CN115381823B - Application of cyclin-dependent kinase 9 inhibitor - Google Patents

Abstract

The present application provides a use of a cyclin-dependent kinase 9 inhibitor. Specifically, the present application provides various uses of a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, a stereoisomer or a prodrug thereof in the treatment of solid tumors, especially in the treatment of liver cancer, breast cancer or prostate cancer, including related pharmaceutical uses, pharmaceutical compositions or treatment methods, etc.

Description

Use of a cyclin-dependent kinase 9 inhibitor

Related Applications

This application claims priority to Chinese Patent Application No. 202110564093.0 filed on May 24, 2021, and the entire contents of that application are incorporated herein by reference in their entirety for all purposes.

Technical Field

The present application relates to the field of medicine, and specifically, to a cyclin-dependent kinase 9 (CDK9) inhibitor or a pharmaceutically acceptable salt thereof, a stereoisomer or a prodrug thereof, a pharmaceutical composition comprising the same, or a drug containing the same, in particular, the inhibitor or a pharmaceutically acceptable salt thereof, a stereoisomer or a prodrug thereof, a pharmaceutical composition comprising the same, or a drug containing the same in the preparation of a drug for treating solid tumor diseases, in particular malignant solid tumors.

Background Art

Cyclin-dependent kinases (CDKs) are a class of serine/threonine protein kinases that play a key role in regulating the cell cycle and transcription. To date, there are about 20 human CDK subtypes and about 30 cell cycle chaperones (Cao et al. 2014). These CDKs can be activated by cell cycle proteins and play different biological functions. CDKs can be divided into two types according to their functions, one controlling the cell cycle and the other regulating cell transcription. For example, CDK1, 2, 3, 4, and 6 directly intervene in the cell cycle; CDK5 does not regulate the cell cycle, but plays a key role in the complex migration of post-mitotic neurons; CDK7 indirectly acts as an activator of these CDKs; CDK9 only plays a role in cell transcription and does not participate in the regulation of the cell cycle.

CDK9 is an important member of the transcription CDKs subfamily, which is a group of kinases whose function is to control the main steps of eukaryotic RNA polymerase II (Pol II) synthesis and processing of mRNA. CDK9 exists in all mammalian cells. The activation of CDK9 in vivo depends on its binding to the corresponding cyclin (Cyclin T/K) to form a heterodimer, namely positive transcription elongation factor b (P-TEFb). When negative transcription elongation factors (NELF, NELFs) participate in the negative regulation of cell transcription, transcription is inhibited, and P-TEFb is recruited to the system where negative transcription elongation factors inhibit transcription elongation, catalyzing the phosphorylation of the carbon terminal domain (CTD) of RNA polymerase II, and at the same time catalyzing the phosphorylation of the SPT5 subunit of NELFs and the RD subunit of NELF, causing the negative transcription elongation factor to detach from the transcription complex, thereby allowing transcription to continue.

Tumors are usually caused by the loss of cyclin-dependent kinase inhibitor (CDKI) expression or the overexpression of cyclins, which leads to unregulated cell proliferation. In view of the above regulatory mechanism, the use of CDK9 inhibitors will be able to prevent P-TEFb from phosphorylating the carbon-terminal domain of RNA polymerase II, further hindering the departure of NEFL, strengthening the negative inhibitory effect, causing transcription to stop, and causing the levels of intracellular mRNA and short-lived proteins to drop rapidly, thereby causing apoptosis of tumor cells. CDK9 has become a potential protein target for the development of effective cancer treatments. Recently, pharmaceutical companies have conducted research on the use of CDK9 inhibitors for cancer treatment. For example, AstraZeneca's AZD4573 and Bayer's BAY-1251152 are both in Phase I clinical trials, and no CDK9 inhibitor has been approved for marketing.

Although some CDK9 inhibitor small molecules have been disclosed (for example, WO2009047359, WO2014076091, etc.), it is still necessary to develop new compounds with good efficacy and good safety so that more patients can benefit clinically.

Summary of the invention

In one aspect, the present application provides a use of a compound as represented by formula (I) or a pharmaceutically acceptable salt thereof, a stereoisomer or a prodrug thereof in the preparation of a medicament for treating solid tumors, especially malignant solid tumors.

On the other hand, the present application provides a compound as shown in formula (I) or a pharmaceutically acceptable salt thereof, a stereoisomer or a prodrug thereof for preparing a drug for treating liver cancer, breast cancer or prostate cancer, especially CDK9-related liver cancer, breast cancer or prostate cancer.

In another aspect, the present application provides a use of compound 45 or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof as a CDK9 inhibitor in the preparation of a medicament for treating solid tumors, especially malignant solid tumors.

On the other hand, the present application provides a use of compound 45 or a pharmaceutically acceptable salt thereof, a stereoisomer or a prodrug thereof as a CDK9 inhibitor for preparing a medicament for treating liver cancer, breast cancer or prostate cancer, in particular CDK9-related liver cancer, breast cancer or prostate cancer.

In another aspect, the present application provides a pharmaceutical composition for treating solid tumors, comprising a compound as shown in formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof.

On the other hand, the present application provides a pharmaceutical composition for treating liver cancer, breast cancer or prostate cancer, comprising a compound as shown in formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof.

In another aspect, the present application provides a pharmaceutical composition for treating solid tumors, comprising compound 45 or a pharmaceutically acceptable salt, a stereoisomer or a prodrug thereof.

In another aspect, the present application provides a pharmaceutical composition for treating liver cancer, breast cancer or prostate cancer, comprising compound 45 or a pharmaceutically acceptable salt thereof, a stereoisomer or a prodrug thereof.

The inventors of the present application previously developed novel compounds with cyclin-dependent kinase 9 (CDK9) inhibitor activity (see publication number WO2021/115335A1 or application number PCT/CN2020/134966, the entire contents of which are incorporated herein by reference for all purposes). On this basis, the inventors of the present application conducted a more in-depth study of the medical use of the compound and obtained the various inventions of the present application.

Specifically, in the first aspect of the present application, there is provided a use of a compound as represented by formula (I) or a pharmaceutically acceptable salt thereof, a stereoisomer or a prodrug thereof in the preparation of a drug for treating solid tumors,

in,

X is selected from Cl, F, wherein F is preferred;

^R1 is selected from substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; the "substituted" in ^R1 refers to being replaced by 1, 2, 3, 4 or 5 groups independently selected from -F, -Cl, -Br, _-NH2 , -OH, -SH, -CN, _{-NO2, -N3 ,} -C≡CH, ^-COOH , ^-R3 , -( _CH2 ) _{wO (} CH2) _nR3 , - _{( CH2} ) _wNH ( _CH2 ) ^nR3 , - _{( CH2} ) _{wNR3 (CH2)nR4, -(CH2)wS (} ^CH2 _{) nR3} , -( ^CH2 ) _wC ( ^O ₎ ⁽ _{CH2 )} nR3, ^- ( _CH2 )wC(O)O( _CH2 ) _nR3 , _- ( _CH2 ) _{w wOC} (O)( _CH2 ) _nR3 , -( _CH2 ) _wC (O)NH ⁽ _CH2 ) _nR3 , -( _CH2 ) _wNHC ( ^O ) ⁽ _CH2 )nR3, -( _{CH2 ) wC} (O) ^NR3 ( _CH2 )nR4, ^- ₍ CH2) _wNR3C (O ₎ ( _CH2 ^{) nR4} , -( _CH2 )wOS(O) ₂ ( _CH2 ) _nR3 or -( _CH2 ) _wS (O) _2O ( _CH2 ) _nR3 ^; wherein w and ⁿ are each independently selected from 0, 1, 2, 3 or 4; ^R3 and ^R4 are independently selected from _substituted or _{unsubstituted} aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted _C1-6 alkyl, substituted or unsubstituted C _{R 3 and R 4 are substituted or unsubstituted C 1-6} haloalkyl, substituted or unsubstituted C _2-6 alkenyl, substituted or unsubstituted C _2-6 alkynyl, substituted or unsubstituted C _1-6 alkoxy, substituted or unsubstituted C _1-6 haloalkoxy, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, or when R ³ and R ⁴ are jointly connected to the same nitrogen atom, R ³ , R ⁴ and the jointly connected nitrogen atom constitute a substituted or unsubstituted heterocycloalkyl; the "substituted" in R ³ and R ⁴ refers to being substituted by 1, 2 or 3 groups independently selected from -F, -Cl, -Br, -NH ₂ , -OH, -SH, -CN, -NO ₂ , -N ₃ , -C≡CH, -COOH, C _1-6 alkyl, C _1-6 haloalkyl, C _1-6 alkoxy, C _1-6 haloalkoxy, etc.;

The A ring is selected from substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl; the "substituted" in the A ring refers to being substituted by 1, 2, 3, 4 or 5 groups independently selected from -F, -Cl, -Br, OH, NH ₂ , SH, CN, NO ₂ , -N ₃ , -C≡CH, COOH, R ⁵ , OR ⁵ , -NHR ⁵ , -NR ⁵ R ⁶ , -SR ⁵ , -NHCOR ⁵ , -CONHR ⁵ , -NHS(O) _{2 R 5 , -S(O) 2 NHR 5 , -NR 5 S(O) 2 R} ^{6 ,} _-S ⁽ O) ₂ NR 5 R ^{6 , or 1, 2 or more -CH 2 - groups in the A ring structure may be optionally replaced by a -C(O)- group; wherein R 5} and R ⁶ are independently C 1-6 alkyl, C 1-6 alkyl, C ^1-6 alkyl, C ^1-6 alkyl, C 1-6 alkyl, C 1-6 alkyl, C 1-6 alkyl, C 1-6 alkyl, C 1-6 alkyl, C 1-6 alkyl, C 1-6 alkyl, C 1-6 alkyl, C 1-6 _alkyl , C 1-6 alkyl, C 1-6 alkyl, C 1-6 alkyl ^, C _1-6 alkyl, C 1-6 alkyl, C _1-6 haloalkyl.

R ² is selected from H, R ⁷ , -(CH ₂ ) _x R ⁷ , -(CH ₂ ) _x NH(CH ₂ ) _y R ⁷ , -(CH ₂ ) _x O(CH ₂ ) _y R ⁷ , -( CH ₂ ) _x NR ⁷ (CH ₂ ) _y R ⁸ , -(CH ₂ ) _x C(O)(CH ₂ ) _y H, -(CH ₂ ) _x C(O)(CH ₂ ) _y R ⁷ , - (CH ₂ ) _x S(O) ₂ (CH ₂ ) _y R ⁷ , -(CH ₂ ) _x C(O)C(O)(CH ₂ ) _y R ⁷ , -(CH ₂ ) _x S(O) ₂ NH ₂ , -(CH ₂ ) _x NHS(O) ₂ H, -(CH ₂ ) _x S(O) ₂ NH(CH ₂ ) _y R ⁷ , -(CH ₂ ) _x NHS(O) ₂ (CH ₂ ) _y R ⁷ , -(CH ₂ ) _x S(O) ₂ NR ⁷ (CH ₂ ) _y R ⁸ , - (CH ₂ ) _x NR ⁷ S(O) ₂ (CH ₂ ) _y R ⁸ , -(CH ₂ ) _x C(O)O(CH ₂ ) _y R ⁷ , -(CH ₂ ) _x OC(O)( CH ₂ ) _y R ⁷ , -(CH ₂ ) _x C(O)NH ₂ , -(CH ₂ ) _x NHC(O)H, -(CH ₂ ) _x C(O)NH(CH ₂ ) _y R ⁷ ,-(CH ₂ ) _x NHC(O)(CH ₂ ) _y R ⁷ ,-(CH ₂ ) _x C(O)NR ⁷ (CH ₂ ) _y R ⁸ or -(CH ₂ ) _x NR ⁷ C(O)(CH ₂ ) _y R ⁸ ; wherein one, two or more -CH ₂ - groups The group may be optionally replaced by a -C(O)- group; x and y are each independently selected from 0, 1, 2, 3 or 4 at each occurrence;

R ⁷ and R ⁸ are independently selected from substituted or unsubstituted R ⁹ , OR ⁹ , -R ¹⁰ -OR ⁹ , -R ¹⁰ -NH-R ⁹ , -R ¹⁰ -C(O)-R ⁹ , -R ¹⁰ -NHC(O)-R ⁹ , -R ¹⁰ -C(O)NH-R ⁹ , -R ¹⁰ -SR ⁹ , -R ¹⁰ -S(O)-R ⁹ , -R ¹⁰ -SC(O)-R ⁹ , cycloalkyl, heterocycloalkyl, aryl, heteroaryl, -R ¹⁰ -aryl, -R ¹⁰ -heteroaryl, -OR ¹⁰ -aryl, -OR ¹⁰ -heteroaryl, -R ¹⁰ -O-aryl, -R ¹⁰ -O-heteroaryl, -cycloalkyl-aryl, -cycloalkyl-heteroaryl, -heterocycloalkyl-aryl, -heterocycloalkyl-heteroaryl, C 2-6 alkene and C _2-6 alkyne, or when R ⁷ and R ⁸ are connected to the same nitrogen atom, R ⁷ and R ⁸ and the nitrogen atom connected to them form a substituted or unsubstituted heterocycloalkyl; wherein R ⁹ is C _1-6 alkyl, R ¹⁰ is C _1-6 alkylene, C _2-6 alkenylene or C _2-6 alkynylene; the "substituted" in R ₇ and R ⁸ refers to being replaced by 1, 2 or 3 groups independently selected from -F, -Cl, -Br, -OH, -NH ₂ , -SH, -CN, -NO ₂ , -N ₃ , ^-C≡CH , -COOH, C _1-6 alkyl, C 1-6 alkoxy, C _1-6 haloalkyl, C _2-6 alkoxy ... The group may be substituted with a group such as -C _1-6 haloalkoxy, -NHCN, -NHCONH ₂ , NHC(O)CH ₃ , N(CH ₃ ) ₂ , N(C ₂ H ₅ ) ₂ , -SC(O)CH ₃ , or -OC(O)-C _1-6 alkyl.

The definitions of “aryl”, “heteroaryl”, “cycloalkyl” and “heterocycloalkyl” in the present application are as follows in the “Definition” section.

In one embodiment, the aryl group preferably contains 6 to 10 carbon atoms, the cycloalkyl group preferably contains 3 to 6 carbon atoms, the heteroaryl group is preferably a 5-10-membered heteroaryl group, and the heterocycloalkyl group is preferably a 3-8-membered heterocyclic group; the heteroaryl group or heterocycloalkyl group preferably contains 1, 2 or 3 heteroatoms independently selected from N, O or S, and the rest are carbon atoms.

Regarding the "w", "n", "x" and "y" in the present application, as mentioned above, they can be independently selected from 0, 1, 2, 3 or 4. When "w", "n", "x" and "y" appear in a group at the same time, specifically, the numerical combination of "w", "n", "x" and "y" can be selected from (0,0), (0,1), (0,2), (0,3), (0,4), (1,0), (1,1), (1,2), (1,3), (1,4), (2,0), (2,1), (2,2), (2,3), (2,4), (3,0), (3,1), (3,2), (3,3), (3,4), (4,0), (4,1), (4,2), (4,3), (4,4), and this numerical combination is applicable to each relevant group in the definition of ^R1 and ^R2 . For example, the definition of ^R1 is -( _CH2 ) _wO ( _CH2 ) _nR ³ is equivalent to disclosing -OR ³ , -OCH ₂ R ³ , -O(CH ₂ ) ₂ R ³ , -O(CH ₂ ) ₃ R ³ , -O(CH ₂ ) ₄ R ³ , -CH ₂ OR ³ , -CH ₂ OCH ₂ R ³ , -CH ₂ O(CH ₂ ) ₂ R ³ , -CH ₂ O(CH ₂ ) ₃ R ³ , -CH ₂ O(CH ₂ ) ₄ R ³ , -(CH ₂ ) ₂ OR ³ , -(CH ₂ ) ₂ OCH ₂ R ³ , -(CH ₂ ) ₂ O(CH ₂ ) ₂ R ³ , -(CH ₂ ) ₂ (CH ₂ ) ₃ R ³ , -(CH ₂ ) ₂ O(CH ₂ ) ₄ R ³ , -(CH ₂ ) ₃ OR ³ , -(CH ₂ ) ₃ OCH ₂ R ³ , -(CH ₂ ) ₃ O(CH ₂ ) ₃ R ³ , -(CH ₂ ) ₃ O(CH ₂ ) ₃ R ³ , -(CH ₂ ) ₃ O(CH ₂ ) ₄ R ³ , -(CH ₂ ) ₄ OR ³ , -(CH ₂ ) ₄ OCH ₂ R ³ , -(CH ₂ ) ₄ O(CH ₂ ) ₄ R ³ , -(CH ₂ ) ₄ O(CH ₂ ) ₃ R ³ , -(CH ₂ ) ₄ O(CH ₂ ) ₄ R ³ and the like. Similarly, other related groups in R ¹ and R ² also disclose such selections, which will not be elaborated one by one.

In one embodiment, the compound represented by formula (I) or its pharmaceutically acceptable salt, stereoisomer or prodrug, wherein the A ring is selected from substituted or unsubstituted 4-6 membered cycloalkyl, substituted or unsubstituted 4-6 membered heterocycloalkyl, or substituted or unsubstituted 5-6 membered cycloalkyl, or substituted unsubstituted 5-6 membered heterocycloalkyl; further preferably, the A ring is selected from substituted or unsubstituted 5-6 membered cycloalkyl, or substituted unsubstituted 5-6 membered heterocycloalkyl; further preferably, the A ring is selected from substituted or unsubstituted 5-6 membered cycloalkyl.

In one embodiment, the A ring is selected from cyclohexyl, tetrahydropyrrolyl, piperidinyl, piperazinyl, cyclopentanyl or morpholinyl, and more preferably, the A ring is selected from cyclohexyl, cyclopentanyl or tetrahydropyrrolyl.

In one embodiment, the compound represented by formula (I) or its pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein the "substituted" in ring A refers to substitution with 1, 2, 3, 4 or ⁵ groups independently selected from -F, -Cl, -Br, OH, _NH2 , SH, CN, ^R5 , OR5, wherein ^R5 is _C1-6 alkyl or _C1-6 alkoxy, and ^R5 can be further selected from _C1-4 alkyl or _C1-4 alkoxy.

In one embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof has a structure represented by formula (II):

Wherein, R ¹ , R ² and X are as shown in formula (I).

In one embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof has a structure represented by formula (IIa):

Wherein, R ¹ , R ² and X are as shown in formula (I).

In one embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof has a structure represented by formula (III):

Wherein, R ¹ , R ² and X are as shown in formula (I).

In one embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof has a structure represented by formula (IIIa):

Wherein, R ¹ , R ² and X are as shown in formula (I).

In one embodiment, the compound represented by formula (I) or its pharmaceutically acceptable salt, stereoisomer or prodrug, wherein ^R1 is selected from substituted or unsubstituted 6-10 membered aryl, or substituted or unsubstituted 5-10 membered heteroaryl; the heteroaryl contains 1 or 2 heteroatoms independently selected from N or O; the number of the substituents is selected from 1, 2 or 3.

In one embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, a stereoisomer or a prodrug thereof, wherein ^R1 is selected from a substituted or unsubstituted benzene ring, a pyridine ring, an indole ring, an indazole ring, a benzofuran ring, and a pyrrolopyridine ring; further preferably, a substituted or unsubstituted benzene ring, a pyridine ring, an indole ring, a benzofuran ring, and a pyrrolopyridine ring; further preferably, a substituted benzene ring, a pyridine ring and an unsubstituted indole ring, a benzofuran ring, and a pyrrolopyridine ring; further preferably, a substituted benzene ring.

In one embodiment, the compound represented by formula (I) or its pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein the substituents on R ¹ are independently selected from -F, -Cl, -OH, -NH ₂ , -R ³ , -(CH ₂ ) _w O(CH ₂ ) _n R ³ or -(CH ₂ ) _w OC(O)(CH ₂ ) _n R ³ ; w and n are independently selected from 0, 1 or 2 at each occurrence, wherein R ³ is defined as described in formula (I). Preferably, the substituents on R ¹ are independently selected from -F-, -Cl, -OH, -R ³ , -(CH ₂ ) _w O(CH ₂ ) _n R ³ ; further preferably, the substituents on R ¹ are independently selected from -F-, -OH, -R ³ , -(CH ₂ ) _w O(CH ₂ ) _n R ³ . For example, when R ¹ is a substituted benzene ring, the substituent is selected from -F, -OH or alkoxy, preferably 1 or 2 fluorine atoms and 1 -OH or alkoxy, preferably 1 or 2 fluorine atoms and 1 alkoxy.

In one embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof has a structure represented by formula (II):

Wherein, R ¹ is a substituted benzene ring, and the substituents are selected from -F, -OH or alkoxy; preferably 1 or 2 fluorine atoms are substituted and 1 -OH or alkoxy is substituted, preferably 1 or 2 fluorine atoms are substituted and 1 alkoxy is substituted;

^R2 and X are as shown in formula (I).

In one embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof has a structure represented by formula (IIa):

Wherein, R ¹ is a substituted benzene ring, the substituents are selected from -F, -OH or alkoxy; preferably 1 or 2 fluorine atoms and 1 -OH or alkoxy, preferably 1 or 2 fluorine atoms and 1 alkoxy;

^R2 and X are as shown in formula (I).

In one embodiment, the compound represented by formula (I) or its pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein ^R3 and ^R4 are independently selected from substituted or unsubstituted 6-membered aryl, substituted or unsubstituted 5-6-membered heteroaryl, substituted or unsubstituted _C1-3 alkyl, substituted or unsubstituted _C1-3 alkoxy, substituted or unsubstituted _C3-6 cycloalkyl, substituted or unsubstituted _C3-6 heterocycloalkyl, or when ^R3 and ^R4 are connected to the same nitrogen atom, ^R3 , ^R4 and the connected nitrogen atom form a 3-7-membered substituted or unsubstituted heterocycloalkyl; the heterocycloalkyl contains 1 or 2 heteroatoms independently selected from N, O or S; the "substituted" in ^R3 and ^R4 refers to being replaced by 1, 2 or 3 atoms independently selected from -F, -Cl, -Br, -OH, -CH3, _-C2H5 , _-OCH3 , _{-OC2H5 , - 5} ; preferably, R ³ and R ⁴ are independently selected from substituted or unsubstituted C _1-3 alkyl, substituted or unsubstituted C _1-3 alkoxy.

In one embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein ^R3 and ^R4 are independently selected from substituted or unsubstituted benzene ring, pyridine ring, methyl, ethyl, propyl, isopropyl, methoxy, ethoxy, propoxy, isopropoxy, cyclopropyl, cyclobutyl, cyclopentyl or cyclohexyl, and the "substituted" in ^R3 and ^R4 means substituted by 1, 2 or ₃ substituents independently selected from -F, -Cl _, -Br, _-OH , -CH3, _-C2H5 , _-OCH3 , _-OC2H5 . Preferably, R ³ and R ⁴ are independently selected from substituted or unsubstituted methyl, ethyl, isopropyl, methoxy, ethoxy, isopropoxy, cyclopropyl, pyridine ring; further preferably, R 3 and R ⁴ are independently selected from substituted or unsubstituted methyl, ethyl, isopropyl, methoxy, ethoxy, isopropoxy; further ^{preferably, R 3 and R 4 are independently} selected from substituted or unsubstituted methyl, methoxy.

In one embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or _prodrug thereof, wherein ^R2 is selected from ^R7 , -( _CH2 ) _xR7 , -( _CH2 ) _xNH ( _CH2 ) ^yR7 , -( _CH2 ) _xC (O ₎ ( _CH2 ) _yR7 , -( _CH2 ) _xS (O ₎₂ ^{( CH2} ) ^yR7 , - _{( CH2} ) _xC (O)C(O)( _CH2 ) _yR7 , -( _CH2 ) ^{xC(O)O(CH2)yR7} _{, -} ⁽ _CH2 ) _xC (O) _{NH(CH2)yR7, -(CH2)xC (} ^O _{) NR7 (} ^{CH2 )} _{yR8 or -} ( _CH2 ) ^xNR7 C(O)(CH ₂ ) _y R ⁸ , wherein R ⁷ and R ⁸ are the same as defined in formula (I). Further preferably, ^R2 is selected from ^{R7 , -(CH2)xR7} _{, -} ( _CH2 ) _xC (O)( _CH2 ) _yR7 , -( ^CH2 ) _xS (O) _{2 (} ^CH2)yR7 _, -( _CH2 ) _xC (O)C(O)( _CH2 ) ^yR7 , -( _CH2 ) _{xC (} O) _O ( _CH2 ) _yR7 , -( _CH2 ) _xC (O)NH( _CH2 ) _yR7 ; further preferably, ^R2 is _selected ^{from R7 ,} -( _CH2 ) ^xR7 , -( _CH2 ) _xC (O)( _CH2 ) _yR7 ; further preferably, ^R2 is ^selected from -( _CH2 ) _xC (O ⁾ ( _CH2 ) ^yR7 _.

In one embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein R ⁷ and R ⁸ are each independently selected from substituted or unsubstituted R ⁹ , OR ⁹ , -R ¹⁰ -OR ⁹ , -R ¹⁰ -NH-R ⁹ , -R ¹⁰ -C(O)-R ⁹ , -R ^{10 -NHC} (O)-R 9, -R ¹⁰ -C(O)NH-R ⁹ , -R ¹⁰ -SR ⁹ , -R ¹⁰ -SC(O)-R ⁹ , C _3-6 cycloalkyl, 3-6 membered heterocycloalkyl, C _6-10 aryl, 5-10 membered heteroaryl, -R ¹⁰ -C _6-10 aryl, -R ¹⁰ -5-10 membered heteroaryl, -OR ¹⁰ -C _6-10 aryl, -OR ¹⁰ -5-10 membered heteroaryl, -R ¹⁰ -OC _6-10 aryl, -R ¹⁰ -O-5-10 membered heteroaryl, C _2-6 alkene and C _2-6 alkyne, or when R ⁷ and R ⁸ are connected to the same nitrogen atom, R ⁷ and R ⁸ and the nitrogen atom connected to them form a substituted or unsubstituted 3-6 membered heterocycloalkyl; wherein R ⁹ is C _1-6 alkyl, R ¹⁰ is C _1-6 alkylene, C _2-6 alkenylene or C _2-6 alkynylene; the "substituted" in R ⁷ and R ⁸ refers to being replaced by 1, 2 or 3 groups independently selected from -F, -Cl, -Br, -OH, -NH ₂ , -SH, -CN, C _1-3 alkyl, C 1-3 alkoxy, C _1-3 haloalkyl, C _1-3 haloalkoxy, -NHCN, -NHCONH ₂ , NHC(O) _{CH 3} , N(CH ₃ ) ₂ , N(C ₂ H ₅ ) ₂ , -SC(O)CH ₃ , -OC(O)-C _1-6 alkyl and the like; preferably, R ⁷ is independently selected from substituted or unsubstituted R ⁹ , OR ⁹ , -R ¹⁰ -OR ⁹ , -R ¹⁰ -NHC(O)-R ⁹ , C _3-6 cycloalkyl, 3-6 membered heterocycloalkyl, C _6-10 aryl, 5-10 membered heteroaryl, C 2-6 alkene and C _2-6 alkyne, or when R 7 and R ⁸ are jointly connected to the same nitrogen atom, R ⁷ and R ⁸ and the jointly connected nitrogen atom constitute a substituted or unsubstituted 3-6 membered heterocycloalkyl; further preferably, R ⁷ is independently selected from substituted or unsubstituted R ⁹ , OR ⁹ , -R ₁₀ -OR ⁹ , C 3-6 cycloalkyl, ^3-6 membered heterocycloalkyl, C 6-10 aryl, ^5-10 membered heteroaryl, C 2-6 alkene and C 2-6 alkyne. Preferably, R ⁷ is independently selected from substituted or unsubstituted R ⁹ , OR ⁹ , -R ¹⁰ -OR ⁹ , C _3-6 cycloalkyl, _3-6 membered heterocycloalkyl, C _6-10 aryl, 5-10 membered heteroaryl; more preferably, R ⁷ is independently selected from substituted or unsubstituted R ⁹ ; the "substituted" in R ⁷ refers to being replaced by 1, 2 or 3 groups independently selected from -F, -Cl, -Br, -OH, -NH ₂ , -SH, -CN, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkyl, C _1-3 haloalkoxy, -NHCN, -NHCONH ₂ , NHC(O)CH ₃ , N(CH ₃ ) ₂ , N(C ₂ H ₅ ) ₂ , -SC(O)CH ₃ , -OC(O)-C _1-6 alkyl, etc.; preferably, the "substituted" in R ⁷ refers to substituted with 1, 2 or 3 groups independently selected from -F, -OH, -NH ₂ , -SH, -CN, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkyl, NHC(O)CH ₃ , N(CH ₃ ) ₂ , -OC(O)-C _1-6 alkyl, etc.; further preferably, the "substituted" in R ⁷ refers to substituted with 1, 2 or 3 groups independently selected from -F, -OH, -NH ₂ , -CN, C _1-3 alkyl, C _1-3 alkoxy, etc.; further preferably, the "substituted" in R ⁷ refers to substituted with 1, 2 or 3 groups independently selected from -OH, -CN, etc.; further preferably, R The "substituted" in ^R7 refers to being replaced by one group independently selected from -OH, -CN, etc.; more preferably, the "substituted" in ^R7 refers to being replaced by one group independently selected from -OH groups.

