4.46(m,5H),4.43-4.32(m,2H) ,3.82-3.72(m,1H),3.65(br dd,J＝3.3,11.0Hz,1H),3.00(br d,J＝11.8Hz,2H),2.94(br d,J＝1.4Hz,2H),2.86-2.76(m,2H),2.75-2.68(m,1H),2.37(s,3H),2.34-2.26(m,2H),2.23(br d,J ＝6.6Hz,2H),2.16-2.07(m,4H),1.88-1.82(m,2H),1.74(br t,J＝12.4Hz,2H),1.55-1.46(m,1H),1.42(br d,J＝7.0Hz,2H),1.32-1.17(m,2H),1.00-0.90(m,9H),0.70(br d,J＝5.4Hz,2H),0.54(br s,2H).

Example 136: Synthesis of Compound 402

(2S,4R)-1-[(2S)-2-[[2-(4-(2S,4R)-1-((S)-2-(2-(4-formylpiperidin-1-yl)acetylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (50 mg, 83.64 μmol) and 2-(3-(isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6 -yl)phenol (26.80 mg, 76.04 μmol) was dissolved in tetrahydrofuran (0.2 mL) and N,N-dimethylformamide (0.2 mL), acetic acid (13.70 mg, 228.12 μmol) was added, the reaction solution was stirred at 25 ° C for 2 hours, and sodium triacetoxyborohydride (48.35 mg, 228.12 μmol) was added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS monitored the reaction to be complete. The reaction solution was concentrated to dryness under reduced pressure and purified by high performance liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia water) and acetonitrile with decreasing polarity (acetonitrile ratio 48%-88%) as eluent) to obtain compound 402 (10.27 mg).

MS m/z (ESI): 934.4 [M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ = 8.96-8.81 (m, 1H), 8.27-8.14 (m, 1H), 8.07-7.93 (m, 1H), 7.54-7.26 (m, 6H), 7.07 -6.90(m,2H),4.61(br s,5H),4.52-4.42(m,2H),4.17-4.02(m,1H),3.90-3.69(m,2H),3.16-3.02(m,4H ),2.97-2.87(m,2H),2.48(br s,3H),2.37(br s,3H),2.28-2.21(m,3H),1.95(br d,J＝2.5Hz,3H),1.90-1.73(m,3H),1.52(br s,3H),1.35(br s,9H),1.29(br s,3H),1.06(br s,9H) .

Example 137: Synthesis of Compound 409

Intermediate 2-1 (40 mg, 67.37 μmol) and 2-fluoro-6-[3-(isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (32.89 mg, 80.84 μmol) were dissolved in tetrahydrofuran (1 mL) and N,N-dimethylformamide (1 mL), and acetic acid (12.14 mg, 202.11 μmol), sodium triacetoxyborohydride (42.84 mg, 202.11 μmol) and sodium acetate (16.58 mg, 202.11 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was complete when monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by HPLC (Phenomenex C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia water) and acetonitrile with decreasing polarity (acetonitrile ratio 63%-83%) as the eluent) to give compound 409 (26.06 mg).

MS m/z (ESI): 948.4 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ )δ=14.78-14.62(m,1H),8.97(s,1H),8.54(s,1H),8.43-8.33(m,1H),7.99-7.93(m ,1H),7.43(br d,J＝8.3Hz,2H),7.39-7.28(m,3H),7.02-6.92(m,2H),6.20-6.05(m,1H),5.16-5.05(m, 1H),4.98-4.88(m,1H),4.64-4.52(m,1H),4.42-4.34(m,1H),4.29(br s,1H),4.09-3.96(m,1H),3.72(br d,J＝6.4Hz,1H),3.673.55(m,3H),3.09-2.94(m,3H),2.81-2.65(m,3H),2.45(s,3H),2.22(br s,3H ),2.13(br d,J＝3.9Hz,3H),1.88-1.72(m,6H),1.46(br d,J＝7.1Hz,1H),1.38(br d,J＝7.1Hz,3H), 1.28 (br d, J＝6.2Hz, 6H), 1.19-1.10 (m, 2H), 0.95 (br d, J＝6.4Hz, 3H), 0.80 (br d, J＝6.1Hz, 3H).

Example 138: Synthesis of Compound 410

Intermediate 2-1 (45 mg, 75.79 μmol) and 4-fluoro-2-(3-(isopropylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (56.54 mg, 83.37 μmol) were dissolved in tetrahydrofuran (1 mL) and N,N-dimethylformamide (1 mL), and acetic acid (13.65 mg, 227.37 μmol), sodium triacetoxyborohydride (48.19 mg, 227.37 μmol) and sodium acetate (18.65 mg, 227.37 μmol) were added. The mixture was stirred at 25° C. for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (Boston Prime C18 column: 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile of decreasing polarity (acetonitrile ratio 63%-83%) as the eluent) to give compound 410 (35.30 mg).

MS m/z (ESI): 948.4 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.86-13.78 (m, 1H), 9.02-8.93 (m, 1H), 8.55-8.47 (m, 1H), 8.43-8.34 (m, 1H), 8.04 -7.95(m,1H),7.43(br d,J＝6.8Hz,2H),7.39-7.33(m,2H),7.26-7.18(m,1H),6.98(br d,J＝6.7Hz,1H),6.93-6.86(m,1H),6.19-6.06(m,1H),5.14-4.99(m,1H),4.96-4.85(m,1H),4.60-4.48( m,1H),4.45-4.24(m,2H),4.12-3.97(m,1H),3.72(br dd,J＝4.3,10.2Hz,1H),3.67-3.59(m,2H),3.59-3.54 (m,1H),3.07-2.94(m,3H),2.84-2.70(m,3H),2.45(br s,3H),2.21(br d,J＝1.5Hz,3H),2.10(br d,J＝6.6Hz,3H),2.02-1.96(m,1H),1.86-1.70(m,6H),1.38(br d,J＝6.5Hz,3H),1.28(br d,J＝5.9Hz ,6H),1.19-1.11(m,2H),0.95(br d,J＝5.3Hz,3H),0.79(br d,J＝6.1Hz,3H).

Example 139: Synthesis of Compound 412

2-[3-[(2-hydroxy-2-methylpropyl)amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (35.28 mg, 58.95 μmol) and intermediate 2-1 (35 mg, 58.95 μmol) were dissolved in N,N-dimethylformamide (1 mL) and anhydrous tetrahydrofuran (1 mL), and sodium acetate (9.67 mg, 117.90 μmol), acetic acid (10.11 μL, 176.85 μmol) and sodium triacetoxyborohydride (37.48 mg, 176.85 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile ratio 11%-51%) as eluent) gave compound 412 (13.0 mg).

MS m/z (ESI): 960.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.05-13.78 (m, 1H), 8.98 (s, 1H), 8.50 (s, 1H), 8.40 (d, J = 7.8Hz, 1H), 8.12 ( d,J＝7.5Hz,1H),7.48-7.42(m,2H),7.40-7.33(m,3H),7.05-6.95(m,2H),6.68-6.59(m,1H),6.16-5.98 ( m,1H),5.11(br s,1H),4.92(br t,J＝7.3Hz,1H),4.70-4.50(m,2H),4.41-4.24(m,2H),3.76-3.53(m,4H),3.39(br d,J＝6.0Hz,4H), 2.99(br d,J＝6.5Hz,2H),2.76(br t,J＝11.3Hz,2H),2.46(s,3H),2.25-1.98(m,9H),1.88-1.72(m,6H) ,1.48-1.36(m,3H),1.20(s,6H),0.99-0.92(m,3H),0.85-0.77(m,3H).

Example 140: Synthesis of Compound 418

Step 1: Synthesis of tert-butyl 4-(2-(methylthio)pyrimidin-5-yl)piperazine-1-carboxylate

Piperazine-1-carboxylic acid tert-butyl ester (6.81 g, 36.57 mmol), 5-bromo-2-(methylthio)pyrimidine (5 g, 24.38 mmol), sodium tert-butoxide (4.68 g, 48.76 mmol) and palladium acetate (546.14 mg, 2.44 mmol) were added to toluene (50 mL). The reaction solution was stirred at 100 ° C for 18 hours under a nitrogen atmosphere. LCMS detected that the reaction was complete. The reaction solution was cooled to room temperature, diluted with 80 mL of water, extracted with ethyl acetate (50 mL * 3), the organic layers were combined and washed with water (40 mL * 2), washed with saturated brine (40 mL), and dried over anhydrous sodium sulfate. The desiccant was filtered off, and the filtrate was concentrated under reduced pressure and slurried with ethyl acetate: petroleum ether = 10: 1 (10 mL) to obtain the title compound (6.1 g).

MS m/z(ESI):311.2[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-(2-(methylsulfonyl)pyrimidin-5-yl)piperazine-1-carboxylate

Tert-butyl 4-(2-(methylthio)pyrimidin-5-yl)piperazine-1-carboxylate (705.88 mg, 1.93 mmol) was added to dichloromethane (5 mL), and m-chloroperbenzoic acid (997.38 mg, 5.80 mmol) was added under an ice-water bath. The reaction solution was stirred at room temperature for 2 h. LCMS showed that the reaction was complete. 30 mL of saturated aqueous sodium sulfite solution was added to the reaction solution to quench, extracted with ethyl acetate (50 mL*3), the organic layers were combined and washed with water (40 mL*2), washed with saturated brine (40 mL), and dried over anhydrous sodium sulfate. The desiccant was filtered off, the filtrate was concentrated under reduced pressure, and the title compound (300 mg) was purified by column chromatography (silicon dioxide, petroleum ether/ethyl acetate = 1/5).

MS m/z(ESI):343.1[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-(2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl)piperazine-1-carboxylate

Piperidin-4-ol (14.77 g, 146.02 mmol), tert-butyl 4-(2-(methylsulfonyl)pyrimidin-5-yl)piperazine-1-carboxylate (2.5 g, 7.30 mmol) and potassium carbonate (10.08 g, 73.01 mmol) were added to N,N-dimethylformamide (10 mL) and acetonitrile (10 mL), and the reaction solution was stirred at 100 ° C for 24 h. LCMS showed that the reaction was complete. The reaction solution was cooled to room temperature, 100 mL of water was added to the reaction solution for dilution, and extracted with ethyl acetate (100 mL*3), the organic layers were combined and washed with water (40 mL*2), washed with saturated brine (40 mL), and dried over anhydrous sodium sulfate. The desiccant was filtered off, the filtrate was concentrated under reduced pressure, and the title compound (320 mg) was purified by column chromatography (silicon dioxide, dichloromethane/methanol=10/1).

MS m/z(ESI):364.3[M+H] ⁺ .

Step 4: Synthesis of tert-butyl 4-(2-(4-((methylsulfonyl)oxy)piperidin-1-yl)pyrimidin-5-yl)piperazine-1-carboxylate

Tert-butyl 4-(2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl)piperazine-1-carboxylate (300 mg, 825.42 μmol) and triethylamine (167.05 mg, 1.65 mmol) were added to dichloromethane (5 mL), and methylsulfonyl chloride (189.10 mg, 1.65 mmol) was added under an ice-water bath. The reaction solution was stirred at room temperature for 1 hour. LCMS showed that the reaction was complete. 30 mL of water was added to the reaction solution for dilution, extracted with ethyl acetate (50 mL*3), the organic layers were combined and washed with water (40 mL*2), washed with saturated brine (40 mL), and dried over anhydrous sodium sulfate. The desiccant was filtered off, the filtrate was concentrated under reduced pressure, and the title compound (170 mg) was purified by column chromatography (silicon dioxide, ethyl acetate/petroleum ether=5/1).

Step 5: Synthesis of tert-butyl 4-(2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)piperazine-1-carboxylate

Tert-butyl 4-(2-(4-((methylsulfonyl)oxy)piperidin-1-yl)pyrimidin-5-yl)piperazine-1-carboxylate (100 mg, 226.48 μmol), 6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-3-amine (77.19 mg, 339.72 μmol) and cesium carbonate (147.21 mg, 452.96 μmol) were added to N,N-dimethylformamide (2 mL). The reaction solution was stirred at 80 ° C for 18 h. LCMS showed that the reaction was complete. 20 mL of water was added to the reaction solution for dilution, extracted with ethyl acetate (20 mL*3), the organic layers were combined and washed with water (20 mL*2), washed with saturated brine (20 mL), and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure and purified by column chromatography (silica, ethyl acetate/petroleum ether = 9/1) to give the title compound (75 mg).

MS m/z(ESI):617.4[M+H] ⁺ .

Step 6: Synthesis of 2-(3-amino-1-(1-(5-(piperazin-1-yl)pyrimidin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

Tert-butyl 4-(2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)piperazine-1-carboxylate (70 mg, 122.24 μmol) was added to methanol (0.3 mL) and dichloromethane (0.3 mL). A 4M HCl solution in dioxane (1 mL) was added to the reaction solution at room temperature. The reaction solution was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated, 30 mL of saturated sodium bicarbonate aqueous solution was added under an ice-water bath to adjust the pH to 8, and extracted with dichloromethane:methanol=10:1 (20 mL*3), the organic layers were combined and washed with water (40 mL*2), washed with saturated brine (40 mL), and dried over anhydrous sodium sulfate. The desiccant was removed by filtration, and the filtrate was concentrated under reduced pressure to obtain the title compound (57 mg).

MS m/z(ESI):473.3[M+H] ⁺ .

Step 7: Synthesis of compound 418

2-(3-amino-1-(1-(5-(piperazin-1-yl)pyrimidin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (25.47 mg, 43.12 μmol), intermediate 2-1 (20 mg, 26.95 μmol) and acetic acid (161.69 μg, 2.69 μmol) were added to N,N-dimethylformamide (1 mL), and sodium triacetoxyborohydride (22.74 mg, 107.79 μmol) was added under ice-water bath. The reaction solution was stirred at 50°C for 2 h. LCMS showed that the reaction was complete. The reaction solution was concentrated and then purified by preparative liquid chromatography (Phenomenex C18, 5 μm silica, 30 mm diameter, 80 mm length; using a mixture of water (containing 0.05% ammonium bicarbonate) and acetonitrile (acetonitrile content: 60%-70%) as the eluent) to give compound 418 (12 mg).

MS m/z (ESI): 1050.5 [M+H] ⁺ ;

¹ H NMR (600 MHz, DMSO-d ₆ )δ13.92(s,1H),9.01–8.97(m,1H),8.54(s,1H),8.43–8.38(m,1H),8.23(s,2H),8.14–8.09(m,1H),7.49–7.42(m,2H),7.39–7.34(m,3H),7.04–6.98(m,2H),6.45(s,2H),6.12(s,1H),5.13–5.09(m,1H),4.96–4.84(m,2H),4.74–4.64(m,2H),4.39–4.34(m,1H),4.31–4.26(m,1H),3.74–3.69(m ,1H),3.65–3.60(m,3H),3.61–3.54(m,1H),3.47–3.40(m,1H),3.07–3.00(m,6H),2.79–2.72(m,3H),2.48–2.44(m,3H),2.27–2.18(m,3H),2.07 –1.87(m,6H),1.83–1.73(m,4H),1.48–1.43(m,1H),1.40–1.36(m,2H),1.25–1.22(m,1H),1.17–1.12(m,2H),0.99–0.93(m,3H),0.87–0.77(m,3 H).

Example 141: Synthesis of Compound 421

Step 1: Synthesis of 4-(methoxymethylene)cyclohexanol

Methoxymethyltriphenylphosphonium chloride (14.42 g, 42.05 mmol) and potassium tert-butoxide (9.83 g, 87.61 mmol) were dissolved in anhydrous tetrahydrofuran (100 mL), stirred at 0°C for 1 hour, and then a solution of 4-hydroxycyclohexanone (4.46 g, 15.81 mmol) in anhydrous tetrahydrofuran (40 mL) was added at 0°C. After stirring at 0°C for 1 hour, the mixture was heated to 25°C and stirred for 14 hours. The reaction was completed by thin layer chromatography (petroleum ether: ethyl acetate = 3:1). The reaction solution was quenched with saturated aqueous ammonium chloride solution (100 mL), extracted with ethyl acetate (80 mL*2), and the organic phase was dried over anhydrous sodium sulfate and purified by column chromatography (silica, petroleum ether: ethyl acetate = 3:1) to obtain the title compound (2.82 g).

Step 2: Synthesis of 4-(methoxymethylene)cyclohexanone

Dissolve 4-(methoxymethylene)cyclohexanol (300 mg, 2.11 mmol) in acetonitrile (10 mL), add 2-iodobenzoic acid (886.16 mg, 3.16 mmol). The reaction solution is white and turbid and stirred at 80°C for 3 hours. The reaction is complete by thin layer chromatography (petroleum ether: ethyl acetate = 3:1). Filter the reaction solution, and concentrate the filtrate to dryness under reduced pressure to obtain the title compound (340 mg).

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 6.06 (s, 1H), 3.58-3.50 (m, 3H), 2.44-2.38 (m, 2H), 2.29-2.24 (m, 6H).

Step 3: Synthesis of (E)-[-4-(methoxymethylene)cyclohex-1-en-1-yl] trifluoromethanesulfonate

Dissolve 4-(Methoxymethylene)cyclohexanone (340 mg, 2.43 mmol) in anhydrous tetrahydrofuran (10 mL), add potassium di(trimethylsilyl)amide (1 M, 2.91 mL) at -78 °C under nitrogen atmosphere, then stir at -78 °C for half an hour, then add N-phenylbis(trifluoromethanesulfonyl)imide (1.04 g, 2.91 mmol) in anhydrous tetrahydrofuran (10 mL) solution at -78 °C under nitrogen atmosphere. The reaction solution was stirred at -78 °C under nitrogen atmosphere for 2 hours. Thin layer chromatography (petroleum ether: ethyl acetate = 3: 1) detected the disappearance of the raw material. The reaction solution was quenched with saturated aqueous ammonium chloride solution (5 mL), extracted with ethyl acetate (5 mL*3) and water (10 mL), and the organic phase was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure, and purified by column chromatography (silica, petroleum ether: ethyl acetate = 3: 1) to obtain the title compound (450 mg).

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 6.13-6.01 (m, 1H), 5.94-5.83 (m, 1H), 3.53 (d, J = 4.0Hz, 3H), 2.85 (brd, J = 1.9 Hz,1H),2.73(br d,J＝2.5Hz,1H),2.41-2.36(m,1H),2.33(br d,J＝4.8Hz,2H),2.27-2.22(m,1H).

Step 4: Synthesis of (E)-2-[4-(methoxymethylene)cyclohex-1-en-1-yl]-4,4,5,5-tetramethyl-1,3,2-dioxaborane

(E)-[-4-(Methoxymethylene)cyclohex-1-ene-1-yl] trifluoromethanesulfonate (450 mg, 1.65 mmol) and bis-naphthalene borate (629.62 mg, 2.48 mmol) were dissolved in anhydrous dioxane (15 mL), and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (67.49 mg, 82.65 μmol) and potassium acetate (324.45 mg, 3.31 mmol) were added. The reaction solution turned black and stirred at 80 ° C for 16 hours under a nitrogen atmosphere. The reaction was completed by thin layer chromatography (petroleum ether: ethyl acetate = 5: 1). The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silicon dioxide, petroleum ether: ethyl acetate = 5: 1) to obtain the title compound (353 mg).

MS m/z(ESI):251.2[M+H] ⁺ .

Step 5: Synthesis of (E)-2-[3-amino-1-[1-[5-[-4-(methoxymethylene)cyclohex-1-en-1-yl]pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

2-[3-amino-1-[1-(5-bromopyrimidin-2-yl)piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (390 mg, 834.54 μmol) and (E)-2-[4-(methoxymethylene)cyclohex-1-ene-1-yl]-4,4,5,5-tetramethyl-1,3,2-dioxaborane (313.13 mg, 1.25 mmol) were dissolved in anhydrous dioxane (12 mL) and water (3 mL), and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (68.15 mg, 83.45 μmol) and cesium carbonate (543.82 mg, 1.67 mmol) were added. The reaction solution was black and stirred at 90°C for 3 hours under a nitrogen atmosphere. The reaction was complete by LCMS detection. The reaction solution was diluted with water (5 mL) and extracted three times with ethyl acetate (20 mL). The organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether:tetrahydrofuran = 1:1) to give the title compound (350 mg).

MS m/z(ESI):511.3[M+H] ⁺ .

Step 6: Synthesis of 2-[3-amino-1-[1-[5-[4-(dimethoxymethyl)cyclohex-1-en-1-yl]pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

(E)-2-[3-amino-1-[1-[5-[-4-(methoxymethylene)cyclohex-1-ene-1-yl]pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (300 mg, 352.53 μmol) was dissolved in anhydrous methanol (10 mL), and p-toluenesulfonic acid (60.71 mg, 352.53 μmol) was added. The reaction solution was stirred at 60°C for 12 hours. LCMS detected that the reaction was complete. The reaction solution was extracted with dichloromethane (6 mL*3) and saturated sodium bicarbonate solution (6 mL), and the organic phase was dried over anhydrous sodium sulfate, concentrated to dryness under reduced pressure, and purified by column chromatography (silicon dioxide, petroleum ether:tetrahydrofuran = 2:1) to obtain the title compound (90 mg).

MS m/z(ESI):543.2[M+H] ⁺ .

Step 7: Synthesis of 2-[3-amino-1-[1-[5-[4-(dimethoxymethyl)cyclohexyl]pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

2-[3-amino-1-[1-[5-[4-(dimethoxymethyl)cyclohex-1-ene-1-yl]pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (90 mg, 165.86 μmol) was dissolved in anhydrous methanol (2 mL) and anhydrous tetrahydrofuran (2 mL), and wet palladium carbon (100 mg, 10%) was added. The reaction solution was stirred at 25°C under a hydrogen (15 psi) atmosphere for 12 hours. LCMS detected that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (90 mg).

MS m/z(ESI):545.4[M+H] ⁺ .

Step 8: Synthesis of 4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]cyclohexane-1-carbaldehyde

2-[3-amino-1-[1-[5-[4-(dimethoxymethyl)cyclohexyl]pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (85 mg, 156.06 μmol) was dissolved in anhydrous methanol (1 mL) and water (1 mL), and pyridine p-toluenesulfonate (78.44 mg, 312.13 μmol) was added. The reaction solution was brown and stirred at 60°C for 12 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness, extracted with ethyl acetate (1 mL*3) and water (5 mL), and the organic phase was dried over anhydrous sodium sulfate and concentrated to dryness under reduced pressure to obtain the title compound (40 mg).

MS m/z(ESI):499.3[M+H] ⁺ .

Step 9: Synthesis of (2S,4R)-1-[2-[3-[4-[[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]cyclohexyl]methyl]piperazin-1-yl]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide

4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]cyclohexane-1-carbaldehyde (30 1-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (18.15 mg, 30.09 μmol) and (2S,4R)-4-hydroxy-1-[3-methyl-2-(3-piperazine-1-yl)isoxazol-5-yl)butyryl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (18.15 mg, 30.09 μmol) were dissolved in N,N-dimethylformamide (0.8 mL) and anhydrous tetrahydrofuran (0.8 mL), and acetic acid (1.72 μL, 30.09 μmol), sodium acetate (4.94 mg, 60.17 μmol) and sodium triacetoxyborohydride (19.13 mg, 90.26 μmol) were added. The reaction solution was stirred at 25 ° C for 3 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. The product was purified by HPLC (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile ratio 30%-50%) as eluent) to give (2S,4R)-1-[2-[3-[4-[[4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]cyclohexyl]methyl]piperazin-1-yl]isoxazol-5-yl]-3-methylbutanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (1.6 mg).

MS m/z(ESI):1049.9[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.91 (br s, 1H), 9.02-8.96 (m, 1H), 8.53 (s, 1H), 8.43-8.20 (m, 3H), 8.11 (d, J＝7.7Hz,1H),7.51-7.29(m,5H),7.06-6.96(m,2H),6.45(s,2H),6.15(d,J＝7.0Hz,1H),5.19-4.97(m ,1H),4.95-4.86(m,2H),4.79(br d,J＝12.3Hz,2H),4.70-4.33(m,1H),4.28(br s,1H),3.70(br d,J＝8.4Hz,1H),3.62-3.50(m,1H),3.20-3.01(m,5H),2.57-2.52(m,3H),2.45(d,J＝2.6Hz,3H),2.44 -2.32(m,5H),2.27(br d,J＝6.6Hz,1H),2.14(br dd,J＝6.6,16.1Hz,2H),2.03(br d,J＝11.9Hz,1H),1.94 (br s,6H),1.79(br d,J＝6.2Hz,3H),1.58(br s,2H),1.46(br d,J＝6.6Hz,2H),1.43-1.33(m,3H), 0.95(br dd,J＝3.0,6.5Hz,3H),0.84-0.75(m,3H).

Example 142: Synthesis of Compound 429

Step 1: Synthesis of benzyl 4-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate

6-Chloro-1H-pyrazolo[4,3-c]pyridazine-3-amine (5 g, 24.27 mmol) was dissolved in N,N-dimethylformamide (100 mL), cesium carbonate (23.73 g, 72.80 mmol) was added, and the mixture was stirred for 20 minutes. Then, 4-methanesulfonyloxypiperidine-1-carboxylic acid benzyl ester (15.21 g, 48.54 mmol) was added, and the mixture was heated to 80°C for 4 hours. After the reaction was completed as monitored by LC-MS, water (100 mL) was added, and the mixture was extracted with ethyl acetate (150 mL*3). The organic phases were combined, dried, filtered, and the filtrate was concentrated. The title compound (7.30 g) was obtained by column chromatography (silica, petroleum ether/ethyl acetate: 1/1).

MS m/z(ESI):387.10[M+H] ⁺

Step 2: Synthesis of benzyl 4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate

Benzyl 4-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidine-1-carboxylate (1.20 g, 3.10 mmol), 2-(methoxymethoxyphenyl)boronic acid (1.13 g, 6.20 mmol) and potassium phosphate (2.63 g, 12.41 mmol) were dissolved in 1,4-dioxane (30 mL) and water (6 mL); after replacing the reaction flask with nitrogen, [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (253 mg, 310.10 μmol) was added, and the temperature was raised to 100°C to react overnight; after the reaction was complete as monitored by LC-MS, the liquids were separated, the organic phase was concentrated to dryness under reduced pressure, and the residue was purified by column chromatography (petroleum ether/ethyl acetate: 1/1) to obtain the title compound (960 mg).

MS m/z(ESI):489.10[M+H] ⁺

Step 3: Synthesis of 6-(2-(methoxymethoxy)phenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-amine

Dissolve 4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidine-1-carboxylic acid benzyl ester (560mg, 1.15mmol) in methanol (10mL), add palladium carbon (56mg, 46.10μmol, 10% purity). The reaction apparatus was evacuated, replaced with hydrogen three times, and reacted at room temperature for 4 hours under a hydrogen atmosphere. After the reaction was complete as monitored by LC-MS, it was filtered and the filtrate was concentrated to obtain a crude product, namely the title compound (421mg).

MS m/z(ESI):355.10[M+H] ⁺

Step 4: Synthesis of tert-butyl 4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)-[1,4'-bipiperidinyl]-1'-carboxylate

6-(2-(Methoxymethoxy)phenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-amine (220 mg, 620.76 μmol) and tert-butyl 4-oxopiperidine-1-carboxylate (495 mg, 2.48 mmol) were dissolved in tetrahydrofuran (6 mL) and N,N-dimethylformamide (6 mL). Acetic acid (111 mg, 1.86 mmol) and sodium acetate (153 mg, 1.86 mmol) were added, and the mixture was stirred at room temperature for 1 hour. Then, sodium triacetoxyborohydride (395 mg, 1.86 mmol) was added and the reaction was allowed to react overnight. After the reaction was completed as monitored by LC-MS, water (6 mL) was added to quench the reaction, and the mixture was extracted with ethyl acetate (10 mL*3). The organic phases were combined, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to dryness and the residue was purified by column chromatography (silica, petroleum ether/ethyl acetate: 1/1) to obtain the title compound (165 mg).

MS m/z(ESI):538.20[M+H] ⁺

Step 5: Synthesis of 2-(1-([1,4'-bipiperidinyl]-4-yl)-3-amino-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

Dissolve tert-butyl 4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)-[1,4'-bipiperidine]-1'-carboxylate (157 mg, 292.01 μmol) in dichloromethane (3 mL) and methanol (1.5 mL), add a solution of hydrogen chloride in 1,4-dioxane (4M, 3.5 mL), and react at room temperature overnight; after the reaction is complete as monitored by LC-MS, concentrate the reaction solution, add dichloromethane (5 mL) and saturated sodium bicarbonate solution (10 mL) in sequence, and adjust the pH to alkaline; then add dichloromethane for extraction (5 mL×3), combine the organic phases, dry, filter, and concentrate the filtrate to obtain the crude product, i.e., the title compound (69 mg).

MS m/z(ESI):394.20[M+H] ⁺

Step 6: Synthesis of compound 429

2-(1-([1,4'-bipiperidinyl]-4-yl)-3-amino-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (40 mg, 101.66 μmol) and (2S,4R)-1-((R)-2-(3-(4-formylpiperidin-1-yl)isoxazol-5-yl)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (24 mg, 40.66 μmol) were dissolved in N,N-dimethylformamide (1.5 mL); acetic acid (609 μg, 10.17 μmol) was added thereto, and the mixture was stirred at room temperature for 10 minutes, and then sodium triacetoxyborohydride (64 mg, 304.97 μmol) was added, and the mixture was stirred at room temperature for 2 hours. After the reaction was completed as monitored by LC-MS, the product was purified by high performance preparative liquid chromatography (Phenomenex C18, 5 μm silica, 30 mm diameter, 80 mm length; using a mixture of water (containing 0.05% ammonium bicarbonate) and acetonitrile (acetonitrile content: 60%-70%) as the eluent) to give compound 429 (10.4 mg).

MS m/z (ESI): 971.50 [M+H] ⁺ ;

¹ H NMR (400MHz, Methanol-d ₄ ) δ8.85–8.64(m,1H),8.27–8.05(m,1H),8.00–7.79(m,1H),7.42–7.17(m,5H),7.00 –6.76(m,2H),6.15–5.86(m,1H),4.52–4.25(m,3H),3.61–3.48(m,3H),3.12–2.92(m,3H),2.83–2. 66(m,2H),2.44–2.33(m,5H),2.26–2.08(m,5H),1.95–1.80(m,6H),1.74–1.65(m,2H),1.58–1.50(m,2H ),1.45–1.41(m,2H),1.23–1.14(m,8H),1.00–0.92(m,3H),0.86–0.73(m,6H).

Example 143: Synthesis of Compound 430

Step 1: Synthesis of tert-butyl 4-((4-(3-(isopropylamino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)methyl)piperidine-1-carboxylate

N-isopropyl-6-(2-(methoxymethoxy)phenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-amine (113 mg, 285.00 μmol) and tert-butyl 4-formylpiperidine-1-carboxylate (91 mg, 427.51 μmol) were dissolved in tetrahydrofuran (5 mL); acetic acid (1.7 mg, 28.50 μmol) was added, followed by sodium triacetoxyborohydride (181 mg, 855.01 μmol) added in batches, and reacted at room temperature for 2 hours; after the reaction was completed as detected by LC-MS, water (5 mL) was added to quench the reaction, and after concentrating to remove tetrahydrofuran, the mixture was extracted and concentrated with dichloromethane (5 mL*3), and the title compound (132 mg) was purified by column chromatography (silica, dichloromethane/methanol: 10/1) to obtain the title compound (132 mg).

MS m/z(ESI):594.40[M+H] ⁺

Step 2: Synthesis of 2-(3-(isopropylamino)-1-(1-(piperidin-4-ylmethyl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

Dissolve tert-butyl 4-((4-(3-(isopropylamino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)methyl)piperidine-1-carboxylate (98 mg, 165.72 μmol) in dichloromethane/methanol (2/1, 2 mL); add hydrochloric acid in 1,4-dioxane (4 M, 2 mL) and stir at room temperature for 2 hours; after the reaction is completed as monitored by LC-MS, concentrate to obtain the crude product, namely the title compound (52 mg).

MS m/z(ESI):450.20[M+H] ⁺

Step 3: Synthesis of compound 430

2-(3-(Isopropylamino)-1-(1-(piperidin-4-ylmethyl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (30 mg, 66.73 μmol) and (2S,4R)-1-((R)-2-(3-(4-formylpiperidin-1-yl)isoxazol-5-yl)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazole- 5-(5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (50 mg, 66.73 μmol) was dissolved in N,N-dimethylformamide (1.5 mL); acetic acid (400 μg, 6.67 μmol) was added, stirred for 10 minutes, and then sodium triacetoxyborohydride (42 mg, 200.18 μmol) was added, and the reaction was reacted at room temperature for 2 hours; after the reaction was completed as monitored by LC-MS, it was purified by high performance liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length; a mixture of water (containing 0.05% ammonia water) and acetonitrile with decreasing polarity (acetonitrile ratio 65%-85%) was used as the eluent) to obtain compound 430 (10 mg).

MS m/z(ESI):1027.50[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.09-13.91 (m, 1H), 8.98 (s, 1H), 8.48 (s, 1H), 8.41 (br d, J = 7.5Hz, 1H), 8.12 (br d,J＝7.8Hz,1H),7.48-7.40(m,2H),7.40-7.30(m,3H),7.03-6.95(m,2H),6.88(br d,J＝7.5Hz,1H ),6.14-5.96(m,1H),5.15-4.99(m,1H),4.96-4.86(m,1H),4.62-4.48(m,1H),4.36(br t,J＝7.9Hz,1H) ,4.28(br s,1H),4.10-3.97(m,1H),3.71(br d,J＝6.6Hz,1H),3.66-3.54(m,3H),2.96(br d,J＝8.3Hz,2H),2.81 (brd,J＝9.5Hz,2H),2.73(br t,J＝11.3Hz,2H),2.45(s,3H),2.29-1.96(m,11H),1.92-1.77(m,5H),1.75 -1.63(m,5H),1.48-1.42(m,1H),1.40-1.33(m,3H),1.27(br d,J＝6.3Hz,6H),1.23-1.21(m,1H),1.11( br d,J＝8.1Hz,3H),0.94(br d,J＝6.3Hz,3H),0.85-0.76(m,3H).

Example 144: Synthesis of Compound 431

Step 1: Synthesis of benzyl 4-((2-chloropyrimidin-5-yl)oxy)piperidine-1-carboxylate

2-Chloro-5-hydroxypyrimidine (2.6 g, 20 mmol) and benzyl 4-(methylsulfonyloxy)piperidine-1-carboxylate (6.3 g, 20 mmol) were dissolved in DMF (50 mL), and potassium carbonate (5.6 g, 40 mmol) was added. The mixture was reacted at 100 °C for 6 h. Water (200 mL) and ethyl acetate (200 mL×3) were added to the reaction solution for extraction. The organic phases were combined and concentrated under reduced pressure to obtain 6.5 g of a crude product of the title compound, which was used directly in the next step without purification.

MS m/z(ESI):348.10[M+H] ⁺ .

Step 2: Synthesis of benzyl 4-((2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl)oxy)piperidine-1-carboxylate

Benzyl 4-((2-chloropyrimidin-5-yl)oxy)piperidine-1-carboxylate (6.5 g, 18.7 mmol) and 4-hydroxypiperidine (3.8 g, 38 mmol) were dissolved in DMF (80 mL), and potassium carbonate (5.6 g, 40 mmol) was added. The mixture was reacted at 100° C. for 12 h. Water (200 mL) and ethyl acetate (200 mL×3) were added to the reaction solution for extraction. The organic phases were combined and concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA=100%-20%) to obtain the title compound (5.4 g).

MS m/z(ESI):413.10[M+H] ⁺ .

Step 3: Synthesis of 1-(5-(piperidin-4-yloxy)pyrimidin-2-yl)piperidin-4-ol

Benzyl 4-((2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl)oxy)piperidine-1-carboxylate (5.4 g) was dissolved in methanol (100 mL), palladium on carbon (500 mg) was added, and the mixture was exchanged with hydrogen three times. The reaction was then continued in a hydrogen atmosphere until the raw material was completely consumed. The reaction solution was filtered and the filtrate was concentrated to obtain the title compound (3.2 g).

MS m/z(ESI):279.10[M+H] ⁺ .

Step 4: Synthesis of 3-(4-((2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl)oxy)piperidin-1-yl)-5-(1-methoxy-3-methyl-1-oxobutan-2-yl)-2-methylisoxazolium trifluoromethanesulfonate

3-Bromo-5-(1-methoxy-3-methyl-1-oxobutan-2-yl)-2-methylisoxazol-2-ium trifluoromethanesulfonate (2.15 g, 5 mmol) was dissolved in methanol (30 mL), N,N-diisopropylethylamine (5 mL) was added, and the mixture was cooled to 0°C. Then, a methanol solution (10 mL) of 1-(5-(piperidin-4-yloxy)pyrimidin-2-yl)piperidin-4-ol (2 g) was added. After the addition was completed, the mixture was heated to room temperature and the reaction was continued for 16 h. The reaction solution was concentrated under reduced pressure to obtain a crude product of the title compound (8 g), which was used directly in the next step without purification.

MS m/z(ESI):474.20[M+H] ⁺ .

Step 5: Synthesis of methyl 2-(3-(4-((2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl)oxy)piperidin-1-yl)isoxazol-5-yl)-3-methylbutanoate

3-(4-((2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl)oxy)piperidin-1-yl)-5-(1-methoxy-3-methyl-1-oxobutan-2-yl)-2-methylisoxazolium trifluoromethanesulfonate (8 g) was added to toluene (100 mL), and the reaction was carried out at 100° C. for 36 h. The reaction solution was concentrated under reduced pressure, and water (100 mL) and ethyl acetate (100 mL×3) were added to the residue for extraction. The organic phases were combined and concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA=100%-60%) to obtain the title compound (1.1 g).

MS m/z(ESI):460.20[M+H] ⁺ .

Step 6: Synthesis of methyl 3-methyl-2-(3-(4-((2-(4-((methylsulfonyl)oxy)piperidin-1-yl)pyrimidin-5-yl)oxy)piperidin-1-yl)isoxazol-5-yl)butanoate

Dissolve methyl 2-(3-(4-((2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl)oxy)piperidin-1-yl)isoxazol-5-yl)-3-methylbutanoate (1.1 g, 2.4 mmol) in DCM (30 mL), add triethylamine (1.1 g, 10 mmol), cool to 0°C, and then slowly add methanesulfonyl chloride (541 mg, 4.8 mmol). After the addition is complete, slowly warm to room temperature and react for 1 h. Then add water (50 mL) and DCM (50 mL×3) for extraction. Combine the organic phases and concentrate under reduced pressure. The residue is purified by column chromatography (PE:EA=100%-30%) to obtain the title compound (1.0 g).

MS m/z(ESI):538.10[M+H] ⁺ .

Step 7: Synthesis of methyl 2-(3-(4-((2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)oxy)piperidin-1-yl)isoxazol-5-yl)-3-methylbutanoate

Methyl 3-methyl-2-(3-(4-((2-(4-((methylsulfonyl)oxy)piperidin-1-yl)pyrimidin-5-yl)oxy)piperidin-1-yl)isoxazol-5-yl)butanoate (600 mg, 1.1 mmol) and 6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-3-amine (300 mg, 1.1 mmol) were dissolved in DMF (5 mL), and then cesium carbonate (750 mg, 2 mmol) was added, and nitrogen was replaced three times. The reaction was carried out at 80° C. for 12 h, and then water (60 mL) and ethyl acetate (60 mL×3) were added to the reaction system for extraction. The organic phases were combined and concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA=100%-0%) to obtain the title compound (90 mg).

MS m/z(ESI):713.30[M+H] ⁺ .

Step 8: Synthesis of 2-(3-(4-((2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)oxy)piperidin-1-yl)isoxazol-5-yl)-3-methylbutanoic acid

Methyl 2-(3-(4-((2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)oxy)piperidin-1-yl)isoxazol-5-yl)-3-methylbutanoate (90 mg, 0.11 mmol) was dissolved in a mixed solvent of THF/ _H2O (3 mL/1 mL), and then an aqueous solution (1 mL) of lithium hydroxide monohydrate (21 mg, 0.5 mmol) was added. The mixture was reacted at room temperature overnight, and 0.1 M dilute hydrochloric acid was added to adjust the pH to 7. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse phase chromatography (chromatographic column: C18 spherical, 40-60 μm, 100A, 40 g; eluent: acetonitrile/water = 5%-95%) to give the title compound (15 mg).

MS m/z(ESI):699.30[M+H] ⁺ .

Step 9: Synthesis of (2S,4R)-1-(2-(3-(4-((2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)oxy)piperidin-1-yl)isoxazol-5-yl)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

2-(3-(4-((2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)oxy)piperidin-1-yl)isoxazol-5-yl)-3-methylbutanoic acid (15 mg, 0.02 mmol) and (2S,4R)-4-hydroxy-N-[(1S)-1-[4-(4-methyl-1,3-thiazol-5-yl)phenyl]ethyl]tetrahydropyrrole-2-carboxamide (14 mg, 0.04 mmol) were dissolved in DMF (1 mL), and HATU (15.4 mg, 0.04 mmol) was added. The mixture was reacted at room temperature overnight. Water (10 mL) and ethyl acetate (10 mL×3) were added to the reaction solution for extraction. The organic phases were combined and concentrated under reduced pressure to obtain the title compound (45 mg), which was used directly in the next step without purification.

MS m/z(ESI):506.8[M+2H] ²⁺ .

Step 10: Synthesis of compound 431

(2S,4R)-1-(2-(3-(4-((2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)oxy)piperidin-1-yl)isoxazol-5-yl)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (45 mg) was dissolved in methanol (1 mL), and then a 4M hydrochloric acid dioxane solution (1 mL) was added and reacted at room temperature for 1 h. The reaction was complete when LC-MS detected that the reaction was complete. The reaction solution was concentrated under reduced pressure and the residue was purified by preparative chromatography (Boston Prime C18 column: 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile of decreasing polarity (acetonitrile ratio 44%-84%) as the eluent) to purify compound 431 (2.2 mg).

MS m/z(ESI):484.7[M+2H] ²⁺ .

¹ H NMR (400MHz, CD ₃ OD) δ8.88-8.90(m,1H),8.32(s,1H),8.22(s,1H),8.20(s,1H),8.03-8.05(m,1H) ,7.44–7.49(m,3H),7.36–7.41(m,2H),7.01–7.04(m,2H),6.16-6.18(m,1H),5.04-5.07(m,1H),4.60-4.62( m,1H),4.53-4.56(m,1H),4.45-4.47(m,1H),3.86-3.88(m,1H),3.72-3.77(m,2H),3.66-3.71(m,2 H),3.64-3.66(m,1H),3.61-3.64(m,2H),3.59–3.61(m,1H),3.43–3.45(m,1H),3.17–3.21(m,2H),2.48- 2.52(m,3H),2.40–2.43(m,1H),2.17–2.21(m,2H),2.05-2.09(m,2H),1.98-2.01(m,2H),1.80-1.84(m,2H ),1.51-1.57(m,3H),1.31-1.33(m,2H),1.06-1.11(m,3H),0.91-0.96(m,3H).

Example 145: Synthesis of Compound 432

Step 1: Synthesis of 2-(4-hydroxypiperidin-1-yl)pyrimidine-5-carbaldehyde

2-Chloropyrimidine-5-carboxaldehyde (2.84 g, 20 mmol) and 4-hydroxypiperidine (2.02 g, 20 mmol) were dissolved in acetonitrile (100 mL), and triethylamine (3.03 g, 30 mmol) was added. The mixture was reacted at 90 °C for 12 h and concentrated under reduced pressure to obtain the title compound (6.5 g), which was used directly in the next step without purification.

MS m/z(ESI):208.10[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-((2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl)methyl)piperazine-1-carboxylate

2-(4-Hydroxypiperidin-1-yl)pyrimidine-5-carboxaldehyde (6.5 g, 20 mmol) and tert-butyl piperazine-1-carboxylate (3.8 g, 20 mmol) were dissolved in DCM (80 mL), and then sodium triacetoxyborohydride (6.4 g, 30 mmol) was added. The mixture was reacted at room temperature for 5 h. Saturated aqueous ammonium chloride solution (200 mL) and DCM (200 mL×3) were added to the reaction solution for extraction. The organic phases were combined and concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA=100%-20%) to obtain the title compound (5.4 g).

MS m/z(ESI):378.20[M+H] ⁺ .

Step 3: Synthesis of 1-(5-(piperazin-1-ylmethyl)pyrimidin-2-yl)piperidin-4-ol

Dissolve tert-butyl 4-((2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl)methyl)piperazine-1-carboxylate (5.4 g) in DCM (50 mL), cool to 0°C, then add 4M dioxane hydrochloride solution (20 mL). After the addition is complete, warm to room temperature and continue to react for 4 hours. The reaction solution is concentrated under reduced pressure to obtain a crude product of the title compound (8.2 g). Use it directly in the next step without purification.

MS m/z(ESI):278.10[M+H] ⁺ .

Step 4: Synthesis of 3-(4-((2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl)methyl)piperazin-1-yl)-5-(1-methoxy-3-methyl-1-oxobutan-2-yl)-2-methylisoxazolium trifluoromethanesulfonate

3-Bromo-5-(1-methoxy-3-methyl-1-oxobutan-2-yl)-2-methylisoxazol-2-ium trifluoromethanesulfonate (2.15 g, 5 mmol) was dissolved in methanol (30 mL), triethylamine (5 mL) was added, and the mixture was cooled to 0°C. Then, a methanol solution (20 mL) of 1-(5-(piperazin-1-ylmethyl)pyrimidin-2-yl)piperidin-4-ol (4 g) was added. After the addition was completed, the mixture was warmed to room temperature and the reaction was continued for 16 h. The reaction solution was concentrated under reduced pressure to obtain the title compound (9.5 g), which was used directly in the next step without purification.

MS m/z(ESI):473.30[M+H] ⁺ .

Step 5: Synthesis of methyl 2-(3-(4-((2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl)methyl)piperazin-1-yl)isoxazol-5-yl)-3-methylbutanoate

3-(4-((2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl)methyl)piperazin-1-yl)-5-(1-methoxy-3-methyl-1-oxobutan-2-yl)-2-methylisoxazolium trifluoromethanesulfonate (9.5 g) was added to toluene (150 mL), and the reaction was carried out at 100° C. for 36 h. The reaction solution was concentrated under reduced pressure, and water (100 mL) and ethyl acetate (100 mL×3) were added to the residue for extraction. The organic phases were combined and concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA=100%-60%) to obtain the title compound (500 mg).

MS m/z(ESI):459.10[M+H] ⁺ .

Step 6: Synthesis of methyl 3-methyl-2-(3-(4-((2-(4-((methylsulfonyl)oxy)piperidin-1-yl)pyrimidin-5-yl)methyl)piperazin-1-yl)isoxazol-5-yl)butanoate

Dissolve methyl 2-(3-(4-((2-(4-hydroxypiperidin-1-yl)pyrimidin-5-yl)methyl)piperazin-1-yl)isoxazol-5-yl)-3-methylbutanoate (500 mg, 1.1 mmol) in DCM (30 mL), add triethylamine (500 mg, 5 mmol), cool to 0°C, and then slowly add methanesulfonyl chloride (270 mg, 2.4 mmol). After the addition is complete, slowly warm to room temperature and react for 1 h. Then add water (50 mL) and DCM (50 mL×3) for extraction. Combine the organic phases and concentrate under reduced pressure. The residue is purified by column chromatography (PE:EA=100%-20%) to obtain the title compound (350 mg).

MS m/z(ESI):537.20[M+H] ⁺ .

Step 7: Synthesis of methyl 2-(3-(4-((2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)methyl)piperazin-1-yl)isoxazol-5-yl)-3-methylbutanoate

3-Methyl-2-(3-(4-((2-(4-((methylsulfonyl)oxy)piperidin-1-yl)pyrimidin-5-yl)methyl)piperazin-1-yl)isoxazol-5-yl)butanoic acid methyl ester (350 mg, 0.65 mmol) and 6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-3-amine (180 mg, 0.66 mmol) were dissolved in DMF (5 mL), and then cesium carbonate (422 mg, 1.3 mmol) was added, and nitrogen was replaced three times. The reaction was carried out at 90° C. for 12 h, and then water (60 mL) and ethyl acetate (60 mL×3) were added to the reaction system for extraction. The organic phases were combined and concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA=100%-0% & DCM:MeOH=10:1) to obtain the title compound (262 mg).

MS m/z(ESI):712.30[M+H] ⁺ .

Step 8: Synthesis of 2-(3-(4-((2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)methyl)piperazin-1-yl)isoxazol-5-yl)-3-methylbutanoic acid

Methyl 2-(3-(4-((2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)methyl)piperazin-1-yl)isoxazol-5-yl)-3-methylbutanoate (262 mg, 0.37 mmol) was dissolved in a mixed solvent of THF/ _H2O (3 mL/1 mL), and then an aqueous solution (1 mL) of lithium hydroxide monohydrate (84 mg, 2 mmol) was added. The mixture was reacted at room temperature overnight, and 0.1 M dilute hydrochloric acid was added to adjust the pH to 7. The reaction solution was concentrated under reduced pressure, and the residue was purified by reverse phase chromatography (chromatographic column: C18 spherical, 40-60 μm, 100A, 40 g; eluent: acetonitrile/water = 5%-95%) to give the title compound (100 mg).

MS m/z(ESI):698.30[M+H] ⁺ .

Step 9: Synthesis of (2S,4R)-1-(2-(3-(4-((2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)methyl)piperazin-1-yl)isoxazol-5-yl)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

2-(3-(4-((2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)methyl)piperazin-1-yl)isoxazol-5-yl)-3-methylbutanoic acid (100 mg, 0.14 mmol) and (2S,4R)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (47 mg, 0.14 mmol) were dissolved in DMF (1 mL). HATU (108 mg, 0.28 mmol) was then added and the mixture was reacted at room temperature overnight. Water (10 mL) and ethyl acetate (10 mL×3) were added to the reaction solution for extraction, and the organic phases were combined and concentrated under reduced pressure to obtain the title compound (70 mg), which was used directly in the next step without purification.

MS m/z(ESI):506.20[M+2H] ²⁺ .

Step 10: Synthesis of compound 432

(2S,4R)-1-(2-(3-(4-((2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)methyl)piperazin-1-yl)isoxazol-5-yl)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (70 mg) was dissolved in methanol (1 mL), and then a 4 M hydrochloric acid solution of dioxane (1 mL) was added. The reaction was carried out at room temperature for 1 h. LC-MS detected that the reaction was complete. The reaction solution was concentrated under reduced pressure. The residue was purified by preparative chromatography to give (2S,4R)-1-(2-(3-(4-((2-(4-(3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrimidin-5-yl)methyl)piperazin-1-yl)isoxazol-5-yl)-3-methylbutanoyl)-4-hydroxy-N-((S)-1-(4)-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (2.5 mg).

MS m/z(ESI):484.20[M+2H] ²⁺ .

¹ H NMR (400MHz, CD ₃ OD) δ8.85-8.88(m,1H),8.32-8.34(m,1H),8.30-8.32(m,1H),8.01-8.03(m,1H),7.40– 7.496(m,3H),7.32–7.39(m,3H),6.98–7.02(m,2H),6.11-6.13(m,1H),5.00-5.04(m,1H),4.57-4.61(m,2H ),4.49-4.53(m,1H),4.42-4.45(m,1H),3.82-3.85(m,1 H),3.69-3.75(m,2H),3.59-3.64(m,2H),3.45-3.47(m,1H),3.41-3.43(m,1H),3.16–3.20(m,2H),2.51- 2.62(m,5H),2.46–2.49(m,2H),2.11–2.20(m,4H),1.94-2.03(m,4H),1.47-1.53(m,3H),1.27-1.31(m,2H ),1.02-1.08(m,3H),0.87-0.93(m,3H).

Example 146: Synthesis of Compound 435

Step 1: Synthesis of tert-butyl 4-(2-chloropyrimidin-4-yl)-3,6-dihydropyridine-1(2H)-carboxylate

2,4-Dichloropyrimidine (300 mg, 2.01 mmol) and tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (622.66 mg, 2.01 mmol) were dissolved in dioxane (6 mL) and water (2 mL). Sodium carbonate (533.58 mg, 5.03 mmol) and 1,1-bis(diphenylphosphino)ferrocenepalladium chloride (147.34 mg, 201.37 μmol) were added under nitrogen atmosphere. The reaction solution was stirred at 80 ° C for 4 hours. LCMS showed that the reaction was completed. The reaction solution was concentrated to dryness under reduced pressure, then extracted with ethyl acetate (5 mL*3) and water (10 mL). The organic phase was concentrated to dryness under reduced pressure, and the concentrate was purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 2/1) to give the title compound (360 mg).

MS m/z(ESI):296.1[M+H] ⁺ ;

Step 2: Synthesis of tert-butyl 4-[2-[4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-4-yl]-3,6-dihydropyridine-1(2H)-carboxylate

Tert-butyl 4-(2-chloropyrimidin-4-yl)-3,6-dihydropyridine-1(2H)-carboxylate (150 mg, 507.16 μmol) and tert-butyl (6-(2-(methoxymethoxy)phenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-yl)carbamate (230.51 mg, 507.16 μmol) were dissolved in dimethyl sulfoxide (4 mL), N,N-diisopropylethylamine (262.18 mg, 2.03 mmol) was added, and the reaction solution was stirred at 100° C. for 12 hours. LCMS showed that the reaction was complete. The reaction solution was extracted with ethyl acetate (50 mL*3) and water (10 mL), and the organic phase was concentrated to dryness under reduced pressure. The concentrate was purified by column chromatography (silica, petroleum ether/tetrahydrofuran=1/0 to 3/2) to obtain the title compound (46 mg).

MS m/z(ESI):614.2[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-[2-[4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-4-yl]piperidine-1-carboxylate

4-[2-[4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]pyrimidin-4-yl]-3,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (45 mg, 73.32 μmol) was dissolved in methanol (1 mL), palladium carbon (30 mg, 73.32 μmol, 10% purity) and triethylsilane (85.26 mg, 733.25 μmol) were added, and the reaction solution was stirred at 25° C. for 12 hours. LCMS showed that the reaction was complete. The reaction solution was filtered and concentrated to give the title compound (45 mg).

MS m/z(ESI):616.2[M+H] ⁺ .

Step 4: Synthesis of 2-[3-amino-1-[1-[4-(piperidin-4-yl)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[2-[4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-4-yl]piperidine-1-carboxylic acid tert-butyl ester (45 mg, 73.08 μmol) was dissolved in methanol (2 mL), dioxane hydrochloride (4M, 182.71 μL) was added, and the reaction solution was stirred at 25° C. for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (35 mg).

MS m/z(ESI):472.2[M+H] ⁺ .

Step 5: Synthesis of compound 435

2-[3-amino-1-[1-[4-(piperidin-4-yl)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (35 mg, 68.90 μmol) and intermediate 2-1 (40.91 mg, 68.90 μmol) were dissolved in N,N-dimethylformamide (1 mL) and tetrahydrofuran (1 mL), and acetic acid (12.41 mg, 206.69 μmol), sodium acetate (16.96 mg, 206.69 μmol) and sodium acetate borohydride (43.81 mg, 206.69 μmol) were added. The reaction solution was reacted at 25 ° C for 1 hour. The reaction was completed by LCMS. The reaction solution was quenched by adding water (0.2 mL) and concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.225% formic acid) and acetonitrile (acetonitrile ratio 15%-55%) as eluent) gave compound 435 (15.7 mg).

MS m/z(ESI):1049.9[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.91 (br s, 1H), 8.98 (s, 1H), 8.54 (s, 1H), 8.43-8.33 (m, 1H), 8.29 (d, J＝ 5.0Hz,1H),8.11(d,J＝7.9Hz,1H),7.48-7.40(m,2H),7.40-7.33(m,3H),7.05-6.97(m,2H),6.58(d,J ＝5.0Hz,1H),6.44(s,2H),6.15-5.97(m,1H),5.11(br s,1H),4.97-4.80(m,4H),4.35(t,J＝7.9Hz,1H ),4.27(br s,1H),3.70(br d,J＝5.6Hz,1H),3.65-3.53(m,4H),3.12-3.00(m,3H),2.93(br d,J＝9.4Hz,2H),2.78 -2.69(m,2H),2.45(s,3H),2.30-2.11(m,4H),1.96(br s,7H),1.84-1.64(m,7H),1.37(br d,J＝7.0Hz ,3H),1.12(br d,J＝10.6Hz,2H),0.94(br d,J＝6.5Hz,3H),0.84-0.75(m,3H).

Example 147: Synthesis of Compound 436

Step 1: Synthesis of tert-butyl 4-(2-chloropyrimidin-4-yl)piperazine-1-carboxylate

2,4-Dichloropyrimidine (1 g, 6.71 mmol) and triethylamine (407.53 mg, 4.03 mmol) were dissolved in dichloromethane (3 mL), and added to a dichloromethane solution (3 mL) of tert-butyl piperazine-1-carboxylate (1.38 g, 7.38 mmol) at 0°C. The reaction solution was stirred at 0°C for 2 hours, and LCMS showed that the reaction was complete. The reaction solution was washed with water (10 mL) and then concentrated to dryness under reduced pressure, and the concentrate was purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 3/1) to obtain the title compound (230 mg).

MS m/z (ESI): 298.9 [M+H] ⁺ ;

Step 2: Synthesis of tert-butyl 4-[2-[4-[3-(tert-butoxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-4-yl]piperazine-1-carboxylate

Tert-butyl 4-(2-chloropyrimidin-4-yl)piperazine-1-carboxylate (72.31 mg, 242.01 μmol) and tert-butyl (6-(2-(methoxymethoxy)phenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-yl)carbamate (100 mg, 220.01 μmol) were dissolved in N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (85.30 mg, 660.04 μmol) was added. The reaction solution was stirred at 110° C. for 12 hours. LCMS showed that the reaction was complete. The reaction solution was extracted with ethyl acetate (30 mL*3) and water (5 mL), and the organic phase was concentrated to dryness under reduced pressure. The concentrate was purified by column chromatography (silica, petroleum ether/tetrahydrofuran=1/0 to 3/2) to obtain the title compound (41 mg).

MS m/z(ESI):717.3[M+H] ⁺ .

Step 3: Synthesis of 2-[3-amino-1-[1-(4-piperazin-1-ylpyrimidin-2-yl)piperidin-4-yl]pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[2-[4-[3-(tert-butyloxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-4-yl]piperazine-1-carboxylic acid tert-butyl ester (41 mg, 57.20 μmol) was dissolved in methanol (1 mL), and dioxane hydrochloride (4M, 142.99 μL) was added. The reaction solution was stirred at 25° C. for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (40 mg).

MS m/z(ESI):473.2[M+H] ⁺ .

Step 4: Synthesis of compound 436

2-[3-amino-1-[1-(4-piperazin-1-ylpyrimidin-2-yl)piperidin-4-yl]pyrazolo[4,3-c]pyridazin-6-yl]phenol (40 mg, 78.58 μmol) and intermediate 2-1 (46.66 mg, 78.58 μmol) were dissolved in N,N-dimethylformamide (1 mL) and tetrahydrofuran (1 mL), and acetic acid (14.16 mg, 235.75 μmol), sodium acetate (12.89 mg, 157.17 μmol) and sodium acetate borohydride (49.97 mg, 235.75 μmol) were added. The reaction solution was reacted at 25 ° C for 1 hour. The reaction was completed by LCMS. The reaction solution was quenched by adding water (0.2 mL) and concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile ratio 45%-85%) as eluent) gave compound 436 (6.2 mg).

MS m/z(ESI):1050.8[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.91 (s, 1H), 8.98 (s, 1H), 8.52 (s, 1H), 8.40 (d, J = 7.4Hz, 1H), 8.11 (d, J＝7.8Hz,1H),7.91(d,J＝6.0Hz,1H),7.48-7.41(m,2H),7.40-7.32(m,3H),7.06-6.96(m,2H),6.43(s ,2H),6.15-5.96(m,2H),5.16-5.00(m,1H),4.96-4.73(m,4H),4.35(t,J＝7.9Hz,1H),4.27(br s,1H),3.75-3.68(m,1H),3.65-3.50(m,8H),2.97(br t,J＝11.4Hz,2H),2.74(br t,J＝11.1Hz,2H),2.45 (s,3H),2.39(br s,4H),2.18(br d,J＝6.1Hz,3H),2.07-1.86(m,5H),1.83-1.65(m,4H),1.47-1.32(m ,3H),1.13(br d,J＝9.8Hz,2H),1.00-0.88(m,3H),0.84-0.74(m,3H).

Example 148: Synthesis of Compound 438

Step 1: Synthesis of trans-4-hydroxymethylcyclohexanecarboxylic acid methyl ester

Trans-1,4-cyclohexanedicarboxylic acid monomethyl ester (500 mg, 2.69 mmol) was dissolved in anhydrous tetrahydrofuran (8 mL), and borane tetrahydrofuran solution (1 M, 4.03 mL) was added at 0 ° C. The reaction solution was stirred at 25 ° C for 2 hours. Thin layer chromatography (petroleum ether: tetrahydrofuran = 1: 1) detected the disappearance of the raw material. The reaction solution was added with 2 mL of methanol at 0 ° C and then stirred at 50 ° C for 30 minutes, concentrated to dryness under reduced pressure, ethyl acetate (40 mL) and water (40 mL) were added, and then extracted three times with ethyl acetate (50 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain trans-4-hydroxymethylcyclohexanecarboxylic acid methyl ester (320 mg).

Step 2: Synthesis of trans-4-formylcyclohexanecarboxylic acid methyl ester

Dimethyl sulfoxide (435.53 μL, 5.57 mmol) was added to dichloromethane (1 mL), and oxalyl chloride (243.97 μL, 2.79 mmol) dichloromethane solution (1 mL) was added at -78 °C under nitrogen atmosphere. The mixture was stirred at -78 °C for 30 minutes, and then trans-4-hydroxymethylcyclohexanecarboxylic acid methyl ester (320 mg, 9.29 mmol) dichloromethane solution (1 mL) was added. The mixture was stirred at -78 °C for 30 minutes, and then triethylamine (1.29 mL, 9.29 mmol) was added. The reaction solution was stirred at -78 °C for 1 hour, then heated to 0 °C and stirred for 1 hour. Thin layer chromatography (petroleum ether: ethyl acetate = 1:1, nitrophenylhydrazine color development) was used to detect the disappearance of the raw material. The reaction solution was quenched by adding hydrochloric acid (1M, 2 mL), extracted with dichloromethane (10 mL) and water (10 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain trans-4-formylcyclohexanecarboxylic acid methyl ester (280 mg).

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 9.61-9.49 (m, 1H), 3.58-3.55 (m, 3H), 2.31-2.22 (m, 2H), 1.94-1.85 (m, 4H), 1.43 -1.34(m,2H),1.21-1.14(m,2H).