In one embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein ^R7 and ^R8 are each independently selected from substituted or unsubstituted methyl, ethyl, propyl, isopropyl, butyl, pentyl, methoxy, ethoxy, propoxy, isopropoxy, -CH2OCH3, _-CH2OCH2CH3 , -CH2CH2OCH3, -CH2CH2OCH2CH3, _cyclopropyl , cyclobutyl, _cyclopentyl , _cyclohexyl , _aziridine , _azetidinyl, aziridine, azidoyl, _{azihexyl , oxiranyl , oxetanyl} , oxolanyl, oxhexyl, phenyl, pyridyl, _pyrazolyl , oxazolyl, isoxazolyl, thienyl, thiazolyl, benzyl, phenethyl, vinyl, propenyl, ethynyl or propynyl. Preferably, R ⁷ is independently selected from substituted or unsubstituted methyl, ethyl, methoxy, ethoxy, cyclopropyl, cyclobutyl, azetidinyl, azetidinyl, oxetanyl, oxetanyl, phenyl, pyridinyl, pyrazolyl, isoxazolyl, thienyl, thiazolyl, benzyl, vinyl, propenyl or ethynyl; further preferably, R ⁷ is independently selected from substituted or unsubstituted methyl, ethyl, methoxy, cyclopropyl, cyclobutyl, azetidinyl, oxetanyl, oxetanyl, pyridinyl, pyrazolyl, isoxazolyl, vinyl, propenyl or ethynyl; further preferably, R ^{7 is independently selected from substituted or unsubstituted methyl, ethyl, cyclopropyl, azetidinyl, oxetanyl, pyrazolyl, vinyl, propenyl or ethynyl; further preferably, R 7} is independently selected from substituted or unsubstituted methyl, ethyl, cyclopropyl ^; further preferably, R ⁷ is independently selected from substituted or unsubstituted methyl. The "substituted" in R ⁷ and R ⁸ refers to being substituted by 1, 2 or 3 groups independently selected from -F, -Cl, -Br, -OH, -NH ₂ , -SH, -CN, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkyl, C _1-3 haloalkoxy, -NHCN, -NHCONH ₂ , NHC(O)CH ₃ , N(CH ₃ ) ₂ , N(C ₂ H ₅ ) ₂ , -SC(O)CH ₃ , -OC(O)-C _1-6 alkyl, etc.; preferably, the "substituted" in R ⁷ and R ⁸ refers to being substituted by 1, 2 or 3 groups independently selected from -F, -OH, -SH, -CN, C _1-3 alkyl, C _1-3 alkoxy, C _1-3 haloalkoxy, NHC(O)CH ₃ , N(CH ₃ ) ₂ , -N(C ₂ H ₅ ) ₂ , -OC(O)-C _1-6 alkyl and the like; preferably, the "substituted" in R ⁷ and R ⁸ refers to being substituted by 1, 2 or 3 groups independently selected from -F, -OH, -NH ₂ , -CN, C _1-3 haloalkoxy and the like; further preferably, the "substituted" in R ⁷ and R ⁸ refers to being substituted by 1, 2 or 3 groups independently selected from -F, -OH, -CN and the like; further preferably, the "substituted" in R ⁷ and R ⁸ refers to being substituted by 1, 2 or 3 groups independently selected from -OH, -CN and the like; further preferably, the "substituted" in R ⁷ and R 8 refers to being substituted by 1 group independently selected from -OH, -CN and the like; further preferably, the "substituted" in R 7 and R ⁸ refers to being substituted by 1 group independently selected from -OH, -CN and the like; further preferably, the "substituted" in R ⁷ and R ⁸ refers to being substituted by 1 group independently selected from -OH group.

In one embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof, wherein R ⁹ is a C _1-4 alkyl group and R ¹⁰ is a C _1-4 alkylene group.

In one embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, a stereoisomer or a prodrug thereof, wherein the compound has the following structure:

In one embodiment, the solid tumor is a malignant solid tumor; preferably an advanced malignant solid tumor; further preferably liver cancer, breast cancer or prostate cancer.

In one embodiment, the liver cancer, breast cancer or prostate cancer is CDK9-related liver cancer, breast cancer or prostate cancer. Preferably, the liver cancer, breast cancer or prostate cancer is liver cancer, breast cancer or prostate cancer caused by overexpression of CDK9.

In one embodiment, the liver cancer is hepatocellular carcinoma; preferably advanced hepatocellular carcinoma; further preferably CDK9-related advanced hepatocellular carcinoma; further preferably advanced hepatocellular carcinoma caused by overexpression of CDK9.

In one embodiment, the breast cancer is triple-negative breast cancer.

In one embodiment, the triple-negative breast cancer is CDK9-related triple-negative breast cancer; preferably, it is triple-negative breast cancer caused by overexpression of CDK9.

In one embodiment, the CDK9 association refers to overexpression of CDK9.

In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof is the only active ingredient in the medicament.

In one embodiment, the compound represented by formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof is combined with one or more other targeted drugs or chemotherapeutic drugs to prepare the drug.

In one embodiment, the drug is formulated into a clinically acceptable formulation. In a preferred embodiment, the formulation is an oral formulation or an injectable formulation.

In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt thereof, a stereoisomer or a prodrug thereof is administered in a daily dosage range of from about 0.001 mg/kg to about 1000 mg/kg; preferably 0.01 mg/kg to about 100 mg/kg, and further preferably 0.02 mg/kg to about 10 mg/kg.

In one embodiment, the compound represented by formula (I) or its pharmaceutically acceptable salt, stereoisomer or prodrug thereof is administered in a daily dosage range of from about 0.001 mg to about 1000 mg; preferably 0.01 mg to about 100 mg, more preferably 0.1 mg to about 80 mg, more preferably 1 mg to about 70 mg, more preferably 1.5 mg to about 60 mg, and more preferably 2 mg to about 50 mg. More preferably, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 8 mg, 10 mg, 12 mg, 16 mg, 18 mg, 20 mg, 24 mg, 25 mg, 30 mg, 32 mg, 36 mg, 40 mg, 42 mg, 45 mg, 50 mg. The frequency of administration is single administration or multiple administration. Preferably, the frequency of administration is once a day, twice a day, three times a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

In one embodiment, the drug contains a therapeutically effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a prodrug thereof. In a preferred embodiment, the therapeutically effective amount is 0.001-1000 mg. In a preferred embodiment, the therapeutically effective amount is 0.01-100 mg. In a preferred embodiment, the therapeutically effective amount is 0.1-50 mg. Further preferably, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 8 mg, 10 mg, 12 mg, 16 mg, 18 mg, 20 mg, 24 mg, 25 mg, 30 mg, 32 mg, 36 mg, 40 mg, 42 mg, 45 mg, 50 mg.

In one embodiment, a single dose of the drug contains 0.01-100 mg of a compound of formula (I) or a pharmaceutically acceptable salt thereof, a stereoisomer or a prodrug thereof; preferably 0.1-80 mg; more preferably 1-60 mg; more preferably 1-50 mg; more preferably 2-20 mg; more preferably 2-15 mg; more preferably 2-10 mg; more preferably 2-8 mg; more preferably 2-6 mg; more preferably 2-5 mg; more preferably 2-4 mg; more preferably 2-3 mg.

In one embodiment, the compound of formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof is administered in a single dose or in divided doses.

In one embodiment, the drug is administered orally or by injection. In a preferred embodiment, the drug is administered orally.

In the second aspect of the present application, a pharmaceutical composition for treating solid tumors is provided, which comprises the compound as shown in formula (I) or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof as described in the first aspect, and optionally comprises a pharmaceutically acceptable carrier.

In the absence of contradictions and conflicts, the technical solutions or technical features described in the first aspect are applicable to the second aspect.

In the third aspect of the present application, there is provided a use of the compound 45 of the present application or a pharmaceutically acceptable salt thereof, a stereoisomer or a prodrug thereof in the preparation of a drug for treating solid tumors.

In one embodiment, the solid tumor is a malignant solid tumor; preferably an advanced malignant solid tumor; further preferably liver cancer, breast cancer or prostate cancer.

In one embodiment, the liver cancer, breast cancer or prostate cancer is CDK9-related liver cancer, breast cancer or prostate cancer. Preferably, the liver cancer, breast cancer or prostate cancer is liver cancer, breast cancer or prostate cancer caused by overexpression of CDK9.

In one embodiment, the liver cancer is hepatocellular carcinoma; preferably advanced hepatocellular carcinoma; further preferably CDK9-related advanced hepatocellular carcinoma; further preferably advanced hepatocellular carcinoma caused by overexpression of CDK9.

In one embodiment, the breast cancer is triple-negative breast cancer.

In one embodiment, the triple-negative breast cancer is CDK9-related triple-negative breast cancer; preferably, it is triple-negative breast cancer caused by overexpression of CDK9.

In one embodiment, the CDK9 association refers to overexpression of CDK9.

In one embodiment, the compound 45 or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof is the sole active ingredient in the medicament.

In one embodiment, the compound 45 or a pharmaceutically acceptable salt thereof, a stereoisomer or a prodrug thereof is used in combination with one or more other targeted drugs or chemotherapeutic drugs for the preparation of the medicament.

In one embodiment, the drug is formulated into a clinically acceptable formulation. In a preferred embodiment, the formulation is an oral formulation or an injectable formulation.

In one embodiment, the compound 45 or a pharmaceutically acceptable salt thereof, a stereoisomer or a prodrug thereof is administered in a daily dosage range of from about 0.001 mg/kg to about 1000 mg/kg; preferably 0.01 mg/kg to about 100 mg/kg, further preferably 0.02 mg/kg to about 10 mg/kg.

In one embodiment, the compound 45 or its pharmaceutically acceptable salt, its stereoisomer or prodrug is administered in a daily dosage range of from about 0.001 mg to about 1000 mg; preferably 0.01 mg to about 100 mg, further preferably 0.1 mg to about 80 mg, further preferably 1 mg to about 70 mg, further preferably 1.5 mg to about 60 mg, further preferably 2 mg to about 50 mg. Further preferably 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 8 mg, 10 mg, 12 mg, 16 mg, 18 mg, 20 mg, 24 mg, 25 mg, 30 mg, 32 mg, 36 mg, 40 mg, 42 mg, 45 mg, 50 mg. The frequency of administration is single administration or multiple administration. Preferably, the frequency of administration is once a day, twice a day, three times a day, once every two days, once every three days, once every four days, once every five days, once every six days, or once every seven days.

In one embodiment, the medicament contains a therapeutically effective amount of compound 45 or a pharmaceutically acceptable salt thereof, a stereoisomer thereof or a prodrug thereof. In a preferred embodiment, the therapeutically effective amount is 0.001-1000 mg. In a preferred embodiment, the therapeutically effective amount is 0.01-100 mg. In a preferred embodiment, the therapeutically effective amount is 0.1-50 mg.

In one embodiment, a single dose of the drug contains 0.01-100 mg of compound 45 or a pharmaceutically acceptable salt thereof, a stereoisomer or a prodrug thereof; preferably 0.1-80 mg; more preferably 1-60 mg; more preferably 1-50 mg; more preferably 2-20 mg; more preferably 2-15 mg; more preferably 2-10 mg; more preferably 2-8 mg; more preferably 2-6 mg; more preferably 2-5 mg; more preferably 2-4 mg; more preferably 2-3 mg.

In one embodiment, the compound 45 or a pharmaceutically acceptable salt, stereoisomer or prodrug thereof is administered in a single dose or in divided doses.

In one embodiment, the drug is administered orally or by injection. In a preferred embodiment, the drug is administered orally.

In the fourth aspect of the present application, a pharmaceutical composition for treating solid tumors is provided, which comprises the compound 45 of the present application or a pharmaceutically acceptable salt, a stereoisomer or a prodrug thereof, and optionally comprises a pharmaceutically acceptable carrier.

In the absence of contradictions and conflicts, the technical solutions or technical features described in the third aspect are applicable to the fourth aspect.

The compounds of the present application are optically active and can be either racemates, optical isomers or mixtures thereof. The synthesis of optical isomers in the compounds of the present application can be prepared by using starting materials of optical isomers or by separation of racemates.

definition

Unless otherwise specified, the following terms used in this application have the following meanings. A particular term should not be considered to be 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 the corresponding commodity or its active ingredient.

The term "optionally" or "optionally" means that the event or situation described subsequently may or may not occur, and the description includes both the occurrence of the event or situation and the non-occurrence of the event or situation. 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 (such as CH ₂ CH ₂ F), polysubstituted (such as CHFCH ₂ F, CH ₂ CHF _2, etc.) or fully substituted (CF ₂ CF ₃ ). 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 and/or cannot be synthesized will be introduced.

Herein, C _mn means that the moiety has an integer number of carbon atoms in a given range. For example, "C _1-6 " means that the group can have 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms.

When any variable (e.g., R) occurs more than once in a compound's composition or structure, its definition at each occurrence is independent. Thus, for example, if a group is substituted with 2 R's, each R has an independent choice.

The term "alkyl" refers to a monovalent saturated aliphatic hydrocarbon group, a straight or branched group containing 1 to 20 carbon atoms, preferably containing 1 to 10 carbon atoms (i.e., C _1-10 alkyl), further preferably containing 1 to 8 carbon atoms (C _1-8 alkyl), and more preferably containing 1 to 6 carbon atoms (i.e., C _1-6 alkyl). For example, "C _1-6 alkyl" means that the group is an alkyl group, and the number of carbon atoms on the carbon chain is between 1 and 6 (specifically 1, 2, 3, 4, 5 or 6). Examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, neopentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, n-heptyl, n-octyl, etc.

The term "cycloalkyl" refers to a monocyclic saturated aliphatic hydrocarbon group having a specific number of carbon atoms, preferably containing 3-12 carbon atoms (i.e., C _3-12 cycloalkyl), more preferably containing 3-10 carbon atoms (C _3-10 cycloalkyl), further preferably 3-6 carbon atoms (C _3-6 cycloalkyl), 4-6 carbon atoms (C _4-6 cycloalkyl), 5-6 carbon atoms (C _5-6 cycloalkyl). Examples include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, methylcyclopropyl, 2-ethyl-cyclopentyl, dimethylcyclobutyl, and the like.

The term "alkoxy" refers to -O-alkyl, and the alkyl is defined as above, i.e., contains 1-20 carbon atoms, preferably, contains 1-10 carbon atoms, preferably 1-8 carbon atoms, and more preferably 1 to 6 carbon atoms (specifically 1, 2, 3, 4, 5 or 6). Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, isopropoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, tert-butoxy, pentyloxy, 1-methylbutoxy, 2-methylbutoxy, 3-methylbutoxy, 1,1-dimethylpropoxy, 1,2-dimethylpropoxy, 2,2-dimethylpropoxy, 1-ethylpropoxy, and the like.

The term "halogen" or "halo" refers to F, Cl, Br, I. The term "haloalkyl" refers to an alkyl group as defined above in which one, two or more hydrogen atoms or all hydrogen atoms are replaced by halogen. Representative examples of haloalkyl include CCl ₃ , CF ₃ , CHCl ₂ , CH ₂ Cl, CH ₂ Br, CH ₂ I, CH ₂ CF ₃ , CF ₂ CF ₃ and the like.

The term "heterocyclyl" refers to a saturated or partially unsaturated monocyclic, bicyclic or polycyclic hydrocarbon substituent, which is a non-aromatic structure and contains 3-20 ring atoms, wherein 1, 2, 3 or more ring atoms are selected from N, O or S, and the remaining ring atoms are C. Preferably, it contains 3-12 ring atoms (C _3-12 heterocyclyl), and more preferably contains 3-10 ring atoms (C _3-10 heterocyclyl), or 3 to 8 ring atoms (C _3-8 heterocyclyl), or 3 to 6 ring atoms (C _3-6 heterocyclyl), or 4 to 6 ring atoms (C _4-6 heterocyclyl), or 5 to 6 ring atoms (C _5-6 heterocyclyl). The number of heteroatoms is preferably 1 to 4, and more preferably 1 to 3 (i.e., 1, 2 or 3). Examples of monocyclic heterocyclic groups include pyrrolidinyl, imidazolidinyl, tetrahydrofuranyl, dihydropyrrolyl, piperidinyl, piperazinyl, pyranyl, etc. Polycyclic heterocyclic groups include spiro, fused and bridged heterocyclic groups.

The term "heterocycloalkyl" refers to a saturated "heterocyclic group" as defined above, containing 3-20 ring atoms, wherein 1, 2, 3 or more ring atoms are selected from N, O or S, and the remaining ring atoms are C. Preferably, it contains 3-12 ring atoms (C _3-12 heterocycloalkyl), further preferably 3-10 ring atoms (C _3-10 heterocycloalkyl), or 3-8 ring atoms (C _3-8 heterocycloalkyl), or 3-7 ring atoms (C _3-7 heterocycloalkyl), or 3-6 ring atoms (C _3-6 heterocycloalkyl), or 4-6 ring atoms (C _4-6 heterocycloalkyl), or 5-6 ring atoms (C _5-6 heterocycloalkyl). The number of heteroatoms is preferably 1-4, more preferably 1 to 3 (i.e. 1, 2 or 3). Examples include aziridine, oxirane, thiirane, azetidinyl, oxetanyl, thietanyl, pyrrolidinyl, tetrahydrofuranyl, oxane, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, dioxanyl, dithioxanyl, oxazolidinyl, thiazolidinyl, pyrazolidinyl, imidazolinidine, and the like.

The term "aryl" refers to a monocyclic, bicyclic and tricyclic aromatic carbocyclic ring system containing 6-16 carbon atoms, or 6-14 carbon atoms, or 6-12 carbon atoms, or 6-10 carbon atoms, preferably 6-10 carbon atoms, and the term "aryl" can be used interchangeably with the term "aromatic ring". Examples of aryl groups may include, but are not limited to, phenyl, naphthyl, anthracenyl, phenanthrenyl or pyrenyl, etc.

The term "heteroaryl" refers to an aromatic monocyclic or polycyclic ring system containing 5-12 members, or preferably 5-10 members, 5-8 members, and more preferably 5-6 members, wherein 1, 2, 3 or more ring atoms are heteroatoms and the remaining atoms are carbon, the heteroatoms are independently selected from O, N or S, and the number of heteroatoms is preferably 1, 2 or 3. Examples of heteroaryl include, but are not limited to, furanyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, oxadiazolyl, thiadiazolyl, pyrrolyl, pyrazolyl, imidazolyl, triazolyl, tetrazolyl, pyridinyl, pyrimidinyl, pyrazinyl, pyridazinyl, thiodiazolyl, triazinyl, phthalazinyl, quinolyl, isoquinolyl, pteridinyl, purinyl, indolyl, isoindolyl, indazolyl, benzofuranyl, benzothienyl, benzopyridinyl, benzopyrimidinyl, benzo pyrazinyl, benzimidazolyl, benzophthalazinyl, pyrrolo[2,3-b]pyridinyl, imidazo[1,2-a]pyridinyl, pyrazolo[1,5-a]pyridinyl, pyrazolo[1,5-a]pyrimidinyl, imidazo[1,2-b]pyridazinyl, [1,2,4]triazolo[4,3-b]pyridazinyl, [1,2,4]triazolo[1,5-a]pyrimidinyl, [1,2,4]triazolo[1,5-a]pyridinyl, and the like.

The term "pharmaceutically acceptable salt" or "pharmaceutically acceptable salt" refers to a salt that is suitable for use in contact with mammalian tissues, especially human tissues, within the scope of sound medical judgment without excessive toxicity, irritation, allergic response, etc., and is commensurate with a reasonable benefit/risk ratio.

The term "salt" includes salts prepared from inorganic acids. If the compound of the present application is acidic, pharmaceutically acceptable non-toxic bases include salts prepared from inorganic bases and organic bases.

The term "stereoisomer" refers to isomers produced by different spatial arrangements of atoms in molecules, including configurational isomers and conformational isomers. Configurational isomers further include geometric isomers (or cis-trans isomers) and optical isomers (including enantiomers and diastereomers).

Geometric isomers may be present in the present compound. The present compound may contain a carbon-carbon double bond or a carbon-nitrogen double bond of E or Z configuration, wherein the term "E" represents a higher order substituent on the opposite side of a carbon-carbon or carbon-nitrogen double bond, and the term "Z" represents a higher order substituent on the same side of a carbon-carbon or carbon-nitrogen double bond (determined using the Cahn-Ingold Prelog priority rule). The present compound may also exist in a mixture of "E" and "Z" isomers. The substituents around cycloalkyl or heterocycloalkyl are referred to as cis or trans configurations.

Optical isomers refer to substances with exactly the same molecular structure, similar physical and chemical properties, but different optical rotations.

The compounds of the present application may contain asymmetrically substituted carbon atoms in the R or S configuration, wherein the terms "R" and "S" are as defined in IUPAC 1974 Recommendations for Section E, Fundamental Stereochemistry, Pure Appl. Chem. (1976) 45, 13-10. Compounds with asymmetrically substituted carbon atoms (having equal numbers of R and S configurations) are racemic at those carbon atoms. Atoms with an excess of one configuration (relative to the other) allow that configuration to be present in a higher number, preferably an excess of about 85%-90%, more preferably an excess of about 95%-99%, and even more preferably an excess of greater than about 99%. Accordingly, the present application includes racemic mixtures, relative and absolute stereoisomers, and mixtures of relative and absolute stereoisomers.

The term "prodrug" or "prodrug" is a derivative of an active drug that is designed to improve some defined, undesirable physical or biological property. Physical properties are usually related to solubility (too high or insufficient lipid or water solubility) or stability, while problematic biological properties include too rapid metabolism or poor bioavailability, which themselves may be related to physicochemical properties.

Prodrugs are generally prepared by a) forming esters, half-esters, carbonates, nitrates, amides, hydroxamic acids, carbamates, imines, Mannich bases, phosphates, phosphate esters and enamines of the active drug, b) functionalizing the drug with azo, glycoside, peptide and ether functional groups, c) using the aminal, hemiaminal, polymer, salt, complex, phosphoramide, acetal, hemiacetal and ketal forms of the drug. For example, see Andrejus Korolkovas's, "Essentials of Medicinal Chemistry", John Wiley-Interscience Publications, John Wiley and Sons, New York (1988), pp. 97-118, all of which are incorporated herein by reference. Esters can be prepared from substrates containing hydroxyl or carboxyl groups using general methods known to those skilled in the art. The typical reaction for these compounds is substitution of one heteroatom with another atom. Amides can be prepared in a similar manner from substrates containing amino or carboxyl groups. Esters can also react with amines or ammonia to form amides. Another way to prepare amides is to heat a carboxylic acid and an amine together.

The term "liver cancer" refers to any proliferative disorder or abnormality of the liver, for example, a malignant tumor originating from hepatocytes, which make up the majority of the liver, and includes all types of malignancies that begin in the liver and metastatic liver cancers that spread to the liver from elsewhere.

The terms "breast cancer" or "breast tumor" refer to any proliferative lesion or proliferative abnormality of the breast, including, for example, benign lesions, premalignant and malignant lesions, solid tumors, and metastatic disease (localized, e.g., stage III, and more extensively, e.g., stage IV), including but not limited to triple-negative breast cancer.

The terms "prostate cancer" or "prostate tumor" refer to any proliferative lesion or proliferative abnormality of the prostate that develops in the prostate and is confirmed histologically or cytologically as a prostate cancer or prostate tumor, including but not limited to primary prostate tumors and metastases of said primary prostate tumors (which may be located anywhere in the body).

The term "treatment" means administering the compounds or preparations described herein to improve or eliminate a disease or one or more symptoms associated with the disease, and includes: (i) inhibiting a disease or disease state, i.e., curbing its development; (ii) alleviating a disease or disease state, i.e., causing the disease or disease state to regress.

The term "therapeutically effective amount" means the amount of the compound of the present application that (i) treats a particular disease, condition or disorder, or (ii) alleviates, ameliorates or eliminates one or more symptoms of a particular disease, condition or disorder. The amount of the compound of the present application that constitutes a "therapeutically effective amount" varies depending on the compound, the disease state and its severity, the mode of administration, and the characteristics of the individual to be treated (e.g., sensitivity, weight, age, etc.), but can be routinely determined by those skilled in the art based on their own knowledge and the present disclosure.

The term "pharmaceutical composition" refers to a mixture of one or more pharmaceutically acceptable salts of the compounds of the present application, their stereoisomers or prodrugs and pharmaceutically acceptable excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the pharmaceutically acceptable salts of the compounds of the present application, their stereoisomers or prodrugs to an organism. The pharmaceutical composition of the present invention can be prepared by conventional methods in the art.

In the context of the present application, the term "pharmaceutically acceptable carrier" or "excipient" or "pharmaceutically acceptable excipient" or "pharmaceutically acceptable excipient" refers to those excipients that have no obvious irritation to the organism and do not damage the biological activity and performance of the active compound. The term "pharmaceutically acceptable excipient" includes: solvents, propellants, solubilizers, cosolvents, emulsifiers, colorants, adhesives, disintegrants, fillers, lubricants, wetting agents, osmotic pressure regulators, stabilizers, glidants, flavoring agents, preservatives, suspending agents, coating materials, fragrances, anti-adhesives, antioxidants, chelating agents, penetration enhancers, pH regulators, buffers, plasticizers, surfactants, foaming agents, defoamers, thickeners, inclusion agents, humectants, absorbents, diluents, flocculants and deflocculating agents, filter aids, release retardants, etc. Those skilled in the art can select specific pharmaceutically acceptable excipients according to actual needs. The knowledge about excipients is well known to those skilled in the art, for example, reference may be made to "Pharmaceutics" (edited by Cui Fude, 5th edition, People's Medical Publishing House, 2003).

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".

In the scope of this application, various options of any feature can be combined with various options of other features to form many different embodiments. This application is intended to include all possible embodiments composed of various options of all technical features.

In addition to the specific embodiments listed below, the compounds of the present application can be prepared by other synthetic methods, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent replacement methods known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present application.

As used herein, the term "individual" or "subject" refers to a cell or a mammal, such as a human, but may also be other mammals, such as livestock or experimental animals.

The above embodiments represent exemplary embodiments of the present application, but the present application is not limited to the above embodiments. In addition, the various technical features in the above embodiments of the present application can be combined with each other to form one or more new technical solutions, which also fall within the scope of the present application, as long as such new technical solutions are technically feasible.