Step 3: Synthesis of trans-4-((4-(3-ethylamino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)methyl)cyclohexanecarboxylic acid methyl ester

Trans-4-formylcyclohexanecarboxylic acid methyl ester (100 mg, 587.53 μmol) and 2-[3-ethylamino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (198.22 mg, 528.77 μmol) were dissolved in anhydrous N,N-dimethylformamide (0.5 mL) and anhydrous tetrahydrofuran (0.5 mL), and sodium acetate (96.39 mg, 1.18 mmol), acetic acid (33.60 μL, 587.53 μmol) and sodium acetate borohydride (373.56 mg, 1.76 mmol) were added. The reaction solution was stirred at 25°C for 3 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silicon dioxide, petroleum ether: tetrahydrofuran = 2:1) to obtain the title compound (120 mg).

MS m/z(ESI):493.2[M+H] ⁺ .

Step 4: Synthesis of trans-4-[[4-[3-ethylamino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]cyclohexanecarboxylic acid

Trans-4-((4-(3-ethylamino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)methyl)cyclohexanecarboxylic acid methyl ester (120 mg, 243.60 μmol) was dissolved in anhydrous methanol (2 mL) and water (2 mL), and sodium hydroxide (19.49 mg, 487.20 μmol) was added. The reaction solution was stirred at 25°C for 3 hours. LCMS detected that the reaction was complete. The reaction solution was adjusted to pH = 7 with hydrochloric acid (1 M) and concentrated to dryness under reduced pressure to obtain the title compound (200 mg).

MS m/z(ESI):479.2[M+H] ⁺ .

Step 5: Synthesis of compound 438

trans-4-[[4-[3-ethylamino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]cyclohexanecarboxylic acid (160 mg, 167.16 μmol) and (2S,4R)-1-[(S)-2-amino-3,3-dimethyl-butanoyl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]cyclohexanecarboxylic acid (160 mg, 167.16 μmol) were added. Phenyl]ethyl]pyrrolidine-2-carboxamide (80.41 mg, 167.16 μmol) was dissolved in anhydrous dimethylformamide (3 mL), O-(7-azabenzotriazole-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (50.85 mg, 133.73 μmol) was added, and then N,N-diisopropylethylamine (87.35 μL, 501.48 μmol) was added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by preparative liquid chromatography (Boston Prime C18 column: 5 μm silica, 150 mm diameter, 30 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile with decreasing polarity (acetonitrile ratio 58%-78%) as eluent) to obtain compound 438 (20.4 mg).

MS m/z (ESI): 905.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.99 (s, 1H), 8.98 (s, 1H), 8.48 (s, 1H), 8.39 (d, J = 7.8Hz, 1H), 8.18-8.06 ( m,1H),7.66(br d,J＝9.0Hz,1H),7.46-7.40(m,2H),7.40-7.32(m,3H),7.07(t,J＝5.5Hz,1H),7.02- 6.95(m,2H),5.12(d,J＝3.5Hz,1H),5.00-4.83(m,1H),4.61-4.47(m,2H),4.43(t,J＝8.0Hz,1H),4.28 (br s,1H),3.66-3.53(m,2H),3.47-3.39(m,4H),3.34(br s,1H),2.96(br d,J＝8.5Hz,2H),2.45(s,3H) ,2.21-1.95(m,8H),1.88-1.61(m,8H),1.46(br d,J＝6.8Hz,1H),1.37(br d,J＝7.0Hz,3H),1.26(t,J =7.0Hz,3H),0.94(s,9H).

Example 149: Synthesis of Compound 442

2-Fluoro-6-[1-(piperidin-4-yl)-3-propylamino-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (20 mg, 49.15 μmol) and intermediate 2-1 (29.18 mg, 49.15 μmol) were dissolved in tetrahydrofuran (0.2 mL) and N,N-dimethylformamide (0.2 mL), and acetic acid (8.86 mg, 147.46 μmol), sodium triacetoxyborohydride (31.25 mg, 147.46 μmol) and sodium acetate (12.10 mg, 147.46 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was complete when monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by HPLC (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile with decreasing polarity (acetonitrile ratio 60%-80%) as the eluent) to give compound 442 (27.2 mg).

MS m/z (ESI): 948.4 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ )δ=14.83-14.65(m,1H),8.98(s,1H),8.55(br s,1H),8.47-8.35(m,1H),8.01-7.93( m,1H),7.44(br d,J＝8.0Hz,2H),7.40-7.31(m,3H),7.22-7.14(m,1H),7.02-6.92(m,1H),6.20-6.04(m,1H),5.18-4.99( m,1H),4.96-4.85(m,1H),4.62-4.52(m,1H),4.39-4.25(m,2H),3.75-3.68(m,1H),3.64(br d,J＝9.0Hz ,2H),3.56(br d,J＝9.9Hz,1H),3.42(br d,J＝10.1Hz,1H),3.03-2.96(m,2H),2.80-2.72(m,2H),2.46( br s,3H),2.21(br s,4H),2.11(br d,J＝1.1Hz,4H),1.84-1.74(m,5H),1.73-1.66(m,3H),1.46(br d,J ＝7.3Hz,1H),1.38(br d,J＝6.1Hz,3H),1.24-1.03(m,3H),0.95(br d,J＝6.5Hz,6H),0.79(br d,J＝6.3 Hz,3H).

Example 150: Synthesis of Compound 443

2-Fluoro-6-[3-[(2-hydroxy-2-methylpropyl)amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (25 mg, 62.43 μmol) and intermediate 2-1 (37.07 mg, 62.4 μmol) were dissolved in tetrahydrofuran (0.5 mL) and N,N-dimethylformamide (0.5 mL), and acetic acid (11.25 mg, 187.29 μmol) and sodium triacetoxyborohydride (39.69 mg, 187.29 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was complete when monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by HPLC (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile with decreasing polarity (acetonitrile ratio 53%-73%) as the eluent) to give compound 443 (10.5 mg).

MS m/z(ESI):978.3[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.75-14.48 (m, 1H), 9.01-8.94 (m, 1H), 8.57 (s, 1H), 8.44-8.35 (m, 1H), 7.96 (br d,J＝7.9Hz,1H),7.49-7.27(m,5H),7.02-6.92(m,1H),6.78-6.65(m,1H),6.15-5.98(m,1H),5.19-4.99( m,1H),4.96-4.86(m,1H),4.71-4.52(m,2H),4.44-4.20(m,2H),3.85-3.44(m,5H),3.04-2.93(m,2H), 2.76(br t,J＝11.8Hz,2H),2.45(s,3H),2.15(br s,9H),1.96-1.53(m,7H),1.47-1.36(m,3H),1.20(s,6H),1.15(br dd,J＝1.7,12.6Hz,2H),0.95(br d, J＝6.3Hz,3H),0.86-0.74(m,3H).

Example 151: Synthesis of Compound 444

Step 1: Synthesis of 2-[3-amino-1-[1-(5-bromopyrimidin-2-yl)piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]-6-fluorophenol

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]-6-fluorophenol (278.4 mg, 763.15 μmol) was dissolved in N,N-dimethylformamide (6 mL), and N-ethyldiisopropylamine (295.89 mg, 2.29 mmol, 398.78 μL) and 5-bromo-2-chloro-pyrimidine (177.14 mg, 915.78 μmol) were added. The reaction solution was stirred at 80 ° C for 2 hours. LCMS detected that the reaction was complete. After filtering, the reaction solution was concentrated to dryness under reduced pressure, diluted with water (20 mL) and extracted three times with ethyl acetate (60 mL), and the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness to obtain the title compound (260.7 mg).

MS m/z(ESI):485.1,487.1[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-[2-[4-[3-amino-6-(3-fluoro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]-3,6-dihydropyridine-1(2H)-carboxylate

2-[3-amino-1-[1-(5-bromopyrimidin-2-yl)piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]-6-fluorophenol (260 mg, 535.74 μmol) and tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (248.48 mg, 803.61 μmol) were dissolved in anhydrous dioxane (4 mL) and water (1 mL), and cesium carbonate (349.11 mg, 1.07 mmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (43.75 mg, 53.57 μmol) were added. The reaction solution was stirred at 100° C. under a nitrogen atmosphere for 2 hours. LCMS detected that the reaction was complete, and the reaction solution was diluted with water (10 mL) and extracted three times with ethyl acetate (30 mL). The organic phase was dried over anhydrous sodium sulfate and filtered, concentrated to dryness under reduced pressure, and purified by column chromatography (silica, petroleum ether:tetrahydrofuran = 1:1) to give the title compound (211.2 mg).

MS m/z(ESI):588.2[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-[2-[4-[3-amino-6-(3-fluoro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidine-1-carboxylate

4-[2-[4-[3-amino-6-(3-fluoro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]pyrimidin-5-yl]-3,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (211 mg, 244.16 μmol) was dissolved in anhydrous tetrahydrofuran (1 mL) and anhydrous methanol (1 mL), palladium/carbon (200 mg, 10% purity) was added, and the reaction solution was stirred at 25°C under a hydrogen atmosphere for 16 hours. LCMS detected that the reaction was complete. The reaction solution was filtered and concentrated to dryness under reduced pressure to obtain the title compound (199.6 mg).

MS m/z(ESI):590.3[M+H] ⁺ .

Step 4: Synthesis of 2-[3-amino-1-[1-[5-(piperidin-4-yl)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]-6-fluoro-phenol

4-[2-[4-[3-amino-6-(3-fluoro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidine-1-carboxylic acid tert-butyl ester (199.6 mg, 338.50 μmol) was dissolved in anhydrous methanol (3 mL), dioxane hydrochloride (4 M, 6.25 μL) was added, and the reaction solution was stirred at 25° C. for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (264 mg).

MS m/z(ESI):490.2[M+H] ⁺ .

Step 6: Synthesis of compound 444

Intermediate 2-1 (40 mg, 7 μmol), 2-[3-amino-1-[1-[5-(piperidin-4-yl)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]-6-fluoro-phenol (56.86 mg, 7.37 μmol) and anhydrous sodium acetate (16.58 mg, 202.11 μmol) were dissolved in anhydrous tetrahydrofuran (1 mL) and anhydrous N,N-dimethylformamide (1 mL), acetic acid (12.14 mg, 202.11 μmol, 11.56 μL) and sodium acetate borohydride (42.84 mg, 202.11 μmol) were added, and the reaction solution was stirred at 25°C for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Compound 444 (3.58 mg) was purified by HPLC (C18-1 column, 5 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile ratio 38%-78%) as eluent).

MS m/z(ESI):534.1[M+2H] ²⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.00-8.98 (m, 1H), 8.61 (s, 1H), 8.42 (d, J = 7.48Hz, 1H), 8.33 (s, 2H), 7.96 ( d,J＝8.14Hz,1H),7.48-7.42(m,2H),7.40-7.31(m,3H),7.00(td,J＝8.03,5.28Hz,1H),6.52(s,2H),6.12 (s,1H),5.13(d,J＝3.74Hz,1H),4.96-4.87(m,2H),4.80(br d,J＝12.32Hz,2H),4.37(t,J＝7.81Hz,1H ),4.29(br d,J＝3.30Hz,1H),3.72(br dd,J＝10.34,3.96Hz,1H),3.66-3.54(m,4H),3.12-3.02(m,2H),2.99-2.91(m,2H ),2.75(br t,J＝11.66Hz,2H),2.45(br s,4H),2.25-2.13(m,3H),2.07-1.90(m,8H),1.85-1.61(m,9H), 1.38(d,J＝6.82Hz,3H), 0.95(br d,J＝6.38Hz,3H), 0.79(d,J＝6.82Hz,3H).

Example 152: Synthesis of Compound 445

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]-6-fluorophenol (60 mg, 164.47 μmol) and intermediate 2-1 (97.65 mg, 164.47 μmol) were dissolved in tetrahydrofuran (0.5 mL) and N,N-dimethylformamide (0.5 mL), and acetic acid (29.63 mg, 493.41 μmol), sodium triacetoxyborohydride (104.57 mg, 493.41 μmol) and sodium acetate (40.48 mg, 493.41 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was complete when monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by HPLC (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia water) and acetonitrile with decreasing polarity (acetonitrile ratio 48%-68%) as the eluent) to give compound 445 (42.29 mg).

MS m/z (ESI): 906.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.81-14.54 (m, 1H), 8.98 (s, 1H), 8.61-8.53 (m, 1H), 8.40 (br d, J = 7.1Hz, 1H) ,7.97(br d,J＝7.9Hz,1H),7.47-7.41(m,2H),7.40-7.31(m,3H),7.03-6.94(m,1H),6.52(br s,2H),6.12 (s,1H),5.16-4.98(m,1H),4.97-4.87(m,1H),4.64-4.51(m,1H),4.36(br t,J＝7.8Hz,1H),4.32-4.24(m,1H),3.72(br dd,J＝3.7,10.4Hz,1H),3.63(br d,J＝11.0Hz,2H),3.56(br d,J＝9.8Hz,1H),3.43(br d,J＝10.1Hz,2H),3.05-2.93(m,2H),2.76(br t,J＝11.3Hz,2H),2.45(s,3H ),2.22(br d,J＝1.3Hz,3H),2.10(br s,3H),2.01(br dd,J＝4.3,8.8Hz,1H),1.87-1.68(m,6H),1.38(br d,J＝7.0Hz,3H),1.19-1.07(m,2H),0.95(brd,J＝6.5Hz,3H),0.83-0.74(m,3H).

Example 153: Synthesis of Compound 446

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]-4-fluorophenol hydrochloride (46.9 mg, 128.56 μmol) and intermediate 2-1 (91.60 mg, 154.27 μmol) were dissolved in anhydrous tetrahydrofuran (1.5 mL) and anhydrous N,N-dimethylformamide (1.5 mL), and sodium acetate borohydride (81.74 mg, 385.69 μmol), acetic acid (23.16 mg, 385.69 μmol) and sodium acetate (31.64 mg, 385.69 μmol) were added. The reaction solution was stirred at 25°C for 2 hours, and the reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile ratio 45%-85%) as eluent) gave compound 446 (33.5 mg).

MS m/z (ESI): 906.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 8.98 (s, 1H), 8.55 (s, 1H), 8.41 (d, J = 7.6Hz, 1H), 8.00 (dd, J = 3.0, 10.5Hz, 1H),7.48-7.41(m,2H),7.39-7.34(m,2H),7.20(br dd,J＝2.8,8.4Hz,1H),7.00(dd,J＝5.1,9.0Hz,1H), 6.48(br s,2H),6.12(s,1H),5.12(d,J＝3.5Hz,1H),4.91(br t,J＝7.1Hz,1H),4.54(br s,1H),4.37(t,J＝7.9Hz,1H),4.28(br s,1H),3.76-3.68(m,1H),3.63(br d,J＝11.5Hz,2H),3.56(d ,J＝9.8Hz,1H),3.42(br dd,J＝5.3,10.2Hz,2H),2.99(br d,J＝4.9Hz,2H),2.76(br t,J＝11.6Hz,2H), 2.27-2.16(m,4H),2.09(br s,4H),1.88-1.72(m,6H),1.46(d,J＝7.0Hz,1H),1.38(d,J＝7.0Hz,3H), 1.14(br d,J＝11.1Hz,3H),0.98-0.91(m,3H),0.83(br d,J＝6.6Hz,1H),0.79(d,J＝6.8Hz,3H).

Example 154: Synthesis of Compound 449

Step 1: Synthesis of tert-butyl 3-(2-chloropyrimidin-5-yl)oxyazetidine-1-carboxylate

2-Chloro-5-hydroxypyrimidine (300 mg, 2.30 mmol), 1-BOC-3-(hydroxy)azetidine (437.89 mg, 2.53 mmol) and triphenylphosphine (904.21 mg, 3.45 mmol) were dissolved in anhydrous toluene (6 mL), and diisopropyl azodicarboxylate (697.10 mg, 3.45 mmol) was added at 0 ° C under a nitrogen atmosphere. The reaction solution was stirred at 100 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (petroleum ether: ethyl acetate = 2: 1) to obtain the title compound (360 mg).

MS m/z(ESI):286.0[M+H] ⁺ ;

Step 2: Synthesis of tert-butyl 3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]oxyazetidine-1-carboxylate

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (132.41 mg, 381.80 μmol) and tert-butyl 3-(2-chloropyrimidin-5-yl)oxyazetidine-1-carboxylate (120 mg, 419.98 μmol) were dissolved in N,N-dimethylformamide (5 mL), and N,N-diisopropylethylamine (148.04 mg, 1.15 mmol) was added. The reaction solution was stirred at 80 ° C for 12 hours. LCMS detected that the reaction was complete. The reaction solution was extracted with ethyl acetate (5 mL) and water (5 mL), and the organic phase was concentrated to dryness under reduced pressure and purified by column chromatography (petroleum ether: ethyl acetate = 1: 1) to obtain the title compound (53 mg).

MS m/z(ESI):560.2[M+H] ⁺ .

Step 3: Synthesis of 2-[3-amino-1-[1-[5-(azetidin-3-yloxy)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

Dissolve tert-butyl 3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]oxyazetidine-1-carboxylate (40 mg, 71.48 μmol) in dichloromethane (0.5 mL), and add trifluoroacetic acid (81.50 mg, 714.77 μmol). The reaction solution was stirred at 25°C for 2 hours. LCMS monitored the reaction to be complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (30 mg).

MS m/z(ESI):460.2[M+H] ⁺ .

Step 4: Synthesis of compound 449

2-[3-amino-1-[1-[5-(azetidin-3-yloxy)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (30 mg, 52.31 μmol) and intermediate 2-1 (31.06 mg, 52.31 μmol) were dissolved in tetrahydrofuran (0.2 mL) and N,N-dimethylformamide (0.2 mL), and acetic acid (9.42 mg, 156.92 μmol), sodium triacetoxyborohydride (33.26 mg, 156.92 μmol) and sodium acetate (12.87 mg, 156.92 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was complete when monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by HPLC (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile with decreasing polarity (acetonitrile ratio 44%-84%) as the eluent) to give compound 449 (19.8 mg).

MS m/z (ESI): 1037.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.95-13.83 (m, 1H), 8.98 (s, 1H), 8.53 (s, 1H), 8.40 (br d, J = 7.8Hz, 1H), 8.18 -8.09(m,3H),7.48-7.41(m,2H),7.37(br d,J＝8.0Hz,3H),7.04-6.95(m,2H),6.44(br s,2H),6.11(s ,1H),5.14-4.99(m,1H),4.96-4.83(m,2H),4.81-4.75(m,1H),4.70(br d,J＝13.5Hz,2H),4.36(br t,J＝7.6Hz,1H),4.28(br s,1H),3.71(br t,J＝6.4Hz,3H),3.64-3.53(m,3H),3.42(br d,J＝10.0Hz, 1H),3.05(br t,J＝10.9Hz,2H),2.97(br t,J＝5.6Hz,2H),2.72(br t,J＝11.8Hz,2H),2.45(s,3H),2.35 (br d,J＝6.5Hz,2H),2.26-2.18(m,1H),2.03-1.89(m,5H),1.83-1.74(m,1H),1.70(br d,J＝11.8Hz,2H ),1.45(br d,J＝6.9Hz,1H),1.38(br d,J＝6.9Hz,3H),1.21-1.10(m,2H),0.94(br d,J＝6.3Hz,3H),0.84-0.74(m ,3H).

Example 155: Synthesis of Compound 453

Step 1: Synthesis of tert-butyl 6-(hydroxymethyl)-2-azaspiro[3.3]heptane-2-carboxylate

2-(tert-Butyloxycarbonyl)-2-azaspiro[3.3]heptane-6-carboxylic acid (150 mg, 621.68 μmol) was dissolved in tetrahydrofuran (4 mL), and borane tetrahydrofuran solution (1 M, 1.87 mL) was added at 0°C. The reaction solution was stirred at 25°C for 2 hours. Thin layer chromatography (petroleum ether: ethyl acetate = 3:1) showed that the reaction was complete. The reaction solution was cooled to 0°C, quenched by slowly adding ethanol (4 mL), then heated to 60°C, stirred for 0.5 hours, concentrated to dryness under reduced pressure, and then extracted with ethyl acetate (20 mL*3) and water (3 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (130 mg).

¹ H NMR (400MHz, CDCl ₃ ) δ4.78 (br s, 1H), 3.92 (s, 2H), 3.82 (s, 2H), 3.55 (d, J = 6.3Hz, 2H), 2.40-2.29 (m ,1H),2.28-2.17(m,2H),1.99-1.89(m,2H),1.42(s,9H).

Step 2: Synthesis of tert-butyl 6-formyl-2-azaspiro[3.3]heptane-2-carboxylate

6-(Hydroxymethyl)-2-azaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (120 mg, 527.94 μmol) was dissolved in dichloromethane (3 mL), and Dess-Martin periodinane (335.88 mg, 791.91 μmol) was added at 0°C. The reaction solution was stirred at 25°C for 2 hours, and thin layer chromatography (petroleum ether: ethyl acetate = 1:1) showed that the reaction was complete. The reaction solution was diluted with dichloromethane (50 mL) and washed with saturated sodium sulfite solution (5 mL*2), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The concentrate was purified by column chromatography (silicon dioxide, petroleum ether/ethyl acetate = 1/0 to 4/1) to obtain the title compound (60 mg).

Step 3: Synthesis of tert-butyl 6-[[4-[3-(ethylamino)-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]-2-azaspiro[3.3]heptane-2-carboxylate

6-Formyl-2-azaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (50 mg, 221.94 μmol) and 2-[3-(ethylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (91.52 mg, 244.14 μmol) were dissolved in N,N-dimethylformamide (2 mL) and tetrahydrofuran (2 mL), and acetic acid (39.98 mg, 665.83 μmol), sodium acetate (36.41 mg, 443.89 μmol) and sodium acetate borohydride (141.12 mg, 665.83 μmol) were added. The reaction solution was reacted at 25° C. for 1 hour. The reaction was completed by LCMS. The reaction solution was extracted with dichloromethane (30 mL*3) and water (8 mL), the organic phase was concentrated to dryness under reduced pressure, and the concentrate was purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 2/1) to give the title compound (87 mg).

MS m/z(ESI):548.3[M+H] ⁺ .

Step 4: Synthesis of 2-[1-[1-(2-azaspiro[3.3]heptane-6-ylmethyl)piperidin-4-yl]-3-ethylamino-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

6-[[4-[3-(ethylamino)-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]-2-azaspiro[3.3]heptane-2-carboxylic acid tert-butyl ester (30 mg, 54.78 μmol) was dissolved in dichloromethane (1 mL), trifluoroacetic acid (124.91 mg, 1.10 mmol) was added, and the reaction solution was stirred at 25° C. for 1 hour. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (24 mg).

MS m/z(ESI):448.2[M+H] ⁺ .

Step 5: Synthesis of phenyl N-[(1S)-1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidine-1-carbonyl]-2,2-dimethylpropyl]carbamate

(2S,4R)-1-[(2S)-2-amino-3,3-dimethyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (200 mg, 416 μmol, monohydrochloride) was dissolved in dichloromethane (2 mL) and pyridine (1 mL), cooled to 0°C, and then a solution of chloroformic acid phenol ester (65.1 mg, 416 μmol) in dichloromethane (2 mL) was slowly added dropwise. The reaction solution was stirred at 25°C for 2 hours. LCMS showed that the reaction was complete. The reaction solution was diluted with water (5 mL) and dichloromethane (5 mL), the aqueous phase was extracted twice with dichloromethane (5 mL), and the organic phase was washed twice with water (5 mL). The organic layer was collected, dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure and purified by thin layer chromatography (silica, petroleum ether/tetrahydrofuran = 1/1) to give the title compound (110 mg).

MS m/z(ESI):565.6[M+H] ⁺ ;

Step 6: Synthesis of compound 453

2-[1-[1-(2-azaspiro[3.3]heptane-6-ylmethyl)piperidin-4-yl]-3-ethylamino-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (24 mg, 53.62 μmol) and [(S)-1-[(2S,4R)-4-hydroxy-2-[[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidin-1-yl]-3,3-dimethyl-1-oxobutan-2-yl]carbamic acid phenyl ester (33.31 mg, 58.98 μmol) were dissolved in N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (34.65 mg, 268.11 μmol) was added. The reaction solution was reacted at 80° C. for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by HPLC (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile of decreasing polarity (acetonitrile ratio 48%-88%) as eluent) to give compound 453 (13.1 mg).

MS m/z (ESI): 918.4 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.98 (s, 1H), 8.50 (s, 1H), 8.40 (br d, J = 7.9Hz, 1H), 8.11 (d, J = 7.8Hz, 1H ),7.47-7.40(m,2H),7.39-7.31(m,3H),7.03(br t,J＝5.5Hz,1H),6.99-6.90(m,2H),5.56(br d,J＝9.4 Hz,1H),4.94-4.87(m,1H),4.56-4.39(m,2H),4.33-4.25(m,2H),3.94-3.80(m,3H),3.74(br d,J＝6.5Hz,2H),3.66-3.55(m,4H),2.92(br d,J＝5.1Hz,2H),2.45(s,3H),2.35(br s,2H),2.23(br s,2H),2.16-1.89(m,6H),1.87-1.70(m,5H),1.37(d,J＝6.9Hz,3H),1.25(t,J＝7.1Hz,3H),0.94-0.88 (m,9H).

Example 156: Synthesis of Compound 460

4-Fluoro-2-[3-[(2-hydroxy-2-methylpropyl)amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (30 mg, 74.92 μmol), (2S,4R)-1-[(2R)-2-[3-(4-formyl-1-piperidinyl)isoxazol-5-yl]-3-methyl-butyryl]-4-hydroxy-N-[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (30 mg, 74.92 μmol), ]ethyl]pyrrolidine-2-carboxamide (44.48 mg, 74.92 μmol) and anhydrous sodium acetate (18.44 mg, 224.75 μmol) were dissolved in anhydrous tetrahydrofuran (1 mL) and anhydrous N,N-dimethylformamide (1 mL), and acetic acid (13.50 mg, 224.75 μmol, 12.85 μL) and sodium acetate borohydride (47.63 mg, 224.75 μmol) were added, and the reaction solution was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. Purification by high performance liquid chromatography (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile with decreasing polarity (acetonitrile ratio 42%-72%) as eluent) gave compound 460 (32.2 mg).

MS m/z(ESI):978.7[M+H]+;

¹ H NMR (400MHz, DMSO-d6) δ13.78-13.73(m,1H),8.98(s,1H),8.54(s,1H),8.43(br d,J＝7.48Hz,1H),8.00( dd,J＝10.56,3.08Hz,1H),7.46-7.42(m,2H),7.39-7.35(m,2H),7.22(td,J＝8.36,3.08Hz,1H),7.00(dd,J＝ 8.80,5.06Hz,1H),6.67(br t,J＝5.83Hz,1H),6.13(s,1H),5.14(d,J＝3.74Hz,1H),4.92(br t,J＝7.26Hz,1H),4.68(s,1H),4.61-4.49(m,1H),4.37(t,J＝7.92Hz,1H),4.29(br s,1H),3.64(br d ,J＝11.88Hz,3H),3.57(br d,J＝9.90Hz,1H),3.00(br d,J＝7.26Hz,2H),2.76(br t,J＝11.33Hz,2H),2.46( s,3H),2.34-1.97(m,10H),1.89-1.64(m,7H),1.39(d,J＝7.04Hz,3H),1.21(s,6H),1.18-1.10(m,2H) ,0.95(br d,J＝6.60Hz,3H),0.85-0.78(m,3H).

Example 157: Synthesis of Compound 461

Step 1: Synthesis of 2-[3-amino-1-[1-(5-bromopyrimidin-2-yl)piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]-4-fluorophenol

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]-4-fluorophenol hydrochloride (390 mg, 1.19 mmol) and 2-chloro-5-bromopyrimidine (298.68 mg, 1.54 mmol) were dissolved in anhydrous dimethylformamide (4 mL), and N,N-diisopropylethylamine (517.22 μL, 2.97 mmol) was added. The reaction solution was stirred at 80°C for 12 hours. LCMS detected that the reaction was complete. The reaction solution was extracted with dichloromethane (50 mL) and water (200 mL), concentrated to dryness under reduced pressure, and purified by column chromatography (silicon dioxide, petroleum ether: tetrahydrofuran = 2:1) to obtain the title compound (380 mg).

MS m/z(ESI):485.0,486.9[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-[2-[4-[3-amino-6-(5-fluoro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]-3,6-dihydropyridine-1(2H)-carboxylate

2-[3-amino-1-[1-(5-bromopyrimidin-2-yl)piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazine-6-yl]-4-fluorophenol (360 mg, 741.79 μmol) and N-Boc-1,2,5,6-tetrahydropyridine-4-boronic acid naphthalene ester (275.24 mg, 890.15 μmol) were dissolved in anhydrous dioxane (8 mL) and water (3.2 mL), and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (60.58 mg, 74.18 μmol) and cesium carbonate (483.38 mg, 1.48 mmol) were added. The reaction solution was stirred at 90 ° C for 3 hours under a nitrogen atmosphere. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether:tetrahydrofuran = 1:1) to give the title compound (410 mg).

MS m/z(ESI):588.3[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-[2-[4-[3-amino-6-(5-fluoro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidine-1-carboxylate

4-[2-[4-[3-amino-6-(5-fluoro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]pyrimidin-5-yl]-3,6-dihydropyridine-1(2H)-carboxylic acid tert-butyl ester (410 mg, 697.70 μmol) was dissolved in anhydrous methanol (10 mL) and anhydrous tetrahydrofuran (10 mL), and wet palladium carbon (700 mg, 10% purity) was added. The reaction solution was stirred at 25°C in a hydrogen atmosphere (15 psi) for 16 hours. LCMS detected that the reaction was complete. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain the title compound (400 mg).

MS m/z(ESI):590.2[M+H] ⁺ .

Step 4: Synthesis of 2-[3-amino-1-[1-[5-(piperidin-4-yl)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]-4-fluoro-phenol hydrochloride

Dissolve tert-butyl 4-[2-[4-[3-amino-6-(5-fluoro-2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]pyrimidin-5-yl]piperidine-1-carboxylate (400 mg, 678.35 μmol) in anhydrous methanol (5 mL), and add dioxane hydrochloride (4M, 1.70 mL). The reaction solution turns red and is stirred at 25°C for 3 hours. LCMS detection of the reaction is complete. The reaction solution is concentrated to dryness under reduced pressure to obtain the title compound (400 mg).

MS m/z(ESI):490.2[M+H] ⁺ .

Step 7: Synthesis of compound 461

2-[3-amino-1-[1-[5-(piperidin-4-yl)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazine-6-yl]-4-fluoro-phenol hydrochloride (20 mg, 38.02 μmol) and intermediate 2-1 (22.58 mg, 38.02 μmol) were dissolved in N,N-dimethylformamide (0.5 mL) and anhydrous tetrahydrofuran (0.5 mL), and acetic acid (2.17 μL, 38.02 μmol), sodium acetate (6.24 mg, 76.04 μmol) and sodium triacetoxyborohydride (24.18 mg, 114.07 μmol) were added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile ratio 51%-91%) as eluent) gave compound 461 (7.8 mg).

MS m/z(ESI):534.5[M+2H] ²⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.73 (br s, 1H), 9.10-8.71 (m, 1H), 8.57 (s, 1H), 8.40 (br d, J = 7.6Hz, 1H), 8.32(s,2H),7.98(dd,J＝2.8,10.4Hz,1H),7.50-7.40(m,2H),7.40-7.33(m,2H),7.22(dt,J＝2.9,8.4Hz, 1H),7.01(dd,J＝5.1,8.9Hz,1H),6.47(br s,2H),6.17-5.95(m,1H),5.21-4.85(m,3H),4.80(br d,J＝12.8Hz,2H),4.71-4.33(m,1H),4.29(br s,1H),3.78-3.68(m,1H),3.67-3.53(m,3H),3.12-3.00(m ,2H),2.94(br d,J＝10.0Hz,2H),2.74(br t,J＝11.6Hz,2H),2.45(s,3H),2.41-2.34(m,1H),2.30-2.04( m,4H),2.03-1.87(m,7H),1.84-1.57(m,8H),1.48-1.34(m,3H),1.13(br d,J＝11.3Hz,2H),0.95(br d,J＝6.5Hz,3H),0.86-0.76(m,3H).

Example 158: Synthesis of Compound 473

Step 1: Synthesis of tert-butyl 3-(trifluoromethanesulfonyloxy)-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylate

N-Boc-nortropinone (2g, 8.88mmol) was dissolved in anhydrous tetrahydrofuran (30mL), potassium di(trimethylsilyl)amide (1M, 10.65mL) was added under a nitrogen atmosphere at -78°C, and then stirred at -78°C for half an hour, and then N-phenylbis(trifluoromethanesulfonyl)imide (3.81g, 10.65mmol) anhydrous tetrahydrofuran (20mL) solution was added under a nitrogen atmosphere at -78°C. The reaction solution was stirred at -78°C for 2 hours under a nitrogen atmosphere. The disappearance of the raw material was detected by thin layer chromatography (petroleum ether: ethyl acetate = 3:1). The reaction solution was quenched with saturated aqueous ammonium chloride solution (10 mL), extracted with ethyl acetate (20 mL*3) and water (60 mL), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether: ethyl acetate = 3:1) to give the title compound (3.5 g).

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 6.33 (d, J = 5.5Hz, 1H), 4.48-4.20 (m, 2H), 2.90 (br d, J = 15.2Hz, 1H), 2.18 (br s,2H),1.91(br s,2H),1.69(br d,J＝12.5Hz,1H),1.39(s,9H).

Step 2: Synthesis of tert-butyl 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylate

3-(Trifluoromethanesulfonyloxy)-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylic acid tert-butyl ester (2.5 g, 7.00 mmol) and bis-pinacol borate (2.66 g, 10.49 mmol) were dissolved in anhydrous dioxane (70 mL), and potassium acetate (1.37 g, 13.99 mmol) and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (285.66 mg, 349.80 μmol) were added. The reaction solution was stirred at 80 ° C for 16 hours under a nitrogen atmosphere. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, diluted with water (300 mL), and extracted with ethyl acetate (100 mL*3). The organic phase was dried over anhydrous sodium sulfate and purified by column chromatography (silicon dioxide, petroleum ether: tetrahydrofuran = 5:1) to obtain the title compound (1.36 g).

MS m/z(ESI):280.0[M+H-56] ⁺ .

Step 3: Synthesis of tert-butyl 3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylate

3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylic acid tert-butyl ester (242.11 mg, 577.76 μmol) and 2-[3-amino-1-[1-(5-bromopyrimidin-2-yl)-4-piperidinyl]pyrazolo[4,3-c]pyridazin-6-yl]phenol (180 mg, 385.17 μmol) were dissolved in anhydrous dioxane (0.8 mL) and water (0.2 mL), and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane (31.45 mg, 38.52 μmol) and cesium carbonate (250.99 mg, 770.35 μmol) were added. The reaction solution was stirred at 90° C. under a nitrogen atmosphere for 16 hours. The reaction was complete after LCMS detection. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silica, petroleum ether:tetrahydrofuran = 1:1) to obtain the title compound (110 mg).

MS m/z(ESI):596.2[M+H] ⁺ .

Step 4: Synthesis of tert-butyl 3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]-8-azabicyclo[3.2.1]octane-8-carboxylate

3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]pyrimidin-5-yl]-8-azabicyclo[3.2.1]oct-2-ene-8-carboxylic acid tert-butyl ester (110 mg, 184.66 μmol) was dissolved in anhydrous tetrahydrofuran (10 mL), and wet palladium carbon (200 mg, 10% purity) was added. The reaction solution was stirred at 40° C. for 8 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (89 mg).

MS m/z(ESI):598.2[M+H] ⁺ .

Step 5: Synthesis of 2-(1-(1-(5-(8-azabicyclo[3.2.1]octan-3-yl)pyrimidin-2-yl)piperidin-4-yl)-3-amino-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

3-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]pyrimidin-5-yl]-8-azabicyclo[3.2.1]octane-8-carboxylic acid tert-butyl ester (89 mg, 148.90 μmol) was dissolved in anhydrous methanol (2 mL), and dioxane hydrochloride (4M, 372.25 μL) was added. The reaction solution was stirred at 25 ° C for 3 hours when it turned red. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (120 mg).

MS m/z(ESI):498.2[M+H] ⁺ .

Step 6: Synthesis of compound 473

2-(1-(1-(5-(8-azabicyclo[3.2.1]octan-3-yl)pyrimidin-2-yl)piperidin-4-yl)-3-amino-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (55.35 mg, 67.37 μmol) and intermediate 2-1 (40 mg, 67.37 μmol) were dissolved in anhydrous tetrahydrofuran (1 mL) and N,N-dimethylformamide (1 mL), and acetic acid (67.37 μmol, 3.85 μL) and sodium acetate (11.05 mg, 134.74 μmol) were added. Ultrasonication was performed until color changed, and sodium triacetoxyborohydride (42.84 mg, 202.11 μmol) was added. The reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by high performance liquid chromatography (Phenomenex Purification by C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile ratio 18%-58%) as eluent) gave compound 473 (7.2 mg).

MS m/z (ESI): 1075.5 [M+H] ⁺ ;

¹ H NMR (400 MHz, DMSO-d ₆ )δ=13.98-13.78(m,1H),9.02-8.94(m,1H),8.84-8.75(m,1H),8.55-8.48(m,1H),8.48-8.31(m,2H),8.14-8.04(m,1H),7.42(s,2H),7.39-7.30(m ,3H),7.06-6.94(m,2H),6.54-6.34(m,2H),6.15-5.95(m,1H),5.21-5.02(m,1H),5.00-4.82(m,4H),4.70-4.32(m,1H),4.32-4.23(m,1H),3.78- 3.66 (m,1H),3.64-3.52(m,4H),3.21-3.10(m,4H),2.85-2.77(m,2H),2.77-2.67(m,2H),2.47-2.42(m,3H),2.28-2.17(m,1H),2.12-1.90(m,7H),1.8 5-1.59(m,8H),1.57-1.47(m,1H),1.46(br s,3H),1.23-1.05(m,4H),0.99-0.89(m,3H),0.84-0.73(m,3H).

Example 159: Synthesis of Compound 475

Step 1: Synthesis of tert-butyl 4-(6-(2-hydroxyphenyl)-3-(propylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl)-[1,4'-bipiperidinyl]-1'-carboxylate

2-[1-(piperidin-4-yl)-3-(propylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (120 mg, 340.49 μmol) and N-tert-butyloxycarbonyl-4-piperidone (135.68 mg, 680.98 μmol) were dissolved in tetrahydrofuran (2 mL) and N,N-dimethylformamide (2 mL), and acetic acid (20.45 mg, 340.49 μmol), sodium triacetoxyborohydride (72.16 mg, 340.49 μmol) and sodium acetate (83.80 mg, 1.02 mmol) were added. The reaction solution was stirred at 25°C for 12 hours. The reaction was complete as monitored by LCMS. Saturated sodium bicarbonate was added to the reaction solution to adjust the pH to about 8-9, and extracted three times with dichloromethane/methanol = 10/1 (15 mL), and the organic phase was dried over anhydrous sodium sulfate. The organic phase was concentrated to dryness under reduced pressure and then purified by column chromatography (petroleum ether/tetrahydrofuran = 1/2) to obtain the title compound (139 mg).

MS m/z(ESI):536.3[M+H] ⁺ .

Step 2: Synthesis of 2-(1-([1,4'-bipiperidinyl]-4-yl)-3-(propylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

Dissolve tert-butyl 4-(6-(2-hydroxyphenyl)-3-(propylamino)-1H-pyrazolo[4,3-c]pyridazine-1-yl)-[1,4'-bipiperidine]-1'-carboxylate (139 mg, 259.48 μmol) in anhydrous methanol (1.5 mL), and add dioxane hydrochloride (4M, 1.30 mL). Stir the reaction solution at 25°C for 2 hours. LCMS monitors the reaction to be complete. The reaction solution is concentrated to dryness under reduced pressure to obtain the title compound (110 mg).

MS m/z(ESI):436.2[M+H] ⁺ .

Step 3: Synthesis of compound 475

2-(1-([1,4'-bipiperidinyl]-4-yl)-3-(propylamino)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (36 mg, 76.27 μmol) and intermediate 2-1 (45.28 mg, 76.27 μmol) were dissolved in tetrahydrofuran (0.5 mL) and N,N-dimethylformamide (0.5 mL), and acetic acid (13.74 mg, 228.80 μmol), sodium triacetoxyborohydride (48.49 mg, 228.80 μmol) and sodium acetate (18.77 mg, 228.80 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. The reaction was complete when monitored by LCMS. The reaction solution was concentrated to dryness under reduced pressure and purified by HPLC (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile with decreasing polarity (acetonitrile ratio 60%-80%) as eluent) to give compound 475 (25.5 mg).

MS m/z(ESI):507.6[M+2H] ²⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.00 (br s, 1H), 8.98 (s, 1H), 8.48 (s, 1H), 8.40 (br d, J = 7.6Hz, 1H), 8.12 ( br d,J＝8.0Hz,1H),7.48-7.40(m,2H),7.39-7.31(m,3H),7.09(br t,J＝5.5Hz,1H),7.03-6.93(m,2H) ,6.11(s,1H),5.16-4.98(m,1H),4.91(quin,J＝6.9Hz,1H),4.58-4.47(m,1H),4.36(br t,J＝7.7Hz,1H) ,4.28(br s,1H),3.76-3.67(m,1H),3.65-3.52(m,3H),3.40(br s,2H),3.01(br d,J＝9.0Hz,2H),2.89(br d,J ＝8.8Hz,2H),2.73(br t,J＝11.6Hz,2H),2.45(s,3H),2.41-2.17(m,5H),2.16-1.99(m,5H),1.93-1.78(m ,5H), 1.77-1.63(m,7H),1.53-1.35(m,5H),1.17-1.03(m,2H),1.01-0.91(m,6H),0.84-0.73(m,3H).

Example 160: Synthesis of Compound 482

Step 1: Synthesis of tert-butyl 4-(6-chloropyridin-3-yl)piperazine-1-carboxylate

2-Chloro-5-iodo-pyridine (2g, 8.35mmol) and tert-butyl piperazine-1-carboxylate (1.56g, 8.35mmol) were dissolved in anhydrous dioxane (40mL), and sodium tert-butoxide (1.61g, 16.71mmol), tris(dibenzylideneacetone)dipalladium (764.88mg, 835.28μmol) and 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (966.62mg, 1.67mmol) were added. The reaction solution was stirred at 90°C for 2 hours under a nitrogen atmosphere. LCMS detected that the reaction was complete. The reaction solution was diluted with water (20mL) and extracted three times with ethyl acetate (50mL). The organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness. The title compound (618.1mg) was purified by column chromatography (silicon dioxide, petroleum ether: tetrahydrofuran = 2:1) to obtain the title compound (618.1mg).

MS m/z(ESI):298.0[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-[6-[4-[3-(tert-butoxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-3-yl]piperazine-1-carboxylate

Tert-butyl 4-(6-chloropyridin-3-yl)piperazine-1-carboxylate (518 mg, 1.74 mmol) and tert-butyl (6-(2-(methoxymethoxy)phenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-yl)carbamate (658.88 mg, 1.45 mmol) were dissolved in dioxane (14 mL), and sodium tert-butoxide (417.94 mg, 4.35 mmol), tris(dibenzylideneacetone)dipalladium (121.24 mg, 144.96 μmol) and 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (167.72 mg, 0.29 mmol) were added. The reaction solution was stirred at 100°C under nitrogen protection for 16 hours. The reaction was completed by thin layer chromatography (silica, petroleum ether:tetrahydrofuran = 1:1). The reaction solution was diluted with water (10 mL) and extracted three times with ethyl acetate (30 mL). The organic phases were combined and dried over anhydrous sodium sulfate. The filtrate was filtered and concentrated to dryness to obtain the title compound (457.7 mg).

MS m/z(ESI):716.3[M+H] ⁺ .

Step 3: Synthesis of 2-(3-amino-1-(1-(5-(piperazin-1-yl)pyridin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

4-[6-[4-[3-(tert-butoxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-3-yl]piperazine-1-carboxylic acid tert-butyl ester (457.7 mg, 639.39 μmol) was dissolved in anhydrous methanol (5 mL), and dioxane hydrochloride (4M, 1.60 mL) was added, and the reaction solution was stirred at 50° C. for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness to obtain the title compound (505.6 mg, crude product).

MS m/z(ESI):472.3[M+H] ⁺ .

Step 4: Synthesis of compound 482

2-(3-amino-1-(1-(5-(piperazin-1-yl)pyridin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (235.30 mg, 463.17 μmol) and intermediate 2-1 (250 mg, 421.06 μmol) were dissolved in anhydrous tetrahydrofuran (4 mL) and anhydrous N,N-dimethylformamide (4 mL), and acetic acid (75.86 mg, 1.26 mmol, 72.31 μL), sodium acetate (103.62 mg, 1.26 mmol) and sodium acetate borohydride (267.72 mg, 1.26 mmol) were added. The reaction solution was stirred at 25°C for 2 hours, and the reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile ratio 43%-83%) as eluent) gave compound 482 (144.4 mg).

MS m/z(ESI):525.5[M+2H] ²⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.93 (br s, 1H), 9.01-8.92 (m, 1H), 8.51 (s, 1H), 8.42 (br d, J = 7.5Hz, 1H), 8.14-8.03(m,1H),7.90-7.80(m,1H),7.48-7.40(m,2H),7.34(br s,3H),7.29(br dd,J＝1.9,8.8Hz,1H), 7.05-6.96(m,2H),6.85(br d,J＝9.3Hz,1H),6.52-6.35(m,2H),6.15-5.95(m,1H),5.19-5.12(m,1H),4.97-4.75(m,2H),4.43-4.24( m,4H),3.76-3.69(m,1H),3.61(br d,J＝11.4Hz,3H),3.56(br d,J＝9.8Hz,4H),3.08-2.88(m,6H),2.81 -2.67(m,2H),2.45(s,3H),2.30-1.94(m,7H),1.87(br d,J＝9.9Hz,2H),1.83-1.61(m,4H),1.49-1.32(m,3H),1.22-1.04(m,2H),1.01-0.90(m,3H),0.84-0.73( m,3H).

Example 161: Synthesis of Compound 493

Step 1: Synthesis of tert-butyl 6-chloro-3',6'-dihydro-[3,4'-bipyridine]-1'(2'H)-carboxylate

2-Chloro-5-iodo-pyridine (500 mg, 2.09 mmol) and tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (710.26 mg, 2.30 mmol) were dissolved in dioxane (16 mL) and water (4 mL). Cesium carbonate (1.36 g, 4.18 mmol) and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (170.53 mg, 208.82 μmol) were added under nitrogen atmosphere. The reaction solution was stirred at 100 ° C for 2 hours. LCMS showed that the reaction was completed. The reaction solution was concentrated to dryness under reduced pressure, then extracted with ethyl acetate (50 mL*3) and water (50 mL). The organic phase was concentrated to dryness under reduced pressure and the concentrate was purified by column chromatography (silica, petroleum ether/ethyl acetate = 1/0 to 5/1) to give the title compound (295 mg).

MS m/z(ESI):295.0[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 6-(4-(3-((tert-butyloxycarbonyl)amino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)-3',6'-dihydro-[3,4'-bipyridine]-1'(2'H)-carboxylate

6-Chloro-3',6'-dihydro-[3,4'-bipyridine]-1'(2'H)-carboxylic acid tert-butyl ester (150 mg, 330.02 μmol) was dissolved in dioxane (6 mL), sodium tert-butoxide (95.15 mg, 990.05 μmol) and methanesulfonic acid-(2-dicyclohexylphosphine-2',6'-diisopropoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium (II) (27.60 mg, 33.00 μmol) were added under nitrogen atmosphere, and the reaction solution was reacted at 100° C. for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, the concentrate was extracted with dichloromethane (15 mL*3) and water (5 mL), the organic phase was concentrated to dryness under reduced pressure, and the concentrate was purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 2/1) to give the title compound (95 mg).

MS m/z(ESI):713.5[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-[6-[4-[3-(tert-butoxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-3-yl]piperidine-1-carboxylate

6-(4-(3-((tert-butyloxycarbonyl)amino)-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl)piperidin-1-yl)-3',6'-dihydro-[3,4'-bipyridine]-1'(2'H)-carboxylic acid tert-butyl ester (95 mg, 133.27 μmol) was dissolved in tetrahydrofuran (2 mL), palladium carbon (30 mg, 133.27 μmol, 10% purity) and triethylsilane (305.52 mg, 1.33 mmol) were added, and the reaction solution was stirred at 25° C. for 2 hours. LCMS showed that the reaction was complete. The reaction solution was filtered and concentrated to give the title compound (90 mg).

MS m/z(ESI):715.2[M+H] ⁺ .

Step 4: Synthesis of 2-(3-amino-1-(1-(5-(piperidin-4-yl)pyridin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

4-[6-[4-[3-(tert-butyloxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridin-3-yl]piperidine-1-carboxylic acid tert-butyl ester (45 mg, 62.95 μmol) was dissolved in methanol (1 mL), and dioxane hydrochloride (4M, 157.38 μL) was added. The reaction solution was stirred at 25° C. for 2 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (30 mg).

MS m/z(ESI):471.1[M+H] ⁺ .

Step 5: Synthesis of compound 493

2-(3-amino-1-(1-(5-(piperidin-4-yl)pyridin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (28.18 mg, 55.58 μmol) and intermediate 2-1 (30 mg, 50.53 μmol) were dissolved in N,N-dimethylformamide (1.5 mL) and tetrahydrofuran (1.5 mL), and acetic acid (9.10 mg, 151.58 μmol), sodium acetate (12.43 mg, 151.58 μmol) and sodium acetate borohydride (32.13 mg, 151.58 μmol) were added. The reaction solution was reacted at 25°C for 1 hour. The reaction was completed by LCMS. The reaction solution was quenched by adding water (0.2 mL) and concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile ratio 50%-90%) as eluent) gave compound 493 (8.9 mg).

MS m/z(ESI):1048.6[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.92 (br s, 1H), 9.03-8.74 (m, 1H), 8.53 (s, 1H), 8.41 (d, J = 7.8Hz, 1H), 8.11 (d,J＝7.5Hz,1H),8.03(d,J＝2.1Hz,1H),7.52-7.41(m,3H),7.39-7.29(m,3H),7.06-6.97(m,2H), 6.88(d,J＝8.9Hz,1H),6.45(s,2H),6.17-5.95(m,1H),5.20-5.00(m,1H),4.96-4.81(m,2H),4.45(br d,J＝13.4Hz,2H),4.37(t,J＝7.8Hz,1H),4.29(br s,1H),3.71(br dd,J＝4.2,10.4Hz,1H),3.66-3.53(m ,3H),3.43(br s,2H),3.07-2.89(m,4H),2.82-2.69(m,2H),2.45(s,3H),2.30-2.13(m,3H),2.07-1.85( m,7H),1.83-1.57(m,8H),1.49-1.34(m,3H),1.13(br d,J＝10.6Hz,2H),1.00-0.91(m,3H),0.86-0.74(m ,3H).

Example 162: Synthesis of Compound 494

Step 1: Synthesis of 1-(6-chloropyridazin-3-yl)piperidin-4-ol

3,6-Dichloropyridazine (2 g, 13.42 mmol) and piperidine-4-ol (1.49 g, 14.77 mmol) were dissolved in ethanol (40 mL) and the temperature was raised to 80°C for overnight reaction. The reaction was complete as monitored by LCMS and concentrated in vacuo to obtain the title compound (3.2 g).

MS m/z(ESI):214.10[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-[6-(4-hydroxypiperidin-1-yl)pyridazin-3-yl]-3,6-dihydropyridine-1(2H)-1-carboxylate

1-(6-chloropyridazine-3-yl)piperidin-4-ol (2.87 g, 13.43 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (6.23 g, 20.15 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (1.10 g, 1.34 mmol) and potassium phosphate (11.39 g, 53.73 mmol) were dissolved in 1,4-dioxane (200 mL) and water (40 mL). The apparatus was replaced with nitrogen and then heated to 100°C for overnight reaction. The reaction was completed as monitored by LCMS. After separation, the organic phase was concentrated in vacuo and purified by column chromatography (silica, dichloromethane/methanol: 20/1) to give the title compound (3.47 g).

MS m/z(ESI):361.20[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-[6-(4-hydroxypiperidin-1-yl)pyridazin-3-yl]piperidine-1-carboxylate

Dissolve tert-butyl 4-[6-(4-hydroxypiperidin-1-yl)pyridazin-3-yl]-3,6-dihydropyridine-1(2H)-1-carboxylate (3.4 g, 9.43 mmol) and palladium on carbon (340 mg, 279.94 μmol, 10% purity) in methanol (50 mL), and heat to 50° C. to react overnight. The reaction was complete as monitored by LCMS, and the title compound (3.2 g) was obtained after filtration and vacuum concentration.

MS m/z(ESI):363.20[M+H] ⁺ .

Step 4: Synthesis of tert-butyl 4-[6-(4-methanesulfonyloxypiperidin-1-yl)pyridazin-3-yl]piperidine-1-carboxylate

Dissolve tert-butyl 4-[6-(4-hydroxypiperidin-1-yl)pyridazin-3-yl]piperidine-1-carboxylate (500 mg, 1.38 mmol) and methanesulfonyl chloride (237 mg, 2.07 mmol) in dichloromethane (15 mL), add triethylamine (418 mg, 4.14 mmol), react at room temperature for 2 hours, and the reaction is complete as monitored by LCMS. After vacuum concentration, purify by column chromatography (silica, petroleum ether/ethyl acetate: 1/1) to obtain the title compound (227 mg).

MS m/z(ESI):441.10[M+H] ⁺ .

Step 5: Synthesis of tert-butyl 4-[6-[4-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl]pyridazin-3-yl]piperidine-1-carboxylate

6-Chloro-1H-pyrazolo[4,3-c]pyridazin-3-amine (133 mg, 646.91 μmol) and cesium carbonate (421 mg, 1.29 mmol) were dissolved in N,N-dimethylformamide (10 mL), stirred for 10 minutes, and then tert-butyl 4-[6-(4-methanesulfonyloxypiperidin-1-yl)pyridazin-3-yl]piperidine-1-carboxylate (190 mg, 431.27 μmol) was added, and the temperature was raised to 80°C to react overnight. The reaction was complete as monitored by LCMS, and the title compound (190 mg) was obtained after vacuum concentration and purification by column chromatography (silica, petroleum ether/ethyl acetate: 1/1).

MS m/z(ESI):514.20[M+H] ⁺ .

Step 6: Synthesis of tert-butyl 4-[6-[4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridazin-3-yl]piperidine-1-carboxylate

Tert-butyl 4-[6-[4-(3-amino-6-chloro-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl]pyridazin-3-yl]piperidine-1-carboxylate (180 mg, 350.18 μmol), 2-(methoxymethoxy)phenylboronic acid (127 mg, 700.36 μmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (28 mg, 35.02 μmol) and potassium phosphate (296 mg, 1.40 mmol) were dissolved in 1,4-dioxane (5 mL) and water (1 mL), the apparatus was purged with nitrogen and then heated to 100°C for 4 hours. The reaction was complete as monitored by LCMS, the organic phase was concentrated after separation, and the title compound (164 mg) was purified by column chromatography (silica, dichloromethane/methanol: 20/1).

MS m/z(ESI):616.30[M+H] ⁺ .

Step 7: Synthesis of 2-[3-amino-1-[1-[6-(piperidin-4-yl)pyridazin-3-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[6-[4-[3-amino-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridazin-3-yl]piperidine-1-carboxylic acid tert-butyl ester (164 mg, 266.35 μmol) was dissolved in dichloromethane (2 mL) and methanol (1 mL), and dioxane hydrochloride (4M, 2 mL) was added. The mixture was reacted at room temperature for 6 hours, and the reaction was complete as monitored by LCMS. After concentration, dichloromethane (5 mL), methanol (0.5 mL) and saturated sodium bicarbonate solution (5 mL) were added, and the pH was adjusted to weak alkaline. After extraction, drying, filtration, and concentration of the filtrate, the crude product, i.e., the title product (110 mg), was obtained.

MS m/z(ESI):472.20[M+H] ⁺ .

Step 8: Synthesis of compound 494

2-[3-Amino-1-[1-[6-(piperidin-4-yl)pyridazin-3-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (50 mg, 106.03 μmol) and intermediate 2-1 (37 mg, 63.62 μmol) were dissolved in N,N-dimethylformamide (2 mL), and acetic acid (636 μg, 10.60 μmol) was added. After stirring at -20°C for 30 minutes, sodium triacetoxyborohydride (67 mg, 318.10 μmol) was added, and the mixture was returned to room temperature for reaction for 4 hours. The reaction was completed as monitored by LCMS and purified by preparative liquid chromatography (YMC-Actus Triart C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.05% formic acid) and acetonitrile with decreasing polarity (acetonitrile ratio 60%-70%) as eluent) to obtain compound 494 (7.6 mg).

MS m/z(ESI):1049.50[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.90(s,1H),8.99(s,1H),8.55(s,1H),8.44–8.36(m,1H),8.16–8.09(m,1H ),7.63–7.53(m,1H),7.48–7.42(m,2H),7.40–7.30(m,4H),7.08–6.96(m,2H),6.46(s,2H),6.13(s,1H ),5.15–5.07(m,1H),4.98–4.86(m,2H),4.60–4.48(m,2H),4.42–4.33(m,1H),4.33–4.26(m,1H), 3.77–3.68(m,1H),3.68–3.53(m,3H),3.48–3.40(m,1H),3.17–3.04(m,2H),3.02–2.91(m,2H),2.83–2.68(m ,3H),2.46(s,3H),2.32–2.14(m,3H),2.12–1.89(m,6H),1.86–1.67(m,6H),1.50–1.42(m,1H),1.42–1.34 (m,2H),1.29–1.05(m,3H),1.03–0.91(m,3H),0.90–0.74(m,3H).

Example 163: Synthesis of Compound 495

Step 1: Synthesis of tert-butyl 4-(5-chloropyrazin-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate

2-Bromo-5-chloropyrazine (1 g, 5.1 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (3.1 g, 10 mmol), potassium carbonate (4.2 g, 30 mmol) and Pd(dppf)Cl ₂ (550 mg, 0.76 mmol) were added to dioxane (50 mL) and water (10 mL), nitrogen exchanged three times, reacted at 85°C for 4 h, and then concentrated under reduced pressure. DCM (100 mL×3) and water (100 mL) were added to the residue for extraction. The organic phases were combined and concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA=1:0 to 1:1) to obtain the title compound (1.3 g).

MS m/z(ESI):240.00[M+H-56] ⁺ .

Step 2: Synthesis of tert-butyl 4-(5-(4-hydroxypiperidin-1-yl)pyrazin-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate

Dissolve tert-butyl 4-(5-chloropyrazin-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (1.1 g, 3.7 mmol) and 4-hydroxypiperidine (740 mg, 7.4 mmol) in DMF (10 mL), then add potassium carbonate (2.1 g, 15 mmol), and react at 100° C. for 12 h. Then add ethyl acetate (100 mL×3) and water (100 mL) for extraction, combine the organic phases, and concentrate under reduced pressure. The residue is purified by column chromatography (PE:EA=100%-10%) to obtain the title compound (700 mg).

MS m/z(ESI):361.20[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-(5-(4-hydroxypiperidin-1-yl)pyrazin-2-yl)piperidine-1-carboxylate

Dissolve tert-butyl 4-(5-(4-hydroxypiperidin-1-yl)pyrazine-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (700 mg, 1.94 mmol) in methanol (10 mL), then add 10% palladium on carbon (70 mg), exchange the hydrogen three times, and then continue to react in a hydrogen atmosphere until the raw material is completely consumed. The reaction solution is filtered and the filtrate is concentrated to obtain the title compound (650 mg).

MS m/z(ESI):363.20[M+H] ⁺ .

Step 4: Synthesis of tert-butyl 4-(5-(4-methanesulfonyloxypiperidin-1-yl)pyrazin-2-yl)piperidine-1-carboxylate

Dissolve tert-butyl 4-(5-(4-hydroxypiperidin-1-yl)pyrazine-2-yl)piperidine-1-carboxylate (650 g, 1.8 mmol) in DCM (10 mL), add triethylamine (500 g, 5 mmol), cool to 0°C, and then slowly add methanesulfonyl chloride (370 mg, 3.2 mmol). After the addition is complete, slowly warm to room temperature and react for 1 h. Then add water (50 mL) and DCM (50 mL×3) for extraction. Combine the organic phases and concentrate under reduced pressure. The residue is purified by column chromatography (PE:EA=100%-50%) to obtain the title compound (520 mg).

MS m/z(ESI):441.10[M+H] ⁺ .

Step 5: Synthesis of tert-butyl 4-(5-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrazin-2-yl)piperidine-1-carboxylate

Tert-butyl 4-(5-(4-methanesulfonyloxypiperidin-1-yl)pyrazine-2-yl)piperidine-1-carboxylate (520 mg, 1.2 mmol) and 6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-3-amine (326 mg, 1.2 mmol) were dissolved in DMF (5 mL), and then cesium carbonate (750 mg, 2 mmol) was added, and nitrogen was replaced three times. The reaction was carried out at 85° C. for 12 h, and then water (60 mL) and ethyl acetate (60 mL×3) were added to the reaction system for extraction. The organic phases were combined and concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA=100%-0%) to obtain the title compound (350 mg).

MS m/z(ESI):616.30[M+H] ⁺ .

Step 6: Synthesis of 2-(3-amino-1-(1-(5-(piperidin-4-yl)pyrazin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

Dissolve tert-butyl 4-(5-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)pyrazin-2-yl)piperidine-1-carboxylate (350 mg, 0.57 mmol) in methanol (3 mL), then add dioxane hydrochloride solution (4 M, 3 mL), react at room temperature for 1 h, concentrate the reaction solution under reduced pressure, add methanol to dissolve the residue, then add triethylamine (1 mL) to neutralize for 30 min, and finally concentrate under reduced pressure to obtain the title compound (260 mg).

MS m/z(ESI):472.20[M+H] ⁺ .

Step 7: Synthesis of compound 495

2-(3-Amino-1-(1-(5-(piperidin-4-yl)pyrazin-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (50 mg, 0.11 mmol) was dissolved in DMF (3 mL), triethylamine (1 mL) was added, followed by the addition of intermediate 2-1 (50 mg, 0.08 mmol), and then sodium triacetoxyborohydride (63 mg, 0.3 mmol) was added and reacted at room temperature for 12 h. The reaction solution was concentrated under reduced pressure and the residue was purified by preparative chromatography to obtain compound 495 (22 mg).

MS m/z(ESI):350.70[1/3M+H] ⁺ ;

¹ H NMR (600MHz, CD ₃ OD) δ8.09(s,1H),8.32(s,1H),8.25(s,1H),8.07(s,1H),8.03-8.05(m,1H), 7.44-7.48(m,3H),7.35-7.38(m,2H),7.01-7.04(m,2H),6.13(s,1H),5.05–5.09(m,2H),4.52-4.57(m,4H ),4.44-4.48(m,2H),3.86–3.88(m,2H),3.70-3.75(m,3H),3.62-3.65(m,3H),3.43–3.46(m,1H),3.17–3.23 (m,4H), 2.88–2.91(m,2H),2.50(s,3H),2.38-2.43(m,2H),2.24-2.27(m,2H),2.19-2.22(m,2H),2.03-2.07(m,2H ),1.93-1.97(m,3H),1.86-1.90(m,2H),1.52-1.56(m,3H),1.29-1.33(m,2H),1.05-1.09(m,3H),0.89-0.93 (m,3H).