The present application has one or more of the following beneficial effects:

In order to prove that the compound represented by formula (I) of the present application is an effective CDK9 inhibitor for solid tumors (e.g., liver cancer, prostate cancer, breast cancer), the present application evaluated the in vitro kinase inhibitory activity of the compound represented by formula (I) against different subtypes of CDK and the proliferation inhibitory activity against a variety of liver cancer, breast cancer and prostate cancer cell lines, and further evaluated the inhibitory effect of the compound represented by formula (I) (especially compound 45) on tumor growth in a liver cancer tumor xenograft model.

The results of in vitro kinase activity tests and cell tests showed that the compounds of the present application have good in vitro kinase inhibitory activity against CDK9 and good selectivity for other CDK isoforms; they have strong inhibitory effects on a variety of liver cancer cell lines, prostate cancer cell lines and breast cancer cell lines.

The results of in vivo tests show that, compared with the control compound, the compound of the present application has better in vivo anti-tumor effect, good tolerability, and higher drugability, providing a better choice for drugs that inhibit the CDK9 target.

The results of in vitro and in vivo safety tests showed that compared with the control compound, the compound represented by formula (I) of the present application had no obvious inhibitory activity on the hERG channel in vitro within the detected concentration range, indicating that the compound of the present application has a lower risk of cardiac toxicity. In addition, when mice were given the drug for 7 consecutive days, no animal deaths were observed as the dose increased, indicating that the animals had good tolerance.

The development of the compounds of the present application expands the selection of drugs for treating cancer.

Example

The present application is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and are not used to limit the scope of the present application. The experimental methods in the following examples that do not specify specific conditions are usually performed under conventional conditions or according to the conditions recommended by the manufacturer. Unless otherwise defined, all professional and scientific terms used in the text have the same meanings as those familiar to professionals in the field. In addition, any methods and materials similar or equivalent to the recorded contents can be applied to the present application method. The preferred implementation methods and materials shown in the text are for demonstration purposes only.

Embodiment 1:

Synthesis of intermediate 1a:

4-Bromo-5-chloropyridin-2-amine (3.00 g, 14.50 mmol) was dissolved in ethylene glycol dimethyl ether (50 mL) and water (10 mL), followed by the addition of 4-fluoro-2-methoxyphenylboronic acid (2.50 g, 14.70 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (1.06 g, 1.45 mmol) and potassium carbonate (6.00 g, 44.10 mmol), and the atmosphere was replaced with nitrogen three times to place the entire system under a nitrogen atmosphere. The system was stirred under reflux at 100 ° C and reacted for 4 hours. TLC monitoring showed that there was no residual raw material. The mixture was concentrated under reduced pressure and purified by column chromatography (dichloromethane: methanol = 50: 1-10: 1) to obtain 1a (3.11 g, yield 85%).

Synthesis of intermediate 1b:

1a (0.89 g, 3.50 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of (1S,3R)-3-[(tert-butyloxycarbonyl)amino]cyclopentanecarboxylic acid (0.85 g, 3.50 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.60 g, 4.20 mmol) and N,N-diisopropylethylamine (0.91 g, 7.00 mmol). The whole system was stirred at room temperature overnight. TLC monitoring showed that no raw material remained. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. 1b (0.96 g, yield 59%) was obtained by column chromatography separation and purification (dichloromethane: methanol = 50: 1-20: 1).

Synthesis of intermediate 1c:

1b (0.96 g, 2.07 mmol) was dissolved in dichloromethane (30 mL), and then trifluoroacetic acid (2 mL) was added under ice bath. The whole system was stirred at room temperature overnight and monitored by TLC until no raw material remained. Water (100 mL) was added to the reaction solution, and the pH was adjusted to 9-10 with saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and separated and purified by column chromatography (dichloromethane: methanol = 50: 1-8: 1) to obtain 1c (0.66 g, yield 88%).

Synthesis of final product 1:

1c (0.66 g, 1.80 mmol) was dissolved in dichloromethane (35 mL), followed by the addition of acetic anhydride (0.92 g, 9.00 mmol) and triethylamine (0.91 g, 9.00 mmol). The system was stirred at room temperature and monitored by TLC until no raw material remained. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain the final product 1 (0.48 g, yield 64%). MS m/z (ESI): 406.1 [M+H] ⁺ .

¹ H NMR (600MHz, CDCl ₃ ) δ9.16 (s, 1H), 8.28-8.25 (m, 2H), 7.55 (s, 1H), 7.18 (d, J = 7.2Hz, 1H), 6.79-6.71 ( m,2H),4.44(s,1H),3.80(s,3H),2.98(t,J＝4.8Hz,1H),2.20-2.17(m,3H),1.97(s,3H),1.88-1.82 (m,3H).

Embodiment 2:

Synthesis of intermediate 2a:

5-Fluoro-4-iodopyridin-2-amine (1.00 g, 4.20 mmol) was dissolved in ethylene glycol dimethyl ether (20 mL) and water (4 mL), followed by the addition of 4-fluoro-2-methoxyphenylboronic acid (0.71 g, 4.20 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (0.31 g, 0.42 mmol) and potassium carbonate (1.70 g, 12.60 mmol), and the atmosphere was replaced with nitrogen three times to place the entire system under a nitrogen atmosphere. The system was stirred under reflux at 100 °C for 4 hours, and no raw material remained after TLC monitoring. The mixture was concentrated under reduced pressure and purified by column chromatography (dichloromethane: methanol = 50: 1-10: 1) to obtain 2a (0.80 g, yield 81%).

Synthesis of intermediate 2b:

2a (0.80 g, 3.40 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of (1S,3R)-3-[(tert-butyloxycarbonyl)amino]cyclopentanecarboxylic acid (0.83 g, 3.40 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.56 g, 4.10 mmol) and N,N-diisopropylethylamine (0.88 g, 6.80 mmol). The whole system was stirred at room temperature overnight. TLC showed that no raw material remained. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. 2b (0.82 g, yield 54%) was obtained by column chromatography separation and purification (dichloromethane: methanol = 50: 1-20: 1).

Synthesis of intermediate 2c:

2b (0.82 g, 1.83 mmol) was dissolved in dichloromethane (30 mL), and then trifluoroacetic acid (2 mL) was added under an ice-water bath. The whole system was stirred at room temperature overnight, and TLC was performed until no raw material remained. Water (100 mL) was added to the reaction solution, and the pH was adjusted to 9-10 with a saturated sodium bicarbonate aqueous solution, and extracted with dichloromethane (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. After separation and purification by column chromatography (dichloromethane: methanol = 50: 1-8: 1), 2c (0.59 g, yield 93%) was obtained.

Synthesis of final product 2:

2c (0.59 g, 1.70 mmol) was dissolved in dichloromethane (35 mL), followed by the addition of acetic anhydride (0.87 g, 8.50 mmol) and triethylamine (0.86 g, 8.50 mmol). The system was stirred at room temperature and monitored by TLC until no raw material remained. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain the final product 2 (0.42 g, yield 63%). MS m/z (ESI): 390.2 [M + H] ⁺ .

¹ H NMR (600MHz, CDCl ₃ ) δ9.20 (s, 1H), 8.32 (s, 1H), 8.12 (s, 1H), 7.30 (d, J = 6.6Hz, 1H), 6.82-6.75 (m, 3H),4.44(s,1H),3.84(s,3H),2.99(q,J＝3.6Hz,1H),2.22-2.15(m,3H),1.99(s,3H),1.89-1.85(m ,3H).

Embodiment 3:

Synthesis of intermediate 3a:

2a (0.80 g, 3.40 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of cis-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (0.83 g, 3.40 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.56 g, 4.10 mmol) and N,N-diisopropylethylamine (0.88 g, 6.80 mmol). The whole system was stirred at room temperature overnight. TLC monitoring showed that no raw material remained. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. 3a (0.90 g, yield 57%) was obtained by column chromatography separation and purification (dichloromethane: methanol = 50: 1-20: 1).

Synthesis of intermediate 3b:

3a (0.90 g, 1.95 mmol) was dissolved in dichloromethane (30 mL), and trifluoroacetic acid (2 mL) was then added under ice bath. The whole system was stirred at room temperature overnight. TLC monitoring showed that no raw material was left. Water (100 mL) was added to the reaction solution, and the pH was adjusted to 9-10 with saturated sodium bicarbonate aqueous solution. The product was extracted with dichloromethane (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-8: 1) to obtain 3b (0.61 g, yield 87%).

Synthesis of final product 3:

3b (0.61 g, 1.69 mmol) was dissolved in dichloromethane (35 mL), followed by the addition of acetic anhydride (0.86 g, 8.40 mmol) and triethylamine (0.85 g, 8.40 mmol). The system was stirred at room temperature, and TLC monitoring showed that there was no residual raw material. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain the final product 3 (0.38 g, yield 56%). MS m/z (ESI): 404.2 [M + H] ⁺ .

¹ H NMR (600MHz, CDCl ₃ ) ¹ H NMR (600MHz, CDCl ₃ ) δ8.88(s,1H),8.29(s,1H),8.10(s,1H),7.28(s,1H),6.77- 6.72(m,3H),3.82(s,3H),2.52-2.49(m,1H),2.24-2.22(m,1H),2.00-1.95(m,4H),1.98(s,3H),1.48- 1.38(m,3H),1.18-1.13(m,1H).

Embodiment 4:

Synthesis of intermediate 4a:

1a (0.40 g, 1.58 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of (1R,3S)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (0.38 g, 1.58 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.72 g, 1.90 mmol) and N,N-diisopropylethylamine (0.41 g, 3.16 mmol), and the system was stirred at room temperature overnight. TLC monitoring showed that no raw material remained. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. 4a (0.45 g, yield 60%) was obtained by column chromatography separation and purification (dichloromethane: methanol = 50: 1-20: 1).

Synthesis of intermediate 4b:

4a (0.45 g, 0.94 mmol) was dissolved in dichloromethane (30 mL), and then 2 mL of trifluoroacetic acid was added under an ice-water bath. The whole system was stirred at room temperature overnight, and TLC was performed until no raw material remained. Water (100 mL) was added to the reaction solution, and the pH was adjusted to 9-10 with saturated sodium bicarbonate aqueous solution, and extracted with dichloromethane (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. After separation and purification by column chromatography (dichloromethane: methanol = 50: 1-8: 1), 4b (0.30 g, yield 85%) was obtained.

Synthesis of final product 4:

4b (0.30 g, 0.80 mmol) was dissolved in dichloromethane (35 mL), followed by the addition of acetic anhydride (0.24 g, 2.39 mmol) and triethylamine (0.24 g, 2.39 mmol), and the system was stirred at room temperature. TLC monitoring showed that there was no residual raw material. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-10: 1) to obtain the final product 4 (0.17 g, yield 51%). MS m/z (ESI): 420.14 [M+H] ⁺ .

¹ H NMR (600MHz, CDCl ₃ ) ¹ H NMR (600MHz, DMSO-d6) δ10.69(s,1H),8.41(s,1H),8.05(s,1H),7.78-7.73(m,1H) ,7.27-7.22(m,1H),7.10-7.08(m,1H),6.91-6.88(m,1H),3.76(s,3H),3.57-3.54(m,1H),2.62-2.59(m, 1H), 1.86 (d, J = 12.6Hz, 1H), 1.76 (s, 6H), 1.31-1.23 (m, 3H), 1.07-1.05 (m, 1H).

Embodiment 5:

Synthesis of intermediate 5a:

2a (0.80 g, 3.40 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of (1S,3R)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (0.83 g, 3.40 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.56 g, 4.10 mmol) and N,N-diisopropylethylamine (0.88 g, 6.80 mmol). The whole system was stirred at room temperature overnight. TLC monitoring showed that there was no residual raw material. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. 5a (0.90 g, yield 58%) was obtained by column chromatography separation and purification (dichloromethane: methanol = 50: 1-20: 1).

Synthesis of intermediate 5b:

5a (0.90 g, 1.95 mmol) was dissolved in dichloromethane (30 mL), and then trifluoroacetic acid (2 mL) was added under an ice-water bath. The whole system was stirred at room temperature overnight, and TLC was performed until no raw material remained. Water (100 mL) was added to the reaction solution, and the pH was adjusted to 9-10 with a saturated sodium bicarbonate aqueous solution, and extracted with dichloromethane (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. After separation and purification by column chromatography (dichloromethane: methanol = 50: 1-8: 1), 5b (0.61 g, yield 87%) was obtained.

Synthesis of final product 5:

5b (0.61 g, 1.69 mmol) was dissolved in dichloromethane (35 mL), followed by the addition of acetic anhydride (0.86 g, 8.40 mmol) and triethylamine (0.85 g, 8.40 mmol), and the system was stirred at room temperature and monitored by TLC until no raw material remained. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain the final product 5 (0.38 g, yield 56%). MS m/z (ESI): 404.2 [M + H] ⁺ .

¹ H NMR (600MHz, CD ₃ OD) δ8.18 (s, 1H), 8.09 (s, 1H), 7.33-7.29 (m, 1H), 6.94 (d, J = 10.8Hz, 1H), 6.84-6.81 (m,1H),3.83(s,3H),3.76-3.72(m,1H),2.60-2.57(m,1H),2.06(d,J＝12.0Hz,1H),

1.96-1.90(m,3H),1.93(s,3H),1.51-1.39(m,3H),1.24-1.21(m,1H).

Embodiment 6:

Synthesis of intermediate 6a:

3,4-difluoro-2-methoxyphenylboronic acid (0.57 g, 3.03 mmol) was dissolved in dioxane (50 mL), followed by the addition of 5-fluoro-4-iodopyridin-2-amine (0.60 g, 2.52 mmol), tetrakistriphenylphosphine palladium (150 mg, 0.13 mmol) and potassium phosphate trihydrate (1.00 g, 3.78 mmol). The system was heated to 100 ° C under nitrogen protection and reacted for 4 hours. TLC tracking monitoring showed that there was no residual raw material. The reaction solution was cooled to room temperature, and the crude product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-1: 1) to obtain 6a (0.40 g, yield 52%).

Synthesis of intermediate 6b:

(1S, 3R)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (348 mg, 1.43 mmol) was dissolved in dichloromethane (50 mL), followed by the addition of pyridine (572 mg, 7.24 mmol) and thionyl chloride (300 mg, 2.52 mmol) for 4 hours at room temperature, and then 6a (400 mg, 1.57 mmol) was directly added to the above reaction solution. The reaction was continued overnight at room temperature, and TLC tracking monitoring showed that there was no residual raw material. Water (30 mL) was added to the reaction solution, and ethyl acetate was extracted (20 mL × 3) was added to the reaction solution. The organic phases were combined, washed with saturated sodium chloride (20 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The mixture was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain 6b (250 mg, yield 33%).

Synthesis of intermediate 6c:

6b (250 mg, 0.52 mmol) was dissolved in dichloromethane (10 mL), followed by the addition of trifluoroacetic acid (1 mL), and the reaction was carried out at room temperature for 1.5 hours. TLC tracking showed that no raw material remained. Saturated sodium bicarbonate (30 mL) was added to the reaction solution, and dichloromethane was extracted (20 mL × 3). The organic phases were combined, washed with saturated sodium chloride (20 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain 6c (130 mg, yield 65%).

Synthesis of final product 6:

6c (130 mg, 0.34 mmol) was dissolved in dichloromethane (10 mL), followed by the addition of acetic anhydride (45 mg, 0.44 mmol) and triethylamine (44 mg, 0.44 mmol), and the reaction was carried out at room temperature for 1.5 hours. TLC tracking showed that no raw material remained. Saturated sodium bicarbonate (30 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (20 mL × 3). The organic phases were combined, washed with saturated sodium chloride (20 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was purified by column chromatography (dichloromethane: methanol = 50: 1-30: 1) to obtain the final product 6 (120 mg, yield 84%). MS m/z (ESI): 422.2 [M + H] ⁺ .

¹ H NMR (600MHz, DMSO-d6) δ10.68(s,1H),8.43(s,1H),8.20(s,1H),7.78(d,J＝7.8Hz,1H),7.33(t,J ＝7.8Hz,1H),7.21(t,J＝7.8Hz,1H),3.95(s,3H),3.59-3.54(m,1H),2.63-2.59(m,1H),1.88-1.84(m, 1H),1.80-1.72(m,3H),1.78(s,3H),1.31-1.25(m,3H),1.10-1.04(m,1H).

Embodiment 7:

Synthesis of intermediate 7a:

2-Amino-5-fluoro-4-iodopyridine (0.50 g, 2.10 mmol) and 5-fluoro-2-ethoxyphenylboronic acid (0.46 g, 2.50 mmol) were dissolved in ethylene glycol dimethyl ether (10 mL) and water (2 mL), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (71 mg, 0.10 mmol) and potassium carbonate (0.87 g, 6.30 mmol) were added. The atmosphere was replaced with nitrogen three times to place the entire system under a nitrogen atmosphere. The system was reacted at 100 ° C for 2 hours. TLC monitoring showed that no raw material remained. After cooling, the solvent was removed by concentration. The crude product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain 7a (0.50 g, yield 95%).

Synthesis of intermediate 7b:

Compound (1S, 3R)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (0.29 g, 1.20 mmol) was dissolved in dichloromethane (10 mL), pyridine (395 mg, 5.00 mmol) and thionyl chloride (202 mg, 1.70 mmol) were added in an ice bath, the system was reacted at room temperature for 2 hours and then concentrated to remove the solvent and excess thionyl chloride. Dichloromethane (10 mL) and compound 7a (250 mg, 1.00 mmol) were then added. The system was reacted at room temperature overnight. TLC monitoring showed that no raw material remained. The system was concentrated, and the crude product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10:1-1:1) to obtain 7b (100 mg, yield 21%).

Synthesis of intermediate 7c:

7b (47 mg, 0.10 mmol) was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (2 mL) was added. The system was reacted at room temperature for 1 hour. TLC monitoring showed that there was no residual raw material. The system was concentrated to obtain 7c (50 mg, crude product). The obtained product was not further purified and was directly used in the next step reaction.

Synthesis of final product 7:

7c (37 mg, 0.10 mmol) was dissolved in dichloromethane (2 mL), and triethylamine (20 mg, 0.20 mmol) and acetic anhydride (20 mg, 0.20 mmol) were added. The system was reacted at room temperature for 1 hour. TLC monitoring showed that no starting material remained. The system was concentrated, and the crude product was separated and purified by thin-plate chromatography (dichloromethane: methanol = 10:1) to obtain the final product 7 (30 mg, yield 72%). MS: (m/z, ESI): 417.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d6) δ10.57 (s, 1H), 8.33 (s, 1H), 8.11 (d, J = 5.4Hz, 1H), 7.79 (d, J = 7.8Hz, 1H), 7.35(d,J＝7.8Hz,1H),7.09(d,J＝11.4Hz,1H),6.90(d,J＝7.8Hz,1H),4. 10-4.07(m,2H),3.57-3.55(m,1H),2.59-2.57(m,1H),1.87-1.84(m,1H),1.77-1.75(m,5H),1.70-1.50(m ,1H),1.31-1.26(m,3H),1.24-1.21(m,3H),1.07-1.05(m,1H).

Embodiment 8:

Synthesis of intermediate 8a:

5-Fluoro-4-iodopyridin-2-amine (1.00 g, 4.20 mmol) was dissolved in ethylene glycol dimethyl ether (20 mL) and water (4 mL), followed by the addition of 4-fluoro-2-isopropoxyphenylboronic acid (0.83 g, 4.20 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (0.31 g, 0.42 mmol) and potassium carbonate (1.74 g, 12.60 mmol), and the atmosphere was replaced with nitrogen three times to place the entire system under a nitrogen atmosphere. The system was stirred under reflux at 100 °C for 4 hours, and no raw material remained after TLC monitoring. The mixture was concentrated under reduced pressure and purified by column chromatography (dichloromethane:methanol=50:1-10:1) to obtain 8a (0.85 g, yield 77%).

Synthesis of intermediate 8b:

8a (0.85 g, 3.20 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of (1S,3R)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (0.78 g, 3.20 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.44 g, 3.80 mmol) and N,N-diisopropylethylamine (0.83 g, 6.40 mmol). The whole system was stirred at room temperature overnight. TLC showed that no raw material remained. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. 8b (0.90 g, yield 57%) was obtained by column chromatography separation and purification (dichloromethane: methanol = 50: 1-20: 1).

Synthesis of intermediate 8c:

8b (0.90 g, 1.84 mmol) was dissolved in dichloromethane (30 mL), and then trifluoroacetic acid (2 mL) was added under an ice-water bath. The whole system was stirred at room temperature overnight and detected by TLC until no raw material remained. Water (100 mL) was added to the reaction solution, and the pH was adjusted to 9-10 with a saturated sodium bicarbonate aqueous solution, and extracted with dichloromethane (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. 8c (0.64 g, yield 89%) was obtained by column chromatography separation and purification (dichloromethane: methanol = 50: 1-8: 1).

Synthesis of final product 8:

8c (0.64 g, 1.64 mmol) was dissolved in dichloromethane (35 mL), followed by the addition of acetic anhydride (0.84 g, 8.20 mmol) and triethylamine (0.83 g, 8.20 mmol). The system was stirred at room temperature and monitored by TLC until no raw material remained. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain the final product 8 (0.42 g, yield 59%). MS: (m/z, ESI): 431.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.57 (s, 1H), 8.32 (s, 1H), 8.12 (s, 1H), 7.78 (d, J = 7.8Hz, 1H), 7.34 (d, J＝7.2Hz,1H),7.10(s,1H),6.88(d,J＝8.4Hz,1H),4.72-4.69(m,1H),3.56(s,1H),2.62-2.58(m,1H ),1.78(s,6H),1.31-1.23(m,4H),1.20(s,6H),1.09-1.04(m,1H).

Embodiment 9:

Synthesis of intermediate 9a:

1-Bromo-2-difluoromethoxy-4-fluorobenzene (1.00 g, 4.15 mmol), bis-pinacol borate (1.26 g, 4.98 mmol), potassium acetate (1.22 g, 12.45 mmol), [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride (0.24 g, 0.33 mmol), ethylene glycol dimethyl ether (30 mL) were added to the reaction bottle, heated to 100 ° C under nitrogen protection for 8 hours, and no raw material remained after TLC monitoring. Stop heating and cool to room temperature. Concentrate under reduced pressure and separate by column chromatography (n-hexane: ethyl acetate = 10: 1) to obtain 9a (0.50 g, yield 42%).

Synthesis of intermediate 9b:

9a (0.50 g, 1.74 mmol), 5-fluoro-4-iodo-pyridin-2-amine (0.33 g, 1.39 mmol), tetrakistriphenylphosphine palladium (0.12 g, 0.10 mmol), tripotassium phosphate trihydrate (0.60 g, 2.26 mmol), and dioxane (30 mL) were added to a reaction flask, heated to 100 ° C under nitrogen protection for 8 hours, and TLC monitoring showed that there was no residual raw material. Heating was stopped and the mixture was cooled to room temperature. Concentrated under reduced pressure and separated by column chromatography (n-hexane: ethyl acetate = 1:1) to obtain 9b (0.39 g, yield 83%).

Synthesis of intermediate 9c:

9b (0.39 g, 1.43 mmol,) (1S, 3R)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (0.35 g, 1.43 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.65 g, 1.72 mmol), N,N-diisopropylethylamine (0.37 g, 2.86 mmol) and N,N-dimethylformamide (20 mL) were added to a reaction bottle and reacted at room temperature for 15 hours. TLC monitoring showed that no raw material remained. Water (30 mL) was added to the reaction solution, and ethyl acetate was extracted (30 mL × 3). The organic phases were combined, washed with saturated sodium chloride (30 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. 9c (0.16 g, yield 23%) was obtained by column chromatography separation and purification (n-hexane: ethyl acetate = 1:1).

Synthesis of intermediate 9d:

9c (0.16 g, 0.33 mmol) was dissolved in dichloromethane (20 mL), trifluoroacetic acid (4 mL) was added, and the reaction was carried out at room temperature for 4 hours. TLC monitoring showed that no raw material remained. The reaction solution was washed with water (20 mL × 3), the aqueous phase was combined, the pH of the aqueous phase was adjusted to 8-9 with sodium carbonate, and dichloromethane was extracted (30 mL × 3), the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain 9d (0.10 g, yield 76%).

Synthesis of final product 9:

9d (0.10 g, 0.26 mmol) was dissolved in dichloromethane (20 mL), and acetic anhydride (0.05 g, 0.52 mmol) and triethylamine (0.05 g, 0.52 mmol) were added to react at room temperature for 2 hours. TLC monitoring showed that there was no residue of the raw material. The reaction solution was directly separated by column chromatography (dichloromethane: methanol = 25: 1) to obtain the final product 9 (0.05 g, yield 44%). MS m/z (ESI): 440.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.67 (s, 1H), 8.41 (s, 1H), 8.12 (d, J = 5.4Hz, 1H), 7.78 (d, J = 7.2Hz, 1H) ,7.58-7.56(m,1H),7.42(s,1H),7.34-7.28(m,1H),3.57-3.56(m,1H),2.61-2.59(m,1H),1.89-1.78(m, 4H),1.77(s,3H),1.31-1.24(m,3H),1.08-1.06(m,1H).

Embodiment 10:

Synthesis of intermediate 10a:

5-Fluoro-4-iodopyridin-2-amine (500 mg, 2.10 mmol) was dissolved in a mixed solvent of DME (20 mL) and water (4 mL), followed by the addition of 2-benzyloxy-4-fluorophenylboronic acid (620 mg, 2.52 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (90 mg, 0.11 mmol) and potassium carbonate (870 mg, 6.30 mmol), and the atmosphere was replaced with nitrogen three times to place the entire system under a nitrogen atmosphere. The system was stirred under reflux at 100 ° C and reacted for 4 hours. TLC monitored the complete reaction of the raw materials. The reaction was directly separated and purified by column chromatography (petroleum ether: ethyl acetate = 5: 1-2: 1) to obtain 10a (0.50 g, yield 76%).

Synthesis of intermediate 10b:

10a (0.30 g, 0.96 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of (1S,3R)-3-[(tert-butoxycarbonyl)amino]cyclohexanecarboxylic acid (0.25 g, 1.01 mmol), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.43 g, 1.15 mmol) and N,N-diisopropylethylamine (0.25 g, 1.92 mmol). The system was stirred at room temperature overnight. TLC monitoring revealed that no starting material remained. Water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed with saturated sodium chloride (50 mL×2), and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and the crude product was purified by column chromatography (petroleum ether:ethyl acetate=10:1-2:1) to give 10b (0.30 g, yield 58%).

Synthesis of intermediate 10c:

10b (0.30 g, 0.56 mmol) was dissolved in dichloromethane (30 mL), and then trifluoroacetic acid (2 mL) was added under an ice-water bath. The system was stirred at room temperature for 2 hours. TLC monitoring showed that there was no residual raw material. Water (100 mL) was added to the reaction solution, and the pH was adjusted to 9-10 with a saturated sodium bicarbonate aqueous solution, and extracted with dichloromethane (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The crude product was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-8: 1) to obtain 10c (0.20 g, yield 82%).

Synthesis of final product 10:

10c (0.20 g, 0.46 mmol) was dissolved in dichloromethane (10 mL), and then acetic anhydride (94 mg, 0.92 mmol) and triethylamine (93 mg, 0.92 mmol) were added. The reaction was carried out at room temperature for 1.5 hours. TLC (ethyl acetate) was used to monitor the complete reaction of the raw materials. Saturated sodium bicarbonate (30 mL) was added to the reaction solution, and dichloromethane was extracted (20 mL × 3). The organic phases were combined, washed with saturated sodium chloride (20 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (dichloromethane: methanol = 50: 1-20: 1) to obtain the final product 10 (150 mg, yield 68%). MS m/z (ESI): 480.2 [M + H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.56 (s, 1H), 8.32 (s, 1H), 8.14 (d, J = 5.4Hz, 1H), 7.76 (d, J = 7.8Hz, 1H) ,7.37(dd,J＝7.8Hz,J＝7.2Hz,1H),7.32-7.27(m,5H),7.16(d,J＝11.4Hz,1H),6.92(dd,J＝8.4Hz,J＝ 8.4Hz,1H),5.15(s,2H),3.55-3.54(m,1H),2.58-2.56(m,1H),1.86-1.84(m,1H),1.75-1.70(m,6H),1.33 -1.15(m,4H).

Embodiment 11:

Synthesis of intermediate 11a:

5 (0.10 g, 0.25 mmol) was dissolved in dichloromethane (20 mL), and boron tribromide (0.12 g, 0.50 mmol) was added. The reaction was allowed to react at room temperature for 4 hours. TLC monitoring showed that no raw material remained. The reaction solution was adjusted to pH 6 with sodium bicarbonate aqueous solution, and the organic phase was separated and dried over anhydrous sodium sulfate. The solvent was evaporated under reduced pressure to obtain 11a (0.08 g, yield 82%).

Synthesis of final product 11:

11a (0.08 g, 0.21 mmol) was dissolved in N,N-dimethylformamide (10 mL), 2-bromoethyl methyl ether (0.03 g, 0.25 mmol) and potassium carbonate (0.06 g, 0.42 mmol) were added, and the reaction was carried out at room temperature for 8 hours. TLC monitoring showed that there was no residual raw material. Water (20 mL) was added to the reaction solution, and ethyl acetate was extracted (20 mL × 3). The organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was separated by column chromatography (dichloromethane: methanol = 25: 1) to obtain the final product 11 (0.05 g, yield 53%). MS m/z (ESI): 448.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.56(s,1H),8.31(s,1H),8.11-8.10(m,1H),7.78-7.58(m,1H),7.36-7.33(m ,1H),7.12-7.01(m,1H),6.92-6.89(m,1H),4.15-4.13(m,2H),3.55-3.54(m,2H),3.53-3.52(m,1H),3.15 (s,3H),2.59-2.58(m,1H),1.85(s,3H),1.83-1.47(m,4H),1.29-1.04(m,4H).

Embodiment 12:

Synthesis of intermediate 12a:

2-Amino-5-fluoro-4-iodopyridine (0.50 g, 2.10 mmol) and 5-chloro-2-methoxyphenylboronic acid (0.47 g, 2.50 mmol) were dissolved in ethylene glycol dimethyl ether (10 mL), and [1,1-bis(diphenylphosphino)ferrocene]dichloropalladium (73 mg, 0.10 mmol), potassium carbonate (0.87 g, 6.3 mmol) and water (2 mL) were added. The system was replaced with nitrogen three times to place the entire system under a nitrogen atmosphere. The system was reacted at 100 ° C for 2 hours. TLC monitoring showed that no raw material remained. After cooling, the solvent was removed by concentration. The crude product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain 12a (0.50 g, yield 95%).

Synthesis of intermediate 12b:

(1S, 3R)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (0.29 g, 1.20 mmol) was dissolved in dichloromethane (10 mL), and pyridine (0.40 g, 5.00 mmol) and thionyl chloride (0.20 g, 1.70 mmol) were added in an ice bath. The system was reacted at room temperature for 2 hours and then concentrated to remove the solvent and excess thionyl chloride. Dichloromethane (10 mL) and compound 12a (0.25 g, 1.00 mmol) were then added. The system was reacted at room temperature overnight. TLC monitoring showed that no raw material remained. The system was concentrated, and the crude product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10:1-1:1) to obtain 12b (0.10 g, yield 21%).

Synthesis of intermediate 12c:

12b (48 mg, 0.10 mmol) was dissolved in dichloromethane (5 mL) and trifluoroacetic acid (2 mL) was added. The system was reacted at room temperature for 1 hour. TLC monitoring showed that there was no residual raw material. The system was concentrated to obtain 12c (50 mg, crude product). The obtained product was not further purified and was directly used in the next step reaction.

Synthesis of final product 12:

12c (50 mg, 0.10 mmol) was dissolved in dichloromethane (2 mL), and triethylamine (20 mg, 0.20 mmol) and acetic anhydride (20 mg, 0.20 mmol) were added. The system was reacted at room temperature for 1 hour. TLC monitoring showed that no starting material remained. The system was concentrated, and the crude product was separated and purified by thin-plate chromatography (dichloromethane: methanol = 10:1) to obtain the final product 12 (30 mg, yield 72%). MS: (m/z, ESI): 420.1 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.60 (s, 1H), 8.34 (s, 1H), 8.08 (d, J = 5.4Hz, 1H), 7.78 (d, J = 7.8Hz, 1H) ,7.33(d,J＝8.4Hz,1H),7.27(s,1H),7.20-7.15(m,1H),3.80(s,3H),3.65-3.55(m,1H),2.68-2.62(m ,1H),1.90-1.85(m,1H),1.76-1.55(m,6H),1.28-1.23(m,3H),1.15-1.10(m,1H).

Embodiment 13:

Synthesis of intermediate 13a:

3-Methoxy-4-bromophenol (3.00 g, 14.80 mmol) was dissolved in acetone (50 mL), followed by the addition of bromomethylcyclopropane (2.20 g, 16.30 mmol), sodium iodide (1.11 g, 7.40 mmol), and cesium carbonate (9.64 g, 29.60 mmol). The mixture was stirred under reflux and reacted for 8 hours. TLC monitoring indicated that no raw material remained. Heating was stopped and the mixture was cooled to room temperature. Acetone was evaporated under reduced pressure, water (50 mL) was added to the residue, and the mixture was extracted with ethyl acetate (30 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to remove the solvent to obtain 13a (3.70 g, yield 97%).

Synthesis of intermediate 13b:

13a (3.20 g, 12.40 mmol) was dissolved in ethylene glycol dimethyl ether (50 mL), followed by the addition of bis-pinacol borate (3.79 g, 14.90 mmol), potassium acetate (3.65 g, 37.2 mmol) and [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride (0.88 g, 1.2 mmol), and the mixture was heated to 100 °C under nitrogen protection for 8 hours. No raw material remained after TLC monitoring. Heating was stopped and the mixture was cooled to room temperature. The solvent was removed under reduced pressure, and 13b (2.50 g, yield 66%) was obtained.

Synthesis of intermediate 13c:

13b (2.19 g, 7.20 mmol) was dissolved in dioxane (50 mL), followed by the addition of 5-fluoro-4-iodo-pyridin-2-amine (1.38 g, 5.80 mmol), tetrakistriphenylphosphine palladium (0.46 g, 0.40 mmol) and potassium phosphate trihydrate (2.50 g, 9.40 mmol), and the mixture was heated to 100 °C under nitrogen protection for 8 hours. TLC monitoring showed that no raw material remained. Heating was stopped and the mixture was cooled to room temperature. The solvent was removed by concentration under reduced pressure, and the mixture was separated and purified by column chromatography (n-hexane: ethyl acetate = 1:1) to obtain 13c (1.90 g, yield 92%).

Synthesis of intermediate 13d:

13c (0.60 g, 2.10 mmol) was dissolved in N,N-dimethylformamide (20 mL), followed by the addition of (1S, 3R)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (0.51 g, 2.10 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.95 g, 2.50 mmol) and N,N-diisopropylethylamine (0.54 g, 4.20 mmol), and the reaction was carried out at room temperature for 15 hours. TLC monitoring showed that no raw material remained. Water (50 mL) was added to the reaction solution, and ethyl acetate was used for extraction (30 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. 13d (0.50 g, yield 46%) was obtained.

Synthesis of intermediate 13e:

13d (0.50 g, 0.97 mmol) was dissolved in dichloromethane (20 mL), trifluoroacetic acid (4 mL) was added, and the reaction was carried out at room temperature for 4 hours. TLC monitoring showed that there was no residual raw material. The reaction solution was washed with water (30 mL × 3), the aqueous phase was combined, the pH of the aqueous phase was adjusted to 8-9 with sodium carbonate, and extracted with dichloromethane (30 mL × 3), the organic phase was combined, washed with saturated sodium chloride (50 mL × 2), and dried over anhydrous sodium sulfate. The solvent was removed by concentration under reduced pressure to obtain 13e (0.35 g, yield 88%).

Synthesis of final product 13:

13e (0.06 g, 0.14 mmol) was dissolved in dichloromethane (20 mL), followed by the addition of acetic anhydride (0.03 g, 0.28 mmol) and triethylamine (0.03 g, 0.28 mmol). The mixture was reacted at room temperature for 2 h. TLC showed that no residue was left. The reaction solution was directly separated by column chromatography (DCM: MeOH = 25: 1) to obtain the final product 13 (0.02 g, yield 31%). MS m/z (ESI): 456.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.52 (s, 1H), 8.28 (s, 1H), 8.06 (d, J = 4.8Hz, 1H), 7.78 (d, J = 7.8Hz, 1H) ,7.18(d,J＝8.4Hz,1H),6.71(s,1H),6.63(d,J＝8.4Hz,1H),3.89(d,J＝7.2Hz,2H),3.76(s,3H ),3.57-3.55(m,1H),2.61-2.60(m,1H),1.88-1.86(m,1H),1.77(s,3H),1.76-1.75(m,3H),1.31-1.23(m ,4H),1.09-1.04(m,1H),0.60-0.59(m,2H),0.35-0.34(m,2H).

Embodiment 14:

Synthesis of intermediate 14a:

4-Bromo-3-methoxyphenol (0.40 g, 2.00 mmol) was dissolved in dioxane (50 mL), followed by the addition of bis-pinacol diborane (0.60 g, 2.40 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (0.12 g, 0.16 mmol) and potassium acetate (0.59 g, 6.00 mmol), the system was protected with nitrogen, stirred at 100°C, reacted for 5 hours, and the reaction of the raw materials was completed by TLC monitoring. Water (100 mL) was added to the reaction solution, extracted with ethyl acetate (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and purified by column chromatography (petroleum ether-petroleum ether: ethyl acetate = 4:1) to obtain 14a (0.36 g, yield 72%).

Synthesis of intermediate 14b:

14a (0.36 g, 1.44 mmol) was dissolved in diethylene glycol dimethyl ether (50 mL) and water (10 mL), followed by the addition of compound 5-fluoro-4-iodo-pyridin-2-amine (0.29 g, 1.20 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (0.44 g, 0.60 mmol) and potassium carbonate (0.50 g, 3.60 mmol). The whole system was stirred at 80 °C for 5 hours, and no raw material remained after TLC detection. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (100 mL × 3). The organic phases were combined, washed with saturated sodium chloride (100 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. 14b (0.25 g, yield 74%) was obtained by column chromatography separation and purification (petroleum ether: ethyl acetate = 4:1-2:3).

Synthesis of intermediate 14c:

14b (0.15 g, 0.64 mmol) was dissolved in N,N-dimethylformamide (50 mL), followed by the addition of (1S,3R)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (0.18 g, 0.72 mmol), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.58 g, 0.72 mmol) and diisopropylethylamine (0.20 mL, 1.20 mmol). The whole system was stirred at 25°C for 15 hours, and TLC monitoring showed that no raw material remained. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (100 mL×3). The organic phases were combined, washed with saturated sodium chloride (100 mL×2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The mixture was separated and purified by column chromatography (petroleum ether: ethyl acetate =

4:1-2:1) to afford 14c (0.15 g, 51% yield).

Synthesis of final product 14:

14c (0.15 g, 0.32 mmol) was dissolved in dichloromethane (50 mL), and then trifluoroacetic acid (2 mL) was added and stirred at room temperature for 4 hours. TLC showed that there was no residual raw material. Saturated sodium carbonate aqueous solution was added to the system, and the pH value of the reaction system was adjusted to 9-10. Dichloromethane extraction (50 mL×3) was performed, and the organic phases were combined and concentrated to a volume of about 50 mL. Triethylamine (2 mL) and acetic anhydride (2 mL) were added and reacted at room temperature for 30 minutes, and then sodium carbonate aqueous solution was added to wash the organic phase. The liquid was separated, and then extracted with dichloromethane (50 mL×3), concentrated under reduced pressure, and separated by column chromatography (ethyl acetate) to obtain the final product 14 (90 mg, yield 63%). MSm/z(ESI):444.2[M+H] ⁺ .

¹ H NMR (600MHz, CD ₃ OD) δ8.16 (s, 1H), 8.10 (s, 1H), 7.29 (d, J = 7.8 Hz, 1H), 6.87 (s, 1H), 6.78 (t, J ＝7.8Hz,1H),3.79(s,3H),3.78-3.73(m,1H),2.72(t,J＝12.0Hz,1H),2.31-2.29(m,1H),2.20-2.10(m, 1H),2.00-1.80(m,6H),1.51-1.38(m,3H),1.27-1.21(m,2H).

Embodiment 15:

Synthesis of intermediate 15a:

3-Methoxy-4-bromophenol (540 mg, 2.66 mmol) was dissolved in N,N-dimethylformamide (50 mL), and potassium carbonate (735 mg, 5.32 mmol) and benzyl bromide (910 mg, 5.32 mmol) were added. The reaction was stirred at room temperature for 15 hours. TLC detected that there was no residual raw material. Water (50 mL) was added to the system, and ethyl acetate was extracted (50 mL×3). The organic phases were combined, washed with saturated sodium chloride aqueous solution (50 mL×2), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and separated and purified by column chromatography (petroleum ether-petroleum ether: ethyl acetate = 4:1) to obtain 15a (662 mg, yield 85%).

Synthesis of intermediate 15b:

Compound 15a (662 mg, 2.27 mmol), bis-pinacol diborane (1.15 g, 4.54 mmol), potassium acetate (667 mg, 6.81 mmol) and [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride (146 mg, 0.20 mmol) were dissolved in anhydrous 1,4-dioxane (100 mL). The reaction system was protected with nitrogen and reacted at 100 ° C for 4 hours. TLC detected that there was no residual raw material. Water (100 mL) was added to the reaction system, and ethyl acetate was extracted (100 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure to remove the solvent, and separated and purified by column chromatography (petroleum ether-petroleum ether: ethyl acetate = 4:1) to obtain 15b (510 mg, yield 65%).

Synthesis of intermediate 15c:

15b (510 mg, 1.50 mmol) was dissolved in diethylene glycol dimethyl ether (150 mL), 5-fluoro-4-iodopyridin-2-amine (536 mg, 2.25 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (110 mg, 0.15 mmol) and potassium carbonate (621 mg, 4.50 mmol) were added at room temperature, and the reaction system was stirred at 80°C for 4 hours. TLC detected that there was no residual raw material. Water (100 mL) was added to the reaction system, and ethyl acetate was extracted (100 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and purified by column chromatography (petroleum ether-petroleum ether: ethyl acetate = 1:1) to obtain 15c (263 mg, yield 54%).

Synthesis of intermediate 15d:

15c (263 mg, 0.81 mmol) and (1S, 3R)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (290 mg, 1.23 mmol) were dissolved in N, N-dimethylformamide (20 mL), and 2-(7-benzotriazole oxide)-N, N, N', N'-tetramethyluronium hexafluorophosphate (470 mg, 1.23 mmol) and diisopropylethylamine (480 mg, 3.69 mmol) were added in sequence. The reaction system was reacted at room temperature for 4 hours. TLC detected that there was no residual raw material. Water (100 mL) was added to the reaction system, and ethyl acetate was extracted (100 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. After purification by column chromatography (petroleum ether: ethyl acetate = 4:1-2:1), 15d (250 mg, yield 56%) was obtained.

Synthesis of intermediate 15e:

15d (250 mg, 0.45 mmol) was dissolved in dichloromethane (50 mL), and then trifluoroacetic acid (5 mL) was added and stirred at room temperature for 4 hours. TLC detected that there was no residual raw material. Saturated sodium carbonate aqueous solution was added to the system to adjust the pH value of the reaction system to 9-10. After separation, dichloromethane was used for extraction (100 mL×3), the organic phases were combined, concentrated to a volume of about 50 mL, triethylamine (2 mL) and acetic anhydride (2 mL) were added, and the organic phase was washed with sodium carbonate aqueous solution, extracted with dichloromethane (20 mL×3), and the organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. 15e (176 mg, yield 79%) was obtained by column chromatography (ethyl acetate).

Synthesis of final product 15:

15e (175 mg, 0.36 mmol) was dissolved in methanol (20 mL), palladium carbon (10 mg) was added, the reaction system was protected with hydrogen, and the reaction was carried out at room temperature for 10 hours. LC-MS detected that there was no residue of the raw material. After the palladium carbon was filtered off, the reaction solution was concentrated under reduced pressure to obtain the final product 15 (117 mg, yield 81%). MS m/z (ESI): 402.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.49 (s, 1H), 10.09 (s, 1H), 8.25 (s, 1H), 8.05 (s, J = 5.4Hz, 1H), 7.78 (d, J＝7.8Hz,1H),7.07(d,J＝8.4Hz,1H),6.55(d,J＝2.4Hz,1H),6.48(s,1H),3.71(s,3H),3.55-3.53( m,1H),2.61-2.57(m,1H),1.87(d,J＝11.4Hz,1H),1.77(s,3H),1.77-1.75(m,3H),1.29-1.27(m,4H) .

Embodiment 16:

Synthesis of intermediate 16a:

2-(3,4-dimethoxyphenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborane (150 mg, 0.57 mmol) and 5-fluoro-4-iodopyridin-2-amine (202 mg, 0.85 mmol) were dissolved in diethylene glycol dimethyl ether (50 mL), and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (37 mg, 0.05 mmol), potassium carbonate (117 mg, 0.85 mmol) and water (10 mL) were added in sequence. The reaction system was protected by nitrogen and reacted at 80°C for 4 hours. TLC detected that there was no residual raw material. Water (50 mL) was added to the reaction solution, and ethyl acetate was extracted (100 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. After purification by column chromatography (petroleum ether: ethyl acetate = 4:1-1:1), 16a (124 mg, yield 88%) was obtained.

Synthesis of intermediate 16b:

Compound 16a (36 mg, 0.15 mmol) was dissolved in N,N-dimethylformamide (10 mL), followed by the addition of (1S,3R)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (71 mg, 0.29 mmol), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (110 mg, 0.29 mmol) and diisopropylethylamine (57 mg, 0.44 mmol), and the whole system was stirred at 25°C for 15 hours, and TLC monitoring showed that there was no residual raw material. Water (15 mL) was added to the reaction solution, and ethyl acetate was extracted (20 mL×3), the organic phases were combined, washed with saturated sodium chloride (50 mL×2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. After separation and purification by column chromatography (petroleum ether:ethyl acetate=4:1-2:1), 16b (35 mg, yield 49%) was obtained.

Synthesis of final product 16:

16b (35 mg, 0.074 mmol) was dissolved in dichloromethane (20 mL), and then trifluoroacetic acid (2 mL) was added and stirred at room temperature for 4 hours. TLC detected that there was no residual raw material. Saturated sodium carbonate aqueous solution was added to the system, and the pH value of the reaction system was adjusted to 9-10. Dichloromethane extraction (50 mL × 3) was performed, and the organic phase was combined and concentrated to a volume of about 20 mL. Triethylamine (2 mL) and acetic anhydride (2 mL) were added. The reaction was carried out at room temperature for 30 minutes, and sodium carbonate aqueous solution was added to wash the organic phase. The aqueous phase was extracted with dichloromethane (20 mL × 3), and the organic phase was combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The final product 16 (15 mg, yield 49%) was obtained by silica gel column chromatography (ethyl acetate). MS m/z (ESI): 416.2 [M + H] ⁺ .

¹ H NMR (600MHz, CD ₃ OD) δ8.12 (s, 1H), 8.08 (d, J = 5.4Hz, 1H), 7.19 (d, J = 8.4Hz, 1H), 6.64 (s, 1H), 6.60(s,1H),3.88(s,3H),3.79(s,3H),3.71(t,J＝4.8Hz,2H),2.71-2.57(m,1H),2.24-2.22(m,1H) ,1.91(s,3H),1.89(s,2H),1.49-1.19(m,6H).

Embodiment 17:

Synthesis of intermediate 17a:

2-Methoxyphenylboronic acid (0.42 g, 2.77 mmol) was dissolved in diethylene glycol dimethyl ether (30 mL) and water (6 mL), followed by the addition of compound 5-fluoro-4-iodopyridin-2-amine (0.60 g, 2.52 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (95 mg, 0.13 mmol) and potassium carbonate (1.04 g, 7.56 mmol). The system was protected with nitrogen, stirred at 100 ° C, and reacted for 4 hours. TLC monitored the reaction of the raw materials without residue. Water (50 mL) was added to the reaction system, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. Purification by column chromatography (petroleum ether: ethyl acetate = 4: 1-2: 3) gave 17a (0.60 g, yield 99%).

Synthesis of intermediate 17b:

17a (0.60 g, 2.77 mmol) was dissolved in N,N-dimethylformamide (50 mL), followed by the addition of compound (1S,3R)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (0.79 g, 3.20 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.22 g, 3.20 mmol) and diisopropylethylamine (1.2 mL, 7.00 mmol). The whole system was stirred at room temperature for 15 hours. TLC showed that no raw material remained. Water (100 mL) was added to the reaction system, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. 17b (0.60 g, yield 49%) was obtained by column chromatography separation and purification (petroleum ether: ethyl acetate = 4:1-2:3).

Synthesis of intermediate 17c:

17b (0.60 g, 1.35 mmol) was dissolved in dichloromethane (50 mL), and then trifluoroacetic acid (3 mL) was added. The whole system was stirred at 25 ° C and monitored by TLC until no raw material remained. Sodium carbonate aqueous solution (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product 17c can be directly subjected to the next step reaction.

Synthesis of final product 17

17c was dissolved in dichloromethane (50 mL), followed by the addition of triethylamine (0.4 mL, 2.70 mmol) and acetic anhydride (0.3 mL, 2.70 mmol) and stirred at room temperature for 30 minutes. The reaction of the raw material was completed by TLC. Saturated sodium carbonate aqueous solution was added to the system, extracted with dichloromethane (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and purified by column chromatography (petroleum ether: ethyl acetate = 4: 1-2: 1) to obtain the final product 17 (0.35 g, yield 68%). MS m/z (ESI): 386.2 [M + H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.55 (s, 1H), 8.30 (s, 1H), 8.07 (d, J = 5.4Hz, 1H), 7.76 (d, J = 7.8Hz, 1H) ,7.46(t,J＝7.8Hz,1H),7.27(d,J＝7.2Hz,1H),7.15(d,J＝8.4Hz,1H),7.06(t,J＝7.8Hz,1H),3.75 (s,3H),3.55-3.54(m,1H),2.56-2.48(m,1H),1.86-1.84(m,1H),1.77-1.74(m,6H),1.31-1.02(m,4H) .

Embodiment 18:

Synthesis of intermediate 18a:

4-(Bromomethyl)pyridine hydrochloride (1.72 g, 6.81 mmol) was dissolved in 20 mL N,N-dimethylformamide, followed by the addition of 2-iodophenol (1.50 g, 6.81 mmol), potassium carbonate (2.83 g, 20.45 mmol), and sodium iodide (1.02 g, 6.81 mmol). The system was stirred at room temperature and reacted for 4 hours. TLC monitoring showed that no raw material remained. The reaction was directly separated and purified by column chromatography (petroleum ether: ethyl acetate = 2:1-petroleum ether: ethyl acetate = 1:1) to obtain 18a (1.90 g, yield 90%).

Synthesis of intermediate 18b:

18a (1.00 g, 3.21 mmol) was dissolved in 15 mL of ethylene glycol dimethyl ether, followed by the addition of bis-pinacol borate (980 mg, 3.85 mmol), [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride (234 mg, 0.32 mmol) and potassium acetate (0.95 g, 9.64 mmol), and replaced with nitrogen three times to place the entire system under a nitrogen atmosphere. The system was stirred under reflux at 100 ° C and reacted for 4 hours. TLC monitoring showed that there was no residual raw material. The reaction was directly separated and purified by column chromatography (petroleum nitrate: ethyl acetate = 2:1-petroleum nitrate: ethyl acetate = 1:1) to obtain compound 18b (0.80 g, yield 80%).

Synthesis of intermediate 18c:

5-Fluoro-4-iodopyridin-2-amine (802 mg, 3.37 mmol) was dissolved in 20 mL of ethylene glycol dimethyl ether, followed by the addition of 18b (700 mg, 2.25 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (168 mg, 0.23 mmol) and potassium carbonate (932 mg, 6.75 mmol), and the atmosphere was replaced with nitrogen three times to place the entire system under a nitrogen atmosphere. The system was stirred under reflux at 100°C and reacted for 4 hours. TLC monitoring showed that there was no residual raw material. The reaction was directly separated and purified by column chromatography (dichloromethane: methanol = 50: 1-dichloromethane: methanol = 10: 1) to obtain 18c (220 mg, yield 33%).

Synthesis of intermediate 18d:

18c (220 mg, 0.75 mmol) was dissolved in 10 mL of N,N-dimethylformamide, followed by the addition of (1S,3R)-3-[(tert-butoxycarbonyl)amino]cyclohexanecarboxylic acid (217 mg, 0.89 mmol), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (340 mg, 0.89 mmol) and N,N-dimethylethylamine (192 mg, 1.49 mmol). The whole system was stirred at room temperature overnight. TLC detected that no raw material remained. 100 mL of water was added to the reaction solution, and the mixture was extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed with saturated sodium chloride (50 mL×2), and dried over anhydrous sodium sulfate. The solvent was then removed under reduced pressure and purified by column chromatography (dichloromethane: methanol = 50:1-dichloromethane: methanol = 20:1) to give 18d (300 mg, yield 77%).

Synthesis of intermediate 18e:

18d (0.30 g, 0.58 mmol) was dissolved in 30 mL of dichloromethane, and then 2 mL of trifluoroacetic acid was added under an ice-water bath. The whole system was stirred at room temperature overnight, and TLC was detected until no raw material remained. 100 mL of water was added to the reaction solution, and the pH was adjusted to 9-10 with a saturated sodium bicarbonate aqueous solution, and extracted with dichloromethane (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. After separation and purification by column chromatography (dichloromethane: methanol = 50: 1-dichloromethane: methanol = 8: 1), 18e (0.16 g, yield 66%) was obtained.

Synthesis of final product 18:

18e (160 mg, 0.38 mmol) was dissolved in dichloromethane (5 mL), followed by the addition of acetic anhydride (116 mg, 1.14 mmol) and triethylamine (115 mg, 1.14 mmol). The system was stirred at room temperature and monitored by TLC tracking until no raw material remained. 50 mL of water was added to the reaction solution, extracted with dichloromethane (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-petroleum ether: ethyl acetate = 2: 1) to obtain the final product 18 (60 mg, yield 34%). MS m/z (ESI): 463.2 [M + H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.61 (s, 1H), 8.50 (d, J = 5.4Hz, 2H), 8.36 (s, 1H), 8.20 (d, J = 5.4Hz, 1H) ,7.80(d,J＝7.8Hz,1H),7.47-7.44(m,1H),7.35(d,J＝7.2Hz,1H),7.30(d,J＝5.4Hz,2H),7.18(d, J＝9.0Hz,1H),7.15(t,J＝7.8Hz,1H),5.22(s,2H),3.58-3.57(m,1H),2.62-2.53(m,1H),1.89-1.75(m ,7H),1.30-1.04(m,4H).

Embodiment 19:

Synthesis of intermediate 19a

7-Bromobenzofuran (600 mg, 3.00 mmol) was dissolved in ethylene glycol dimethyl ether (50 mL), followed by the addition of pinacol diboron (928 mg, 3.70 mmol), [1,1'-bis(diphenylphosphino)ferrocene] dichloropalladium dichloromethane complex (163 mg, 0.20 mmol) and potassium acetate (892 mg, 9.10 mmol), the system was heated to 100°C under nitrogen protection, reacted for 4 hours, and TLC monitored the complete reaction of the raw materials. The reaction solution was cooled to room temperature, and the crude product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 100: 1-50: 1) to obtain 19a (450 mg, yield 62%).