Example 164: Synthesis of Compound 496

Step 1: Synthesis of 1-(5-bromothiazol-2-yl)piperidin-4-ol

Dissolve 2,5-dibromothiazole (3 g, 12.5 mmol), 4-hydroxypiperidine (3 g, 30 mmol) and triethylamine (6 g, 60 mmol) in dioxane (100 mL), react at 90° C. for 48 h, and then concentrate under reduced pressure. The residue is purified by column chromatography (PE:EA=100%-20%) to obtain the title compound (2.6 g).

MS m/z(ESI):264.90[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-(2-(4-hydroxypiperidin-1-yl)thiazol-5-yl)-3,6-dihydropyridine-1(2H)-carboxylate

1-(5-bromothiazol-2-yl)piperidin-4-ol (2 g, 7.6 mmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (4.6 g, 15.2 mmol), potassium carbonate (4.2 g, 30 mmol) and Pd(dppf)Cl ₂ (550 mg, 0.76 mmol) were added to dioxane (100 mL) and water (20 mL), nitrogen exchanged three times, and reacted at 85° C. for 4 h. Then, the mixture was concentrated under reduced pressure, DCM (100 mL×3) and water (100 mL) were added to the residue for extraction, the organic phases were combined and concentrated under reduced pressure, and the residue was purified by column chromatography (PE:EA=100%-50%) to obtain the target compound (1.8 g).

MS m/z(ESI):366.10[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-(2-(4-hydroxypiperidin-1-yl)thiazol-5-yl)piperidine-1-carboxylate

Dissolve tert-butyl 4-(2-(4-hydroxypiperidin-1-yl)thiazol-5-yl)-3,6-dihydropyridine-1(2H)-carboxylate (1.6 g, 4.4 mmol) in methanol (100 mL), then add 10% palladium on carbon (320 mg), exchange hydrogen three times, and then continue to react in a hydrogen atmosphere until the raw material is completely consumed. The reaction solution is filtered and the filtrate is concentrated to obtain the title compound (1.3 g, yield is 81.3%).

MS m/z(ESI):368.20[M+H] ⁺ .

Step 4: Synthesis of tert-butyl 4-(2-(4-methanesulfonyloxypiperidin-1-yl)thiazol-5-yl)piperidine-1-carboxylate

Dissolve tert-butyl 4-(2-(4-hydroxypiperidin-1-yl)thiazol-5-yl)piperidine-1-carboxylate (1.2 g, 3.2 mmol) in DCM (30 mL), add triethylamine (1.1 g, 10 mmol), cool to 0°C, then slowly add methanesulfonyl chloride (741 mg, 6.4 mmol). After the addition is complete, slowly warm to room temperature and react for 1 h. Then add water (50 mL) and DCM (50 mL×3) for extraction. Combine the organic phases and concentrate under reduced pressure. The residue is purified by column chromatography (PE:EA=100%-50%) to obtain the title compound (1.4 g).

MS m/z(ESI):446.10[M+H] ⁺ .

Step 5: Synthesis of tert-butyl 4-(2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)thiazol-5-yl)piperidine-1-carboxylate

Tert-butyl 4-(2-(4-methanesulfonyloxypiperidin-1-yl)thiazol-5-yl)piperidine-1-carboxylate (446 mg, 1 mmol) and 6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-3-amine (272 mg, 1 mmol) were dissolved in DMF (5 mL), and then cesium carbonate (750 mg, 2 mmol) was added. The atmosphere was replaced with nitrogen three times and reacted at 85° C. for 12 h. Water (60 mL) and ethyl acetate (60 mL×3) were added to the reaction system for extraction. The organic phases were combined and concentrated under reduced pressure. The residue was purified by column chromatography (PE:EA=100%-0%) to obtain the title compound (385 mg).

MS m/z(ESI):621.20[M+H] ⁺ .

Step 6: Synthesis of 2-(3-amino-1-(1-(5-(piperidin-4-yl)thiazol-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol

Tert-butyl 4-(2-(4-(3-amino-6-(2-(methoxymethoxy)phenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl)piperidin-1-yl)thiazol-5-yl)piperidine-1-carboxylate (385 mg, 0.62 mmol) was dissolved in methanol (3 mL), and then a dioxane hydrochloride solution (4 M, 3 mL) was added. The mixture was reacted at room temperature for 1 h. The reaction solution was concentrated under reduced pressure, and methanol was added to dissolve the residue. Triethylamine (1 mL) was then added to neutralize the mixture for 30 min. The mixture was finally concentrated under reduced pressure to give 2-(3-amino-1-(1-(5-(piperidin-4-yl)thiazol-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (300 mg).

MS m/z(ESI):477.20[M+H] ⁺ .

Step 7: Synthesis of Compound 496

2-(3-Amino-1-(1-(5-(piperidin-4-yl)thiazol-2-yl)piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (50 mg, 0.11 mmol) was dissolved in DMF (3 mL), triethylamine (1 mL) was added, followed by the addition of intermediate 2-1 (50 mg, 0.08 mmol), and then sodium triacetoxyborohydride (63 mg, 0.3 mmol) was added and reacted at room temperature for 12 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative chromatography (YMCC18, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 5 mmol ammonium bicarbonate) and acetonitrile (acetonitrile ratio 30%-60%) as eluent) to give compound 496 (20 mg).

MS m/z(ESI):527.70[M+2H] ²⁺ ;

¹ H NMR (400MHz, CD ₃ OD) δ8.89(s,1H),8.30(s,1H),8.00-8.05(m,1H),7.40–7.49(m,4H),7.34–7.39(m, 1H),6.99–7.06(m,2H),6.87-6.91(m,1H),6.11(s,1H),5.03-5.08(m,1H),4.50-4.57(m,1H),4.44-4.49( m,1H),4.07-4.15(m,2H),3.84-3.90(m,1H),3.6 7-3.74(m,2H),3.60-3.66(m,2H),2.98-3.05(m,2H),2.83-2.91(m,2H),2.72–2.79(m,1H),2.50(s,3H ),2.25–2.44(m,6H),1.94–2.23(m,9H),1.69-1.90(m,6H),1.52-1.61(m,3H),1.25-1.33(m,2H),1.03-1.10 (m,3H),0.88-0.95(m,3H).

Example 165: Synthesis of Compound 499

2-[3-[(2-Hydroxy-2-methylpropyl)amino]-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (20 mg, 47.74 μmol), (2S,4R)-4-hydroxy-1-[3-methyl-2-[3-(piperazin-1-yl)isoxazol-5-yl]butanoyl]-N-[(S)-1-[4-(4-methylthiazole-5 -yl)phenyl]ethyl]pyrrolidine-2-carboxamide (28.80 mg, 47.74 μmol) and 1,2-dibromoethane (9.87 mg, 52.52 μmol) were dissolved in N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (30.85 mg, 238.71 μmol) was added. The reaction solution was reacted at 60°C for 12 hours, and the reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Purification by high performance liquid chromatography (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile with decreasing polarity (acetonitrile ratio 36%-76%) as eluent) gave compound 499 (3.9 mg).

MS m/z (ESI): 975.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.90 (br s, 1H), 9.02-8.96 (m, 1H), 8.49 (s, 1H), 8.23 (m, 1H), 8.12 (d, J＝ 7.7Hz,1H),7.49-7.41(m,2H),7.40-7.30(m,3H),7.05-6.95(m,2H),6.65(br s,1H),6.15(d,J＝4.8Hz, 1H),5.22-4.99(m,1H),4.90(br dd,J＝6.9,12.9Hz,1H),4.65(s,1H),4.55(br s,1H),4.45-4.34(m,1H) ,4.28(br s, 1H),3.69(br d,J＝9.2Hz,1H),3.59(m,2H),3.57-3.45(m,8H),3.34-3.30(m,2H),3.15(m,4H),3.02( br s,2H),2.45(d,J＝4.2Hz,3H),2.30-1.98(m,6H),1.87-1.71(m,3H),1.45-1.32(m,3H),1.19(s,6H ),1.01-0.91(m,3H),0.85-0.76(m,3H).

Example 166: Synthesis of Compound 504

5-Fluoro-2-(3-((2-hydroxy-2-methylpropyl)amino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl)phenol (50 mg, 0.11 mmol) was dissolved in DMF (3 mL), triethylamine (1 mL) and intermediate 2-1 (50 mg, 0.08 mmol) were added, and then sodium triacetoxyborohydride (63 mg, 0.3 mmol) was added. The reaction was carried out at room temperature for 12 h. The reaction solution was concentrated under reduced pressure, and the residue was purified by preparative chromatography (YMCC18, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 5 mmol ammonium bicarbonate) and acetonitrile (acetonitrile content 30%-60%) as eluent) to obtain compound 504 (7 mg).

MS m/z(ESI):489.80[M+2H] ²⁺ ;

¹ H NMR (400MHz, CD ₃ OD) δ8.89(s,1H),8.20(s,1H),8.04-8.09(m,2H),7.44–7.48(m,3H),6.73–6.77(m, 2H),6.13(m,1H),5.05-5.07(m,1H),4.53-4.55(m,1H),4.45-4.47(m,1H),3.85-3.89(m,2H),3.70-3.75( m,3H),3.62-3.66(m,2H),3.55-3.57(m,2H),3.1 3-3.17(m,2H),2.87-2.91(m,2H),2.50(s,3H),2.35–2.40(m,4H),2.27–2.30(m,1H),2.19–2.22(m,1H ),1.95–2.01(m,3H),1.87–1.91(m,2H),1.81-1.86(m,2H),1.53-1.57(m,3H),1.29-1.36(m,8H),1.06-1.09 (m,3H),0.90-0.93(m,3H).

Example 167: Synthesis of Compound 515

Step 1: Synthesis of tert-butyl 4-((2-chloropyrimidin-5-yl)oxy)piperidine-1-carboxylate

Dissolve 2-chloropyrimidine-5-ol (500 mg, 3.83 mmol) and tert-butyl 4-hydroxypiperidine-1-carboxylate (1.16 g, 5.75 mmol) in anhydrous toluene (30 mL), add diisopropyl azodicarboxylate (929.46 mg, 4.60 mmol, 893.71 μL) and triphenylphosphine (1.21 g, 4.60 mmol). The reaction solution was stirred at 100 ° C for 2 hours under nitrogen protection. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silicon dioxide, petroleum ether: tetrahydrofuran = 2: 1) to obtain the title compound (768 mg).

MS m/z(ESI):258.0[M+H-56] ⁺ .

Step 2: Synthesis of tert-butyl 4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]oxypiperidine-1-carboxylate

Tert-butyl 4-((2-chloropyrimidin-5-yl)oxy)piperidine-1-carboxylate (500 mg, 1.59 mmol) and 2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (494.54 mg, 1.59 mmol) were dissolved in dimethyl sulfoxide (10 mL), and N,N-diisopropylethylamine (617.84 mg, 4.78 mmol, 832.67 μL) was added, and the reaction solution was stirred at 140° C. for 14 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (160 mg).

MS m/z(ESI):588.6[M+H] ⁺ .

Step 3: Synthesis of 2-[3-amino-1-[1-[5-(piperidin-4-yloxy)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

Dissolve tert-butyl 4-[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]pyrimidin-5-yl]oxypiperidine-1-carboxylate (130 mg, 221.21 μmol) in anhydrous methanol (2 mL), and add dioxane hydrochloride (2.21 mmol, 553.03 μL, 4 M). The reaction solution was stirred at 25° C. for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (112.2 mg).

MS m/z(ESI):488.3[M+H] ⁺ .

Step 4: Synthesis of compound 515

2-[3-amino-1-[1-[5-(piperidin-4-yloxy)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (90 mg, 184.59 μmol) and intermediate 2-1 (109.60 mg, 184.59 μmol) were dissolved in anhydrous tetrahydrofuran (1 mL) and N,N-dimethylformamide (1 mL), and acetic acid (11.09 mg, 184.59 μmol, 10.56 μL), sodium acetate (30.29 mg, 369.19 μmol) and sodium triacetoxyborohydride (117.37 mg, 553.78 μmol) were added. The reaction solution was stirred at 25 ° C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Boston Prime C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile ratio 47%-87%) as eluent) gave Compound 515 (19.6 mg).

MS m/z (ESI): 1065.6 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 14.02-13.79 (m, 1H), 9.07-8.88 (m, 1H), 8.57-8.48 (m, 1H), 8.47-8.35 (m, 1H), 8.29 -8.16(m,2H),8.14-8.03(m,1H),7.47-7.39(m,2H),7.39-7.27(m,3H),7.08-6.90(m,2H),6.54-6.34(m, 2H),6.14-5.97(m,1H),5.15(br d,J＝3.0Hz,1H),5.08-4.81(m,3H),4.75-4.67(m,2H),4.38(brt,J＝7.8Hz,1H),4.33-4.26(m,1H),4.22 -4.14(m,1H),3.72(br d,J＝6.1Hz,2H),3.61-3.54(m,4H),3.05(br t,J＝11.4Hz,2H),2.78-2.62(m,4H ),2.46-2.38(m,3H),2.24-2.07(m,5H),2.01-1.84(m,6H),1.71(br d,J＝11.9Hz,2H),1.59(br d,J＝8.9Hz,3H),1.47-1.33(m,3H),1.15-1.03(m,2H),1.01-0.89(m,3H),0.83(br d,J＝6.6Hz,3H).

Example 168: Synthesis of Compound 516

Step 1: Synthesis of tert-butyl 3-((2-chloropyrimidin-5-yl)oxy)pyrrolidine-1-carboxylate

2-Chloropyrimidine-5-ol (200 mg, 1.53 mmol) and tert-butyl 3-hydroxypyrrolidine-1-carboxylate (430.32 mg, 2.30 mmol) were dissolved in anhydrous tetrahydrofuran (8 mL), and triphenylphosphine (602.82 mg, 2.30 mmol) and diisopropyl azodicarboxylate (464.74 mg, 2.30 mmol, 446.86 μL) were added. The reaction solution was stirred at 25 ° C for 2 hours under a nitrogen atmosphere. LCMS detected that the reaction was complete. The reaction solution was quenched with water (2 mL), then extracted three times with ethyl acetate (15 mL), the organic phases were combined and dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness. The title compound (900 mg, crude product) was purified by column chromatography (silicon dioxide, petroleum ether: tetrahydrofuran = 1:1).

MS m/z(ESI):300.10[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 3-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]oxypyrrolidine-1-carboxylate

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (100 mg, 322.21 μmol) and tert-butyl 3-((2-chloropyrimidin-5-yl)oxy)pyrrolidine-1-carboxylate (231.80 mg, 386.66 μmol, 50% purity) were dissolved in anhydrous N,N-dimethylformamide (2 mL), and N,N-diisopropylethylamine (124.93 mg, 966.64 μmol, 168.37 μL) was added. The reaction solution was stirred at 100 ° C for 16 hours. The reaction was detected by thin layer chromatography (silica, petroleum ether: ethyl acetate = 1:2). The reaction solution was filtered and concentrated to obtain the title compound (55.6 mg).

Step 3: Synthesis of 2-[3-amino-1-[1-(5-pyrrolidin-3-yloxy)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

3-[2-[4-[3-amino-6-(2-hydroxyphenyl)pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidin-5-yl]oxypyrrolidine-1-carboxylic acid tert-butyl ester (50 mg, 87.16 μmol) was dissolved in anhydrous dichloromethane (1 mL), dioxane hydrochloride (4M, 217.90 μL) was added, and the reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness to obtain the title compound (57.4 mg, crude product).

MS m/z(ESI):474.1[M+H] ⁺ .

Step 4: Synthesis of compound 516

2-[3-amino-1-[1-(5-pyrrolidin-3-yloxy)pyrimidin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (25 mg, 49.02 μmol) and intermediate 2-1 (29.11 mg, 49.02 μmol) were dissolved in anhydrous tetrahydrofuran (0.5 mL) and anhydrous N,N-dimethylformamide (0.5 mL), and acetic acid (8.83 mg, 147.06 μmol, 8.41 μL), sodium acetate (12.06 mg, 147.06 μmol) and sodium acetate borohydride (31.17 mg, 147.06 μmol) were added. The reaction solution was stirred at 25°C for 2 hours, and the reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex Luna C18 column, 3 μm silica, 30 mm diameter, 150 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile ratio 53%-73%) as eluent) gave Compound 516 (23.7 mg).

MS m/z(ESI):526.4[M+2H] ²⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.90 (s, 1H), 8.99-8.95 (m, 1H), 8.54-8.51 (m, 1H), 8.40 (d, J = 7.6Hz, 1H), 8.20(s,2H),8.13-8.08(m,1H),7.48-7.40(m,2H),7.39-7.34(m,3H),7.04-6.95(m,2H),6.48-6.39(m,2H ),5.99(s,1H),5.14-4.99(m,1H),4.97-4.79(m,3H),4.75-4.67(m,2H),4.41-4.32(m,1H),4.31-4.24(m ,1H),3.76-3.67(m,1H),3.65-3.52(m,3H),3.49-3.41(m,1H),3.10-2.99(m,2H),2.85-2.61(m,5H),2.45 (s,3H),2.43-2.38(m,1H),2.31-2.14(m,4H),2.05-1.88(m,5H),1.84-1.69(m,4H),1.68-1.55(m,1H) ,1.38(d,J=7.0Hz,3H),1.19-1.07(m,2H),1.00-0.90(m,3H),0.85-0.75(m,3H).

Example 169: Synthesis of Compound 518

Step 1: Synthesis of tert-butyl 4-(5-chloropyrazin-2-yl)piperazine-1-carboxylate

2,5-Dichloropyrazine (200 mg, 1.34 mmol) and 1-Boc-piperazine (275.04 mg, 1.48 mmol) were dissolved in dimethyl sulfoxide (5 mL), and potassium carbonate (556.62 mg, 4.03 mmol) was added. The reaction solution was stirred at 80 ° C for 16 hours. LCMS monitored the reaction to be complete. Water (20 mL) was added to the reaction solution and extracted three times with dichloromethane (30 mL). The organic phases were combined and washed three times with water (30 mL) and then dried over anhydrous sodium sulfate. After filtering, the filtrate was concentrated to dryness under reduced pressure and purified by column chromatography (silicon dioxide, petroleum ether/tetrahydrofuran = 1/0 to 13/1) to obtain the title compound (244 mg).

MS m/z(ESI):199.1[M+H-Boc] ⁺ .

Step 2: Synthesis of tert-butyl 4-[5-[4-[3-(tert-butoxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrazin-2-yl]piperazine-1-carboxylate

Tert-butyl 4-(5-chloropyrazin-2-yl)piperazine-1-carboxylate (98.60 mg, 330.02 μmol) and tert-butyl (6-(2-(methoxymethoxy)phenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-yl)carbamate (100 mg, 220.01 μmol) were dissolved in 1,4-dioxane (3 mL), and sodium tert-butoxide (63.43 mg, 660.04 μmol) and (2-dicyclohexylphosphino-2,6-diisopropoxy-1,1-biphenyl)[2-(2-amino-1,1-biphenyl)]palladium(II) (18.40 mg, 22.00 μmol) were added. The reaction solution was replaced with nitrogen and stirred at 100°C for 16 hours. The reaction was completed by LCMS monitoring. Water (20 mL) was added to the reaction solution and extracted three times with dichloromethane (60 mL). The organic phases were combined, washed three times with water (60 mL), dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The title compound (80 mg) was obtained by purification by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 3/1).

MS m/z(ESI):717.5[M+H] ⁺ .

Step 3: Synthesis of 2-[3-amino-1-[1-(5-piperazin-1-yl)pyrazin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

Dissolve tert-butyl 4-[5-[4-[3-(tert-butyloxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrazin-2-yl]piperazine-1-carboxylate (80 mg, 111.60 μmol) in methanol (1 mL), and add dioxane hydrochloride solution (4M, 279.01 μL). The reaction solution was stirred at 25°C for 16 hours. LCMS monitored the reaction to be complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (90 mg, 60% purity).

MS m/z(ESI):473.2[M+H] ⁺ .

Step 4: Synthesis of compound 518

2-[3-amino-1-[1-(5-piperazin-1-yl)pyrazin-2-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (30 mg, 35.36 μmol, 60% pure hydrochloride) and intermediate 2-1 (21.00 mg, 35.36 μmol) were dissolved in N,N-dimethylformamide (0.5 mL) and tetrahydrofuran (0.5 mL), and sodium acetate (8.70 mg, 106.09 μmol), acetic acid (2.12 mg, 35.36 μmol) and sodium acetate borohydride (14.99 mg, 70.73 μmol) were added. The reaction solution was stirred at 25°C for 1 hour. The reaction was complete when monitored by LCMS. The reaction solution was concentrated under reduced pressure to remove tetrahydrofuran, and then purified by HPLC (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (0.05% ammonia water) and acetonitrile with decreasing polarity (acetonitrile ratio 44%-84%) as the eluent) to give compound 518 (6.3 mg).

MS m/z (ESI): 1050.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ 13.95-13.85 (m, 1H), 8.97 (s, 1H), 8.52 (s, 1H), 8.42 (d, J = 7.5Hz, 1H), 8.10 ( d,J＝7.3Hz,1H),8.01(s,1H),7.92(s,1H),7.49-7.40(m,2H),7.36(d,J＝7.9Hz,3H),7.05-6.95(m ,2H),6.45(s,2H),6.12(s,1H),5.14(d,J＝3.3Hz,1H),4.91(s,2H),4. 41-4.23(m,4H),3.70(s,2H),3.65-3.48(m,12H),3.04-2.89(m,2H),2.81-2.71(m,2H),2.45(s,3H), 2.19(s,3H),2.10-2.00(m,2H),1.92(s,2H),1.75(d,J＝11.7Hz,4H),1.38(d,J＝7.5Hz,3H),1.15(s ,2H),0.94(d,J＝6.6Hz,3H),0.85-0.75(m,3H).

Example 170: Synthesis of Compound 519

Step 1: Synthesis of tert-butyl 4-(6-chloropyridazin-3-yl)piperazine-1-carboxylate

3,6-Dichloropyridazine (1 g, 6.71 mmol) and tert-butyl piperazine-1-carboxylate (1.38 g, 7.38 mmol) were dissolved in dioxane (15 mL), N,N-diisopropylethylamine (1.30 g, 10.07 mmol) was added, and the reaction solution was stirred at 80°C for 12 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure, and the concentrate was purified by column chromatography (silicon dioxide, petroleum ether/tetrahydrofuran = 1/0 to 3/1) to obtain the title compound (1 g).

MS m/z(ESI):298.8[M+H] ⁺ .

Step 2: Synthesis of tert-butyl 4-[6-[4-[3-(tert-butoxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridazin-3-yl]piperazine-1-carboxylate

Tert-butyl (6-(2-(methoxymethoxy)phenyl)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-3-yl)carbamate (200 mg, 440.02 μmol) and tert-butyl 4-(6-chloropyridazin-3-yl)piperazine-1-carboxylate (170.90 mg, 572.03 μmol) were dissolved in dioxane (6 mL), and sodium tert-butoxide (126.86 mg, 1.32 mmol) and methanesulfonic acid (2-dicyclohexylphosphine-2',6'-diisopropoxy-1,1'-biphenyl)(2'-amino-1,1'-biphenyl-2-yl)palladium (II) (36.80 mg, 44.00 μmol) were added under nitrogen atmosphere. The reaction solution was stirred at 120° C. for 12 hours, and LCMS showed that the reaction was completed. The reaction solution was concentrated to dryness under reduced pressure, and the concentrate was extracted with dichloromethane (10 mL*3) and water (10 mL). The organic phase was concentrated to dryness under reduced pressure, and the concentrate was purified by column chromatography (silica, petroleum ether/tetrahydrofuran = 1/0 to 3/2) to give the title compound (135 mg).

MS m/z(ESI):717.0[M+H] ⁺ .

Step 3: Synthesis of 2-[3-amino-1-[1-(6-piperazin-1-yl)pyridazin-3-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol

4-[6-[4-[3-(tert-butyloxycarbonylamino)-6-[2-(methoxymethoxy)phenyl]-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyridazin-3-yl]piperazine-1-carboxylic acid tert-butyl ester (40 mg, 55.80 μmol) was dissolved in methanol (1 mL), and dioxane hydrochloride (4M, 139.50 μL) was added. The reaction solution was stirred at 25° C. for 12 hours, and LCMS showed that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (30 mg).

MS m/z(ESI):473.1[M+H] ⁺ .

Step 4: Synthesis of compound 519

2-[3-amino-1-[1-(6-piperazine-1-yl)pyridazine-3-yl]piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (30 mg, 58.94 μmol) and intermediate 2-1 (34.99 mg, 58.94 μmol) were dissolved in N,N-dimethylformamide (1 mL) and tetrahydrofuran (1 mL), and acetic acid (10.62 mg, 176.82 μmol), sodium acetate (14.50 mg, 176.82 μmol) and sodium acetate borohydride (37.47 mg, 176.82 μmol) were added. The reaction solution was reacted at 25 ° C for 1 hour. The reaction was completed by LCMS. The reaction solution was quenched by adding water (0.2 mL) and concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile ratio 39%-79%) as eluent) gave Compound 519 (22.8 mg).

MS m/z(ESI):1050.6[M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.93 (s, 1H), 9.04-8.93 (m, 1H), 8.53 (s, 1H), 8.41 (br d, J = 7.3Hz, 1H), 8.11 (d,J＝7.9Hz,1H),7.50-7.41(m,2H),7.40-7.21(m,5H),7.06-6.95(m,2H),6.46(br s,2H),6.19-5.97(m,1H),5.12(d,J＝3.5Hz,1H),4.98-4.81(m,2H),4.42-4.22(m,4H),3.78-3.54(m, 4H),3.45-3.37(m,7H),3.02(br t,J＝12.3Hz,2H),2.76(br t,J＝11.9Hz,2H),2.45(s,5H),2.20(br d, J＝4.6Hz,3H),2.14-1.97(m,3H),1.91(br d,J＝9.7Hz,2H),1.77(br d,J＝13.4Hz,4H),1.45-1.33(m,3H ),1.15(br d,J＝9.7Hz,2H),0.95(br d,J＝6.6Hz,3H),0.84-0.74(m,3H).

Example 171: Synthesis of Compound 523

Step 1: Synthesis of methyl 2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidine-5-carboxylate

2-[3-amino-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazine-6-yl]phenol (200 mg, 519.01 μmol) and methyl 2-chloropyrimidine-5-carboxylate (134.35 mg, 778.52 μmol) were dissolved in dimethyl sulfoxide (4 mL), and N,N-diisopropylethylamine (201.24 mg, 1.56 mmol, 271.21 μL) was added. The reaction solution was stirred at 140°C for 2 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure and purified by column chromatography (silicon dioxide, petroleum ether:tetrahydrofuran = 2:1, then dichloromethane:tetrahydrofuran = 5:1) to obtain the title compound (150 mg).

MS m/z(ESI):447.2[M+H] ⁺ .

Step 2: Synthesis of 2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidine-5-carboxylic acid

Dissolve 2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]pyrimidine-5-carboxylic acid methyl ester (135 mg, 302.38 μmol) in water (0.5 mL) and tetrahydrofuran (0.5 mL), and add an aqueous solution (10 mL) of lithium hydroxide (10.86 mg). The reaction solution was stirred at 25°C for 14 hours. LCMS detected that the reaction was complete. The reaction solution was adjusted to pH 5 with 1M hydrochloric acid, and then concentrated to dryness under reduced pressure to obtain the title compound (320 mg).

MS m/z(ESI):433.2[M+H] ⁺ .

Step 3: Synthesis of tert-butyl 4-[[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]pyrimidine-5-amido]methyl]piperidine-1-carboxylate

2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]pyrimidine-5-carboxylic acid (290 mg, 272.13 μmol) and tert-butyl 4-(aminomethyl)piperidin-1-carboxylate (64.15 mg, 299.34 μmol) were dissolved in N,N-dimethylformamide (5 mL), and O-(7-azabenzotriazol-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonate (124.17 mg, 326.55 μmol) and N,N-diisopropylethylamine (105.51 mg, 816.39 μmol, 142.20 μL) were added. The reaction solution was stirred at 25° C. for 2 hours. The reaction was completed by LCMS. A small amount of water was added to quench the mixture, and the mixture was extracted with ethyl acetate (30 mL*3) and water (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness under reduced pressure. The title compound (130 mg) was obtained after purification by column chromatography (silicon dioxide, petroleum ether:tetrahydrofuran = 1:2).

MS m/z(ESI):629.5[M+H] ⁺ .

Step 4: Synthesis of 2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-N-(piperidin-4-ylmethyl)pyrimidine-5-carboxamide

Dissolve tert-butyl 4-[[2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]pyrimidine-5-amido]methyl]piperidine-1-carboxylate (130 mg, 206.77 μmol) in anhydrous methanol (4 mL), and add dioxane hydrochloride (56.92 μL, 4 M). The reaction solution was stirred at 25° C. for 16 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (100 mg).

MS m/z(ESI):529.3[M+H] ⁺ .

Step 5: Synthesis of compound 523

2-[4-[3-amino-6-(2-hydroxyphenyl)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]-N-(piperidin-4-ylmethyl)pyrimidine-5-carboxamide (90 mg, 170.26 μmol) and intermediate 2-1 (101.09 mg, 170.26 μmol) were dissolved in anhydrous tetrahydrofuran (2 mL) and N,N-dimethylformamide (2 mL). Acetic acid (10.22 mg, 170.26 μmol, 9.75 μL) and sodium acetate (27.93 mg, 340.52 μmol) were first added, and ultrasonication was performed until the solution changed color. Sodium acetate borohydride (108.25 mg, 510.78 μmol) was then added, and ultrasonication was performed again until the solution became a brown suspension, and the reaction was carried out at 25°C for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. Compound 523 (15 mg) was obtained by purification by HPLC (Boston Prime C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile ratio 42%-82%) as eluent).

MS m/z (ESI): 1106.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.98-13.78 (m, 1H), 9.02-8.94 (m, 1H), 8.84-8.75 (m, 2H), 8.55-8.48 (m, 1H), 8.48 -8.31(m,2H),8.14-8.04(m,1H),7.42(s,2H),7.39-7.30(m,3H),7.06-6.94(m,2H),6.54-6.34(m,2H) ,6.15-5.95(m,1H),5.21-5.02(m,1H),5.00-4.82(m ,4H),4.70-4.32(m,1H),4.32-4.23(m,1H),3.78-3.66(m,1H),3.64-3.52(m,4H),3.21-3.10(m,4H),2.85 -2.77(m,2H),2.77-2.67(m,2H),2.47-2.42(m,3H),2.28-2.17(m,1H),2.12-1.90(m,7H),1.85-1.59(m, 8H),1.57-1.47(m,1H),1.46(br s,3H),1.23-1.05(m,4H),0.99-0.89(m,3H),0.84-0.73(m,3H).

Example 172: Synthesis of Compound 529

Step 1: Synthesis of ethyl 2-[4-(methoxymethylene)cyclohexyl]acetate

Methoxymethyltriphenylphosphine chloride (2.79 g, 8.14 mmol) was dissolved in tetrahydrofuran (30 mL), potassium tert-butoxide (1.52 g, 13.57 mmol) was added in batches at 0°C, and the reaction solution was stirred at 0°C for 1 hour. Then a tetrahydrofuran solution (10 mL) of ethyl 4-oxocyclohexyl acetate (1 g, 5.43 mmol) was added dropwise at 0°C, the reaction solution was stirred at 0°C for 1 hour, and then the temperature was raised to 25°C and stirred for 10 hours. Thin layer chromatography (petroleum ether: tetrahydrofuran = 3:1) showed that the reaction was complete. The reaction solution was quenched with saturated aqueous ammonium chloride solution (50 mL), extracted with ethyl acetate (50 mL*2), the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to dryness. The concentrate was purified by column chromatography (silicon dioxide, petroleum ether/tetrahydrofuran = 0-10%) to obtain the title compound (140 mg).