Synthesis of intermediate 19b

19a (450 mg, 1.84 mmol) was dissolved in ethylene glycol dimethyl ether (50 mL) and water (10 mL), followed by the addition of 5-fluoro-4-iodopyridin-2-amine (350 mg, 1.47 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (110 mg, 0.15 mmol) and potassium carbonate (405 mg, 2.94 mmol), the system was heated to 100 ° C under nitrogen protection, reacted for 4 hours, and TLC monitored the complete reaction of the raw materials. The reaction solution was cooled to room temperature, and the crude product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain 19b (300 mg, yield 71%).

Synthesis of intermediate 19c

(1S,3R)-3-[(tert-Butyloxycarbonyl)amino]cyclohexanecarboxylic acid (292 mg, 1.20 mmol) was dissolved in dichloromethane (50 mL), followed by the addition of pyridine (474 mg, 6.00 mmol) and thionyl chloride (242 mg, 2.04 mmol), and the reaction was carried out at room temperature for 4 hours. Then 19b (300 mg, 1.32 mmol) was added to the above reaction solution, and the reaction was continued at room temperature overnight. TLC monitoring showed that there was no residue of the raw material. The reaction solution was diluted with water (30 mL), the organic phase was extracted and collected, and concentrated under reduced pressure. The crude product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain 19c (200 mg, yield 39%).

Synthesis of intermediate 19d

19c (200 mg, 0.47 mmol) was dissolved in dichloromethane (10 mL), followed by the addition of trifluoroacetic acid (1 mL), and the reaction was carried out at room temperature for 1.5 hours. The reaction mixture was monitored by TLC to confirm the complete reaction of the raw material. Saturated sodium bicarbonate (30 mL) was added to the reaction solution, and the mixture was extracted with dichloromethane (20 mL×3). The organic phases were combined, washed with saturated sodium chloride (20 mL×2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain 19d (128 mg, yield 83%).

Synthesis of final product 19

19d (128 mg, 0.39 mmol) was dissolved in dichloromethane (10 mL), followed by the addition of acetic anhydride (48 mg, 0.47 mmol) and triethylamine (47 mg, 0.47 mmol), and the reaction was carried out at room temperature for 1.5 hours. TLC monitored the complete reaction of the raw material. Saturated sodium bicarbonate (30 mL) was added to the reaction solution, and dichloromethane was extracted (20 mL × 3). The organic phases were combined, washed with saturated sodium chloride (20 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (dichloromethane: methanol = 50: 1-20: 1) to obtain the final product 19 (117 mg, yield 76%). MS m/z (ESI): 396.2 [M + H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.68 (s, 1H), 8.48 (s, 1H), 8.41 (d, J = 5.4Hz, 1H), 8.09 (s, 1H), 7.83-7.78 ( m,2H),7.46(d,J＝7.2Hz,1H),7.41(dd,J＝7.2Hz,J＝7.2Hz,1H),7.09(s,1H),3.58-3.56(m,1H), 2.64-2.60(m,1H),1.90-1.84(m,1H),1.78-1.76(m,6H),1.31-1.25(m,3H),1.10-1.06(m,1H).

Embodiment 20:

Synthesis of intermediate 20a:

2,6-difluoropyridine-3-boric acid (550 mg, 3.14 mmol) and compound 5-fluoro-4-iodopyridin-2-amine (898 mg, 3.77 mmol) were dissolved in 1,4-dioxane (15 mL), and [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (219 mg, 0.30 mmol) and potassium carbonate (1.30 g, 9.42 mmol) were added in sequence, and then water (6 mL) was added. The system was protected with nitrogen, stirred at 100 ° C, and reacted for 5 hours. TLC monitoring showed that there was no residual raw material. Water (100 mL) was added to the reaction solution, extracted with ethyl acetate (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 4: 1-2: 3) to obtain 20a (700 mg, yield 99%).

Synthesis of intermediate 20b:

20a (700 mg, 3.14 mmol) was dissolved in acetonitrile (50 mL), followed by the addition of compound (1S, 3R)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (778 mg, 3.20 mmol), tetramethylchlorouronium hexafluorophosphate (898 mg, 3.20 mmol) and N-methylimidazole (0.87 mL, 11.00 mmol). The whole system was stirred at room temperature for 15 hours, and TLC showed that there was no residual raw material. After concentrating the solvent, column chromatography was used for separation and purification (petroleum ether: ethyl acetate = 4:1-2:3) to obtain 20b (1.40 g, yield 99%)

Synthesis of final product 20:

20b (1.40 g, 3.14 mmol) was dissolved in dichloromethane (50 mL), and 5 mL of trifluoroacetic acid was added at room temperature. The reaction was allowed to react for 4 hours. TLC detected that there was no residual raw material. The reaction system was washed with saturated sodium carbonate aqueous solution until it was weakly alkaline, extracted with dichloromethane (30 mL × 3), and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was concentrated to 50 mL, and 2 mL of triethylamine and 2 mL of acetic anhydride were added. The mixture was stirred at room temperature for 30 minutes. TLC monitored that there was no residual raw material. It was washed with anhydrous sodium carbonate solution, extracted with dichloromethane (30 mL × 3), and the organic phases were combined. The solvent was concentrated under reduced pressure to remove the solvent. The crude product was separated by column chromatography to obtain the final product 20 (750 mg, yield 61%). MS m/z (ESI): 393.2 [M + H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.74 (s, 1H), 8.50 (s, 1H), 8.40 (dd, J = 16.8Hz, J = 7.8Hz, 1H), 8.26 (d, J = 6.0Hz,1H),7.79(d,J＝7.8Hz,1H),7.41(dd,J＝7.8Hz,J＝2.4Hz,1H),3.60-3.55(m,1H),2.64-2.60(m, 1H),1.91-1.88(m,1H),1.78-1.76(m,6H),1.29-1.27(m,3H),1.09-1.06(m,1H).

Embodiment 21:

Synthesis of intermediate 21a:

5-Fluoro-4-iodopyridin-2-amine (1.00 g, 4.20 mmol) was dissolved in ethylene glycol dimethyl ether (20 mL) and water (4 mL), followed by the addition of indazole-4-boric acid (0.68 g, 4.20 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (0.34 g, 0.42 mmol) and potassium carbonate (1.74 g, 12.60 mmol), and the atmosphere was replaced with nitrogen three times to place the entire system under a nitrogen atmosphere. The system was stirred under reflux at 100°C for 4 hours, and no raw material remained after TLC monitoring. The mixture was concentrated under reduced pressure and purified by column chromatography (dichloromethane:methanol=50:1-10:1) to obtain 21a (0.77 g, yield 80%).

Synthesis of intermediate 21b:

21a (0.75 g, 3.30 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of (1S,3R)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (0.80 g, 3.30 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.56 g, 4.10 mmol) and N,N-diisopropylethylamine (0.85 g, 6.60 mmol). The whole system was stirred at room temperature overnight. TLC monitoring showed that no raw material remained. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. 21b (0.91 g, yield 60%) was obtained by column chromatography separation and purification (dichloromethane: methanol = 50: 1-20: 1).

Synthesis of intermediate 21c:

21b (0.91 g, 2.00 mmol) was dissolved in dichloromethane (30 mL), and then 2 mL of trifluoroacetic acid was added under an ice-water bath. The whole system was stirred at room temperature overnight. TLC showed that there was no residual raw material. Water (100 mL) was added to the reaction solution, and the pH was adjusted to 9-10 with a saturated sodium bicarbonate aqueous solution. The mixture was extracted with dichloromethane (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. After separation and purification by column chromatography (dichloromethane: methanol = 50: 1-8: 1), 21c (0.61 g, yield 86%) was obtained.

Synthesis of final product 21:

21c (0.61 g, 1.72 mmol) was dissolved in dichloromethane (35 mL), followed by the addition of acetic anhydride (0.53 g, 5.20 mmol) and triethylamine (0.52 g, 5.20 mmol), and the system was stirred at room temperature. TLC monitoring showed that there was no residual raw material. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain the final product 21 (0.32 g, yield 47%). MS m/z (ESI): 396.1 [M + H] ⁺ .

¹ H NMR (600MHz, CDCl ₃ ) δ8.60 (s, 1H), 8.27 (s, 1H), 8.14 (s, 1H), 7.63 (d, J＝7.8Hz, 1H), 7.51 (d, J＝ 7.8Hz,1H),7.39(d,J＝6.6Hz,1H),7.15(d,J＝7.2Hz,1H),3.89-3.87(m,1H),2.54-2.50(m,1H),2.29( d,J＝12.0Hz,1H),1.79-1.77(m,6H),1.44-1.28(m,3H),1.12-1.09(m,1H).

Embodiment 22:

Synthesis of intermediate 22a:

5-Fluoro-4-iodopyridin-2-amine (1.00 g, 4.20 mmol) was dissolved in ethylene glycol dimethyl ether (20 mL) and water (4 mL), followed by the addition of indole-4-boric acid (0.68 g, 4.20 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (0.34 g, 0.42 mmol) and potassium carbonate (1.74 g, 12.60 mmol), and the atmosphere was replaced with nitrogen three times to place the entire system under a nitrogen atmosphere. The system was stirred under reflux at 100 °C for 4 hours, and no raw material remained after TLC monitoring. The mixture was concentrated under reduced pressure and purified by column chromatography (dichloromethane: methanol = 50: 1-10: 1) to obtain 22a (0.78 g, yield 82%).

Synthesis of intermediate 22b:

22a (0.78 g, 3.40 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of (1S,3R)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (0.83 g, 3.40 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (1.56 g, 4.10 mmol) and N,N-diisopropylethylamine (0.88 g, 6.80 mmol). The whole system was stirred at room temperature overnight. TLC monitoring showed that no raw material remained. Water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed with saturated sodium chloride (50 mL×2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The mixture was separated and purified by column chromatography (dichloromethane:methanol=50:1-20:1) to obtain 22b (0.92 g, yield 59%).

Synthesis of intermediate 22c:

22b (0.92 g, 2.03 mmol) was dissolved in dichloromethane (30 mL), and then 2 mL of trifluoroacetic acid was added under an ice-water bath. The whole system was stirred at room temperature overnight. TLC showed that no raw material remained. Water (100 mL) was added to the reaction solution, and the pH was adjusted to 9-10 with saturated sodium bicarbonate aqueous solution. The mixture was extracted with dichloromethane (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. After separation and purification by column chromatography (dichloromethane: methanol = 50: 1-8: 1), 22c (0.61 g, yield 85%) was obtained.

Synthesis of final product 22:

22c (0.61 g, 1.73 mmol) was dissolved in dichloromethane (35 mL), followed by the addition of acetic anhydride (0.53 g, 5.20 mmol) and triethylamine (0.52 g, 5.20 mmol), and the system was stirred at room temperature. TLC monitoring showed that there was no residual raw material. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain the final product 22 (0.35 g, yield 51%). MS m/z (ESI): 395.1 [M + H] ⁺ .

¹ H NMR (600 MHz, DMSO-d ₆ )δ11.38(s,1H),10.59(s,1H),8.42(s,1H),8.36(d,J＝6.0Hz,1H),7.77(d,J＝7.8Hz,1H),7.54(d,J＝8.4Hz,1H),7.46(d,J＝3.0Hz,1H),7.23(d,J＝8.4Hz ,1H),7.15(d,J＝7.2Hz,1H),6.41(s,1H),3.60-3.54(m,1H),2.64-2.60(m,1H),1.89(d,J＝12.0Hz,1H),1.77-1.76(m,6H),1.34-1.23(m,3H),1.10 -1.08(m,1H).

Embodiment 23:

Synthesis of intermediate 23a:

5-Fluoro-4-iodopyridin-2-amine (1.00 g, 4.20 mmol) was dissolved in N,N-dimethylformamide (20 mL), followed by the addition of (1S,3R)-3-[(tert-butoxycarbonyl)amino]cyclohexanecarboxylic acid (1.32 g, 5.40 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (2.39 g, 6.29 mmol) and N,N-dimethylethylamine (2.08 mL, 12.6 mmol). The whole system was stirred at room temperature overnight. TLC detection revealed no residual starting material. Water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed with saturated sodium chloride (50 mL×2), and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure and purified by column chromatography (dichloromethane:methanol=50:1-20:1) to give 23a (806 mg, yield 41%).

Synthesis of intermediate 23b:

23a (806 mg, 1.74 mmol) was dissolved in dichloromethane (10 mL), and then 5 mL of trifluoroacetic acid was added under an ice-water bath. The whole system was stirred at room temperature for 2 hours. TLC detected that there was no residual raw material. Water (100 mL) was added to the reaction solution, and then the pH was adjusted to 9-10 with a saturated sodium bicarbonate aqueous solution, and extracted with dichloromethane (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure to obtain 23b (770 mg, crude product).

Synthesis of intermediate 23c:

23b (770 mg, crude product) was dissolved in dichloromethane (10 mL), followed by the addition of triethylamine (883 μL, 6.35 mmol) and acetic anhydride (297 μL, 3.18 mmol). The system was stirred at room temperature, and TLC monitoring showed that there was no residue of raw materials. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. 23c (490 mg, yield 69%) was obtained.

Synthesis of intermediate 23d:

23c (100 mg, 0.25 mmol) was dissolved in 1,4-dioxane (10 mL) and water (5 mL), followed by the addition of tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine-1-carboxylate (167 mg, 0.49 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (15 mg, 0.02 mmol) and potassium carbonate (68 mg, 0.49 mmol), and replaced with nitrogen three times to place the entire system under a nitrogen atmosphere. The system was stirred under reflux at 100 ° C and reacted for 4 hours. TLC monitoring showed that there was no residual raw material. The reaction was directly separated and purified by column chromatography (dichloromethane: methanol = 50: 1-10: 1) to obtain 23d (122 mg, yield 99%).

Synthesis of final product 23:

23d (122 mg, 0.24 mmol) was dissolved in dichloromethane (10 mL), and then 5 mL of trifluoroacetic acid was added under an ice-water bath. The whole system was stirred at room temperature for 2 hours. TLC detected that there was no residual raw material. Water (100 mL) was added to the reaction solution, and then the pH was adjusted to 9-10 with a saturated sodium bicarbonate aqueous solution, extracted with dichloromethane (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The final product 23 (57 mg, yield 59%) was obtained by column chromatography separation and purification (dichloromethane: methanol = 50: 1-10: 1). MS m/z (ESI): 396.2 [M + H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ ) δ11.97 (s, 1H), 10.70 (s, 1H), 8.42 (s, 1H), 8.41 (s, 1H), 8.36 (d, J = 4.8Hz, 1H),7.79(d,J＝7.8Hz,1H),7.63-7.62(m,1H),7.23(d,J＝4.2Hz,1H),6.47(s,1H),3.58-3.56(m,1H ),2.62-2.61(m,1H),1.99(d,J=4.8Hz,1H),1.91-1.89(m,6H),1.39-1.21(m,3H),1.20-1.09(m,1H).

Embodiment 24:

Synthesis of intermediate 24a:

(1-Methyl-1H-pyrrolo[2,3-b]pyridin-4-yl)boronic acid (176 mg, 1.00 mmol) and 23a (461 mg, 1.00 mmol) were dissolved in 20 mL of 1,4-dioxane, and [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (73 mg, 0.10 mmol) and potassium carbonate (414 mg, 3.00 mmol) were added in sequence. The system was protected with nitrogen and stirred at 100°C for 4.5 hours. TLC monitored the completion of the reaction of the raw materials. The reaction was stopped and water (50 mL) was added to the reaction system, extracted with ethyl acetate (50 mL×3), the organic phases were combined, washed with saturated sodium chloride (50 mL×2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The mixture was separated and purified by column chromatography (petroleum ether: ethyl acetate = 4:1-petroleum ether: ethyl acetate = 1:1) to obtain 24a (400 mg, yield 86%).

Synthesis of final product 24:

24a (400 mg, 0.86 mmol) was dissolved in dichloromethane (25 mL), trifluoroacetic acid (3 mL) was added at room temperature, and the reaction was carried out at room temperature for 4 hours. TLC monitoring showed that there was no residual raw material. The mixture was washed with a saturated sodium carbonate solution until weakly alkaline. The aqueous phase was extracted with dichloromethane (30 mL × 3), and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was concentrated to a volume of 15 mL, triethylamine (2 mL) and acetic anhydride (2 mL) were added and reacted at room temperature for 30 minutes. TLC monitoring showed that there was no residual raw material. The reaction system was washed with a saturated sodium carbonate solution, the solvent was concentrated, and the final product 24 (169 mg, yield 48%) was obtained after separation by silica gel column chromatography (dichloromethane: methanol = 50: 1-10: 1). MS m/z (ESI): 410.2 [M + H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.69(s,1H),8.50(s,1H),8.41(s,1H),8.40(s,1H),7.77(d,J＝7.8Hz, 1H),7.68(d,J＝3.6Hz,1H),7.26(d,J＝4.8Hz,1H),6.47(d,J＝2.4Hz,1H),3.89(s,3H),3.58-3.56( m,1H),2.64-2.60(m,1H),1.91-1.89(m,1H),1.79-1.77(m,6H),1.30-1.26(m,3H),1.09-1.07(m,1H).

Embodiment 25:

Synthesis of final product 25:

5b (0.61 g, 1.69 mmol) was dissolved in dichloromethane (35 mL), followed by the addition of methylsulfonyl chloride (0.29 g, 2.54 mmol) and triethylamine (0.34 g, 3.38 mmol). The system was stirred at room temperature and monitored by TLC until no raw material remained. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain the final product 25 (0.41 g, yield 55%). MS m/z (ESI): 440.1 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.60 (s, 1H), 8.33 (s, 1H), 8.08 (d, J = 4.8Hz, 1H), 7.34 (d, J = 7.2Hz, 1H) ,7.10(d,J＝7.8Hz,2H),6.92(d,J＝8.4Hz,1H),3.80(s,3H),3.13-3.11(m,1H),2.92(s,3H),2.62- 2.57(m,1H),2.03(d,J＝12Hz,1H),2.25(d,J＝12Hz,1H),1.77-1.73(m,2H),1.36-1.11(m,4H).

Embodiment 26:

Synthesis of intermediate 26a:

5-Fluoro-4-iodopyridin-2-amine (0.50 g, 2.10 mmol) was dissolved in ethylene glycol dimethyl ether (20 mL) and water (4 mL), followed by the addition of 4,5-difluoro-2-methoxyphenylboronic acid (0.39 g, 2.10 mmol), [1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (0.15 g, 0.21 mmol) and potassium carbonate (0.87 g, 6.3 mmol), and the atmosphere was replaced with nitrogen three times to place the entire system under a nitrogen atmosphere. The system was stirred under reflux at 100 ° C and reacted for 4 hours. TLC monitoring showed that there was no residual raw material. The reaction was directly separated and purified by column chromatography (dichloromethane: methanol = 50: 1-10: 1) to obtain 26a (0.40 g, yield 75%).

Synthesis of intermediate 26b:

26a (0.30 g, 1.18 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of (1S,3R)-3-[(tert-butoxycarbonyl)amino]cyclohexanecarboxylic acid (0.35 g, 1.42 mmol), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.54 g, 1.42 mmol) and N,N-dimethylethylamine (0.30 g, 2.36 mmol). The whole system was stirred at room temperature overnight. TLC detection showed that no starting material remained. Water (100 mL) was added to the reaction solution, and the mixture was extracted with ethyl acetate (50 mL×3). The organic phases were combined, washed with saturated sodium chloride (50 mL×2), and dried over anhydrous sodium sulfate. The solvent was then removed under reduced pressure and purified by column chromatography (dichloromethane:methanol=50:1-20:1) to give 26b (0.33 g, yield 58%).

Synthesis of intermediate 26c:

26b (0.20 g, 0.42 mmol) was dissolved in dichloromethane (30 mL), followed by the addition of trifluoroacetic acid (2 mL) under an ice-water bath, and the whole system was stirred at room temperature overnight, and TLC was performed until no raw material remained. Water (100 mL) was added to the reaction solution, and the pH was adjusted to 9-10 with a saturated sodium bicarbonate aqueous solution, and extracted with dichloromethane (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure, and separated and purified by column chromatography (dichloromethane: methanol = 50: 1-8: 1) to obtain 26c (0.11 g, yield 70%).

Synthesis of final product 26:

26c (0.11 g, 0.29 mmol) was dissolved in dichloromethane (35 mL), followed by the addition of methanesulfonyl chloride (0.40 g, 0.35 mmol) and triethylamine (44 mg, 0.46 mmol). The system was stirred at room temperature and monitored by TLC until no raw material remained. Water (50 mL) was added to the reaction solution, extracted with dichloromethane (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain the final product 26 (40 mg, yield 30%). MS m/z (ESI): 458.1 [M + H] ⁺ .

¹ H NMR (600MHz, CDCl ₃ ) δ8.59 (s, 1H), 8.31 (s, 1H), 8.14 (d, J = 10.8Hz, 1H), 7.17 (d, J = 9.0Hz, 1H), 6.85 -6.82(m,1H),5.46(s,1H),3.88(s,1H),3.83(s,3H),2.82(s,1H),2.48(s,1H),2.27-2.25(m,1H ),2.05-1.94(m,5H),1.45-1.16(m,4H).

Embodiment 27:

Synthesis of final product 27:

5b (0.61 g, 1.69 mmol) was dissolved in dichloromethane (35 mL), followed by the addition of thiophenesulfonyl chloride (0.46 g, 2.53 mmol) and triethylamine (0.34 g, 3.38 mmol). The system was stirred at room temperature overnight and monitored by TLC until no raw material remained. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain the final product 27 (0.52 g, yield 61%). MS m/z (ESI): 508.1 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.55 (s, 1H), 8.31 (s, 1H), 8.03 (s, 1H), 7.96 (d, J = 7.2Hz, 1H), 7.89 (s, 1H),7.57(s,1H),7.32(d,J＝7.2Hz,1H),7.15(s,1H),7.08(d,J＝11.4Hz,1H),6.89(d,J＝8.4Hz, 1H),4.02(s,3H),3.04-3.03(m,1H),1.78-1.62(m,5H),1.17-1.08(m,4H).

Embodiment 28:

Synthesis of final product 28:

5b (79 mg, 0.22 mmol) was dissolved in acetonitrile (5 mL), and sodium carbonate (47 mg, 0.44 mmol) and benzyl bromide (37 mg, 0.22 mmol) were added. The system was left to react overnight at room temperature. TLC monitoring showed that no starting material remained. The solvent was removed by concentration, and the crude product was separated and purified by thin-plate chromatography (dichloromethane: methanol = 10:1) to obtain the final product 28 (30 mg, yield 30%). MS: (m/z, ESI): 451.2 [M+H] ⁺ .

¹ H NMR (600MHz, CD ₃ OD) δ8.16 (s, 1H), 8.09 (d, J = 4.8Hz, 1H), 7.37-7.32 (m, 4H), 7.30-7.26 (m, 2H), 6.93 (d,J＝11.4Hz,1H),6.80(dd,J＝8.4Hz,1H),3.86(s,2H),3.81(s,3H),2.71-2.67(m,1H),2.51-2.47( m,1H),2.20-2.18(m,1H),2.06-2.04(m,1H),1.89-1.85(m,2H),1.49-1.39(m,3H),1.21-1.16(m,1H).

Embodiment 29:

Synthesis of final product 29:

5b (0.61 g, 1.69 mmol) was dissolved in N,N-dimethylformamide (35 mL), followed by the addition of bromoethyl methyl ether (0.26 g, 1.86 mmol) and potassium carbonate (0.47 g, 3.38 mmol). The system was stirred at room temperature, and TLC monitoring showed that no raw material remained. Water (50 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-20: 1) to obtain the final product 29 (0.42 g, yield 59%). MS m/z (ESI): 420.2 [M+H] ⁺ .

¹ H NMR (600MHz, CDCl ₃ )δ8.25(d,J＝5.4Hz,1H),8.12(s,1H),7.25(d,J＝6.6Hz,1H),6.75-6.70(m,2H),3.80(s,3H), 3.57(t,J＝4.8Hz,2H),3.36(s, 3H),2.90(t,J＝4.8Hz,2H),2.70(s,1H),2.37(t,J＝5.4Hz,1H),2.26(d,J＝12.0Hz,1H),2.05-1.90( m,3H),1.52-1.22(m,4H).

Embodiment 30:

Synthesis of final product 30:

5b (155 mg, 0.43 mmol) was dissolved in acetonitrile (5 mL), and triethylamine (86 mg, 0.86 mmol), bromoethanol (54 mg, 0.43 mmol) and sodium iodide (10 mg, 0.06 mmol) were added. The system was reacted at 60 °C for 12 hours. TLC monitoring showed that no raw material remained. After cooling, the solvent was removed by concentration. The crude product was separated and purified by thin plate chromatography (dichloromethane: methanol: triethylamine = 40:2:1) to obtain the final product 30 (30 mg, yield 17%). MS m/z (ESI): 406.2 [M+H] ⁺ .

¹ H NMR (600MHz, CD ₃ OD)δ8.18(s,1H),8.08(d,J＝4.8Hz,1H),7.28(d,J＝7.2Hz,1H),6.92(d,J＝11.4Hz,1H),6.79(d ,J＝8.4,1H),3.82-3.80(m,5H),3.20-3.25(m ,1H),3.20-3.18(m,2H),2.65-2.62(m,1H),2.31-2.29(m,1H),2.18- 2.16(m,1H),1.99-1.91(m,2H),1.70-1.64(m,1H),1.53-1.34(m,3H).

Embodiment 31:

Synthesis of final product 31:

5b (79 mg, 0.22 mmol) was dissolved in dioxane (5 mL), and then compound 2-bromo-6-methoxypyridine (40 mg, 0.22 mmol), tris(dibenzylideneacetone)dipalladium (20 mg, 0.022 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethylxanthene (12.7 mg, 0.022 mmol) and cesium carbonate (215 mg, 0.66 mmol) were added. The system was replaced with nitrogen three times to keep the whole system under nitrogen atmosphere. The system was reacted at 120°C for 2 hours. TLC monitoring showed that there was no residual raw material. After cooling, the mixture was concentrated under reduced pressure, and the crude product was separated and purified by thin plate chromatography (dichloromethane: methanol = 10: 1) to obtain the final product 31 (30 mg, yield 29%). MS: (m/z, ESI): 469.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.56(s,1H),8.31(s,1H),8.07(d,J＝5.4Hz,1H),7.32(d,J＝7.2Hz,1H),7.24(d,J＝7.8Hz, 1H),7.08(d,J＝11.4Hz,1H),6.89(d,J＝8.4Hz,1H),6.33(d,J＝7.2Hz,1H),5.97(d,J＝7.8Hz,1 H),5.80(d,J＝7.8Hz,1H),3.76(s,3H),3.72(s,3H),3.63-3.61(m,1H),2.64-2.62(m,1H),2. 06-2.04(m,1H),1.97-1.95(m,1H),1.79-1.77(m,2H),1.36-1.30(m,3H),1.11-1.09(m,1H).

Embodiment 32:

Synthesis of final product 32:

5b (61 mg, 0.17 mmol) was dissolved in N,N-dimethylformamide (5 mL), followed by the addition of cyanoacetic acid (16 mg, 0.18 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (69 mg, 0.18 mmol) and N,N-diisopropylethylamine (83 uL, 0.50 mmol). The whole system was stirred at room temperature and reacted for 10 hours. TLC monitoring and tracking was performed until no raw material remained. Water (50 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The final product 32 (50 mg, yield 71%) was obtained. MS m/z (ESI): 429.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.58(s,1H),8.31(s,1H),8.20(d,J＝7.8Hz,1H),8.06(d,J＝5.4Hz,1H),7.32(t,J＝7.2Hz, 1H),7.08(d,J＝11.4 Hz,1H),6.90(t,J＝8.4Hz,1H),3.77(s,3H),3.56(m,3H),2.61-2.57(m,1H),1.89-1.76(m,4H),1.30 -1.07(m,4H).

Embodiment 33:

Synthesis of final product 33:

5b (0.16 g, 0.43 mmol) was dissolved in N, N-dimethylformamide (30 mL), followed by the addition of 1-cyano-1-cyclopropanecarboxylic acid (72 mg, 0.65 mmol), 2-(7-benzotriazole oxide)-N, N, N', N'-tetramethyluronium hexafluorophosphate (178 mg, 0.47 mmol) and N, N-diisopropylethylamine (111 mg, 0.86 mmol), and the system was stirred at room temperature overnight. TLC monitoring showed that no raw material remained. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The crude product was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-20: 1) to obtain the final product 33 (96 mg, yield 49%). MS m/z(ESI):455.2[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.56(s,1H),8.31(s,1H),8.05(d,J＝4.8Hz,1H),8.09(d,J＝7.8Hz,1H),7.32(dd,J＝7.8Hz, J＝7.2Hz,1H),7.09(d,J＝11.4Hz,1H),6.90 (dd,J＝8.4Hz,J＝7.8Hz,1H),3.77(s,3H),3.68-3.56(m,1H),3.59-3.55(m,1H),1.81-1.67(m,4H), 1.53-1.46(m,5H)),1.28-1.23(m,3H).

Embodiment 34:

Synthesis of final product 34:

5b (0.16 g, 0.43 mmol) was dissolved in N, N-dimethylformamide (30 mL), followed by the addition of 1-cyanocyclobutanecarboxylic acid (81 mg, 0.65 mmol), 2-(7-benzotriazole oxide)-N, N, N', N'-tetramethyluronium hexafluorophosphate (178 mg, 0.47 mmol) and N, N-diisopropylethylamine (111 mg, 0.86 mmol), and the system was stirred at room temperature overnight. TLC monitoring showed that no raw material remained. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The crude product was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-20: 1) to obtain the final product 34 (119 mg, yield 59%). MS m/z(ESI):469.2[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d6) δ10.56(s,1H),8.31(s,1H),8.05(d,J＝4.8Hz,1H),8.09(d,J＝7.8Hz,1H),7.34- 7.31(m,1H),7.09(s,1H) ,6.91-6.88(m,1H),3.77(s,3H),3.68-3.56(m,1H),2.59-2.55(m,1H),1.81-1.67(m,4H),1.53-1.46(m, 5H),1.30-1.23(m,5H).

Embodiment 35:

Synthesis of intermediate 35a:

2,2'-Dibromodiethyl ether (12.85 g, 55.60 mmol) was dissolved in N,N-dimethylformamide (10 mL), followed by the addition of methyl cyanoacetate (5.00 g, 50.45 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (11.50 g, 75.68 mmol), and the atmosphere was replaced with nitrogen three times to place the entire system under a nitrogen atmosphere. The system was stirred at 85°C and reacted for 4 hours. TLC monitoring showed that no raw material remained. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and purified by column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain 35a (5.00 g, yield 59%).

Synthesis of intermediate 35b:

35a (2.37 g, 14.00 mmol) was dissolved in ethanol (36 mL) and water (5 mL), followed by the addition of sodium hydroxide (2.24 g, 56.00 mmol), and the whole system was stirred at room temperature for 4 hours. TLC showed that no raw material remained. Water (100 mL) was added to the reaction solution, and the pH was adjusted to 8 with aqueous sodium bicarbonate solution, and extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain 35b (1.50 g, yield 69%).

Synthesis of final product 35:

35b (123 mg, 0.79 mmol) was dissolved in N,N-dimethylformamide (5 mL), followed by the addition of (1S,3R)-3-amino-N-(5-fluoro-4-(4-fluoro-2-methoxyphenyl)pyridin-2-yl)cyclohexanamide (260 mg, 0.72 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (548 mg, 1.44 mmol) and N,N-diisopropylethylamine (357 μL, 2.16 mmol). The whole system was stirred at room temperature and reacted for 10 hours. TLC monitoring revealed that no raw material remained. Water (50 mL) was added to the reaction solution, extracted with ethyl acetate (50 mL×3), the organic phases were combined, washed with saturated sodium chloride (50 mL×2), dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and purified by column chromatography (dichloromethane: methanol = 40:1-20:1) to obtain the final product 35 (214 mg, yield 60%). MS m/z (ESI): 499.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.58(s,1H),8.33(s,1H),8.17(d,J＝7.8Hz,1H),8.08(d,J＝6.0Hz,1H),7.35-7.33(m,1H), 7.11-7.09(m,1H),6.93-6.89(m,1H),3.8 8-3.86(m,2H),3.78(s,3H),3.66-3.63(m,1H),3.52-3.51(m,2H),2.63(m,1H),1.97-1.96(m,4H), 1.85-1.75(m,4H),1.38-1.21(m,4H).

Embodiment 36:

Synthesis of final product 36:

Triphosgene (33 mg, 0.11 mmol) was dissolved in dichloromethane (2 mL), and then a solution of 5b (61 mg, 0.17 mmol) and triethylamine (22 mg, 0.22 mmol) dissolved in dichloromethane (2 mL) was added dropwise. The whole system was stirred at room temperature and reacted for 2 hours. Then, a methanol solution (2 mL) was added and the reaction was continued for 3 hours. TLC monitoring was performed until no raw material remained. Water (50 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The final product 36 (30 mg, yield 42%) was obtained. MS m/z (ESI): 420.2 [M + H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.56(s,1H),8.31(s,1H),8.06(d,J＝5.4Hz,1H),7.33(d,J＝7.8Hz,1H),7.10-7.07(m,2H), 6.90(d,J =8.4Hz,1H),3.77(s,3H),3.48(s,3H),3.32(m,1H),2.56(m,1H),1.87-1.71(m,4H),1.31-1.06(m, 4H).

Embodiment 37:

Synthesis of final product 37:

Triphosgene (33 mg, 0.11 mmol) was dissolved in dichloromethane (2 mL), and then a dichloromethane solution containing 5b (61 mg, 0.17 mmol) and triethylamine (22 mg, 0.22 mmol) was added dropwise. The whole system was stirred at room temperature and reacted for 2 hours. Then 3 mL of 33% methylamine alcohol solution was added and the reaction was continued for 3 hours. TLC monitoring was performed until no raw material remained. Water (50 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The final product 37 (15 mg, yield 21%) was obtained. MS m/z (ESI): 419.2 [M + H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.56(s,1H),8.31(s,1H),8.06(d,J＝5.4Hz,1H),7.33(t,J＝8.4Hz,1H),7.10-7.07(m,1H), 6.91-6.88(m,1H),5.78(d,J＝8.4Hz ,1H),5.56(d,J=4.8Hz,1H),3.76(s,3H),3.36-3.34(m,1H),2.65-2.55(m,1H),2.51(s,3H),1.88- 1.73(m,4H),1.28-0.82(m,4H).

Embodiment 38:

Synthesis of final product 38:

5b (0.61 g, 1.69 mmol) was dissolved in dichloromethane (35 mL), followed by the addition of dimethylaminoformyl chloride (0.27 g, 2.54 mmol) and triethylamine (0.34 g, 3.38 mmol). The system was stirred at room temperature overnight, and TLC monitoring showed that no raw material remained. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (dichloromethane: methanol = 100: 1-40: 1) to obtain the final product 38 (0.38 g, yield 53%). MS m/z (ESI): 433.1 [M+H] ⁺ .

¹ H NMR (600MHz, CDCl ₃ )δ8.28-8.25(m,2H),8.11(s,1H),7.27-7.24(m,2H),6.76-6.70(m,2H),3.81(s,3H),3.76-3.73(m ,1H),2.88(s,6H),2.47-2.43(m,1H),2.30(d,J=12.0Hz,1H),2.02-1.90(m,3H),1.47-1.12(m,4H).

Embodiment 39:

Synthesis of intermediate 39a:

5b (0.12 g, 0.33 mmol) was dissolved in N,N-dimethylformamide (10 mL), and (S)-2-((tert-butyloxycarbonyl)amino)-2-(4-hydroxyphenyl)acetic acid (0.10 g, 0.36 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.15 g, 0.40 mmol), and N,N-diisopropylethylamine (0.09 g, 0.66 mmol) were added. The reaction was carried out at room temperature for 8 hours. TLC monitoring showed that there was no residual raw material. Water (20 mL) was added to the reaction solution, and ethyl acetate was used for extraction (15 mL×3). The organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was directly separated by column chromatography (dichloromethane:methanol=10:1) to obtain 39a (0.12 g, yield 60%).

Synthesis of final product 39:

39a (0.12 g, 0.20 mmol) was dissolved in dichloromethane (20 mL), trifluoroacetic acid (4 mL) was added, and the reaction was carried out at room temperature for 4 hours. TLC monitoring showed that there was no residual raw material. The reaction solution was washed with water (20 mL × 3), the aqueous phase was combined, the pH of the aqueous phase was adjusted to 8-9 with sodium carbonate, and the dichloromethane was extracted (30 mL × 3), the organic phase was combined, and dried over anhydrous sodium sulfate. The organic phase was evaporated under reduced pressure to obtain the final product 39 (0.04 g, yield 39%). MS m/z (ESI): 511.2 [M+H] ⁺ .

¹ H NMR (600 MHz, DMSO-d ₆ )δ10.56(s,1H),9.23(s,1H),8.32(s,1H),8.08(d,J＝5.4Hz,1H),7.90-7.89 (m,1H),7.36-7.34(m,1H),7.17-7.15(m,2H),7.12-7.10(m,1H),6.93-6.91 (m,1H),6.68-6.66(m,2H),4.19(s,1H),4.04(d,J＝7.2Hz,2H),3.80(s,3H), 3.58-3.57(m,1H),2.59-2.57(m,1H),1.80-1.76(m,4H),1.33-1.25(m,4H).

Embodiment 40:

Synthesis of final product 40:

5b (0.20 g, 0.55 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of N,N-dimethylglycine (67 mg, 0.65 mmol), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (178 mg, 0.47 mmol) and N,N-diisopropylethylamine (111 mg, 0.86 mmol), and the system was stirred at room temperature overnight. TLC monitoring showed that no raw material remained. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The crude product was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-20: 1) to obtain the final product 40 (100 mg, yield 41%). MS m/z(ESI):447.2[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.56(s,1H),8.31(s,1H),8.06(d,J＝4.2Hz,1H),7.58(d,J＝8.4Hz,1H),7.32( dd,J＝7.8Hz,J＝7.2Hz,1H),7.08(d,J＝10.8Hz,1H),6.90(dd,J＝7.8Hz,J＝7.8Hz,1 H),3.76(s,3H),3.65-3.63(m,1H),2.84-2.77(m,2H),2.66-2.60(m,1H),2.16(s ,6H),1.85-1.84(m,1H),1.75-1.69(m,3H),1.41-1.35(m,1H),1.32-1.23(m,3H).

Embodiment 41:

Synthesis of compound 41:

Dissolve triphosgene (0.027 g, 0.09 mmol) in anhydrous tetrahydrofuran (20 mL), add a tetrahydrofuran solution of 3-methylisoxazole-5-amine (0.027 g, 0.28 mmol), triethylamine (0.028 g, 0.28 mmol), and react at room temperature for 6 hours. Add a tetrahydrofuran solution of 5b (0.10 g, 0.28 mmol) and triethylamine (0.028 g, 0.28 mmol) to the reaction solution, heat to 70 ° C, and react for 6 hours. TLC monitoring shows that there is no residual raw material, stop the reaction, cool to room temperature, and separate the reaction solution by column chromatography (petroleum ether: ethyl acetate = 1: 1) to obtain the final product 41 (45 mg, yield 33%). MS m/z (ESI): 486.2 [M + H] ⁺ .

¹ H NMR (600MHz, CDCl ₃ )δ8.65(s,1H),8.29(s,1H),8.12(s,1H),6.76-6.71(m,2H),6.23(s,2H),5.43(s,1H),4.03-4.01 (m,1H ),3.81(s,3H),2.54-2.53(m,1H),2.35(s,3H),2.36-2.24(m,2H),2.05-1.94(m,3H),1.56-1.50(m,3H ).

Embodiment 42:

Synthesis of final product 42:

5b (100 mg, 0.28 mmol) was dissolved in dichloromethane (10 mL), and then trifluoroacetic anhydride (56 mg, 0.55 mmol) and triethylamine (56 mg, 0.55 mmol) were added. The reaction was carried out at room temperature for 1.5 hours. TLC monitoring showed that there was no residual raw material. Saturated sodium bicarbonate (30 mL) was added to the reaction solution, and dichloromethane was extracted (20 mL × 3). The organic phases were combined, washed with saturated sodium chloride (20 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (dichloromethane: methanol = 50: 1-20: 1) to obtain the final product 42 (100 mg, yield 78%). MS m/z (ESI): 458.1 [M + H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.59(s,1H),9.33(d,J＝8.4Hz,1H),8.31(s,1H),8.05(d,J＝6Hz,1H),7.32(dd,J＝7.8Hz,J ＝7.2Hz,1H),7.08(m,1H),6.89(m,1H ),3.76(s,3H),3.70-3.68(m,1H),2.63-2.59(m,1H),1.86-1.83(m,1H),1.80-1.76(m,2H),1.51-1.44(m ,1H),1.33-1.24(m,4H).

Embodiment 43:

Synthesis of compound 43:

5b (0.11 g, 0.30 mmol) and 2-pyridinecarboxylic acid (0.04 g, 0.33 mmol) were dissolved in N,N-dimethylformamide (15 mL), and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.14 g, 0.36 mmol) and N,N-diisopropylethylamine (0.08 g, 0.6 mmol) were added. The mixture was reacted at room temperature for 8 hours. TLC monitoring showed that no raw material remained. Water (30 mL) was added to the reaction solution, and a solid was precipitated. The solid was filtered to obtain the final product 43 (0.07 g, 50%). MS m/z (ESI): 467.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.63(s,1H),8.63-8.62(m,2H),8.31(s,1H),8.07(d,J＝5.4Hz,1H),8.01(d ,J＝7.8Hz,1H),7.98-7.95(m,1H),7.58-7.56(m,1H),7.34-7.31(m,1H),7.09-7 .07(m,1H),6.91-6.88(m,1H),3.89-3.87(m,1H),3.77(s,3H),2.71-2.63(m,1 H),1.92-1.91(m,1H),1.82-1.77(m,3H),1.61-1.55(m,1H),1.45-1.28(m,3H).

Embodiment 44:

Synthesis of final product compound 44:

5b (188 mg, 0.52 mmol) and 1-ethyl-3-methyl-1H-pyrazole-4-carboxylic acid (161 mg, 1.04 mmol) were dissolved in N,N-dimethylformamide (5 mL), and 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (395 mg, 1.04 mmol) and diisopropylethylamine (0.26 mL, 1.56 mmol) were added in sequence. The reaction system was reacted at room temperature for 15 hours. TLC detected that there was no residual raw material. Water (50 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL×3). The organic phases were combined, washed with saturated sodium chloride (50 mL×2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 4:1-2:1) to obtain the final product 44 (230 mg, yield 88%). MS m/z(ESI):498.2[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.63(s,1H),8.34(s,1H),8.22(d,J＝8.4Hz,1H),8.09(d,J＝6.0Hz,1H),7.35(m,1H),7.11( m,1H),6.92(m,1H),6.59(s,1H),4.38(d d,J＝7.2Hz,J＝3.0Hz,2H),3.79(s,4H),2.15(s,3H),2.00(d,J＝11.4Hz,1H),1.84-1.80(m,4H), 1.49-1.46(m,2H), δ1.32-1.30(m,5H).

Embodiment 45:

Synthesis of final product 45:

5b (50 mg, 0.138 mmol) was dissolved in N,N-dimethylformamide (5 mL), followed by the addition of glycolic acid (16 mg, 0.21 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (80 mg, 0.21 mmol) and N,N-diisopropylethylamine (69 μL, 0.42 mmol). The whole system was stirred at room temperature and reacted for 10 hours. TLC monitoring was performed until no raw material remained. Water (50 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The final product 45 (30 mg, yield 52%) was obtained. MS m/z (ESI): 420.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.56(s,1H),8.31(s,1H),8.06(d,J＝5.4Hz,1H),7.56(d,J＝9.0Hz,1H),7.34-7.31(m,1H), 7.09-7.07(m,1H),6.91-6.8 8(m,1H),5.38-5.36(m,1H),3.77-3.75(m,5H),3.66-3.63(m,1H),2.61-2.57(m,1H),1.83-1.68(m,4H ),1.34-1.26(m,4H).

Embodiment 46:

Synthesis of final product 46:

5b (0.20 g, 0.55 mmol) was dissolved in N, N-dimethylformamide (30 mL), followed by the addition of ethoxyacetic acid (68 mg, 0.65 mmol), 2-(7-benzotriazole oxide)-N, N, N', N'-tetramethyluronium hexafluorophosphate (178 mg, 0.47 mmol) and N, N-diisopropylethylamine (111 mg, 0.86 mmol), and the system was stirred at room temperature overnight. TLC monitoring showed that there was no residual raw material. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The crude product was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-20: 1) to obtain the final product 46 (96 mg, yield 39%). MS m/z (ESI): 448.2 [M + H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.58(s,1H),8.33(s,1H),8.07(d,J＝5.4Hz,1H),7.59(d,J＝8.4Hz,1H),7.35-7.33(m,1H), 7.11-7.09(m,1H),6.93-6.90(m,1H), 3.78-3.76(m,5H),3.69-3.67(m,1H),3.47-3.44(m,2H),2.63-2.61(m,1H),1.84-1.69(m,4H),1.47-1.43(m ,1H),1.30-1.28(m,6H).

Embodiment 47:

Synthesis of final product 47:

45 (0.20 g, 0.48 mmol) was dissolved in dichloromethane (30 mL), followed by the addition of acetyl chloride (45 mg, 0.57 mmol) and triethylamine (97 mg, 0.96 mmol). The system was stirred at room temperature for 1.5 hours. TLC monitoring revealed no residue. The solvent was removed under reduced pressure, and the crude product was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-20: 1) to obtain the final product 47 (100 mg, yield 45%). MS m/z (ESI): 462.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.58(s,1H),8.33(s,1H),8.07(d,J＝5.4Hz,1H),7.59(d,J＝8.4Hz,1 H),7.35-7.34(m,1H),7.33-7.11(m,1H),7.09-6.90(m,1H),4.39(s,2H ),3.78(s,3H),3.65-3.63(m,1H),2.66-2.60(m,1H),2.58(s,3H),1.85 -1.84(m,1H),1.75-1.69(m,3H),1.39-1.24(m,3H),1.16-1.14(m,1H).

Embodiment 48:

Synthesis of final product 48:

5b (0.20 g, 0.55 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of L-lactic acid (58.5 mg, 0.65 mmol), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (178 mg, 0.47 mmol) and N,N-diisopropylethylamine (111 mg, 0.86 mmol), and the system was stirred at room temperature overnight. TLC monitoring showed that no raw material remained. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The crude product was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-20: 1) to obtain the final product 48 (96 mg, yield 40%). MS m/z(ESI):434.2[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.57(s,1H),8.31(s,1H),8.05(d,J＝6Hz,1H),7.51(d,J＝8.4Hz,1H),7.34-7 .31(m,1H),7.10-7.07(m,1H),6.91-6.88(m,1H),5.38(d,J＝5.4Hz,1H),3.92-3. 88(m,1H),3.77(s,3H),3.62-3.59(m,1H),2.60-2.56(m,1H),1.83-1.81(m,1H), 1.77-1.72(m,2H),1.41-1.35(m,1H),1.31-1.26(m,4H),1.23(d,J=13.2Hz,3H).

Embodiment 49:

Synthesis of final product 49:

5b (0.20 g, 0.55 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of D-lactic acid (58.5 mg, 0.65 mmol), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (178 mg, 0.47 mmol) and N,N-diisopropylethylamine (111 mg, 0.86 mmol), and the system was stirred at room temperature overnight. TLC monitoring showed that no raw material remained. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The crude product was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-20: 1) to obtain the final product 49 (100 mg, yield 42%). MS m/z(ESI):434.2[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.57(s,1H),8.30(s,1H),8.05(d,J＝4.8Hz,1H),7.51(d,J＝7.8Hz,1H),7.33(t,J＝7. 8Hz,1H),7.08(d,J＝11.4Hz,1H),6.89(t,J＝7.8Hz,1H),5.38(d,J＝5.4Hz,1H),3.92-3.8 8(m,1H),3.77(s,3H),3.62-3.59(m,1H),2.60-2.56(m,1H),1.81(d,J＝12Hz,1H),1.77- 1.72(m,2H),1.67(d,J＝11.4Hz,1H),1.41-1.35(m,1H),1.31-1.25(m,4H),1.23(s,3H).

Embodiment 50:

Synthesis of final product 50:

5b (50 mg, 0.138 mmol) was dissolved in N, N-dimethylformamide (5 mL), followed by the addition of 3-hydroxypropionic acid (19 mg, 0.21 mmol), 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (79 mg, 0.21 mmol) and N, N-diisopropylethylamine (69 μL, 0.42 mmol). The whole system was stirred at room temperature and reacted for 10 hours. TLC monitoring was performed until no raw material remained. Water (50 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The final product 50 (32 mg, yield 53%) was obtained. MS m/z (ESI): 434.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.56(s,1H),8.31(s,1H),8.06-8.05(d,J＝5.4Hz,1H),7.57-7.55(d,J＝9.0Hz,1H),7.34-7.31(m ,1H),7.09-7.07(m,1H),6.91-6. 88(m,1H),5.38-5.36(t,J＝5.4Hz,1H),3.77-3.75(m,5H),3.66-3.63(m,1H),2.61-2.56(m,3H),1.83- 1.68(m,4H),1.34-1.26(m,4H).

Embodiment 51:

Synthesis of final product 51:

5b (50 mg, 0.138 mmol) was dissolved in N, N-dimethylformamide (5 mL), followed by the addition of 1-hydroxycyclopropanecarboxylic acid (21 mg, 0.21 mmol), 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (79 mg, 0.21 mmol) and N, N-diisopropylethylamine (69 μL, 0.42 mmol). The whole system was stirred at room temperature and reacted for 10 hours. TLC monitoring was performed until no raw material remained. Water (50 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The final product 51 (36 mg, yield 59%) was obtained. MS m/z (ESI): 446.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.61(s,1H),8.34(s,1H),8.09-8.08(m,1H),7.73-7.71(m,1H),7.37-7.34(m,1H),7.13-7.10(m, 1H),6.95-6.91(m,1H),3 .80(s,3H),2.61-2.55(m,2H),1.85-1.72(m,4H),1.50-1.47(m,1H),1.32-1.30(m,3H),1.00-0.98(m, 2H),0.81-0.80(m,2H).

Embodiment 52:

Synthesis of final product 52:

5b (50 mg, 0.138 mmol) was dissolved in N, N-dimethylformamide (5 mL), followed by the addition of 3-oxetanecarboxylic acid (21 mg, 0.21 mmol), 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (79 mg, 0.21 mmol) and N, N-diisopropylethylamine (69 μL, 0.42 mmol), and the whole system was stirred at room temperature for 10 hours, and TLC monitoring was performed until no raw material remained. Water (50 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and purified by column chromatography (dichloromethane: methanol = 40: 1-20: 1) to obtain the final product 52 (36 mg, yield 59%). MS m/z(ESI):446.2[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.59(s,1H),8.33(s,1H),8.07-8.06(m,1H),7.98-7.97(m,1H),7.35 -7.32(m,1H),7.10-7.08(m,1H),6.93-6.91(m,1H),4.61-4.56(m,2H),3 .78(s,3H),3.69-3.67(m,2H),3.42-3.40(m,1H),2.69-2.55(m,2H),1.8 9-1.87(m,1H),1.77-1.75(m,3H),1.29-1.25(m,3H),1.18-1.09(m,1H).

Embodiment 53:

Synthesis of final product 53:

5b (50 mg, 0.138 mmol) was dissolved in N, N-dimethylformamide (5 mL), followed by the addition of 1-methylcyclopropane-1-carboxylic acid (21 mg, 0.21 mmol), 2-(7-benzotriazole oxide)-N, N, N', N'-tetramethyluronium hexafluorophosphate (79 mg, 0.21 mmol) and N, N-diisopropylethylamine (69 μL, 0.42 mmol), and the whole system was stirred at room temperature for 10 hours, and TLC monitoring was performed until no raw material remained. Water (50 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and purified by column chromatography (dichloromethane: methanol = 40: 1-20: 1) to obtain the final product 53 (40 mg, yield 65%). MS m/z(ESI):444.2[M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.57(s,1H),8.32(s,1H),8.08-8.06(m,1H),7.34-7.32(m,1H),7.24-7.22(m,1H),7.11-7.09(m, 1H),6.93-6.91(m,1H),3.78(s,3H ),3.66-3.58(m,1H),2.59-2.50(m,1H),1.80-1.69(m,4H),1.47-1.44(m,1H),1.30-1.22(m,6H),0.92-0.90 (m,2H),0.46-0.44(m,2H).

Embodiment 54:

Synthesis of final product 54:

5b (50 mg, 0.138 mmol) was dissolved in N, N-dimethylformamide (5 mL), followed by the addition of 1-fluorocyclopropanecarboxylic acid (22 mg, 0.21 mmol), 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (79 mg, 0.21 mmol) and N, N-diisopropylethylamine (69 μL, 0.42 mmol). The whole system was stirred at room temperature and reacted for 10 hours. TLC monitoring was performed until no raw material remained. Water (50 mL) was added to the reaction solution, extracted with ethyl acetate (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and purified by column chromatography (dichloromethane: methanol = 40: 1-20: 1) to obtain the final product 54 (37 mg, yield 60%). MS m/z (ESI): 448.2 [M + H] ⁺ .

¹ H NMR (600MHz, CDCl ₃ )δ8.23-8.20(m,1H),8.13(s,1H),7.86(s,1H),7.27-7.25(m,1H),6.80-6.70(m,2H),6.40-6.25(m, 1H),4.00-3.85(m,1H),3 .82(s,3H),2.50-2.40(m,1H),2.35-2.25(m,1H),2.10-1.90(m,3H),1.55-1.45(m,3H),1.40-1.30(m, 2H),1.25-1.15(m,3H).

Embodiment 55:

Synthesis of final product 55:

5b (50 mg, 0.138 mmol) was dissolved in N, N-dimethylformamide (5 mL), followed by the addition of 1-trifluoromethylcyclopropane-1-carboxylic acid (32 mg, 0.21 mmol), 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (79 mg, 0.21 mmol) and N, N-diisopropylethylamine (69 μL, 0.42 mmol), and the whole system was stirred at room temperature for 10 hours, and TLC monitoring was performed until no raw material remained. Water (50 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and purified by column chromatography (dichloromethane: methanol = 40: 1-20: 1) to obtain the final product 55 (45 mg, yield 65%). MS m/z(ESI):498.2[M+H] ⁺ .

¹ H NMR (600MHz, CDCl ₃ )δ8.23-8.20(m,1H),8.13(s,1H),7.86(s,1H),7.27-7.25(m,1H),6.79-6.69(m,2H),6.05-5.95(m, 1H),4.00-3.86 (m,1H),3.82(s,3H),2.50-2.35(m,1H),2.30-2.20(m,1H),2.05-1.90(m,3H),1.56-1.35(m,5H),1.21 -1.15(m,3H).

Embodiment 56:

Synthesis of intermediate 56a:

Glycine (500 mg, 6.70 mmol) was dissolved in dioxane (10 mL) and water (10 mL), and triethylamine (1.40 g, 14.40 mmol) and di-tert-butyl dicarbonate (1.74 g, 8.00 mmol) were added. The system was left to react overnight at room temperature. TLC monitoring showed that the raw material had reacted completely. The system was concentrated, and an aqueous sodium carbonate solution (100 mL) was added, and the mixture was washed with ethyl acetate (100 mL×3). The aqueous phase was adjusted to pH 3 with dilute hydrochloric acid, and extracted with ethyl acetate (100 mL×3). The organic phases were combined and dried over anhydrous sodium sulfate. After filtration, the filtrate was concentrated to obtain 56a (1.00 g, yield 85%).