Step 2: Synthesis of ethyl 2-(4-formylcyclohexyl)acetate

Ethyl 2-[4-(methoxymethylene)cyclohexyl]acetate (50 mg, 235.53 μmol) was dissolved in tetrahydrofuran (1 mL), and hydrochloric acid solution (2M, 1.18 mL) was added. The reaction solution was stirred at 25 ° C for 1 hour. Thin layer chromatography (petroleum ether: tetrahydrofuran = 3:1) showed that the reaction was complete. The reaction solution was extracted with ethyl acetate (20 mL*2) and water (5 mL), and the organic phase was concentrated to dryness under reduced pressure to obtain the title compound (45 mg).

Step 3: Synthesis of ethyl 2-[4-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]cyclohexyl]acetate

Ethyl 2-(4-formylcyclohexyl)acetate (20 mg, 100.88 μmol) and 2-[3-(methylamino)-1-(piperidin-4-yl)-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (40.04 mg, 110.97 μmol) were dissolved in N,N-dimethylformamide (1.5 mL) and tetrahydrofuran (1.5 mL), and acetic acid (18.17 mg, 302.64 μmol), sodium acetate (24.83 mg, 302.64 μmol) and sodium acetate borohydride (64.14 mg, 302.64 μmol) were added. The reaction solution was reacted at 25° C. for 1 hour. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure, the concentrate was extracted with dichloromethane (20 mL*3) and water (5 mL), the organic phase was concentrated to dryness under reduced pressure, and the concentrate was purified by thin layer chromatography (silica, petroleum ether:tetrahydrofuran = 2/1) to give the title compound (30 mg).

MS m/z(ESI):507.1[M+H] ⁺ .

Step 4: Synthesis of 2-[4-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]cyclohexyl]acetic acid

Ethyl 2-[4-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]cyclohexyl]acetate (30 mg, 59.21 μmol) was dissolved in tetrahydrofuran (1 mL) and water (1 mL), sodium hydroxide (7.11 mg, 177.64 μmol) was added, and the reaction solution was reacted at 25° C. for 12 hours. LCMS showed that the reaction was complete. The reaction solution was adjusted to pH 5-6 with 1M hydrochloric acid solution, and then concentrated to dryness under reduced pressure to obtain the title compound (30 mg).

MS m/z(ESI):479.3[M+H] ⁺ .

Step 5: Synthesis of compound 529

2-[4-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)-1H-pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]cyclohexyl]acetic acid (30 mg, 62.68 μmol) and (2S,4R)-1-[(S)-2-amino-3,3-dimethyl-butyryl]-4-hydroxy-N-[(S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]pyrrolidine-2-carboxamide (33.17 mg, 68.95 μmol) were dissolved in N,N-dimethylformamide (1.5 mL), and N,N-diisopropylethyl was added. Amine (40.51 mg, 313.42 μmol) and O-(7-azabenzotriazole-1-yl)-N,N,N,N-tetramethyluronium hexafluorophosphonium salt (28.60 mg, 75.22 μmol), the reaction solution was reacted at 25°C for 1 hour. LCMS showed that the reaction was complete, the reaction solution was quenched by adding water (0.2 mL), and then concentrated to dryness under reduced pressure, and the concentrate was purified by high performance liquid chromatography (Phenomenex C18, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile with decreasing polarity (acetonitrile ratio 48%-88%) as eluent) to obtain compound 529 (13.1 mg).

MS m/z (ESI): 906.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.98(br s,1H),8.98(br s,1H),8.49(br s,1H),8.39(br s,1H),8.12(br d, J＝6.6Hz,1H),7.77(br d,J＝9.2Hz,1H),7.40(br d,J＝15.0Hz,5H),7.12-6.93(m,3H),5.10(br s,1H) ,4.91(br s,1H),4.63-4.38(m,3H),4.28(br s,1H),3.61(br s,2H),2.96(br s,5H),2.45(br s,3H),2.34-1.94(m,8H),1.86-1.55(m,7H),1.38(br s,7H),0.94(br s,12H).

Example 173: Synthesis of Compound 530

2-[3-amino-1-[1-(piperidin-4-ylmethyl)piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (20 mg, 45.05 μmol) and intermediate 2-1 (26.75 mg, 45.05 μmol) were dissolved in N,N-dimethylformamide (1 mL) and anhydrous tetrahydrofuran (1 mL), and acetic acid (2.58 μL, 45.05 μmol), sodium acetate (7.39 mg, 90.10 μmol) and sodium triacetoxyborohydride (28.64 mg, 135.14 μmol) were added. The reaction solution was stirred at 25 ° C for 3 hours. LCMS detected that the reaction was complete. The reaction solution was concentrated to dryness under reduced pressure. Purification by HPLC (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using decreasingly polar mixtures of water (containing 0.05% ammonia) and acetonitrile (acetonitrile ratio 46%-86%) as eluent) gave Compound 530 (11.63 mg).

MS m/z (ESI): 985.5 [M+H] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 13.97 (br s, 1H), 9.00-8.95 (m, 1H), 8.82-8.38 (m, 2H), 8.16-8.08 (m, 1H), 7.48- 7.41(m,2H),7.39-7.32(m,3H),7.04-6.93(m,2H),6.44(br s,2H),6.15-5.91(m,1H),5.20-4.99(m,1H) ,4.98-4.60(m,1H),4.54(br s,1H),4.36(t,J＝7.8Hz,1H),4.28(br s,1H),3.71(br dd,J＝4.2,10.3Hz,1H),3.66-3.52(m,4H),2.94(br d,J＝4.6Hz,2H),2.80(br d,J＝10.6Hz,2H),2.73(br t,J＝11.4Hz,2H),2.45(s,3H),2.20-2.02(m,10H),1.82(br s,5H),1.74-1.64(m,5H),1.45-1.36(m,3H ),1.23-1.01(m,5H),1.00-0.91(m,3H),0.85-0.75(m,3H).

Example 174: Synthesis of Compound 531

2-[3-amino-1-[1-(piperidin-4-ylmethyl)piperidin-4-yl]-1H-pyrazolo[4,3-c]pyridazin-6-yl]phenol (22.08 mg, 49.74 μmol), (2S,4R)-4-hydroxy-1-[3-methyl-2-(3-piperazin-1-yl)isoxazol-5-yl)butanoyl]-N-[(S)-1-[4-(4-methylthiazol-5-yl)thiazol-5-yl] ... )phenyl]ethyl]pyrrolidine-2-carboxamide (30.00 mg, 49.74 μmol) and 1,2-dibromoethane (14.02 mg, 74.61 μmol) were dissolved in N,N-dimethylformamide (1.5 mL), and N,N-diisopropylethylamine (32.14 mg, 248.68 μmol) was added. The reaction solution was reacted at 60°C for 12 hours, and the reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure. It was purified by high performance liquid chromatography (Phenomenex C18 column, 3 μm silica, 30 mm diameter, 75 mm length; using a mixture of water (containing 0.05% ammonia water) and acetonitrile with decreasing polarity (acetonitrile ratio 40%-80%) as eluent) to obtain compound 531 (3.4 mg).

MS m/z(ESI):500.9[M+2H] ²⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ13.97(br s,1H),9.02-8.88(m,1H),8.54-8.47(m,1H),8.44-8.18(m,1H),8.12( d,J＝7.0Hz,1H),7.51-7.26(m,5H),7.03-6.95(m,2H),6.44(br s,2H),6.20-6.09(m,1H),5.32-4.99(m ,1H),4.98-4.82(m,1H),4.60-4.48(m,1H),4.47-4.32(m,1H),4.28(br s,1H),3.76-3.65(m,1H),3.60-3.51(m,1H),3.46-3.36(m,2H),3.31-3.23(m,2H),3.12(d,J＝16.5Hz, 4H),2.95(d,J＝5.1Hz,2H),2.85(d,J＝7.1Hz,2H),2.46-2.36(m,8H),2.28-1.99(m,9H),1.95-1.76(m ,5H),1.67(br d,J＝12.1Hz,2H),1.47-1.33(m,3H),1.16-1.03(m,2H),0.86-0.75(m,3H),1.00-0.72(m, 3H).

Example 175: Synthesis of N-[(1S)-1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidine-1-carbonyl]-2,2-dimethylpropyl]-7-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]-2-azaspiro[3.5]nonane-2-carboxamide (Compound 532)

Step 1: Synthesis of tert-butyl 7-(hydroxymethyl)-2-azaspiro[3.5]nonane-2-carboxylate

2-tert-Butyloxycarbonyl-2-azaspiro[3.5]nonane-7-carboxylic acid (200 mg, 742.57 μmol) was dissolved in anhydrous tetrahydrofuran (5 mL), and borane tetrahydrofuran (1 M, 2.23 mL) was added at 0°C. The reaction solution was stirred at 25°C for 2 hours. The reaction was completed by thin layer chromatography (petroleum ether: tetrahydrofuran = 2:1), the reaction solution was cooled to 0°C, anhydrous ethanol (3 mL) was added to quench, and stirred at 60°C for 0.5 hours. The reaction solution was concentrated to dryness under reduced pressure, diluted with water (10 mL) and extracted three times with ethyl acetate (3 mL), and the organic phase was dried and filtered over anhydrous sodium sulfate, and concentrated to dryness under reduced pressure to obtain the title compound (165 mg).

¹ H NMR (400MHz, CDCl ₃ ) δ3.61 (s, 2H), 3.57 (s, 2H), 3.47 (d, J = 6.16Hz, 2H), 1.93 (br d, J = 13.20Hz, 2H), 1.78-1.80(m,2H),1.50-1.42(m,12H),1.05-0.91(m,2H).

Step 2: Synthesis of tert-butyl 7-formyl-2-azaspiro[3.5]nonane-2-carboxylate

Dissolve tert-butyl 7-(hydroxymethyl)-2-azaspiro[3.5]nonane-2-carboxylate (60 mg, 234.97 μmol) in anhydrous dichloromethane (10 mL), add pyridinium chlorochromate (75.97 mg, 352.45 μmol) and silicon dioxide (70.59 mg, 1.17 mmol). The reaction solution was stirred at 25 ° C for 2 hours. The reaction was completed by thin layer chromatography (silicon dioxide, petroleum ether: tetrahydrofuran = 2: 1). After filtering the reaction solution, it was concentrated to dryness under reduced pressure to obtain the title compound (100 mg).

¹ H NMR (400MHz, CDCl ₃ ) δ9.65(br s,1H),3.63-3.53(m,4H),2.15(s,1H),1.96-1.86(m,4H),1.55-1.44(m, 13H).

Step 3: Synthesis of tert-butyl 7-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]-2-azaspiro[3.5]nonane-2-carboxylate

7-Formyl-2-azaspiro[3.5]nonane-2-carboxylic acid tert-butyl ester (90 mg, 213.16 μmol) and 2-[3-(methylamino)-1-(piperidin-4-yl)pyrazolo[4,3-c]pyridazin-6-yl]phenol (69.14 mg, 213.16 μmol) were dissolved in anhydrous tetrahydrofuran (2 mL) and anhydrous N,N-dimethylformamide (2 mL), and anhydrous sodium acetate (52.46 mg, 639.47 μmol), acetic acid (38.40 mg, 639.47 μmol, 36.61 μL) and sodium acetate borohydride (135.53 mg, 639.47 μmol) were added. The reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete, and the reaction solution was concentrated to dryness under reduced pressure, diluted with water (10 mL) and extracted three times with ethyl acetate (30 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated to dryness, and purified by thin layer chromatography (silicon dioxide, petroleum ether:tetrahydrofuran = 2:1) to obtain the title compound (24 mg).

MS m/z(ESI):562.2[M+H] ⁺ .

Step 4: Synthesis of 2-[1-[1-(2-azaspiro[3.5]nonyl-7-ylmethyl)-4-piperidinyl]-3-(methylamino)pyrazolo[4,3-c]pyridazin-6-yl]phenol

7-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)pyrazolo[4,3-c]pyridazine-1-yl]piperidin-1-yl]methyl]-2-azaspiro[3.5]nonane-2-carboxylic acid tert-butyl ester (24 mg, 42.73 μmol) was dissolved in anhydrous methanol (1 mL), and dioxane hydrochloride (4M, 106.82 μL) was added. The reaction solution was stirred at 25°C for 2 hours. LCMS detected that the reaction was complete, and the reaction solution was concentrated to dryness under reduced pressure to obtain the title compound (24 mg).

MS m/z(ESI):462.4[M+H] ⁺ .

Step 5: Synthesis of N-[(1S)-1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidine-1-carbonyl]-2,2-dimethylpropyl]-7-[[4-[6-(2-hydroxyphenyl)-3-(methylamino)pyrazolo[4,3-c]pyridazin-1-yl]piperidin-1-yl]methyl]-2-azaspiro[3.5]nonane-2-carboxamide

2-[1-[1-(2-azaspiro[3.5]nonyl-7-ylmethyl)-4-piperidinyl]-3-(methylamino)pyrazolo[4,3-c]pyridazin-6-yl]phenol (20 mg, 40.16 μmol) and phenyl N-[(1S)-1-[(2S,4R)-4-hydroxy-2-[[(1S)-1-[4-(4-methylthiazol-5-yl)phenyl]ethyl]carbamoyl]pyrrolidine-1-carbonyl]-2,2-dimethylpropyl]carbamate (22.68 mg, 40.16 μmol) were dissolved in anhydrous N,N-dimethylformamide (1 mL), and N,N-diisopropylethylamine (25.95 mg, 200.78 μmol, 34.97 μL) was added, and the reaction solution was stirred at 60° C. for 2 hours. The reaction was completed by LCMS. The reaction solution was concentrated to dryness under reduced pressure, and the residue was purified by HPLC (Boston Prime C18 column, 5 μm silica, 30 mm diameter, 150 mm length; using a mixture of water (containing 0.05% ammonia) and acetonitrile of decreasing polarity (acetonitrile ratio 55%-75%) as the eluent) to give compound 532 (10.1 mg).

MS m/z (ESI): 954.6 [M+Na] ⁺ ;

¹ H NMR (400MHz, DMSO-d ₆ ) δ = 8.99 (s, 1H), 8.50 (s, 1H), 8.41 (d, J = 7.6Hz, 1H), 8.15-8.11 (m, 1H), 7.46- 7.42(m,2H),7.40-7.33(m,3H),7.09(br d,J＝5.0Hz,1H),7.02-6.97(m,2H),5.55(br d,J＝9.4Hz,1H) ,5.13(d,J＝3.5Hz,1H),4.91(br t,J＝7.2Hz,1H),4.62-4.51(m,1H),4.43(t,J＝8.1Hz,1H),4.34-4.26(m,2H),3.64-3.55(m,3H),3.51 (br d,J＝7.5Hz,2H),3.46(br d,J＝7.8Hz,1H),2.97(br d,J＝4.9Hz,5H),2.46(s,3H),2.20-1.97(m ,8H),1.87-1.66(m,8H),1.45(br d,J＝3.9Hz,1H),1.43-1.35(m,5H),0.93(s,9H).

Biological activity and related properties test examples

Test Example 1: TR-FRET Binding Assay

The experiment was tested using the SMARCA2TR-FRET Assay Kit (BPS Bioscience, cat#40342). BRM, Bromodomain Ligand 2, Tb-labeled donor and Dye-labeled acceptor were dissolved in 1X BRD TR-FRET Assay buffer. PFI-3 (Selleck, CAS No.1819363-80-8) was used as a positive control drug, the DMSO group was used as a negative control group, and the group without BRM was used as a blank control group. The test compound was prepared into a 10mM stock solution with DMSO, and then the stock solution of the test compound was diluted three times with ddH ₂ O. 2μL of the diluted test compound solution and 3μL of BRM were added to a 96-well white plate (Cisbio, cat#66PL96025) and incubated at room temperature for 10 minutes. Then, 5 μL Bromodomain Ligand 2, 5 μL Tb-labeled donor and 5 μL Dye-labeled acceptor were added, and the mixture was shaken on an oscillator for 30 seconds and then briefly centrifuged at 1000 rpm/min. After incubation at room temperature for 30 minutes, the mixture was tested using an Envision multifunctional microplate reader (purchased from PerkinElmer). The TR-FRET ratio (655 nm/622 nm) was analyzed using GraphPad software. The concentration-effect curve was fitted using a nonlinear four-parameter curve, and the EC ₅₀ of the compound was calculated as follows:

in:

FRET _Sample : FRET value of each well in the compound addition group;

FRET _Blank : FRET value of blank control group;

FRET _DMSO : FRET value of negative control group;

The binding activity (EC ₅₀ ) of the compounds of the present disclosure with BRM was tested according to the above test method, and some of the results are summarized in Table 1.

Table 1

Compound _EC50 (nM) 2a 672

3a 506 4a 26 5a 186 11a 78 19a 151 20a 99 21a 86 22a 86 23a 161

Test Example 2: Degradation Activity Test

Experimental principle: In-Cell Western technology is used to detect the effects of compounds on the degradation of BRM and BRG1.

Test method:

Adjust the cell concentration (A549: 5000 cells/well, SW1573: 8000 cells/well, both purchased from ATCC) and seed in a 384-well plate, and culture overnight at 37°C and 5% _CO2 ; dissolve the compound in DMSO (initial concentration 10 mM), dilute the compound with DMSO and culture medium (DMEM culture medium (Thermo fisher, product number 11995073), containing 10% FBS (Thermo fisher, product number 11995073) and 1% Penicillin-Streptomycin Solution (Thermo fisher, product number 15140-122)) and transfer to the cell plate, the final concentration of the dilution is 10 μM, 3-fold dilution, a total of 10 dose concentrations. Continue to culture at 37°C and 5% _CO2 for 16-18 hours.

Add 50 μL of pre-cooled PBS (purchased from Hyclone, catalog number SH30256.01) to each well of the 384-well plate for washing; discard the solution, add 50 μL of 4% paraformaldehyde fixative (purchased from Biosharp, catalog number BL539A), and incubate at room temperature for 20 minutes; discard the fixative, add 50 μL of 0.1% Triton X-100 (purchased from SIGMA, catalog number 93443-100mL) in PBS solution, and incubate at room temperature for 30 minutes; discard the solution, add 50 μL of Li-Cor blocking Buffer (purchased from Li-Cor, catalog number 927-70001), incubate at room temperature for 1 hour; discard the blocking solution, add 50 μL primary antibody (rabbit anti-BRM/BRG1 (1:500; BRM antibody purchased from CST, catalog number 11966S; BRG1 antibody purchased from CST, catalog number 49360S); mouse anti-a-Tubulin (1:2000, purchased from CST, catalog number 49360S)), and incubate at 4°C overnight.

Discard the primary antibody, add 50μL PBST (purchased from medicago, catalog number 09-9410-100) to wash 4 times; discard the solution, add 50μL secondary antibody (goatα-rabbitIRDye 800CW (purchased from CST, catalog number 5366P); goatα-mouse IRDye 680RD (purchased from CST, catalog number 5151P) (1:5000)), incubate at room temperature in the dark for 1-1.5 hours; discard the secondary antibody, add 50μL PBST to wash 4 times, and discard the solution. Fluorescence scanning was performed using the Sapphire dual-mode multispectral laser imaging system (purchased from Azure Biosystems); Azure Spot analysis software was used to convert the fluorescence intensity of each well into a numerical value, and XLfit was used to calculate the inhibition of BRM and BRG1 protein degradation by each concentration of the compound based on the concentration and fluorescence intensity value of the compound.

The degradation activity of the compounds of the present disclosure on BRM and BRG1 was tested according to the test method described above, and some activity data are summarized in Table 2.

Table 2

Note: “-” means not tested; “NDE” means no degradation was observed at the tested concentration.

Test Results The maximum BRM degradation rate (D _max ) of the control compound ACBI1 (purchased from MCE, catalog number HY-128359-1 mg, CAS No.: 2375564-55-7) was set as Negative control (100% degradation), and the cell group with only DMSO added was set as Positive control (0% degradation) to calculate the DC ₅₀ and D _max of the compounds of the present disclosure.

The results showed that compounds 004, 004-1 and 005 can effectively degrade BRM of A549 cells and SW1573 cells. However, the activity of compounds 004, 004-1 and 005 in degrading BRG1 of SW1573 cells was relatively low.

In addition, the tested compounds can efficiently degrade BRM of A549 cells, such as compounds 004, 005, 011 and 027, which have a DC ₅₀ of less than 10 nM and a D _max of greater than 90% for degrading BRM of A549 cells. Compounds 047, 227, 301, 302, 303, 313, 375, 409 and 410, which have a DC ₅₀ of less than 1 nM and a D _max of greater than 75% for degrading BRM of A549 cells. The tested compounds can efficiently degrade BRM of SW1573, but the activity of degrading BRG1 is weak or no obvious degradation activity is shown. For example, the DC ₅₀ of degrading BRM of SW1573 cells by compounds 010, 030, 037, 042, 048, 049, 054, 058 and 059 is less than 10nM and the D _max is about 90%. At the same time, these compounds do not show obvious BRG1 degradation activity or the degradation rate is less than 50% on SW1573 cells at 10μM, showing good selectivity. The DC ₅₀ of degrading BRM of SW1573 cells by compounds 004, 005, 011, 017, 025, 027, 028 and 056 is less than 10nM and the D _max is greater than 90%, and the DC ₅₀ of degrading BRG1 of SW1573 cells is greater than 10nM and the D _max is less than 90%, showing good selectivity. Compound 047 has a DC ₅₀ of 0.21 nM and a D _max of 98% for degrading BRM of SW1573 cells, and a DC ₅₀ of 3.4 nM and a D _max of 84% for degrading BRG1 of SW1573 cells, with a selectivity of about 16 times. Compounds 227, 278, 301, 303, 304, 306, 309, 311, 318, 318-1, 319, 322, and 323-2 have a DC ₅₀ of less than 1 nM and a D _{max of} greater than or equal to 80% for degrading BRM of SW1573 cells, and a DC ₅₀ of greater than 1 nM and a D _max of less than or equal to 80% for degrading BRG1 of SW1573 cells, indicating a selectivity of more than 10 times. The DC ₅₀ of compounds 310 and 313 for degrading BRM of SW1573 cells is less than 0.1nM and D _max is about 80%, and the DC ₅₀ for degrading BRG1 of SW1573 cells is greater than 0.6nM and D _max is about 70%, both of which have a selectivity of about 10 times. The DC ₅₀ of compounds 375, 378, 398, 401, 409, 410 and 412 for degrading BRM of SW1573 cells is less than 1nM and D _max is greater than 90%, and the DC ₅₀ for degrading BRG1 of SW1573 cells is greater than 3nM and D _max is less than 90%, all of which show a selectivity of more than 9 times. The above results show that the compounds of the present disclosure can selectively degrade BRM, but have little or no effect on BRG1.

Test Example 3: Detection of the degradation activity of compounds on BRM and BRG1 proteins using HiBiT detection technology

1. Cell line construction:

HiBiT detection technology is used to detect the effect of compounds on the degradation of BRM and BRG1 proteins. The HiBiT tag is inserted after the start codon or before the stop codon of the BRM and BRG1 proteins. When the compound degrades the BRM or BRG1 protein, the degradation of the BRM and BRG1 proteins can be quantified by detecting the expression of the HiBiT tag. According to the insertion site of HiBiT, sgRNA and donor DNA of BRM and BRG1 were designed; Cas9 transfection reagent (TrueCut ^TM Cas9Protein v2, A36498, Invitrogen; Lipofectamine ^TM CRISPRMAX ^TM Cas9 transfection reagent, CMAX00003, Invitrogen; Opti-MEM ^TM I Reduced Serum Medium, 31985070, Thermofisher) was prepared, and the required amounts of various reagents, sgRNA and donor DNA were calculated according to the corresponding amounts. The cells were transfected on SW1573 cells according to the Cas9 transfection protocol, and the medium was changed to normal medium after 24 hours; the cells were cultured for 3 days after transfection, and each group of cells was amplified, and the expression level of HiBiT tag in the pool was detected by Nano Glo HiBiT Lytic Detection System (N3050, Promega); and the cell pool with high expression level of HiBiT tag was amplified in 96-well plate in the form of monoclonal clones, and the cells were amplified by Nano Glo HiBiT Lytic Detection System (N3050, Promega) detects the expression of HiBiT tags in monoclonal cells; if the fluorescence value of HiBiT tags amplified by monoclonal cells is very high, it means that this group of cells may have successfully connected to HiBiT tags. These monoclonal cells are amplified and sequenced to confirm that the HiBiT sequence is successfully connected and homozygous, and can be used for subsequent degradation experiments.

2. Cell seeding plate:

Adjust the cell concentration (SW1573 BRM/BRG1 knock in cell line: 9000 cells/well) and seed in a 384-well plate, and culture overnight at 37°C, 5% CO ₂ ; dissolve the compound in DMSO, dilute the compound with DMSO and culture medium in turn and transfer to the cell plate, the final concentration is 1μM, 3-fold dilution. Continue to culture at 37°C, 5% CO ₂ for 16-18 hours.

3. Protein degradation detection:

15 μL of reaction solution (Nano Glo HiBiT Lytic Detection System (N3050, Promega) configured according to the protocol) was added to each well of the 384-well plate, incubated at room temperature for 10 min, and scanned and analyzed using an Envision multi-function microplate reader (purchased from PerkinElmer).

4. Degradation DC ₅₀ calculation

The final drug concentration was 1000 nM, with 3-fold dilution and 10 concentration points. The protein degradation rate of each concentration was calculated using EXCEL XLfit5.4.0, and each concentration point had 2 replicates.

Protein degradation rate (%) = (High control - corresponding well reading) / (High control - Low control) * 100

High control = 0.1% DMSO, defined as 0% inhibition

Low control = PBS (no cells), defined as 100% inhibition

The degradation curve was generated by the degradation rates at 10 concentration points, and the degradation DC ₅₀ and D _max values were calculated.

Table 3

The test results show that the tested compounds can efficiently degrade BRM of SW1573, but the activity of degrading BRG1 is weak or no obvious degradation activity is shown. For example, the DC ₅₀ of compounds 429, 430, 432, 475, 482, 494, 530 and 532 for degrading BRM of SW1573 cells is less than 1nM and the D _max is greater than 90%. At the same time, the DC ₅₀ of these compounds for degrading BRG1 of SW1573 cells is greater than 5nM and the D _max is less than 90%, all showing more than 15 times selectivity. The DC ₅₀ of compounds 211, 334-1, 394, 438, 444, 445, 449, 461, 515 and 518 for degrading BRM of SW1573 cells is less than 10nM and the D _max is greater than 80%. At the same time, the DC ₅₀ of these compounds for degrading BRG1 of SW1573 cells at 1000nM is less than 50%, showing more than 100 times selectivity.

Test Example 4: Antiproliferation Test

Experimental principle: CELL TITER-GLO luminescence method was used to detect the effect of the test compound on the proliferation of A549 and MIA PaCa-2 cell lines.

Test method:

Adjust the cell concentration (A549 & MIA PaCa-2 (both purchased from ATCC): 300 cells/well) in a 96-well plate and culture overnight at 37°C, 5% CO ₂ ; dissolve the compound (initial concentration 10 mM) in DMSO (purchased from Sigma, product number D2650-100 mL), dilute the compound with DMSO and culture medium (DMEM culture medium (Thermo fisher, product number 11995073), containing 10% FBS (Thermo fisher, product number 10099141C) and 1% Penicillin-Streptomycin Solution (Thermo fisher, product number 15140-122)) and transfer to the cell plate, the final concentration of the dilution is 10 μM, 3-fold dilution, a total of 10 dose concentrations. Continue to culture at 37°C, 5% CO ₂ for 7 days. The cell plate was removed, 50 μL of CellTiter-Glo ^™ (purchased from Promega, catalog number G7573) reagent was added to each well, incubated at room temperature for 10 min, and scanned and analyzed using Envision multifunctional microplate reader (purchased from PerkinElmer), the luminescent signal value was read, and the inhibitory activity IC ₅₀ of each compound was calculated using XLfit based on the compound concentration and luminescent signal value.

The anti-proliferative activity of the compounds of the present disclosure on A549 and MIA PaCa-2 cells was tested according to the test method described above. Part of the activity data is summarized in Table 4.

Table 4

Compound IC ₅₀ (nM, A549 cells) IC ₅₀ (nM, MIA PaCa-2 cells) 004 15 - 004-1 141 - 005 20 >10000 007 236 >10000 010 7.7 >10000 011 3.2 >10000 012 340 >10000 013 220 >10000 014 418 >10000 015 746 4305 016 631 >10000 017 38 >10000 018 232 >10000 019 51 2397 021 112 4850 023 151 3867 025 32 3412

027 2.3 2064 028 27 >10000 030 18 >10000 031 95 4793 032-1 72 >10000 033 98 >10000 037 27 >10000 040 15 >1000 042 31 4238 043 26 5372 047 <1.5 >10000 048 27 >10000 049 76 - 053 121 >10000 054 38 >10000 056 37 - 058 97 - 059 70 - 211 39 - 223 38 - 227 1.1 - 228 16 - 229 13 - 278 5.4 - 281 18 - 284 13 - 301 0.73 - 302 0.6 - 303 2.1 - 304 0.6 - 306 12 - 308 74 - 309 5.3 - 310 0.76 - 311 1.4 - 313 1.5 - 318 4.9 - 318-1 8.1 - 319 2.1 - 322 1.9 - 323-1 9.2 - 323-2 4.3 - 332 10 - 334-2 31 - 375 4.0 - 378 5.3 - 398 11 - 400 twenty one - 401 11 - 402 twenty one - 409 3.8 - 410 3.7 - 412 5.0 - 443 16 - 449 25 - 473 14 -

Note: “-” means not tested.

From the above results, it can be seen that the test compound has a stronger inhibitory activity against A549 cells (BRG1 mutation), but has a weaker inhibitory activity against MIA PaCa-2 cells (BRM mutation) or shows no obvious inhibitory activity. For example, compounds 004, 004-1 and 005 have strong inhibitory activity against A549 cells (BRG1 mutation), and compound 005 does not show obvious inhibitory activity against MIA PaCa-2 cells (BRM mutation); compounds 005, 010, 011, 017, 028, 030, 031, 032-1 and 033 have IC _50s of less than 100 nM for A549 cells, and IC _50s of greater than 100 nM for MIA PaCa-2 cells; compounds 019, 025, 027, 031 and 042 have IC _50s of less than 100 nM for A549 cells, and IC _50s of greater than 2000 nM for MIA PaCa-2 cells, and the selectivity is greater than 50 times. The above results show that the compounds of the present disclosure can inhibit the proliferation of tumor cells with BRG1 mutations by selectively degrading BRM, producing a synthetic lethal effect.

Test Example 5: Antiproliferation Test

Experimental principle: CELL TITER-GLO luminescence assay was used to detect the effects of the test compounds on the proliferation of SW1573 and NCI-H1581 cell lines.