Synthesis of intermediate 56b:

56a (46 mg, 0.26 mmol) was dissolved in N,N-dimethylformamide (3 mL), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (125 mg, 0.33 mmol) and diisopropylethylamine (57 mg, 0.44 mmol) were added, and the mixture was stirred for 10 minutes, and 5b (78 mg, 0.22 mmol) was added. The system was left to react overnight at room temperature. TLC monitoring showed that the raw material had reacted completely. Water (100 mL) was added to the system, and ethyl acetate was extracted (100 mL×3). After the organic phases were combined, they were washed with saturated brine (50 mL×3). Drying was performed over anhydrous sodium sulfate, and the filtrate was concentrated after filtration. The crude product was separated and purified by thin plate chromatography (dichloromethane: methanol = 10:1) to obtain 56b (80 mg, yield 70%).

Synthesis of final product 56:

56b (80 mg, 0.22 mmol) was dissolved in dichloromethane (4 mL), and trifluoroacetic acid (1 mL) was added. The system was reacted at room temperature for 2 hours, and TLC monitoring showed that the reaction was complete. The system was concentrated, and the crude product was separated and purified by thin plate chromatography (dichloromethane: methanol = 10:1) to obtain the final product 56 (40 mg, yield 43%). MS m/z (ESI): 419.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.59(s,1H),8.31(s,1H),8.05(d,J＝6.0Hz,1H),7.94(br,2H),7.32-7.30(m,1H),7.09-7.07(m ,1H),6.91-6.88(m,1H),3.76(s,3H), 3.64-3.59(m,1H),3.15(d,J＝4.8Hz,2H),2.62-2.59(m,1H),1.89-1.87(m,1H),1.79-1.77(m,3H),1.34- 1.28(m,3H),1.18-1.10(m,1H).

Embodiment 57:

Synthesis of final product 57:

Compound 56 (205 mg, 0.49 mmol) was dissolved in dichloromethane (35 mL), followed by the addition of acetic anhydride (149 mg, 1.47 mmol) and triethylamine (148 mg, 1.47 mmol), and the system was stirred at room temperature for 4 hours. TLC monitoring showed that no raw material remained. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain the final product 57 (138 mg, yield 61%). MS m/z (ESI): 461.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.60(s,1H),8.33(s,1H),8.07(d,J＝6.0Hz,1H),7.95-7.92(m,2H),7.35-7.33(m,1H),7.11(d ,J＝1.8Hz,1H),6.93-6.90 (m,1H),4.09(s,2H),3.79(s,3H),3.58-3.56(m,1H),2.59-2.50(m,1H),1.86-1.73(m,4H),1.83(s ,3H),1.39-1.14(m,4H).

Embodiment 58:

Synthesis of final product 58:

5b (0.20 g, 0.55 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of N-acetyl-L-leucine (112 mg, 0.65 mmol), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (178 mg, 0.47 mmol) and N,N-diisopropylethylamine (111 mg, 0.86 mmol), and the system was stirred at room temperature overnight. TLC monitoring showed that there was no residual raw material. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The crude product was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-20: 1) to obtain the final product 58 (190 mg, yield 67%). MS m/z(ESI):517.2[M+H] ⁺ .

¹ H NMR (600 MHz, DMSO-d ₆ )δ10.60(s,1H),8.33(s,1H),8.07(d,J＝6.0Hz,1H),7.94(dd,J＝10.2,Hz,J＝8.4 Hz,2H),7.35(dd,J＝7.8Hz,J＝7.2Hz,1H),7.11-7.10(m,1H),6.93-6.90(m,1H), 4.24-4.23(m,1H),3.79(s,3H),3.58-3.56(m,1H),2.59-2.50(m,1H),1.83-1.7 1(m,7H),1.53-1.51(m,1H),1.38-1.36(m,2H),1.31-1.28(m,6H),0.91(m,6H).

Embodiment 59:

Synthesis of final product 59:

5b (200 mg, 0.55 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of N-acetyl-D-leucine (112 mg, 0.65 mmol), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (178 mg, 0.47 mmol) and N,N-diisopropylethylamine (111 mg, 0.86 mmol), and the system was stirred at room temperature overnight. TLC monitoring showed that there was no residual raw material. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The crude product was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-20: 1) to obtain the final product 59 (181 mg, yield 64%). MS m/z(ESI):517.2[M+H] ⁺ .

¹ H NMR (600 MHz, DMSO-d ₆ )δ10.59(s,1H),8.33(s,1H),8.07(d,J＝6.0Hz,1H),7.93(dd,J＝10.2,Hz,J＝8.4 Hz,2H),7.34(dd,J＝7.8Hz,J＝7.2Hz,1H),7.11-7.10(m,1H),6.92-6.89(m,1H), 4.24-4.23(m,1H),3.80(s,3H),3.57-3.55(m,1H),2.59-2.50(m,1H),1.83-1.7 1(m,7H),1.53-1.51(m,1H),1.38-1.36(m,2H),1.30-1.27(m,6H),0.91(m,6H).

Embodiment 60:

Synthesis of intermediate 60a:

5b (500 mg, 1.385 mmol) was dissolved in N, N-dimethylformamide (15 mL), followed by the addition of 1-Boc-4-piperidinic acid (380 mg, 1.66 mmol), 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (787 mg, 2.07 mmol) and N, N-diisopropylethylamine (687 uL, 4.15 mmol), the whole system was stirred at room temperature, reacted for 10 hours, and TLC monitoring showed that there was no residual raw material. Water (50 mL) was added to the reaction solution, stirred for 30 minutes, filtered, and dried over anhydrous sodium sulfate to obtain 60a (623 mg, yield 79%).

Synthesis of intermediate 60b:

60a (623 mg, 1.09 mmol) was dissolved in dichloromethane (20 mL), followed by the addition of trifluoroacetic acid (5 mL), and the whole system was stirred at room temperature for 2 hours. TLC showed that no raw material remained. Water (100 mL) was added to the reaction solution, and the pH was adjusted to 8 with sodium bicarbonate, and extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure to obtain 60b (500 mg, yield 97%).

Synthesis of final product 60:

60b (300 mg, 0.63 mmol) was dissolved in methanol (10 mL), followed by the addition of 1 mL of formaldehyde aqueous solution, sodium triacetoxyborohydride (269 mg, 1.27 mmol), and 1 drop of acetic acid. The whole system was stirred at room temperature and reacted for 10 hours. TLC monitoring showed that there was no residual raw material. Water (50 mL) was added to the reaction solution, and the pH was adjusted to 8 with sodium bicarbonate. The mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the final product 60 (214 mg, yield 70%). MS m/z (ESI): 487.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.59(s,1H),8.34(s,1H),8.08(d,J＝4.8Hz,1H),7.68(d,J＝7.8Hz,1H),7.35(d,J ＝7.8Hz,1H),7.11(d,J＝11.4Hz,1H),6.93(d,J＝8.4Hz,1H),3.79(s,3H),3.58-3.56(m ,1H),2.76-2.74(m,2H),2.62-2.58(m,1H),2.51(s,3H),1.99-1.96(m,1H),1.86-1.8 4(m,1H),1.81-1.72(m,5H),1.57-1.53(m,4H),1.31-1.24(m,3H),1.18-1.08(m,1H).

Example 61:

Synthesis of final product 61:

5b (0.07 g, 0.19 mmol) was dissolved in N,N-dimethylformamide (10 mL), and cyclopropylacetic acid (0.02 g, 0.21 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.09 g, 0.23 mmol) and N,N-diisopropylethylamine (0.05 g, 0.38 mmol) were added. The mixture was reacted at room temperature for 8 hours. TLC monitoring showed that no raw material remained. Water (20 mL) was added to the reaction solution, and ethyl acetate was extracted (15 mL×3). The organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (dichloromethane: methanol = 20:1) to obtain the final product 61 (0.04 g, yield 47%). MS m/z (ESI): 444.2 [M+H] ⁺ .

¹ H NMR (600 MHz, DMSO-d ₆ )δ10.58(s,1H),δ8.33(s,1H),8.07(d,J＝5.4Hz,1H),7.63(d,J＝8.4Hz,1H ),7.35-7.33(m,1H),7.11-7.01(m,1H),6.93-6.90(m,1H),3.79(s,3H),3. 60-3.57(m,1H),2.60-2.61(m,1H),1.94-1.93(m,2H),1.88-1.75(m,4H),1 .32-1.23(m,4H),1.10-1.08(m,1H),0.54-0.45(m,2H),0.19-0.15(m,2H).

Embodiment 62:

Synthesis of final product 62:

5b (0.07 g, 0.19 mmol) was dissolved in dichloromethane (20 mL), and cyclopropylcarboxylic acid (0.02 g, 0.21 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (0.07 g, 0.38 mmol) and 4-dimethylaminopyridine (2.3 mg, 0.019 mmol) were added. The reaction was carried out at room temperature for 8 hours. TLC monitoring showed that there was no residual raw material. The reaction solution was washed with water and sodium bicarbonate aqueous solution in turn, and dried over anhydrous sodium sulfate. The organic phase was directly separated by column chromatography (dichloromethane: methanol = 50: 1) to obtain the final product 62 (0.04 g, yield 48%). MS m/z (ESI): 430.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.58(s,1H),8.32(s,1H),8.08(d,J＝5.4Hz,1H),7.99(d,J＝8.4Hz,1H),7.36-7.33(m,1H), 7.11-7.09(m,1H),6.93-6.90(m,1H),3.79(s ,3H),3.60-3.58(m,1H),2.59-2.50(m,1H),1.89-1.77(m,4H),1.50- 1.47(m,1H),1.34-1.26(m,3H),1.10-1.08(m,1H),0.64-0.60(m,4H).

Embodiment 63:

Synthesis of final product 63:

5b (0.10 g, 0.28 mmol) was dissolved in N,N-dimethylformamide (25 mL), followed by the addition of 2-cyclopropyl-2-carbonylacetic acid (35 mg, 0.31 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (118 mg, 0.31 mmol) and N,N-diisopropylethylamine (0.07 g, 0.55 mmol). The system was stirred at room temperature and monitored by TLC until no raw material remained. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The final product 63 (54 mg, yield 43%) was obtained by column chromatography separation and purification (dichloromethane: methanol = 100: 1-40: 1). MS m/z(ESI):458.1[M+H] ⁺ .

¹ H NMR (600MHz, CDCl ₃ )δ8.55(s,1H),8.32(d,J＝6.0Hz,1H),8.11(s,1H),7.28-7.25(m,1H),6.94(d,J＝8.4Hz,1H), 6.77-6.71(m,2H),3.86-3.82(m,1H),3.81(s,3 H),3.10-3.08(m,1H),2.48-2.46(m,1H),2.25(d,J＝12.0Hz,1H),2.04 -1.95(m,3H),1.53-1.47(m,3H),1.26-1.24(m,1H),1.18-1.15(m,4H).

Embodiment 64:

Synthesis of final product 64:

5b (0.16 g, 0.44 mmol) was dissolved in dichloromethane (20 mL), triethylamine (0.05 g, 0.53 mmol) was added, acryloyl chloride (0.05 g, 0.53 mmol) was added under ice bath, and the reaction was carried out at room temperature for 4 hours. TLC monitoring showed that there was no residual raw material. Water (30 mL) was added to the reaction solution, and dichloromethane was extracted (20 mL × 3). The organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The crude product was separated by column chromatography (dichloromethane: methanol = 50: 1) to obtain the final product 64 (0.10 g, yield 55%). MS m/z (ESI): 416.2 [M + H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.59(s,1H),8.31(s,1H),8.06(d,J＝6.0Hz,1H),8.02(d,J＝7.8Hz,1H),7.34-7.31(m,1H), 7.10-7.07(m,1H),6.91-6.89(m,1H),6.18-6 .14(m,1H),6.06-6.03(m,1H),5.55-5.53(m,1H),3.77(s,3H),3.65- 3.63(m,1H),2.61-2.60(m,1H),1.90-1.77(m,4H),1.34-1.08(m,4H).

Embodiment 65:

Synthesis of final product 65:

5b (180 mg, 0.50 mmol) and propiolic acid (70 mg, 1.00 mmol) were dissolved in N, N-dimethylformamide (20 mL), and 2-(7-benzotriazole oxide)-N, N, N', N'-tetramethyluronium hexafluorophosphate (380 mg, 1.00 mmol) and N, N-diisopropylethylamine (0.25 mL, 1.50 mmol) were added in sequence. The reaction system was reacted at room temperature for 15 hours. TLC detected that there was no residual raw material. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by column chromatography (petroleum ether: ethyl acetate = 4: 1-2: 1) to obtain the final product 65 (200 mg, yield 97%). MS m/z (ESI): 414.2 [M + H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.60(s,1H),8.73(d,J＝7.8Hz,1H),8.33(s,1H),8.08(d,J＝5.4Hz,1H),7.35(dd,J＝8.4Hz, J＝7.8Hz,1H),7.11(dd,J＝11.4Hz,J＝2 .4Hz,1H),6.92-6.90(m,1H),4.12(s,1H),3.79(s,3H),3.64-3.62(m,1H),2.61-2.57(m,1H),1.85-1.74 (m,4H),1.35-1.23(m,4H).

Embodiment 66:

Synthesis of final product 66:

5b (0.25 g, 0.69 mmol) was dissolved in N,N-dimethylformamide (20 mL), and trans-4-dimethylaminocroton hydrochloride (0.13 g, 0.76 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.32 g, 0.83 mmol), N,N-diisopropylethylamine (0.27 g, 2.07 mmol) were added. The reaction was carried out at room temperature for 8 hours. TLC monitoring showed that there was no residual raw material. Water (30 mL) was added to the reaction solution, and ethyl acetate was extracted (20 mL×5). The organic phases were combined and dried over anhydrous sodium sulfate. The organic phase was directly separated by column chromatography (dichloromethane: methanol = 10:1) to obtain the final product 66 (0.18 g, yield 55%). MSm/z (ESI): 472.2 [M+H] ⁺ .

¹ H NMR (600 MHz, DMSO-d ₆ )δ10.61(s,1H),8.33(s,1H),8.08(d,J＝6.0Hz,1H),8.01(d,J＝6.0Hz,1H), 7.35-7.33(m,1H),7.11-7.09(m,1H),6.93-6.90(m,1H),6.56-6.51(m,1H), 6.04(d,J＝15.0Hz,1H),3.78(s,3H),3.66(m,1H),3.14(s,2H),2.64-2.60(m ,1H),2.25(s,6H),1.91-1.90(m,1H),1.84-1.78(m,3H),1.35-1.13(m,4H).

Embodiment 67:

Synthesis of final product 67:

26c (99 mg, 0.26 mmol) was dissolved in N, N-dimethylformamide (5 mL), followed by the addition of 1-cyano-1-cyclopropanecarboxylic acid (44 mg, 0.40 mmol), 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (152 mg, 0.40 mmol) and N, N-diisopropylethylamine (131 μL, 0.79 mmol). The whole system was stirred at room temperature overnight. TLC showed that there was no residual raw material. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The final product 67 (50 mg, yield 41%) was obtained by column chromatography separation and purification (dichloromethane: methanol = 50: 1-20: 1). MS m/z(ESI):473.2[M+H] ⁺ .

¹ H NMR (600MHz, CDCl ₃ )δ8.32-8.31(m,1H),8.13(s,1H),7.18-7.15(m,1H),6.84-6.81(m,1H),6.38-6.37(m,1H),3.90-3.85( m,1H),3.79(s ,3H),2.49-2.47(m,1H),2.27-2.25(m,1H),2.01-1.94(m,3H),1.70-1.62(m,2H),1.60-48(m,5H),1.32 -1.23(m,1H).

Embodiment 68:

Synthesis of final product 68:

26c (99 mg, 0.26 mmol) was dissolved in N, N-dimethylformamide (5 mL), followed by the addition of glycolic acid (20 mg, 0.26 mmol), 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (152 mg, 0.40 mmol) and N, N-diisopropylethylamine (131 μL, 0.79 mmol). The whole system was stirred at room temperature overnight. TLC showed that there was no residual raw material. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The final product 68 (50 mg, yield 44%) was obtained by column chromatography separation and purification (dichloromethane: methanol = 50: 1-20: 1). MS m/z (ESI): 438.2 [M + H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.60(s,1H),8.35(s,1H),8.08(d,J＝6.0Hz,1H),7.58-7.56(m,1H),7.53-7.51(m,1H),7.37-7.34 (m,1H),5 .38(t,J＝6.0Hz,1H),3.82(s,5H),3.74-3.63(m,1H),2.69-2.61(m,1H),1.85-1.70(m,4H),1.33-1.22 (m,4H).

Embodiment 69:

Synthesis of intermediate 69a:

5b (0.07 g, 0.19 mmol) was dissolved in anhydrous ethanol (20 mL), triethylamine (0.02 g, 0.19 mmol) and carbon disulfide (0.02 g, 0.26 mmol) were added, and the mixture was reacted at room temperature for 30 minutes. Di-tert-butyl dicarbonate (0.04 g, 0.19 mmol) and 4-dimethylaminopyridine (2.3 mg, 0.019 mmol) were added to the reaction solution, and the mixture was reacted at room temperature for 4 hours. TLC monitoring showed that no raw material remained. The ethanol was evaporated under reduced pressure, and the residue was directly separated by column chromatography (dichloromethane: methanol = 50: 1) to obtain 69a (0.03 g, yield 39%).

Synthesis of final product 69:

69a (0.03 g, 0.07 mmol) was dissolved in anhydrous ethanol (10 mL), and 2.0 M ethanolic ammonia solution (0.004 g, 0.28 mmol) was added, heated to 90 ° C, and the tube was sealed for reaction for 4 hours. TLC monitoring showed that there was no residual raw material. The solvent was evaporated under reduced pressure, and the residue was dissolved in anhydrous ethanol (10 mL). 40% dichloroacetaldehyde aqueous solution (0.005 g, 0.07 mmol) was added, and the mixture was heated to 90 ° C for reaction for 8 hours. TLC monitoring showed that there was no residual raw material. The anhydrous ethanol was evaporated under reduced pressure, and the residue was directly separated by column chromatography (dichloromethane: methanol = 25: 1) to obtain the final product 69 (0.02 g, yield 64%). MS m/z (ESI): 445.1 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ )δ10.67(s,1H),8.33(s,1H),8.33-8.08(m,1H),7.50(d,J＝7.8Hz,1H),7.35-7 .33(m,1H),7.11(d,J＝2.4Hz,1H),7.09(d,J＝2.4Hz,1H),6.99-6.90(m,1H),6.5 8(d,J＝4.2Hz,1H),3.79(s,3H),3.52-3.50(m,1H),2.63(m,1H),2.14-2.12(m,1 H),2.02-2.00(m,1H),1.82-1.78(m,2H),1.36-1.30(m,3H),1.14-1.12(m,1H).

Embodiment 70:

Synthesis of intermediate 70a:

2a (160 mg, 0.68 mmol) was dissolved in tetrahydrofuran (10 mL), followed by the addition of phenyl chloroformate (191 mg, 1.22 mmol) and potassium carbonate (187 mg, 1.35 mmol), and the whole system was stirred overnight. TLC monitoring showed that no raw material remained. The reaction was directly separated and purified by column chromatography (petroleum ether: ethyl acetate = 10: 1-2: 1) to obtain 70a (200 mg, yield 83%).

Synthesis of intermediate 70b:

(R)-1-tert-Butyloxycarbonyl-3-aminopiperidine (1.00 g, 5.00 mmol) was dissolved in dichloromethane (30 mL), followed by the addition of triethylamine (1.52 g, 15.0 mmol) and acetic anhydride (701 μL, 7.50 mmol). The system was stirred at room temperature overnight, and TLC monitoring showed that no raw material remained. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride aqueous solution (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain 70b (1.17 g, yield 97%).

Synthesis of intermediate 70c:

70b (1.17 g, 4.83 mmol) was dissolved in dichloromethane (30 mL), and then 10 mL of trifluoroacetic acid was added under ice-water bath, and the whole system was stirred at room temperature for 3 hours. The solvent was removed under reduced pressure to obtain 70c (1.50 g), which was directly used in the next step.

Synthesis of final product 70:

70a (235 mg, 0.66 mmol) was dissolved in tetrahydrofuran (10 mL), followed by the addition of 70c (339 mg, 1.32 mmol) and triethylamine (458 μL, 3.30 mmol). The system was stirred at room temperature for 10 hours, and TLC monitoring showed that there was no residue of the raw material. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL×3). The organic phases were combined, washed with saturated sodium chloride (50 mL×2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (dichloromethane: methanol = 40:1-20:1) to obtain the final product 70 (80 mg, yield 30%). MS m/z (ESI): 405.2 [M+H] ⁺ .

¹ H NMR (600 MHz, DMSO-d ₆ )δ9.22(s,1H),8.25(s,1H),7.84(d,J＝7.2Hz,1H),7.75(d,J＝5.4Hz,1H),7.33 (t,J＝7.8Hz,1H),7.10(d,J＝11.4Hz,1H),6.92(t,J＝7.8Hz,1H),3.93(d,J＝12.6 Hz,1H),3.83(d,J＝12.6Hz,1H),3.80(s,3H),3.64-3.59(m,1H),2.98-2.93(m,1 H),2.79-2.76(m,1H),1.82-1.79(m,4H),1.71-1.69(m,1H),1.45-1.36(m,2H).

Embodiment 71:

Synthesis of intermediate 71a:

1a (0.40 g, 1.58 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of (1S,3R)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (0.38 g, 1.58 mmol), 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.72 g, 1.90 mmol) and N,N-diisopropylethylamine (0.41 g, 3.16 mmol). The whole system was stirred at room temperature overnight. TLC monitoring showed that no raw material remained. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The mixture was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-20: 1) to obtain 71a (0.45 g, yield 60%).

Synthesis of intermediate 71b:

71a (0.45 g, 0.94 mmol) was dissolved in dichloromethane (30 mL), and then 2 mL of trifluoroacetic acid was added under an ice-water bath. The whole system was stirred at room temperature overnight. TLC detection showed that no raw material was left. Water (100 mL) was added to the reaction solution, and the pH was adjusted to 9-10 with saturated sodium bicarbonate aqueous solution. The solution was extracted with dichloromethane (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. After separation and purification by column chromatography (dichloromethane: methanol = 50: 1-8: 1), 71b (0.31 g, yield 87%) was obtained.

Synthesis of final product 71:

71b (0.31 g, 0.82 mmol) was dissolved in dichloromethane (35 mL), followed by the addition of acetic anhydride (0.25 g, 2.47 mmol) and triethylamine (0.25 g, 2.47 mmol), and the system was stirred at room temperature. TLC monitoring showed that there was no residual raw material. Water (50 mL) was added to the reaction solution, and dichloromethane was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-10: 1) to obtain the final product 71 (0.18 g, yield 52%). MS m/z (ESI): 420.1 [M+H] ⁺ . ¹ H NMR (600MHz, CDCl ₃ )δ ¹ H NMR (600MHz, DMSO-d ₆ )δ10.69(s,1H),8.41(s,1H),8.05(s,1H),7.78(d,J＝7.8Hz,1H),7.25(d,J＝7.2Hz,1H),7.09(d,J＝2.4Hz,1H),6.91-6.88(m,1H) ,3.76(s,3H),3.57-3.54(m,1H),2.62-2.59(m,1H),1.86-1.84(m,1H),1.76-1.74(m,6H),1.31-1.23(m,3H),1.07-1.05(m,1H).

Embodiment 72:

Synthesis of intermediate 72a:

6-Bromo-1-methyl-1H-indazole (211 mg, 1.00 mmol), bis-pinacol diborane (508 mg, 2.00 mmol), potassium acetate (294 mg, 3.00 mmol) and [1,1'-bis(diphenylphosphino)ferrocene] palladium dichloride (73 mg, 0.10 mmol) were dissolved in 1,4-dioxane (20 mL). The reaction system was protected with nitrogen and reacted at 100 °C for 4 hours. TLC detected that there was no residual raw material and the heating was stopped. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (100 mL × 3). The organic phases were combined, washed with saturated sodium chloride (100 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. 72a (240 mg, yield 93%) was obtained by column chromatography separation and purification (petroleum ether-petroleum ether: ethyl acetate = 4:1).

Synthesis of intermediate 72b:

72a (258 mg, 1.00 mmol) was dissolved in diethylene glycol dimethyl ether (40 mL), and 5-fluoro-4-iodopyridin-2-amine (286 mg, 1.20 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (73 mg, 0.10 mmol), potassium carbonate (414 mg, 3.00 mmol) and 10 mL of water were added at room temperature. The reaction system was stirred at 80°C for 4 hours. TLC detected that there was no residual raw material and the reaction was stopped. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (100 mL×3). The organic phases were combined, washed with saturated sodium chloride (100 mL×2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. 72b (175 mg, yield 72%) was obtained by column chromatography separation and purification (petroleum ether-petroleum ether: ethyl acetate = 1:1).

Synthesis of intermediate 72c:

72b (175 mg, 0.72 mmol) and (1S, 3R)-3-[(tert-butoxycarbonyl)amino]cyclohexanecarboxylic acid (350 mg, 1.44 mmol) were dissolved in N,N-dimethylformamide (20 mL), and 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (548 mg, 1.44 mmol) and diisopropylethylamine (0.36 mL, 2.16 mmol) were added in sequence. The reaction system was reacted at room temperature for 4 hours. TLC detected that there was no residual raw material. Water (50 mL) was added to the reaction solution, and ethyl acetate was extracted (100 mL×3). The organic phases were combined, washed with saturated sodium chloride (100 mL×2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The product was separated and purified by column chromatography (petroleum ether:ethyl acetate=4:1-2:1) to obtain 72c (200 mg, yield 56%).

Synthesis of final product 72:

72c (200 mg, 0.40 mmol) was dissolved in dichloromethane (50 mL), and then trifluoroacetic acid (5.00 mL) was added and stirred at room temperature for 4 hours. TLC detected that there was no residual raw material. Saturated sodium carbonate aqueous solution was added to the system to adjust the pH value of the reaction system to weak alkalinity. After separation, dichloromethane extraction (100 mL × 3) was performed, and the organic phase was combined and concentrated to a volume of about 50 mL. Triethylamine (2.00 mL) and acetic anhydride (2.00 mL) were added and reacted at room temperature for 30 minutes, and then sodium carbonate aqueous solution was added to wash the organic phase. The liquid was separated, and then extracted with dichloromethane (20 mL × 3), the organic phase was combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The final product 72 (135 mg, yield 83%) was obtained by column chromatography (ethyl acetate). MS m/z (ESI): 410.2 [M + H] ⁺ .

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.64(s,1H),8.44(s,1H),8.36(s,1H),8.14(s,1H),7.96(s,1H),7.90( s,1H),7.79(d,J＝7.8Hz,1H),7.33(d,J＝7.8Hz,1H),4.12(s,3H),3.59-3.58(m,1H),2.99-2.95(m ,1H),1.91(s,3H),1.82-1.78(m,4H),1.14-1.12(m,4H).