Test method:

The cell concentration was adjusted (SW1573 & NCI-H1581 (both purchased from ATCC); SW1573: 2000 cells/well, NCI-H1581: 3000 cells/well) and seeded in a 96-well plate, and cultured overnight at 37°C, 5% _CO2 ; DMSO (purchased from Sigma, product number D2650-100mL) was used to dissolve the compound, and DMSO and the corresponding cell culture medium (SW1573 cell culture medium DMEM, NCI-H1581 cell culture medium RPMI 1640, both containing 10% FBS and 1% double antibody; both purchased from Thermo Fisher, DMEM culture medium product number 11965092, RPMI 1640 medium, catalog number 11875119, FBS catalog number 10099141C, double antibody catalog number 15140-122) were used to dilute the compound and transfer it to the cell plate, with a final concentration of 10 μM, diluted 3 times. The cells were cultured for 7 days at 37°C and 5% CO _2. The cell plate was removed, 50 μL of CellTiter-Glo ^TM (purchased from Promega, catalog number G7573) reagent was added to each well, incubated at room temperature for 10 minutes, and scanned and analyzed using an Envision multi-function microplate reader (purchased from PerkinElmer).

Calculate the proliferation inhibition IC50:

The final drug concentration was 10000 nM, and the drug was diluted 3 times, with a total of 10 doses. The cell proliferation inhibition rate of each concentration was calculated using EXCEL XLfit5.4.0, and each concentration point had 2 replicates.

Cell proliferation inhibition efficiency (%) = (High control - corresponding well reading) / (High control - Low control) * 100

High control＝0.1% DMSO, set as 0% Inhibition

Low control＝PBS(no cells),set as 100%Inhibition

The proliferation inhibition curve was generated by the cell proliferation inhibition rate at 10 concentration points, and the IC ₅₀ value was calculated.

Table 5

Compound IC ₅₀ (nM, SW1573 cells) IC ₅₀ (nM, NCI-H1581 cells) 010 - >10000 318 >10000 2044 482 >10000 >10000

From the above results, it can be seen that compounds 010, 318 and 482 that can selectively degrade BRM (they have strong activity in degrading BRM of SW1573 cells, with DC ₅₀ less than 7nM and D _max greater than 85%; at the same time, these compounds have weak activity in degrading BRG1 of SW1573 cells, with DC ₅₀ greater than 70nM and D _max less than 65%, and the selectivity is greater than 100 times.) have weak inhibitory activity against NCI-H1581 cells (BRM-DEL and BRG1-DEL), and IC ₅₀ is greater than 2μM, indicating that the above compounds have no significant off-target activity. Compounds 318 and 482 did not show obvious inhibitory activity against SW1573 cells (BRM-WT and BRG1-WT), and no obvious inhibitory effect on SW1573 cells was observed at a concentration of 10μM, indicating that the above compounds have good selectivity for BRG1.

Test Example 6: Pharmacokinetics test in mice

1. Test Materials

Balbc nude mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

NMP (N-methylpyrrolidone), Solutol HS15 (polyethylene glycol 15-hydroxystearate), PEG400 (polyethylene glycol), HP-β-CD (hydroxypropyl-β-cyclodextrin), and glipizide were purchased from Sigma, and methanol, acetonitrile, and formic acid were purchased from Merck (USA). _{K 2} EDTA anticoagulation blood collection tubes were purchased from Jiangsu Xinkang Medical Instrument Co., Ltd.

2. Test methods

1. Animal testing

For each compound to be tested, three female Balbc nude mice (20-30 g, 4-6 weeks) were selected and administered with the compound to be tested (the solvent of compounds 303, 313, 318, 323-2, 331, 375 and 412 was 30% PEG400 + 70% HP-β-CD (50%, w/v) aqueous solution, the solvent of compound 482 was 5% NMP + 15% PEG400 + 80% HP-β-CD (20%, w/v) aqueous solution, and the solvent of the remaining compounds was 5% NMP + 15% PEG400 + 80% Solutol HS15 (20%, w/v) aqueous solution). For compounds with a dosage of 10 mg/kg, the compound was dissolved in the corresponding solvent to prepare a 2 mg/mL solution, and the administration volume for each mouse was 5 ml/kg. For compound 011, the compound was prepared into a 0.6 mg/mL solution, and the administration volume for each mouse was 5 ml/kg. For compounds 278, 306, and 311, the compound was prepared into a 0.2 mg/mL solution, and the administration volume for each mouse was 5 ml/kg. Before the test, the animals were fed and watered normally. During the test, venous blood was collected from each group of mice before and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours after administration. The collected whole blood samples were placed in K ₂ EDTA anticoagulant tubes, centrifuged for 5 minutes (4000 rpm, 4°C), and plasma was collected for testing.

2. Sample processing and bioanalysis

Take 10 μL of mouse plasma sample, add 150 μL of methanol/acetonitrile (1:1, v/v) solvent (containing internal standard glipizide) to precipitate protein, vortex for 5 minutes, centrifuge for 5 minutes (14000rpm, 4°C), take the supernatant and dilute it 2 times with water containing 0.1% formic acid (v/v), and use LC-MS/MS system (AB Sciex Triple Quad 6500+) for quantitative detection. When determining the sample concentration, female Balbc nude mouse plasma standard curve and quality control sample are carried out. For the 20x diluted sample, take 2 μL of sample and add 38 μL of blank plasma. After vortexing for 1 minute, add 600 μL of methanol/acetonitrile (1:1, v/v) solvent (containing internal standard glipizide) to precipitate protein, and the rest of the processing steps are the same as above.

3. Data processing

The pharmacokinetic parameters were calculated using the non-compartmental statistical moment method using Phoenix WinNonlin 8.0 software (Certara, USA).

Table 6 PK of compounds of the present disclosure in mice

Test Example 7: Pharmacokinetics test in rats

1. Test Materials

SD rats were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

NMP (N-methylpyrrolidone), Solutol HS15 (polyethylene glycol 15-hydroxystearate), PEG400 (polyethylene glycol), HP-β-CD (hydroxypropyl-β-cyclodextrin), and glipizide were purchased from Sigma, and methanol, acetonitrile, and formic acid were purchased from Merck (USA). _{K 2} EDTA anticoagulation blood collection tubes were purchased from Jiangsu Xinkang Medical Instrument Co., Ltd.

2. Test methods

1. Animal testing

For each test compound, three male SD rats (200-300 g, 6-8 weeks) were selected and administered with the test compound via tail vein injection (the solvent for compounds 010 and 318 was 30% PEG400 + 70% HP-β-CD (50%, w/v) aqueous solution, the solvent for compound 304 was 5% NMP + 15% PEG400 + 80% Solutol HS15 (20%, w/v) aqueous solution, the solvent for compounds 303 and 412 was 5% NMP + 15% PEG400 + 80% Solutol HS15 (10%, w/v) aqueous solution, and the solvent for compound 482 was 5% NMP + 15% PEG400 + 80% HP-β-CD (20%, w/v) aqueous solution.). For compounds with a dosage of 1, 5, 10 or 40 mg/kg, the compounds were dissolved in the corresponding solvents to prepare solutions of 0.2, 1, 2 or 8 mg/mL, respectively, and the dosage volume for each mouse was 5 mL/kg. Before the experiment, the animals were fed and watered normally. During the experiment, venous blood was collected from each group of rats before administration and at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24, 48 (only compounds 303 and 318) and 72 (only compound 303) h after administration. The collected whole blood samples were placed in K ₂ EDTA anticoagulant tubes, centrifuged for 5 min (4000 rpm, 4°C), and plasma was collected for testing.

2. Sample processing and bioanalysis

Take 10 μL of rat plasma sample, add 150 μL of methanol/acetonitrile (1:1, v/v) solvent (containing internal standard glipizide) to precipitate protein, vortex for 5 minutes, centrifuge for 5 minutes (14000rpm, 4°C), take the supernatant and dilute it 2 times with water containing 0.1% formic acid (v/v), and use LC-MS/MS system (AB Sciex Triple Quad 6500+) for quantitative detection. When determining the sample concentration, the male SD rat plasma standard curve and quality control sample are carried out. For the 20x diluted sample, take 2 μL of the sample and add 38 μL of blank plasma. After vortexing for 1 minute, add 600 μL of methanol/acetonitrile (1:1, v/v) solvent (containing internal standard glipizide) to precipitate protein, and the rest of the processing steps are the same as the above steps.

3. Data processing

The pharmacokinetic parameters were calculated using the non-compartmental statistical moment method using Phoenix WinNonlin 8.0 software (Certara, USA).

Table 7 Rat PK of compounds of the present disclosure

Test Example 8: Canine Pharmacokinetics Test

1. Test Materials

Beagle dogs were purchased from Jiangsu Zhaoshengyuan Biotechnology Co., Ltd.

NMP (N-methylpyrrolidone), PEG400 (polyethylene glycol), HP-β-CD (hydroxypropyl-β-cyclodextrin), and glipizide were purchased from Sigma, and methanol, acetonitrile, and formic acid were purchased from Merck (USA). _{K 2} EDTA anticoagulation blood collection tubes were purchased from Jiangsu Xinkang Medical Instrument Co., Ltd.

2. Test methods

1. Animal testing

For each test compound, three male Beagle dogs (8-12 kg, age>6 months) were selected and intravenously injected with the test compound (the solvent for compounds 303 and 309 was 30% PEG400 + 70% HP-β-CD (50%, w/v) aqueous solution, and the solvent for the remaining compounds was 5% NMP + 15% PEG400 + 80% HP-β-CD (20%, w/v) aqueous solution.). For compounds with a dosage of 1 or 5 mg/kg, the compound was dissolved in the corresponding solvent to prepare a solution of 0.5 or 2.5 mg/mL, respectively, and the dosage volume for each dog was 2 mL/kg. Before the experiment, the animals were fed and watered normally. During the experiment, venous blood was collected from each group of dogs before and 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24, 48 (only 303 and 318) h after administration. The collected whole blood samples were placed in K ₂ EDTA anticoagulant tubes, centrifuged for 5 min (4000 rpm, 4° C.), and plasma was collected for testing.

2. Sample processing and bioanalysis

Take 20 μL of canine plasma sample, add 300 μL of methanol/acetonitrile (1:1, v/v) solvent (containing internal standard glipizide) to precipitate protein, vortex for 5 minutes, centrifuge for 5 minutes (14000rpm, 4°C), take the supernatant and dilute it 2 times with water containing 0.1% formic acid (v/v), and use LC-MS/MS system (AB Sciex Triple Quad 6500+) for quantitative detection. When determining the sample concentration, a male Beagle dog plasma standard curve and quality control sample are carried out. For the 20x diluted sample, take 2 μL of the sample and add 38 μL of blank plasma. After vortexing for 1 minute, add 600 μL of methanol/acetonitrile (1:1, v/v) solvent (containing internal standard glipizide) to precipitate protein, and the rest of the processing steps are the same as the above steps.

3. Data processing

The pharmacokinetic parameters were calculated using the non-compartmental statistical moment method using Phoenix WinNonlin 8.0 software (Certara, USA).

Table 8 Canine PK of Compounds of the Disclosure

Test Example 9: Monkey Pharmacokinetics Test

1. Test Materials

Cynomolgus monkeys were purchased from Guangxi Guidong Primate Development Experiment Co., Ltd.

NMP (N-methylpyrrolidone), PEG400 (polyethylene glycol), HP-β-CD (hydroxypropyl-β-cyclodextrin) were purchased from Sinopharm Chemical Reagent Co., Ltd., glipizide was purchased from Sigma, methanol, acetonitrile and formic acid were purchased from Merck (USA). _{K 2} EDTA anticoagulant blood collection tubes were purchased from Shanghai Aoxiang Medical Technology Co., Ltd.

2. Test methods

1. Animal testing

For each test compound, two male cynomolgus monkeys (4.5-6.0 kg, age>3 years) were selected and intravenously injected with the test compound (the solvent was 5% NMP + 15% PEG400 + 80% HP-β-CD (20%, w/v) aqueous solution). For compounds with a dose of 1 or 5 mg/kg, the compound was dissolved in the corresponding solvent to prepare a 0.5 or 2.5 mg/mL solution, and the volume of each monkey was 2 mL/kg. During the experiment, venous blood was collected from each group of monkeys before and 0.033, 0.083, 0.25, 0.5, 1, 2, 4, 6, 8 and 24 hours after administration. The collected whole blood samples were placed in K ₂ EDTA anticoagulant tubes, centrifuged for 10 minutes (3000g, 4°C), and plasma was collected for testing.

2. Sample processing and bioanalysis

Take 20 μL of monkey plasma sample, add 300 μL of methanol/acetonitrile (1:1, v/v) solvent (containing internal standard glipizide) to precipitate protein, vortex for 5 minutes, centrifuge for 5 minutes (14000rpm, 4°C), take the supernatant and dilute it 2 times with water containing 0.1% formic acid (v/v), and use LC-MS/MS system (AB Sciex Triple Quad 6500+) for quantitative detection. When determining the sample concentration, the male cynomolgus monkey plasma standard curve and quality control sample are carried out. For the 10x diluted sample, take 2 μL of sample and add 18 μL of blank plasma. After vortexing for 1 minute, add 300 μL of methanol/acetonitrile (1:1, v/v) solvent (containing internal standard glipizide) to precipitate protein, and the rest of the processing steps are the same as above.

3. Data processing

The pharmacokinetic parameters were calculated using the non-compartmental statistical moment method using Phoenix WinNonlin 8.0 software (Certara, USA).

Table 9 Monkey PK of Compounds of the Disclosure

Test Example 10: Determination of metabolic stability of the compounds of the present disclosure in liver microsomes

1. Test materials and instruments

1. Source of liver microsomes: human liver microsomes (Corning 452117), male CD-1 mouse liver microsomes (Corning 452701), male Beagle dog liver microsomes (Corning 452601), male Sprague-Dawley rats (Corning 452501), male cynomolgus monkey liver microsomes (Corning 452413)

2. NADPH (Solarbio 1216C022)

3. Positive control compound Verapamil (Sigma MKBV4993V)

4. AB Sciex Triple Quad 4000 LC/MS

2. Test steps

1. Preparation of 100 mM phosphate buffer (PBS): Weigh 7.098 g Na ₂ HPO ₄ and add 500 mL pure water to dissolve by ultrasonic to prepare solution A. Weigh 3.400 g KH ₂ PO ₄ and add 250 mL pure water to dissolve by ultrasonic to prepare solution B. Place solution A on a stirrer and slowly add solution B until the pH value reaches 7.4 to prepare 100 mM PBS buffer.

2. Preparation of reaction system

Prepare the reaction system according to the following table:

3. Pre-incubate the reaction system in a 37°C water bath for 10 minutes. Add 40 μL of 10 mM NADPH solution (NADPH is dissolved in 100 mM phosphate buffer) to the reaction system, and the final concentration of NADPH is 1 mM. Use 40 μL of phosphate buffer instead of NADPH solution as a negative control. The role of the negative control is to exclude the influence of the chemical stability of the compound itself.

4. Add 4 μL of 100 μM (the solvent is DMSO) of the compound of the present disclosure and the positive control compound verapamil to the reaction system to start the reaction. The final concentration of the compound is 1 μM.

5. After vortexing and mixing thoroughly at 0.5, 5, 15, 30 and 60 minutes, take out 50 μL of the incubation sample and terminate the reaction with 4 times of ice-cold acetonitrile containing internal standard. Centrifuge the sample at 3220g for 45 minutes. After centrifugation, transfer 90 μL of the supernatant to the injection plate, add 90 μL of ultrapure water and mix well for LC-MS/MS analysis.

All data were calculated using Microsoft Excel. Peak areas were detected by extracting ion spectra. The in vitro half-life (t _1/2 ) of the parent drug was detected by linear fitting the natural logarithm of the parent drug elimination percentage with time.

The in vitro half-life (t _1/2 ) was calculated from the slope:

in vitro t _1/2 =0.693/k

In vitro intrinsic clearance (CL _int ) was calculated by the following formula:

in vitro CL _int = (0.693/t _1/2 )*(incubation volume/protein amount)

(Note: The unit of incubation volume is μl; the unit of protein amount is mg)

The in vitro half-life and in vitro intrinsic clearance values calculated by the above formula are shown in Table 10.

Table 10 In vitro half-life and in vitro intrinsic clearance of compounds of the present disclosure in liver microsome stability

Test Example 11: Determination of the plasma protein binding rate of the compounds of the present disclosure to humans, dogs, rats, mice and monkeys

1. Test materials and instruments

1. Reagents and Instruments

2. Plasma (EDTA K ₂ , stored at -80°C)

Species Strain/Sex batch number source people Mixed HUMANPLK2PNNV20210806 After ethical approval, the samples were collected at local hospitals. dog Beagle; mixed sex BGL119670 BioIVT Rat SD; mixed sex RAT476542 BioIVT Mouse CD-1; mixed sex M220080 BioIVT

monkey Cynomolgus monkey; mixed sex PH-Monkey-20220828 Beijing Rixin Technology Co., Ltd.

2. Experimental steps

1. Preparation of buffer solution with concentration of 100 mM sodium phosphate and 150 mM NaCl

Prepare an alkaline solution with a concentration of 14.2 g/L sodium dihydrogen phosphate and 8.77 g/L sodium chloride with ultrapure water. The alkaline solution can be stored at 4°C for 7 days. Prepare an acidic solution with a concentration of 12.0 g/L sodium dihydrogen phosphate and 8.77 g/L sodium chloride with ultrapure water. The acidic solution can be stored at 4°C for 7 days. Titrate the alkaline solution with the acidic solution to a pH of 7.4. The buffer can be stored at 4°C for 7 days. Test the pH of the buffer on the day of the experiment. If it is outside the range of 7.4 ± 0.1, adjust its pH.

2. Preparation of Dialysis Membrane

The dialysis membrane was soaked in ultrapure water for 60 minutes to separate the membrane into two pieces, then soaked in 20% ethanol for 20 minutes, and finally soaked in a buffer used for dialysis for 20 minutes.

3. Preparation of Plasma

Frozen plasma was rapidly thawed at room temperature.

The plasma was centrifuged at 3,220 g for 10 minutes at 4°C to remove clots, and the supernatant was collected into a new centrifuge tube. The pH value of the plasma was measured and recorded.

Note: a) Only use plasma that has been thawed no more than twice. b) Only use plasma with a pH of 7 to 8.

4. Preparation of stock solution and working solution

Prepare 10mM DMSO stock solutions of the test substance and control drug warfarin sodium. Dilute 2μL of the stock solution (10mM) with 98μL DMSO to obtain a working solution (200μM). Take 3μL of the working solution and add 597μL of human, dog, rat, mouse or monkey plasma to a final concentration of 1μM (0.5% DMSO), and vortex thoroughly.

5. Equilibrium Dialysis Step

Assemble the dialysis device according to the operating instructions. Add 120 μL of the drug-doped plasma sample to one side of the dialysis membrane and an equal volume of dialysate (phosphate buffer) to the other side. The experiment is in parallel. Seal the dialysis plate and place it in the incubator. Incubate for 6 hours at 37°C, 5% CO ₂ , and approximately 100 rpm. After the incubation, remove the seal and pipette 50 μL from the buffer and plasma side of each well into different wells of a new plate.

6. Sample Analysis Steps

Add 50 μL of blank plasma to the phosphate buffer sample, and add an equal volume of blank phosphate buffer to the plasma sample. Precipitate the protein by adding 300 μL of room temperature quencher (containing internal standard acetonitrile (IS, 500 nM labetalol, 100 nM alprazolam, and 2 μM ketoprofen). Vortex for 5 minutes. Centrifuge the sample plate at 3,220 g for 30 minutes at 4°C. Transfer 100 μL of supernatant to a new plate. Depending on the HPLC response signal and peak shape of the analyte, the supernatant may be diluted with 100 μL or 200 μL of water. Mix well and analyze the sample by HPLC.

3. Data Analysis

Determine the peak areas of the test substance and the control drug on the buffer side and the plasma side, and calculate the plasma protein binding rate of the test substance and the control drug as follows:

Free rate % = (the ratio of the peak area of the test substance or control drug to the peak area of the internal standard _{on the buffer side} / the ratio of the peak area of the test substance or control drug to the peak area of the internal standard on _{the plasma side} ) * 100%

Binding rate % = 100% - free rate %

All data were calculated using Microsoft Excel software. The calculated plasma protein binding values of the compounds of the present disclosure are shown in Table 11.

Table 11 Protein binding rate values of compounds of the present disclosure in plasma of different species

Test Example 12: Inhibitory Effects of the Compounds of the Disclosure on Cytochrome P450 Enzymes CYP2C9, CYP2D6 and CYP3A4 I. Test Materials and Test Equipment

1. Reagents

2. Liver microsomes

3. Test equipment

2. Experimental steps

1. Prepare the test substance powder into a 30mM DMSO stock solution, and dilute it with DMSO to obtain the following concentrations: 0, 4, 20, 100, 400, 2000 and 10000 μM. The concentration of the test substance in the final incubation system is 0, 0.02, 0.1, 0.5, 2, 10 and 50 μM. The concentration of the organic solvent introduced into the test system with the test substance is 0.5%. The final concentration information of the positive inhibitor in the reaction system is shown in Table 13 below.

Table 12 Working solution concentration of positive inhibitors

Table 13 Final concentration of positive inhibitors in the reaction system

2. Preparation of Substrate Stock Solution

The specific preparation of the substrate stock solution and the incubation time of each enzyme subtype are shown in Table 14. After the stock solution is prepared, it is stored in a -20°C refrigerator and thawed at room temperature before use.

Table 14 Substrate stock solution information

3. Preparation of phosphate buffered saline (100 mM, pH 7.4)

First, weigh 7.098g of disodium hydrogen phosphate, add 500mL of pure water and ultrasonically dissolve it as solution A. Weigh 3.400g of potassium dihydrogen phosphate, add 250mL of pure water and ultrasonically dissolve it as solution B. Place solution A on a stirrer and slowly add solution B until the pH value reaches 7.4. Phosphate buffer is stored at 4°C for future use.

4. Preparation of 10mM NADPH

Before the test, weigh an appropriate amount of NADPH and prepare a working solution with a concentration of 10 mM using phosphate solution. The final concentration of NADPH in the test system is 1 mM.

5. Preparation of incubation system

The incubation system was prepared as shown in the table below and preheated in a 37°C water bath for 15 minutes before use.

6. Test methods

The entire incubation process will be carried out in a 96-well deep-well plate. First, add 179μL of the incubation system to the deep-well plate, and then add 1μL of the compound solution or solvent (DMSO). Before starting the reaction with 20μL of 10mM NADPH solution, preheat the incubation system at 37℃ for 15 minutes. After adding NADPH to start the reaction, incubate at 37℃ for the corresponding time. The test samples are prepared in duplicate.

At the corresponding time, 400 μL of ice methanol (IS, 500 nM Labetalol, 100 nM Alprazolam and 2 μM Ketoprofen) was added to terminate the reaction. After vortexing, the deep-well plate was centrifuged at 3220 g and 4 °C for 40 minutes. 100 μL of supernatant was transferred to a new plate, 100 μL of pure water was added to mix, and then used for LC-MS/MS analysis.

3. Data Analysis

The generated metabolites were analyzed by LC-MS/MS. The reduction of metabolite generation in the treated group compared with the control group was compared by the peak area ratio of the sample to the internal standard, and the _IC50 value was calculated based on the residual activity percentage using Excel XLfit 5.3.1.3.

The remaining activity percentage was calculated using the following formula:

Remaining activity percentage (%) = ratio of metabolite peak area to internal standard peak area of _{test substance} /ratio of metabolite peak area to internal standard peak area of _{blank solvent} × 100%.

The test results are shown in Table 15.

Table 15 Inhibitory effects of compounds of the present disclosure on cytochrome P450 enzymes

Test Example 13: Using manual patch clamp technique to evaluate the effect of test substances on hERG potassium channel current

1. Materials and Instruments

2. Experimental steps

1. Cell Lines and Cell Culture

HEK293 cell line (Cat. No. K1236) stably expressing hERG ion channel was purchased from Invitrogen. The cell line was cultured in a medium containing 85% DMEM, 10% dialyzed fetal bovine serum, 0.1 mM non-essential amino acid solution, 100 U/mL penicillin-streptomycin solution, 25 mM HEPES, 5 μg/mL blasticidin and 400 μg/mL geneticin. When the cell density grew to 40% to 80% of the bottom area of the culture dish, trypsin was used for digestion and passage, and passaged three times a week. Before the experiment, the cells were cultured in a 6 cm culture dish at a density of 5×10 ⁵ , induced by adding 1 μg/mL doxycycline for 48 hours, and then the cells were digested and inoculated on glass slides for subsequent manual patch clamp experiments (Note: the cell generation number used for safety evaluation experiments is <70 generations).

2. Solution Preparation

1) Extracellular solution (in mM): 132 NaCl, 4 KCl, 3 CaCl, 0.5 MgCl, 11.1 Glucose, and 10 HEPES (pH adjusted to 7.35 with NaOH).

2) Intracellular solution (in mM): 140 KCl, 2 MgCl2, 10 EGTA, 5 ATP-Mg salt and 10 HEPES (pH adjusted to 7.35 with KOH).

Note: The osmotic pressure of the solution is controlled between 280 and 300 mOsmol/kg. The solution needs to be filtered and stored at 4°C before use. ATP-magnesium salt is first prepared into a 100mM stock solution and stored in a -20°C refrigerator. On the day of the experiment, a certain amount is added to the intracellular solution and used immediately after preparation.

3. Preparation of test compound solution

1) According to the standard operating procedure SOP-ADMET-MAN-007, the test compound was dissolved in DMSO and prepared into a stock solution with a final concentration of 10 mM.

2) The stock solution was diluted with DMSO in a gradient ratio of 1:3 to form three other intermediate concentration solutions, with concentrations of 3.33 mM, 1.11 mM and 0.37 mM, respectively.

3) Before the experiment, the stock solution and intermediate solution of the test compound were diluted 1000 times with extracellular fluid to obtain a series of working solutions with concentrations of 10μM, 3.33μM, 1.11μM and 0.37μM. At the same time, the 10mM stock solution was diluted 333.33 times with extracellular fluid to obtain a 30μM working solution. The content of DMSO in the working solution was 0.1-0.3% (volume ratio) (Note: The content of DMSO in the working solution should be controlled within 1% (volume ratio) to avoid cytotoxicity).

4) After the working solution is prepared, observe with the naked eye whether there is precipitation or turbidity in the working solution. If there is, it may be due to the poor solubility of the compound in physiological solution. It can be further ultrasonicated in a water bath for 30 minutes to improve the clarity of the solution.

5) The potential inhibitory effect of the test substance on hERG channel at five concentrations of 30 μM, 10 μM, 3.33 μM, 1.11 μM and 0.37 μM was determined, and the dose-effect curve was fitted and the IC ₅₀ was calculated.

4. Patch clamp operation

1) Place the small glass slide containing HEK293 cells in the culture dish into the perfusion tank of the microscope operation table.

2) Place the appropriate cell in the center of the field of view under an Olympus IX51, IX71 or IX73 inverted microscope, use a ×10 objective to find the tip of the glass electrode and place it in the center of the field of view. Then use the micromanipulator to move the electrode down while adjusting the coarse focusing knob to slowly bring the electrode closer to the cell.

3) When approaching the cell, switch to a ×40 objective lens for observation and use the micromanipulator to fine-tune the gear to gradually bring the electrode closer to the cell surface.

4) Apply negative pressure to form a seal with a resistance higher than 1 GΩ between the electrode tip and the cell membrane.

5) Compensate the instantaneous capacitance current Cfast in voltage clamp mode, then repeatedly apply short negative pressure to break the membrane, and finally form a whole-cell recording mode.

6) Under the condition that the membrane potential is clamped at -60 mV, the slow capacitive current Cslow, cell membrane capacitance (Cm) and input membrane resistance (Ra) are compensated respectively.

7) After the cells are stable, the clamping voltage is changed to -90mV, the sampling frequency is set to 20kHz, and the filtering frequency is 10kHz. The detection conditions of the leakage current are as follows: the clamping voltage is changed to -80mV and the time course is 500ms.

8) The hERG current test method is as follows: apply a 4.8-second depolarization command voltage to depolarize the membrane potential from -80mV to +30mV, then instantly apply a 5.2-second repolarization voltage to reduce the membrane potential to -50mV to remove channel inactivation, so that the hERG tail current can be observed. The peak value of the tail current is the magnitude of the hERG current.

9) The hERG current used to detect the test compound was recorded for 120 seconds before administration to evaluate the stability of the hERG current generated by the test cells. Only stable cells within the acceptable range of the evaluation criteria can enter the subsequent compound testing.

10) Determine the inhibitory effect of the test compound on the hERG current: First, the hERG current measured in the extracellular fluid containing 0.1% DMSO is used as the detection baseline. After the hERG current remains stable for at least 5 minutes, the solution containing the test compound is perfused around the cells in sequence from low concentration to high concentration. After each perfusion, wait for about 5 minutes to allow the compound to fully act on the cells and record the hERG current synchronously. After the recorded current tends to stabilize, record the last 5 hERG current values, and take the average value as the final current value at a specific concentration. After testing the compound, add 150nM dofelide to the same cell to completely inhibit its current as a positive control for the cell. At the same time, the positive compound dofelide is synchronously detected using the same patch clamp system before and after the test compound experiment to ensure the reliability and sensitivity of the entire detection system.

3. Data Analysis

The data were exported by PatchMaster software and analyzed according to the following steps:

1) After perfusing the blank solvent or compound gradient solution, the average of the five consecutive current values obtained was calculated and used as the "tail current size _blank " and "tail current size _compound "respectively;

2) The current suppression percentage is calculated by the following formula:

The dose-effect curve was fitted by Graphpad Prism 8.0 software and the IC ₅₀ value was calculated. The results are shown in Table 16.

Table 16 Effects of the compounds of the present disclosure on hERG potassium channel current

Compound IC ₅₀ (μM) Compound IC ₅₀ (μM) 010 >30 318 >30 303 >30 323-2 >30 304 5.7 482 >30 309 12.3

Test Example 14: Growth inhibition experiment of compound on A549 subcutaneous xenograft tumor

1. Experimental Reagents

Human non-small cell lung cancer A549 cells (Shanghai Institutes for Biological Studies, Chinese Academy of Sciences); F-12K culture medium (Gibco, Catalog No. 21127-022); fetal bovine serum (Gibco, Catalog No. 10091-148); penicillin-streptomycin (Gibco, Catalog No. 15140-122); 0.25% trypsin-EDTA (Gibco, Catalog No. 25200-072); phosphate buffered saline (DPBS) (Hyclone, Catalog No. SH30256.01); Matrigel (Corning, Catalog No. 356234).

2. Experimental Methods

Animal information: Balb/c nude mice, female, 7-9 weeks old, weighing approximately 18-22 grams, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. The mice were housed in an SPF-grade environment with separate ventilation in each cage. All animals had free access to standard certified commercial laboratory diets and free drinking water.

Cell culture: Human non-small cell lung cancer A549 cell line was cultured in vitro in F-12K medium with 10% fetal bovine serum, Penicillin-streptomycin, and incubated at 37°C and 5% CO _2. The cells were routinely digested and passaged twice a week using 0.25% trypsin-EDTA digestion solution. When the cell confluence reached 85%-95% and the number reached the required level, the cells were harvested and counted.

Cell inoculation: 0.1 ml/(containing 5×10 ⁶ ) A549 cell suspension (DPBS plus Matrigel, volume ratio of 1:1) was subcutaneously inoculated on the right back of each mouse. On the 16th day after cell inoculation, the mice were randomly divided into groups and administered drugs according to the tumor volume, and the day of grouping was Day 0.

Administration: Group 1 was given vehicle, 5 ml/kg, twice a week x 2 weeks; Group 2 was given compound 304, the dosage was 40 mg/kg, once on Day 0 and Day 3; Group 3 was given compound 047, the dosage was 40 mg/kg, once a week (QW) x 2 weeks; Group 4 was given compound 047, the dosage was 40 mg/kg, twice a week (BIW) x 2 weeks; Group 5 was given compound 027, the dosage was 40 mg/kg, twice a week (BIW) x 2 weeks. There were 6 mice in each group, and the administration method was tail vein injection.

Tumor Measurement and Experimental Indicators

The diameter of the tumor was measured with a vernier caliper twice a week. The formula for calculating the tumor volume was: V = 0.5axb ² , where a and b represent the long diameter and short diameter of the tumor, respectively. The body weight of the mice was measured twice a week.

The anti-tumor efficacy of the compound was evaluated by tumor growth inhibition rate (TGI).

TGI (%) = [(1-(average tumor volume of a treatment group at the end of drug administration-average tumor volume of the treatment group at the beginning of drug administration)/(average tumor volume of the solvent control group at the end of treatment-average tumor volume of the solvent control group at the beginning of treatment)] x 100%.

IV. Experimental Results

As shown in Table 17 and Figure 1, during the administration of the mouse subcutaneous transplanted tumor A549 model, the weight of mice in Group 2 fluctuated significantly, but the weight of mice was able to recover after drug withdrawal. The mice in other groups were all tolerant under their respective dosing regimens. All the dosing groups were able to significantly inhibit tumor growth relative to the vehicle control group (P<0.0001); the tumor inhibition effect of 40 mg/kg compound 047 given twice a week was more significant than that of the same dose of the compound given once a week (P<0.01).

Table 17 Tumor volume of A549 subcutaneous tumor model

Unless expressly excluded or otherwise limited, each document cited herein, including any cross-referenced patent or patent application, any patent application or patent to which this application claims priority, is hereby incorporated by reference in its entirety. In addition, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

Although specific embodiments of the present disclosure have been illustrated and described, those skilled in the art will appreciate that, where feasible, the technical features described in one embodiment may be applied to another embodiment, or combined with the technical features described in another embodiment. Therefore, those skilled in the art will be able to make various changes and modifications to the embodiments of the present disclosure without departing from the essence and scope of the present disclosure.

Claims (31)

1. A compound represented by the formula (I) or a pharmaceutically acceptable salt thereof,

   TL-Linker-DIM

   (I)

   Wherein:

   The DIM is a ligand compound capable of binding to E3 ubiquitin ligase;

   The Linker is a linking group covalently binding at least one TL and at least one DIM;

   TL is a group as shown below:

   wherein:

   selected from: c=c-N, wherein X ⁴ is N; or N-c=c, wherein X ⁴ is C;

   X ³ is selected from CR ³ or N;

   Y ¹、Y ²、Y ³、Y ⁴、X ¹ is selected from one of five cases:

   (1) Y ¹ is CR, Y ² is CR ^2a or N, Y ³ is CR ^3a or N, Y ⁴ is CR ^4a or N, and X ¹ is CR ¹ or N;

   (2) Y ¹ is selected from CR ^1a or N, Y ² is CR, Y ³ is selected from CR ^3a or N, Y ⁴ is selected from CR ^4a or N, and X ¹ is selected from CR ¹ or N;

   (3) Y ¹ is selected from CR ^1a or N, Y ² is selected from CR ^2a or N, Y ³ is CR, Y ⁴ is selected from CR ^4a or N, and X ¹ is selected from CR ¹ or N;

   (4) Y ¹ is selected from CR ^1a or N, Y ² is selected from CR ^2a or N, Y ³ is selected from CR ^3a or N, Y ⁴ is CR, and X ¹ is selected from CR ¹ or N;

   (5) Y ¹ is selected from CR ^1a or N, Y ² is selected from CR ^2a or N, Y ³ is selected from CR ^3a or N, Y ⁴ is selected from CR ^4a or N, and X ¹ is CR;

   the R is Wherein n is selected from 0, 1,2,3 or 4; r ^a is selected from halogen, OH, CN, NO ₂、C ₁-C ₆ alkyl, C ₁-C ₆ alkoxy, C ₃-C ₆ cycloalkyl, NH ₂、NH(C ₁-C ₆ alkyl) or N (C ₁-C ₆ alkyl) ₂, wherein said C ₁-C ₆ alkyl, C ₃-C ₆ cycloalkyl, C ₁-C ₆ alkoxy is optionally substituted with R ^b; r ^b is selected from halogen, OH, = O, NH ₂, CN or COOH;

   The R ^1a、R ^2a、R ^3a、R ^4a、R ¹ is independently selected from H, halogen, NH ₂、NO ₂、CN、COOH、C ₁-C ₆ alkyl, C ₁-C ₆ alkoxy, C ₃-C ₆ cycloalkyl, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, NH (C ₁-C ₆ alkyl), N (C ₁-C ₆ alkyl) ₂、NH(C ₃-C ₆ cycloalkyl), NH-4-8 membered heterocyclyl, NH-5-10 membered heteroaryl, NH- (C ₆-C ₁₀ aryl), NHC (O) -4-8 membered heterocyclyl, NHC (O) (C ₁-C ₆ alkyl), NHC (O) -O (C ₁-C ₆ alkyl), N (C ₁-C ₆ alkyl) C (O) -O (C ₁-C ₆ alkyl), NHC (O) (C ₃-C ₆ cycloalkyl), NHC (O) - (C ₆-C ₁₀ aryl), NHC (O) -5-10 membered heteroaryl, C (O) - (C ₃-C ₆ cycloalkyl), C (O) - (C ₆-C ₁₀ aryl), C (O) -5-10 membered heteroaryl, C (O) NH ₂、C(O)NH(C ₁-C ₆ alkyl), C (O) N (C ₁-C ₆ alkyl) ₂、C(O)NH(C ₃-C ₆ cycloalkyl), C (O) NH- (C ₆-C ₁₀ aryl), C (O) NH-5-10 membered heteroaryl or C (O) NH-4-8 membered heterocyclyl, wherein said C ₁-C ₆ alkyl, C ₁-C ₆ alkoxy, C ₃-C ₆ cycloalkyl, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl optionally substituted with R ⁵;

   The R ³、R ⁴ is independently selected from H, OH, halogen, NH ₂、C ₁-C ₆ alkyl, C ₂-C ₆ alkenyl, C ₂-C ₆ alkynyl, C ₁-C ₆ alkoxy, C ₃-C ₆ cycloalkyl, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, C ₃-C ₆ cycloalkyloxy, NH (C ₁-C ₆ alkyl), N (C ₁-C ₆ alkyl) ₂、NH(C ₃-C ₆ cycloalkyl), NHC (O) (C ₁-C ₆ alkyl), NHC (O) (C ₃-C ₆ cycloalkyl), NHC (O) - (C ₆-C ₁₀ aryl), NHC (O) -5-10 membered heteroaryl, NHC (O) -4-8 membered heterocyclyl, C (O) NH (C ₁-C ₆ alkyl), c (O) NH (C ₃-C ₆ cycloalkyl), C (O) NH- (C ₆-C ₁₀ aryl), C (O) NH-5-10 membered heteroaryl or C (O) NH-4-8 membered heterocyclyl, wherein said C ₁-C ₆ alkyl, C ₂-C ₆ alkenyl, C ₂-C ₆ alkynyl, C ₁-C ₆ alkoxy, C ₃-C ₆ cycloalkyl, C ₆-C ₁₀ aryl, A 5-10 membered heteroaryl, 4-8 membered heterocyclyl or C ₃-C ₆ cycloalkyloxy group optionally substituted with R ⁶;

   Or R ^1a and R ³ together with the atoms to which they are each attached form a C ₅-C ₁₀ cycloalkenyl or a 5-14 membered heterocyclyl, which C ₅-C ₁₀ cycloalkenyl or 5-14 membered heterocyclyl is optionally substituted with R ⁷;

   Or R ^4a and R ⁴ together with the atoms to which they are each attached form a C ₅-C ₁₀ cycloalkenyl or a 5-14 membered heterocyclyl, which C ₅-C ₁₀ cycloalkenyl or 5-14 membered heterocyclyl is optionally substituted with R ⁸;

   Or R ^2a and R ^3a together with the atoms to which they are each attached form a C ₅-C ₁₀ cycloalkenyl, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, or 5-14 membered heterocyclyl, the C ₅-C ₁₀ cycloalkenyl, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, or 5-14 membered heterocyclyl being optionally substituted with R ⁹;

   Or R ¹ and R ⁴ together with the atoms to which they are each attached form a C ₅-C ₁₀ cycloalkenyl or a 5-14 membered heterocyclyl, which C ₅-C ₁₀ cycloalkenyl or 5-14 membered heterocyclyl is optionally substituted with R ¹⁰;

   R ⁵、R ⁶、R ⁷、R ⁸、R ⁹、R ¹⁰ is independently selected from deuterium, halogen, OH, = O, NH ₂、NH(C ₁-C ₆ alkyl), N (C ₁-C ₆ alkyl) ₂、CN、C ₁-C ₆ alkyl, C ₃-C ₆ cycloalkyl, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, or 4-8 membered heterocyclyl, said C ₁-C ₆ alkyl, C ₃-C ₆ cycloalkyl, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, or 4-8 membered heterocyclyl being further optionally substituted with R ^5a;

   Said R ^5a is selected from halogen, OH, NH ₂, =o or C ₁-C ₃ alkyl;

   The conditions are as follows: at least one of R ^1a、R ^2a、R ^3a、R ^4a、R ¹、R ³、R ⁴ is other than H; and when R ⁴ is H, R ³ is not optionally substituted Or optionally substituted
2. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein,Selected from c=c-N, wherein X ⁴ is N.
3. The compound of formula (I) or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein,Selected from N-c=c, wherein X ⁴ is C.
4. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3, wherein R ^1a、R ^2a、R ^3a、R ^4a、R ¹ is independently selected from H, halogen, NH ₂、CN、C ₁-C ₆ alkyl, C ₁-C ₆ alkoxy, C ₃-C ₆ cycloalkyl, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl, 4-8 membered heterocyclyl, NH (C ₁-C ₆ alkyl), N (C ₁-C ₆ alkyl) ₂、NH(C ₃-C ₆ cycloalkyl), NH-4-8 membered heterocyclyl, NH-5-10 membered heteroaryl, NH- (C ₆-C ₁₀ aryl), NHC (O) (C ₃-C ₆ cycloalkyl), NHC (O) -4-8 membered heterocyclyl, NHC (O) - (C ₆-C ₁₀ aryl), NHC (O) -5-10 membered heteroaryl, C (O) NH (C ₃-C ₆ cycloalkyl), C (O) NH- (C ₆-C ₁₀ aryl), C (O) NH-5-10 membered heteroaryl or C (O) NH-4-8 membered heterocyclyl, wherein said C ₁-C ₆ alkyl, C ₁-C ₆ alkoxy, C ₃-C ₆ cycloalkyl, C ₆-C ₁₀ aryl, 5-10 membered heteroaryl or 4-8 membered heterocyclyl are optionally substituted by R ⁵.
5. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 4, wherein R ^1a and R ³ together with the atoms to which they are each attached form a 5-14 membered heterocyclyl optionally substituted by R ⁷.
6. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 5, wherein R ^4a and R ⁴ together with the atoms to which they are each attached form a 6-14 membered heterocyclyl optionally substituted by R ⁸.
7. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 6, wherein R ^2a and R ^3a together with the atoms to which they are each attached form a 5-10 membered heterocyclyl optionally substituted by R ⁹.
8. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 7, wherein R ¹ and R ⁴ together with the atoms to which they are each attached form a 5-10 membered heterocyclyl optionally substituted by R ¹⁰.
9. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 8, wherein R ^a is selected from halogen, OH, NH ₂ or C ₁-C ₆ alkyl optionally substituted by R ^b and n is selected from 0, 1 or 2.
10. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 9, wherein TL isWherein R ⁴ is connected with the Linker.
11. A compound of formula (I) according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof, wherein TL isWherein R ³ is connected with the Linker.
12. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 11, wherein TL isWherein R ^3a is connected with the Linker.
13. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 12, wherein TL isWherein R ^4a is connected with the Linker.
14. A compound of formula (I) according to any one of claims 1 to 13, or a pharmaceutically acceptable salt thereof, wherein TL is a group of the structure:

    Wherein: r ^a, Y ²、Y ³、Y ⁴、X ¹、X ³、R ⁴ and n are as defined in claim 1.
15. A compound of formula (I) according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof, wherein TL is a group of the structure:

    Wherein: r ^a, Y ¹、Y ³、Y ⁴、X ¹、X ³、R ⁴ and n are as defined in claim 1.
16. A compound of formula (I) according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof, wherein TL is a group of the structure:

    Wherein: r ^a, Y ¹、Y ²、Y ⁴、X ¹、X ³、R ⁴ and n are as defined in claim 1.
17. A compound of formula (I) according to any one of claims 1 to 16, or a pharmaceutically acceptable salt thereof, wherein TL is a group of the structure:

    Wherein: r ^a, Y ¹、Y ²、Y ³、X ¹、X ³、R ⁴ and n are as defined in claim 1.
18. A compound of formula (I) according to any one of claims 1 to 17, or a pharmaceutically acceptable salt thereof, wherein TL is a group of the structure:

    Wherein: r ^a, Y ¹、Y ²、Y ³、Y ⁴、X ³、R ⁴ and n are as defined in claim 1.
19. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 18, wherein TL is selected from one of the following structures:

    more preferably one of the following structures:
20. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 19, wherein the Linker is a linking group covalently bonding one TL and one DIM.
21. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 20, wherein the Linker is selected from-L ^A-、-L ^B-、-R ^1L-、-R ^2L-、-Q ¹-、-Q ² -,

    Wherein:

    -L ^A-、-L ^B -is independently from each other selected from the group consisting of bond, -O-, -S-, -NR ^3'-、-CR ^4'R ^5'-、-C(O)-、-S(O)-、-S(O) ₂ -, -C (S) -, -C (O) O-, or-C (O) NR ^6' -;

    R ^1L and R ^2L are independently selected from the group consisting of a bond, an alkylene, a heteroalkylene, an alkenylene, and an alkynylene, wherein the alkylene, heteroalkylene, alkenylene, and alkynylene are optionally substituted with a group selected from the group consisting of: halogen, alkyl, alkoxy, haloalkyl, OH, hydroxyalkyl, CN, NH ₂, =o, cycloalkyl, heterocyclyl, aryl, heteroaryl;

    Q ¹、Q ²、Q ³ and Q ⁴ are independently selected from cycloalkyl, heterocyclyl, aryl, heteroaryl, or cycloalkenyl, wherein each of said cycloalkyl, heterocyclyl, aryl, heteroaryl, and cycloalkenyl is independently optionally substituted with a group selected from the group consisting of: halogen, alkyl, alkoxy, haloalkyl, OH, hydroxyalkyl, CN, NH ₂, =o, cycloalkyl, heterocyclyl, aryl, heteroaryl;

    R ^3' is selected from H, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, and heteroaryl;

    R ^4' and R ^5' are each independently selected from H, halogen, alkyl, alkoxy, haloalkyl, OH, hydroxyalkyl, CN, NH ₂, =o, cycloalkyl, heterocyclyl, aryl or heteroaryl;

    R ^6' is selected from H, alkyl, heteroalkyl, haloalkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl.
22. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 21, wherein the DIM is selected from a VHL ligand or a CRBN ligand.
23. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 22, wherein DIM is selected from structures of formula (DIM-1) or (DIM-2):

    wherein:

    Selected from the group consisting of

    Y is a bond, or Y is selected from Y _A、O、NH、NR _E、C(O)O、C(O)NR _E'、NR _E'C(O)、Y _A-NH、Y _A-NR _E、Y _A-C(O)、Y _A-C(O)O、Y _A-OC(O)、Y _A-C(O)NR _E' or Y _A-NR _E' C (O), wherein the Y _A is selected from C ₁-C ₆ alkylene, C ₂-C ₆ alkenylene, or C ₂-C ₆ alkynylene;

    X is selected from C (O) or C (R _A) ₂;X _A-X _B is selected from C (R _A) =n or C (R _A) ₂-C(R _A) ₂;

    Each R _A is independently selected from H or C ₁-C ₃ alkyl, said C ₁-C ₃ alkyl optionally substituted with C ₆-C ₁₀ aryl or 5-10 membered heteroaryl;

    Each R _A' is independently selected from C ₁-C ₃ alkyl;

    Each R _B is independently selected from H or C ₁-C ₃ alkyl, or two R _B together with the atoms to which they are attached form C (O), C ₃-C ₆ cycloalkyl, C ₃-C ₆ cycloalkenyl, or a 4-6 membered heterocyclyl;

    R _C is selected from H, halogen or C ₁-C ₃ alkyl;

    Each R _D is independently selected from halogen, NO ₂、NH ₂、OH、COOH、C ₁-C ₆ alkyl, or C ₁-C ₆ alkoxy;

    Each R _E is independently selected from C ₁-C ₆ alkyl, C ₂-C ₆ alkenyl, C ₃-C ₈ cycloalkyl, 3-8 membered heterocycloalkyl, C (O) -C ₁-C ₆ alkyl, C (O) -C ₂-C ₆ alkenyl, C (O) -C ₃-C ₈ cycloalkyl, or C (O) -3-8 membered heterocycloalkyl, said R _E being optionally substituted with a group selected from: halogen, N (R _a) ₂、NHC(O)R _a、NHC(O)OR _a、OR _b、C ₃-C ₈ cycloalkyl, 3-8 membered heterocycloalkyl, C ₆-C ₁₀ aryl, or 5-10 membered heteroaryl, wherein said C ₃-C ₈ cycloalkyl, 3-8 membered heterocycloalkyl, C ₆-C ₁₀ aryl, or 5-10 membered heteroaryl is optionally further substituted with a group selected from halogen, NH ₂、CN、NO ₂、OH、COOH、C ₁-C ₆ alkyl, C ₁-C ₆ haloalkyl, C ₁-C ₆ alkoxy, or C ₁-C ₆ haloalkoxy;

    R _E' is selected from H, C ₁-C ₆ alkyl, C ₂-C ₆ alkenyl, C ₃-C ₈ cycloalkyl or 3-8 membered heterocycloalkyl, said C ₁-C ₆ alkyl, C ₂-C ₆ alkenyl, C ₃-C ₈ cycloalkyl or 3-8 membered heterocycloalkyl being optionally substituted with a group selected from: halogen, N (R _a) ₂、NHC(O)R _a、NHC(O)OR _a、OR _b、C ₃-C ₈ cycloalkyl, 3-8 membered heterocycloalkyl, C ₆-C ₁₀ aryl, or 5-10 membered heteroaryl, wherein said C ₃-C ₈ cycloalkyl, 3-8 membered heterocycloalkyl, C ₆-C ₁₀ aryl, or 5-10 membered heteroaryl is optionally further substituted with a group selected from halogen, NH ₂、CN、NO ₂、OH、COOH、C ₁-C ₆ alkyl, C ₁-C ₆ haloalkyl, C ₁-C ₆ alkoxy, or C ₁-C ₆ haloalkoxy;

    Each R _a is independently selected from H or C ₁-C ₆ alkyl;

    R _b is selected from H or p-toluenesulfonyl;

    t is selected from 0 or 1;

    m is selected from 0, 1, 2 or 3;

    p is selected from 0, 1 or 2.
24. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 23, wherein the DIM is selected from structures of formula (DIM-11):

    wherein:

    X _C is selected from bond 、-CH ₂-、-CHCF ₃-、-SO ₂-、-S(O)-、-P(O)R'-、-P(O)OR'-、-P(O)NR' ₂-、-C(O)-、-C(S)- or X _D is selected from C, N or Si;

    X _E is selected from the group consisting of a bond, -CR ' ₂ -, -NR ' -, -O-, -S-, or-SiR ' ₂ -;

    r _F is absent, or R _F is selected from H, deuterium, halogen 、CN、-OR'-、-SR'-、-S(O)R'-、-S(O) ₂R'-、-NR' ₂-、-P(O)(OR') ₂、-P(O)(NR' ₂)OR'-、-P(O)(NR' ₂) ₂、-Si(OH) ₂R'、-Si(OH)R' ₂、-SiR' ₃, or C ₁-C ₄ alkyl;

    Each R _G is independently selected from H, deuterium, R _H, halogen 、CN、-NO ₂、-OR'、-SR'、-NR' ₂、-SiR' ₃、-S(O) ₂R'、-S(O) ₂NR' ₂、-S(O)R'、-C(O)R'、-C(O)OR'、-C(O)NR' ₂、-C(O)N(R')OR'、-C(R') ₂N(R')C(O)R'、-C(R') ₂N(R')C(O)NR' ₂、-OC(O)R'、-OC(O)NR' ₂、-OP(O)R' ₂、-OP(O)(OR') ₂、-OP(O)(OR')NR' ₂、-OP(O)(NR' ₂) ₂、-N(R')C(O)OR'、-N(R')C(O)R'、-N(R')C(O)NR' ₂、-N(R')S(O) ₂R'、-NP(O)R' ₂、-N(R')P(O)(OR') ₂、-N(R')P(O)(OR')NR' ₂, or-N (R ') P (O) (NR' ₂) ₂;

    Each R _H is independently selected from C ₁-C ₆ alkyl, phenyl, 4-7 membered heterocyclyl, or 5-6 membered heteroaryl;

    Ring E, ring F, ring G are independently selected from phenyl, 6 membered heteroaryl, C ₅-C ₇ cycloalkyl, C ₅-C ₇ cycloalkenyl, 5-7 membered heterocyclyl or 5-6 membered heteroaryl, wherein ring E, ring F and ring G are each optionally further substituted with =o;

    L ¹ is selected from a bond, C ₁-C ₃ alkylene, C ₂-C ₃ alkenylene, or C ₂-C ₃ alkynylene, wherein any 1 or 2 methylene groups of the C ₁-C ₃ alkylene, C ₂-C ₃ alkenylene, or C ₂-C ₃ alkynylene are optionally substituted with: -O-, -C (O) -, -C (S) -, -C (R ') ₂-、-CH(R')-、-C(F) ₂ -, -N (R') -, S-, or-S (O) ₂ -;

    Each R 'is independently selected from H, C ₁-C ₆ alkyl, phenyl, 4-7 membered heterocyclyl, or 5-6 membered heteroaryl, or two R' together with the atoms to which they are attached form 4-7 membered heterocyclyl or 5-6 membered heteroaryl;

    q is selected from 0, 1,2,3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16.
25. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 24, wherein DIM is selected from structures of formula (DIM-13), (DIM-14), (DIM-15), (DIM-16) or (DIM-17):

    wherein:

    r _J is selected from optionally substituted C ₁-C ₆ alkyl, optionally substituted- (CH ₂) _j OH), optionally substituted- (CH ₂) _j SH), Optionally substituted- (CH ₂) _j-O-(C ₁-C ₆) alkyl, optionally substituted- (CH ₂) _j-CH ₂OCH ₂-(C ₀-C ₆) alkyl, optionally substituted- (CH ₂) _j COOH, optionally substituted- (CH ₂) _jC(O)-(C ₁-C ₆) alkyl), Optionally substituted- (CH ₂) _jNR _cR _d, optionally substituted- (CH ₂) _jNHC(O)-R _c), optionally substituted- (CH ₂) _jC(O)-NR _cR _d), optionally substituted- (CH ₂) _jOC(O)-NR _cR _d、-(CH ₂O) _j H), optionally substituted- (CH ₂) _jOC(O)-(C ₁-C ₆ alkyl), optionally substituted- (CH ₂) _jC(O)-O-(C ₁-C ₆ alkyl), optionally substituted- (CH ₂O) _j COOH, optionally substituted- (OCH ₂) _jO-(C ₁-C ₆ alkyl), Optionally substituted- (CH ₂O) _jC(O)-(C ₁-C ₆ alkyl), optionally substituted- (OCH ₂) _jNHC(O)-R _c, optionally substituted- (CH ₂O) _jC(O)-NR _cR _d、-(CH ₂CH ₂O) _j H), optionally substituted- (CH ₂CH ₂O) _j COOH), Optionally substituted- (OCH ₂CH ₂) _jO-(C ₁-C ₆ alkyl), optionally substituted- (CH ₂CH ₂O) _jC(O)-(C ₁-C ₆ alkyl), optionally substituted- (OCH ₂CH ₂) _jNHC(O)-R _c), optionally substituted- (CH ₂CH ₂O) _jC(O)-NR _cR _d), Optionally substituted-SO ₂R _s, optionally substituted-S (O) R _s、NO ₂, CN or halogen;

    R _c and R _d are independently selected from H or C ₁-C ₆ alkyl, said C ₁-C ₆ alkyl optionally substituted with OH or halogen;

    R _s is selected from C ₁-C ₆ alkyl, optionally substituted aryl, optionally substituted heteroaryl, optionally substituted heterocyclyl or- (CH ₂) _gNR _cR _d);

    X _F and X _G are independently selected from c= O, C = S, S (O) or S (O) ₂;

    R _L is selected from optionally substituted -(CH ₂) _j-(C＝O) _u(NR _c) _v(SO ₂) _w(C ₁-C ₆) alkyl, optionally substituted -(CH ₂) _j-(C＝O) _u(NR _c) _v(SO ₂) _wNR _1NR _2N、 optionally substituted- (CH ₂) _j-(C＝O) _u(NR _c) _v(SO ₂) _w -aryl, optionally substituted- (CH ₂) _j-(C＝O) _u(NR _c) _v(SO ₂) _w -heteroaryl, optionally substituted- (CH ₂) _j-(C＝O) _vNR _c(SO ₂) _w -heterocyclyl, optionally substituted -NR _e-(CH ₂) _j-C(O) _u(NR _c) _v(SO ₂) _w-C ₁-C ₆ alkyl, optionally substituted -NR _e-(CH ₂) _j-C(O) _u(NR _c) _v(SO ₂) _w-NR _1NR _2N、 optionally substituted -NR _e-(CH ₂) _j-C(O) _u(NR _c) _v(SO ₂) _w-NR _cC(O)R _1N、 optionally substituted -NR _e-(CH ₂) _j-(C＝O) _u(NR _c) _v(SO ₂) _w- aryl, optionally substituted -NR _e-(CH ₂) _j-(C＝O) _u(NR _c) _v(SO ₂) _w- heteroaryl or optionally substituted-NR _e-(CH ₂) _j-(C＝O) _vNR _c(SO ₂) _w -heterocyclyl, optionally substituted-X ^R2'-C ₁-C ₆ alkyl, optionally substituted-X ^R2' -aryl, optionally substituted-X ^R2' -heteroaryl or optionally substituted-X ^R2' -heterocyclyl;

    R _M is selected from optionally substituted C ₁-C ₆ alkyl, optionally substituted -(CH ₂) _j-C(O) _u(NR _c) _v(SO ₂) _w-C ₁-C ₆ alkyl, Optionally substituted -(CH ₂) _j-C(O) _u(NR _c) _v(SO ₂) _w-NR _1NR _2N、 optionally substituted -(CH ₂) _j-C(O) _u(NR _c) _v(SO ₂) _w-NR _cC(O)R _1N、 optionally substituted -(CH ₂) _j-C(O) _u(NR _c) _v(SO ₂) _w-C(O)NR _cR _d、 optionally substituted- (CH ₂) _j-C(O) _u(NR _c) _v(SO ₂) _w -aryl), Optionally substituted- (CH ₂) _j-C(O) _u(NR _c) _v(SO ₂) _w -heteroaryl), optionally substituted- (CH ₂) _j-C(O) _u(NR _c) _v(SO ₂) _w -heterocycle, optionally substituted -NR _e-(CH ₂) _j-C(O) _u(NR _c) _v(SO ₂) _w-C ₁-C ₆ alkyl, optionally substituted -NR _e-(CH ₂) _j-C(O) _u(NR _c) _v(SO ₂) _w-NR _1NR _2N、 optionally substituted -NR _e-(CH ₂) _j-C(O) _u(NR _c) _v(SO ₂) _w-NR _cC(O)R _1N、 optionally substituted -NR _e-(CH ₂) _j-C(O) _u(NR _c) _v(SO ₂) _w- aryl, Optionally substituted -NR _e-(CH ₂) _j-C(O) _u(NR _c) _v(SO ₂) _w- heteroaryl, optionally substituted -NR _e-(CH ₂) _j-C(O) _u(NR _c) _v(SO ₂) _w- heterocycle, optionally substituted -O-(CH ₂) _j-(C＝O) _u(NR _c) _v(SO ₂) _w-C ₁-C ₆ alkyl, optionally substituted -O-(CH ₂) _j-(C＝O) _u(NR _c) _v(SO ₂) _w-NR _1NR _2N、 optionally substituted -O-(CH ₂) _j-(C＝O) _u(NR _c) _v(SO ₂) _w-NR _cC(O)R _1N、 optionally substituted-O- (CH ₂) _j-(C＝O) _u(NR _c) _v(SO ₂) _w -aryl), Optionally substituted-O- (CH ₂) _j-(C＝O) _u(NR _c) _v(SO ₂) _w -heteroaryl or optionally substituted-O- (CH ₂) _j-(C＝O) _u(NR _c) _v(SO ₂) _w -heterocycle, optionally substituted -(CH ₂) _j-(V) _n'-(CH ₂) _j-(V) _n'-C ₁-C ₆ -alkyl, optionally substituted- (CH ₂) _j-(V) _n'-(CH ₂) _n-(V) _n' -aryl), Optionally substituted- (CH ₂) _j-(V) _n'-(CH ₂) _j-(V) _n' -heteroaryl, optionally substituted- (CH ₂) _j-(V) _n'-(CH ₂) _j-(V) _n' -heterocyclyl, optionally substituted- (CH ₂) _j-N(R _1')(C＝O) _m'-(V) _n'-C ₁-C ₆ alkyl), optionally substituted- (CH ₂) _j-N(R _e)(C＝O) _m'-(V) _n' -aryl), Optionally substituted- (CH ₂) _j-N(R _e)(C＝O) _m'-(V) _n' -heteroaryl, optionally substituted- (CH ₂) _j-N(R _e)(C＝O) _m'-(V) _n' -heterocyclyl, optionally substituted-X ^R3'-C ₁-C ₆ alkyl, optionally substituted-X ^R3' -aryl), Optionally substituted-X ^R3' -heteroaryl or optionally substituted-X ^R3' -heterocyclyl;

    Wherein R _1N and R _2N are each independently H, C ₁-C ₆ alkyl optionally substituted with OH and halogen, or optionally substituted- (CH ₂) _j -aryl, - (CH ₂) _j -heteroaryl or- (CH ₂) _j -heterocyclyl);

    V is O, S or NR _c;

    R _e is independently selected from H or C ₁-C ₃ alkyl;

    X ^R2' and X ^R3' are each independently selected from optionally substituted -(CH ₂) _j-、-(CH ₂) _j-C(X ^v)＝C(X ^v)-、-(CH ₂) _j-C≡C-、-(CH ₂CH ₂O) _j- or C ₃-C ₆ cycloalkyl, wherein X ^v is H, halogen or optionally substituted C ₁-C ₃ alkyl;

    g is independently 0, 1, 2,3, 4, 5, 6;

    j is independently 0, 1, 2,3, 4, 5 or 6;

    m' is independently 0 or 1;

    n' is independently 0 or 1;

    u is independently 0 or 1;

    v is independently 0 or 1;

    w is independently 0 or 1.
26. A compound of formula (I) or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 25, wherein the compound has a structure selected from one of:
27. a pharmaceutical composition comprising a compound of any one of claims 1-26, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable adjuvant.
28. Use of a compound according to any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 27, in the manufacture of a medicament for the prevention or treatment of a BRM-mediated disease, preferably a tumour.
29. A compound according to any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 27, for use in the prevention or treatment of a BRM mediated disease, preferably a tumour.
30. Use of a compound according to any one of claims 1 to 26, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to claim 27, for the prevention or treatment of a BRM-mediated disease, preferably a tumor.
31. A method of treating a BRM-mediated disease comprising administering to a subject in need thereof a therapeutically effective amount of a compound of any one of claims 1-26, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition of claim 27, wherein the BRM-mediated disease is preferably a tumor, optionally wherein the subject is a mammal, preferably a human.