Embodiment 73:

Synthesis of final product 73:

23c (101 mg, 0.25 mmol) was dissolved in 1,4-dioxane (10 mL) and water (5 mL), followed by the addition of 1-isopropyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (106 mg, 0.37 mmol), [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (15 mg, 0.02 mmol) and potassium carbonate (68 mg, 0.49 mmol), and the atmosphere was replaced with nitrogen three times to place the entire system under a nitrogen atmosphere. The system was stirred under reflux at 100°C for 4 hours, and no raw material remained after TLC monitoring. The reaction was directly separated and purified by column chromatography (dichloromethane: methanol = 50: 1-10: 1) to obtain the final product 73 (60 mg, yield 55%). MS m/z(ESI):438.2[M+H] ⁺

¹ H NMR (600MHz, DMSO-d6) δ10.70 (s, 1H), 8.51 (s, 1H), 8.41-8.38 (m, 2H), 7.83 (d, J = 3.6Hz, 1H), 7.79 (d ,J＝7.8Hz,1H),7.25-7.24(m,1H),6.50-6.49(m,1H),5.17-5.15(m,1H),3.58-3.56(m,1H),2.64-2.60(m ,1H),1.90-1.88(m,1H),1.77-1.76(m,6H),1.51(s,3H),1.49(s,3H),1.40-1.25(m,3H),1.15-1.05(m ,1H).

Embodiment 74:

Synthesis of intermediate 74a:

2,3-Dichloropyridine-4-boronic acid (1.05 g, 5.50 mmol) was dissolved in dioxane (30 mL) and water (5 mL), followed by the addition of 5-fluoro-4-iodo-pyridine-2-amine (1.00 g, 4.20 mmol), 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium dichloromethane complex (0.34 g, 0.42 mmol), potassium carbonate (1.74 g, 12.6 mmol), and the mixture was heated to 100 °C under nitrogen protection for 8 hours. TLC monitoring showed that there was no residual raw material. Heating was stopped and the mixture was cooled to room temperature. The reaction solution was directly separated by column chromatography (n-hexane: ethyl acetate = 1:1) to obtain intermediate 74a (0.40 g, yield 37%).

Synthesis of intermediate 74b:

74a (0.13 g, 0.50 mmol) was dissolved in acetonitrile (10 mL), followed by the addition of (1S, 3R)-3-[(tert-butyloxycarbonyl)amino]cyclohexanecarboxylic acid (0.16 g, 0.65 mmol), N,N,N',N'-tetramethylchloroformamidine hexafluorophosphate (0.17 g, 0.60 mmol) and N-methylimidazole (0.14 g, 1.75 mmol), and the reaction was carried out at room temperature for 12 hours. TLC monitoring showed that no raw material remained. The reaction solution was directly separated and purified by column chromatography (n-hexane: ethyl acetate = 2:1) to obtain 74b (0.12 g, yield 50%).

Synthesis of intermediate 74c:

74b (0.12 g, 0.25 mmol) was dissolved in dichloromethane (20 ml), 4 mL of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 4 hours. TLC monitoring showed that there was no residual raw material. The reaction solution was washed with water (30 mL × 3), the aqueous phase was combined, the pH of the aqueous phase was adjusted to 8-9 with sodium carbonate, and the dichloromethane was extracted (30 mL × 3), the organic phases were combined, washed with saturated sodium chloride (50 mL × 2), and dried over anhydrous sodium sulfate. The solvent was removed by concentration under reduced pressure to obtain 74c (0.08 g, yield 84%).

Synthesis of final product 74:

74c (0.08 g, 0.21 mmol) was dissolved in dichloromethane (20 mL), followed by the addition of acetic anhydride (0.04 g, 0.42 mmol) and triethylamine (0.04 g, 0.42 mmol). The mixture was reacted at room temperature for 2 hours. TLC monitoring revealed that no residue was left. The reaction solution was directly separated by column chromatography (dichloromethane: methanol = 50: 1) to obtain the final product 74 (0.02 g, yield 22%). MS m/z (ESI): 426.2 [M+H] ⁺ .

¹ H NMR (600MHz, DMSO-d6) δ10.81 (s, 1H), 8.55-8.54 (m, 2H), 8.18 (d, J = 5.4Hz, 1H), 7.80 (d, J = 7.8Hz, 1H ),7.6(d,J＝5.4Hz,1H),3.60-3.54(m,1H),2.64-2.60(m,1H),1.90-1.88(m,1H),1.77-1.76(m,6H), 1.40-1.20(m,3H),1.19-1.05(m,1H).

Embodiment 76:

Synthesis of intermediate 76a:

5b (500 mg, 1.385 mmol) was dissolved in N, N-dimethylformamide (15 mL), followed by the addition of 1-[(tert-butoxy)carbonyl]-3-cyanoazetidine-3-carboxylic acid (375 mg, 1.66 mmol), 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (787 mg, 2.07 mmol) and N, N-diisopropylethylamine (687 uL, 4.15 mmol), the whole system was stirred at room temperature, reacted for 10 hours, and TLC monitoring showed that there was no residual raw material. Water (50 mL) was added to the reaction solution, stirred for 30 minutes, filtered, and dried over anhydrous sodium sulfate to obtain 76a (497 mg, yield 63%).

Synthesis of final product 76:

76a (497 mg, 0.87 mmol) was dissolved in dichloromethane (20 mL), followed by the addition of trifluoroacetic acid (5 mL), and the whole system was stirred at room temperature for 2 hours. TLC showed that no raw material remained. Water (100 mL) was added to the reaction solution, and the pH was adjusted to 8 by sodium bicarbonate. The mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The mixture was separated by column chromatography (dichloromethane: methanol = 40: 1) to obtain the final product 76 (396 mg, yield 97%). MS m/z (ESI): 470.2 [M+H] ⁺ . ¹ H NMR (600MHz, CD ₃ OD) δ8.38 (s, 1H), 7.75 (d, J = 5.4Hz, 1H), 7.50-7.42 (m, 1H), 7.04 (d, J = 10.8Hz, 1H), 6.96-6.86 (m, 1H), 4.60-4.32 (m, 4H), 3.93-3. 76(m,4H),2.70(m,1H),2.23(m,1H),2.00(m,3H),1.71-1.46(m,3H),1.43-1.27(m,1H).

Embodiment 77:

Synthesis of final product 77:

76 (296 mg, 0.63 mmol) was dissolved in methanol (10 mL), followed by the addition of 1 mL of formaldehyde aqueous solution, sodium triacetoxyborohydride (249 mg, 1.27 mmol), and 1 drop of acetic acid. The whole system was stirred at room temperature and reacted for 10 hours. TLC monitoring showed that there was no residual raw material. Water (50 mL) was added to the reaction solution, and the pH was adjusted to 8 with sodium bicarbonate. The mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the final product 77 (186 mg, yield 61%). MS m/z (ESI): 484.2 [M + H] ⁺

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.56 (s, 1H), 8.32 (s, 1H), 8.05 (d, J = 4.8Hz, 1H), 8.10 (d, J = 7.8Hz, 1H) ,7.34-7.31(m,1H),7.09(s,1H),6.91-6.88(m,1H),3.79(s,3H),3.68-3.56(m,1H),2.98-2.73(m,4H) ,2.59-2.55(m,1H),2.42(s,3H),1.81-1.67(m,4H),1.30-1.09(m,4H).

Embodiment 78:

Synthesis of intermediate 78a:

5b (500 mg, 1.385 mmol) was dissolved in N, N-dimethylformamide (15 mL), followed by the addition of 1-[(tert-butoxy)carbonyl]-3-cyanoazetidine-3-carboxylic acid (375 mg, 1.66 mmol), 2-(7-azobenzotriazole)-N, N, N', N'-tetramethyluronium hexafluorophosphate (787 mg, 2.07 mmol) and N, N-diisopropylethylamine (687 uL, 4.15 mmol), the whole system was stirred at room temperature, reacted for 10 hours, and TLC monitoring showed that there was no residual raw material. Water (50 mL) was added to the reaction solution, stirred for 30 minutes, filtered, and dried over anhydrous sodium sulfate to obtain 78a (504 mg, yield 61%).

Synthesis of final product 78:

78a (504 mg, 0.84 mmol) was dissolved in dichloromethane (20 mL), followed by the addition of trifluoroacetic acid (5 mL), and the whole system was stirred at room temperature for 2 hours. TLC showed that no raw material remained. Water (100 mL) was added to the reaction solution, and the pH was adjusted to 8 by sodium bicarbonate. The mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The mixture was separated by column chromatography (dichloromethane: methanol = 40: 1) to obtain the final product 78 (384 mg, yield 92%). MS m/z (ESI): 498.7 [M+H] ⁺ . ^1. 30-3.28(m,2H),2.99-2.97(m,2H),2.62-2.52(m,1H),2.30-2.22(m,4H),1.79-1.77(m,4H),1.46-1.43(m,1H),1.29-1.26(m,3H).

Embodiment 79:

Synthesis of final product 79:

5b (0.16 g, 0.43 mmol) was dissolved in N,N-dimethylformamide (30 mL), followed by the addition of N,N-diethylglycine hydrochloride (109 mg, 0.65 mmol), 2-(7-benzotriazole oxide)-N,N,N',N'-tetramethyluronium hexafluorophosphate (178 mg, 0.47 mmol) and N,N-diisopropylethylamine (111 mg, 0.86 mmol), and the system was stirred at room temperature overnight. TLC monitoring showed that there was no residual raw material. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The crude product was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-20: 1) to obtain the final product 40 (116 mg, yield 57%). MS m/z(ESI):475.3[M+H] ⁺

¹ H NMR (600MHz, CDCl ₃ ) δ8.23 (d, J = 7.8Hz, 1H).8.13 (s, 1H), 8.07 (s, 1H), 7.40 (m, 1H), 7.277.22 (m, 1H),6.87-6.60(m,2H),3.94-3.84(m,1H),3.81(s,3H),3.01(s,2H),2.54(m,4H),2.45(m,1H),2.22 (m,1H),1.96(m,3H),1.56-1.36(m,3H),1.26-1.12(m,1H),1.01(m,6H).

Embodiment 80:

Synthesis of intermediate 80a:

5b (0.30 g, 0.83 mmol) was dissolved in dichloromethane (10 mL), and then triethylamine (420 mg, 4.15 mmol) and chloroacetyl chloride (186 mg, 1.66 mmol) were added at 0°C. The system was stirred overnight at room temperature. TLC monitoring showed that no raw material remained. Water (20 mL) was added to the reaction solution, and ethyl acetate was extracted (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride (10 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The crude product was separated and purified by column chromatography (n-hexane: ethyl acetate = 1:0-0:1) to obtain 80a (200 mg, yield 55%).

Synthesis of final product 80:

80a (200 mg, 0.46 mmol) was dissolved in N,N-dimethylformamide (10 mL), and potassium thioacetate (78.25 mg, 0.69 mmol) was added. The reaction was allowed to react at room temperature for 2 hours. TLC showed that no raw material remained. The reaction solution was added to a 1M HCl aqueous solution (10 mL), and water (10 mL) and dichloromethane (15 mL) were added. The mixture was extracted with dichloromethane (10 mL × 3), the organic phases were combined, washed with saturated sodium chloride (10 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The crude product was separated and purified by column chromatography (n-hexane: ethyl acetate = 1:0-0:1) to obtain the final product 80 (200 mg, yield 91%). MS m/z (ESI): 478.2 [M + H] ⁺

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.58 (s, 1H), 8.33 (s, 1H), 8.07 (d, J = 5.4Hz, 1H), 7.59 (d, J = 8.4Hz, 1H) ,7.35-7.34(m,1H),7.33-7.11(m,1H),7.09-6.90(m,1H),4.39(s,2H),3.78(s,3H),3.65-3.63(m,1H) ,2.66-2.60(m,1H),2.58(s,3H),1.85-1.69(m,4H),1.24-1.14(m,4H).

Embodiment 81:

Synthesis of final product 81:

80 (220 mg, 0.46 mmol) was dissolved in methanol (5 mL), and potassium carbonate (317 mg, 2.30 mmol) was added to react at room temperature for 20 minutes, and then the temperature was raised to 55 ° C for 40 minutes. TLC monitoring showed that there was no residual raw material. Water (20 mL) was added to the reaction solution, and ethyl acetate was extracted (10 mL × 3). The organic phases were combined, washed with saturated sodium chloride (10 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The crude product was separated and purified by column chromatography (n-hexane: ethyl acetate = 1:0-0:1) to obtain the final product 81 (41 mg, yield 20%). MS m/z (ESI): 436.2 [M + H] ⁺

¹ H NMR (600MHz, DMSO-d ₆ ) δ10.55 (s, 1H), 8.31 (s, 1H), 8.07 (d, J = 5.4Hz, 1H), 7.56 (d, J = 9.0Hz, 1H) ,7.35-7.32(m,1H),7.09-7.07(m,1H),6.91-6.88(m,1H),3.89-3.84(m,4H),3.66-3.63(m,1H),3.24-3.22( m,2H),2.61-2.57(m,1H),1.83-1.68(m,4H),1.34-1.26(m,4H).

Embodiment 82:

Synthesis of final product 82:

78 (300 mg, 0.60 mmol) was dissolved in methanol (10 mL), followed by the addition of 1 mL of aqueous formaldehyde solution, sodium triacetoxyborohydride (235 mg, 1.20 mmol), and 1 drop of acetic acid. The whole system was stirred at room temperature and reacted for 10 hours. TLC monitoring showed that there was no residual raw material. Water (50 mL) was added to the reaction solution, and the pH was adjusted to 8 with sodium bicarbonate. The mixture was extracted with ethyl acetate (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure to obtain the final product 82 (205 mg, yield 67%). MS m/z (ESI): 512.2 [M + H] ⁺

¹ H NMR (600 MHz, DMSO-d ₆ )δ10.28(s,1H),8.28(s,1H),8.22-8.09(m,1H),8.05(d,J＝8.4Hz,1H),7.32-7.30(m,1H),7.09-7.02(m,1H),6.91-6.87(m,1H),3.80(s,3H),3.75 -3.66(m,1H),3.57-3.52(m,1H),3.16-2.96(m,2H),2.81-2.59(m,4H),2.50-2.48(m,2H),2.43-2.31(m,3H),2.00-1.74(m,4H),1.52-1.48(m,1H ),1.41-1.22(m,3H).

Embodiment 83:

Synthesis of final product 49:

5b (0.20 g, 0.55 mmol) was dissolved in N, N-dimethylformamide (30 mL), followed by the addition of 2-hydroxyisobutyric acid (68 mg, 0.65 mmol), 2-(7-benzotriazole oxide)-N, N, N', N'-tetramethyluronium hexafluorophosphate (178 mg, 0.47 mmol) and N, N-diisopropylethylamine (111 mg, 0.86 mmol). The system was stirred at room temperature overnight, and TLC monitoring showed that there was no residual raw material. Water (100 mL) was added to the reaction solution, and ethyl acetate was extracted (50 mL × 3). The organic phases were combined, washed with saturated sodium chloride (50 mL × 2), dried over anhydrous sodium sulfate, and then the solvent was removed under reduced pressure. The crude product was separated and purified by column chromatography (dichloromethane: methanol = 50: 1-20: 1) to obtain the final product 83 (81 mg, yield 33%). MS m/z (ESI): 448.2 [M + H] ⁺

¹ H NMR (600MHz, CDCl ₃ ) δ8.31 (s, 1H), 8.24 (d, J = 8.4Hz, 1H), 8.12 (s, 1H), 7.26-7.24 (m, 1H), 6.76-6.73 ( m,2H),6.63(d,J＝12.6Hz,1H),3.90-3.82(m,1H),3.81(s,3H),2.65(s,1H),2.56-2.42(m,1H),2.26 -2.23(m,1H),2.00-1.89(m,3H),1.61-1.49(m,2H),1.45(d,J＝4.8Hz,6H),1.30-1.18(m,1H).

Embodiment 75,84-90:

According to the method of Examples 1-72, corresponding raw materials were selected to synthesize compounds 75, 84-90, whose structures are as follows:

Test Example 1: Test on the inhibitory effect of the compounds of the present application on CDK9, CDK1, CDK2, CDK4, CDK5, CDK6 and CDK7

1. Purpose of the experiment

The inhibitory effects of the compounds on CDK1/2/4/5/6/7/9 kinases were tested and effective IC ₅₀ values were calculated.

2.CDKs detection family

CDK1/CDK2/CDK4/CDK5/CDK6/CDK7/CDK9

Table 1: Information about kinases, substrates and ATP in in vitro assays

3. Testing process

3.1 Compound dilution

The compounds were diluted with DMSO to 11 concentrations, 3-fold dilutions, and the highest concentration of the tested compounds was 10 μM.

3.2 Enzyme reaction

The compound (50 nL) dissolved in DMSO was transferred to the enzyme reaction plate using the sonication technique (Echo). 5 μL of the LCDK enzyme dilution was added to the enzyme reaction plate, centrifuged and incubated at room temperature for 10 minutes. 5 μL of the substrate premix was added to the plate. The final concentrations of substrate and ATP in each well are shown in Table 1. After centrifugation, the reaction was allowed to proceed at 30°C for 120 minutes.

3.3 Termination reaction and signal detection

10 μL of stop solution was added to each well, and the cells were incubated at room temperature for 120 minutes after centrifugation, and then incubated overnight at 4°C. The signal values were read using the HTRF program on the Envision instrument, and data analysis was performed. The IC ₅₀ (inhibitory concentration at 50% of the maximum effect) value was expressed in nM. The results are shown in Table 2.

Table 2 Inhibitory effects of the compounds of the present application on CDK1, 2, 4, 5, 6, 7 and 9 (IC ₅₀ , nM)

The above tests indicate that the compounds of the present application have a selective inhibitory effect on CDK9.

Experimental Example 2: In vitro inhibitory effect of CDK9 inhibitor on proliferation of human liver cancer cells

1. Purpose of the experiment

The inhibitory effects of the synthesized compounds on the proliferation of human hepatoma cells in vitro were investigated.

2. Test Principle

MTT, also known as Thiazole Blue, is a tetrazolium salt of a dye that can accept hydrogen atoms. The amber dehydrogenase in the mitochondria of living cells can reduce exogenous MTT to insoluble blue-purple crystals and deposit them in cells, while dead cells do not have this function. Dimethyl sulfoxide can dissolve the blue-purple complex in cells, and its light absorption value can be measured at a wavelength of 490-550nm using an enzyme-linked immunosorbent assay, which can indirectly reflect the number of cells. Within a certain range of cell numbers, the amount of MTT crystals formed is proportional to the number of cells. The drug to be tested is diluted to different concentrations in turn and added to a 96-well plate. After the drug has acted for a certain period of time, its OD value is measured. The size of the OD value can reflect the number of living cells, and its IC ₅₀ value is calculated using SPSS19.0.

3. Test equipment

Model 371 CO ₂ Incubator: Thermo

IX70-142 inverted fluorescence microscope: Olympus

HFsafe-1500 Biological Safety Cabinet: Shanghai Lishen Scientific Instrument Co., Ltd.

Varloskan flash microplate reader: Thermo

Precision electronic balance: Mettler AL204

TD6 centrifuge: Changsha Xiangrui Centrifuge Co., Ltd.

4. Test materials:

4.1 Cells and culture medium

Note: The cell culture environment is 37°C and 5% CO ₂ .

4.2 Test materials

name Specification Manufacturer Fetal bovine serum 500mL/bottle Gibco PBS 500mL/bottle Gibco DMSO 500mL/bottle Kermiou Chemical Reagent Co., Ltd. MTT 5g/bottle Amresco 0.25%Trypsin-EDTA 500mL/bottle Gibco MEM NEAA 100mL/bottle Gibco Sodium Pyruvate 100mL/bottle Gibco Puromycin 1mL Gibco

4.3 Reagent preparation

5 mg/mL MTT working solution: Weigh 0.5 g of MTT and dissolve it in 100 mL of PBS, filter it through a 0.22 μm microporous filter to sterilize it, and place it in a 4°C refrigerator (for use within two weeks) or -20°C for long-term storage.

5. Test methods

Cells in the logarithmic growth phase were inoculated in a certain number in a 96-well plate (100 μL/well). After 24 hours of attachment, 100 μL of culture medium containing different concentration gradients of the test compound was added to each well. Three replicate wells were set for each drug concentration, and corresponding blank wells (only culture medium) and normal wells (drug concentration was 0) were set. After 72 hours of drug action, MTT working solution (5 mg/mL) was added, 20 μL per well; 37°C for 4 hours, the plate was shaken to remove the supernatant, and 150 μL of DMSO (analytical grade) was added; the microporous oscillator was shaken to mix, the plate was wiped clean, and the optical density value (OD) was detected at 550nm by an enzyme marker.

The inhibition rate of cell growth was calculated using the following formula: Inhibition rate (%) = (OD value of normal well - OD value of drug-administered well) / (OD value of normal well - OD value of blank well) × 100%

According to the inhibition rate of each concentration, the half inhibition concentration IC ₅₀ of the drug was calculated using SPSS19.0. The results of the cell proliferation inhibition of the compounds are shown in Table 3-1 and Table 3-2:

Table 3-1 Cell proliferation inhibition test data of the compounds of the present application (IC ₅₀ , nM)

Cell Type Compound 45 Compound 68 BAY1251152 Hep3B-Luc 71 55 310 SMMC7721-Luc 119 145 182 PLC/PRF/5 292 203 808

Table 3-2 Cell proliferation inhibition test data of the compounds of the present application (IC ₅₀ , nM)

Cell Type Compound 45 Lenvatinib HepG2-Luc 29.2 1881 Hep3B-Luc 450 9826 HuH7-Luc 122 1858 SMMC7721-Luc 28.2 6349 BEL7402-Luc 85.7 8595 SK-Hep-1 73.4 >100μM Hep3B 856 14733 PLC/PRF/5 1124 1172

Experimental conclusion: According to the data in Tables 3-1 and 3-2, the compounds of the present application have a strong inhibitory effect on a variety of liver cancer cells, and the in vitro cell inhibition activity IC ₅₀ is basically below 1000nM, and the optimal value can reach tens of nM.

Experimental Example 3: In vitro inhibitory effect of compounds on prostate cancer and breast cancer cell proliferation

1. Test equipment

Model 371 CO ₂ Incubator: Thermo

IX70-142 inverted fluorescence microscope: Olympus

HFsafe-1500 Biological Safety Cabinet: Shanghai Lishen Scientific Instrument Co., Ltd.

Varloskan flash microplate reader: Thermo

Precision electronic balance: Mettler MS105

TD6 centrifuge: Changsha Xiangrui Centrifuge Co., Ltd.

2. Test materials:

2.1 Cells and culture medium

Note: The cell culture environment is 37°C and 5% CO ₂ .

2.2 Test materials

name Specification Manufacturer Fetal bovine serum 500mL/bottle Lanzhou Minhai Company PBS 500mL Gibco DMSO 500mL/bottle Kermiou Chemical Reagent Co., Ltd. MTT 5g/bottle Amresco

3. Test methods:

Cells in logarithmic growth phase were inoculated in a certain number in 96-well plates (100 μL/well). After adherent cells adhered for 24 hours, 100 μL of culture medium containing different concentrations of the test compound was added to each well. On the day of suspension cell inoculation, 100 μL of culture medium containing different concentrations of the test compound was added. Three replicate wells were set for each drug concentration, and corresponding blank wells (only culture medium) and normal wells (drug concentration was 0) were set. After the drug acted for 72 hours, MTT working solution (5 mg/mL) was added, 20 μL per well; acted at 37°C for 4 hours, the supernatant was removed, and 150 μL of DMSO (analytical grade) was added; the microporous oscillator was shaken and mixed, the plate was wiped clean, and the optical density value (OD) was detected at 550nm by an enzyme marker.

The inhibition rate of cell growth was calculated using the following formula: Inhibition rate (%) = (OD value of _{normal well} - OD value of _{drug-administered well} ) / (OD value of _{normal well} - OD value of _{blank well} ) × 100%

According to the inhibition rate of each concentration, the half maximal inhibition concentration IC ₅₀ of the drug was calculated using SPSS19.0.

The results of cell proliferation inhibition by the compounds are shown in Table 4:

Table 4 Cell proliferation inhibition test data of the compounds of the present application (IC ₅₀ , nM)

Cell Name Compound 45 DU145 85.5 MDA-MB-231 64.4

DU145: human prostate cancer cells; MDA-MB-231: human triple-negative breast cancer cells.

Test conclusion: According to the data in Table 4, the compounds of the present application have strong inhibitory effects on human prostate cancer cell lines and breast cancer cell lines, and the in vitro cell inhibitory activities are basically all below 100 nM.

Experimental Example 4: Investigation of the in vivo anti-tumor activity of the drug - In vivo pharmacodynamics of the present compound in a subcutaneous xenograft tumor model of human liver cancer Huh7 cells

Cell culture: high-glucose DMEM medium containing 10% fetal bovine serum (FBS), 37°C, 5% CO ₂ .

BALB/c Nude mice, female, weighing about 18-22 grams, each mouse was inoculated with 0.1 mL (5×10 ⁶ cells) of Huh7 cells in the subcutaneous tissue of the axilla of the forelimb. When the average tumor volume reached 150 cubic millimeters, the drug administration began. The dosage and method of administration are shown in the table below. The tumor volume was measured twice a week, and the volume was measured in cubic millimeters. When the average tumor volume of the solvent group grew to more than 800 cubic millimeters, the drug administration was terminated to compare the difference in average tumor volume between the test compound group and the solvent group. The anti-tumor efficacy of the compound was evaluated by TGI (%). TGI (%) reflects the tumor growth inhibition rate.

The solvent composition of the solvent group and the drug administration group: DMSO: HP-β-CD (0.5 g/mL): water in a ratio of 2%: 20%: 78% (v/v/v)

Calculation of 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)/(average tumor volume of the solvent control group at the end of drug administration-average tumor volume of the solvent control group at the beginning)] × 100%.

The results are shown in Tables 5 and 6.

Table 5 In vivo tumor inhibition test data

Table 6 In vivo tumor inhibition test data

Test conclusion:

The compound of the present application exhibited good in vivo efficacy and tolerability in a subcutaneous xenograft tumor model of human liver cancer Huh7 cells, and had a significant tumor inhibition effect and better tolerability compared with BAY1251152 (two animals died at a dose of 5 mg/kg, with poor tolerability).

Experimental Example 5: Investigation of the in vivo anti-tumor activity of the drug - In vivo pharmacodynamics of the compound of the present application in a subcutaneous xenograft tumor model of human liver cancer SMMC-7721 cells

Cell culture: RPMI-1640 medium containing 10% fetal bovine serum (FBS), 37°C, 5% CO ₂ .

BALB/c Nude mice, female, 6-8 weeks, weighing about 18-22 grams, each mouse was inoculated with 0.1 mL (1×10 ⁸ cells) of SMMC-7721 cells in the subcutaneous tissue of the axilla of the forelimb. When the average tumor volume reached 150 cubic millimeters, the drug administration began. The dosage and method of administration are shown in the table below. The tumor volume was measured twice a week, and the volume was measured in cubic millimeters. When the average tumor volume of the solvent group grew to more than 800 cubic millimeters, the drug administration was terminated to compare the difference in average tumor volume between the test compound group and the solvent group. The anti-tumor efficacy of the compound was evaluated by TGI (%). TGI (%) reflects the tumor growth inhibition rate.

The solvent composition of the solvent group and the drug administration group: DMSO: HP-β-CD (0.5 g/mL): water in a ratio of 2%: 20%: 78% (v/v/v)

Calculation of 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)/(average tumor volume of the solvent control group at the end of drug administration-average tumor volume of the solvent control group at the beginning)] × 100%.

The results are shown in Table 7.

Table 7 In vivo tumor inhibition test data

Test conclusion:

The compound of the present application showed good in vivo efficacy and tolerability in a subcutaneous xenograft tumor model of human liver cancer SMMC-7721 cells, and the tolerance of mice was significantly better than that of the control drug. At the same dose, 4 animals died in the BAY1251152 group, and the animals showed a significant decrease in weight, indicating very poor tolerance.

Experimental Example 6: Investigation of the in vivo anti-tumor activity of the drug - In vivo efficacy of the compound of the present application in a subcutaneous xenograft tumor model of human liver cancer HepG2-Luc cells.

BALB/c Nude mice, weighing approximately 17-20 g.

Resuscitate the in vitro passaged cells to the required cell number, dilute the cells with serum-free medium, count them under a microscope, adjust the number of tumor cells, add matrix gel and mix well, so that the final cell number is about 5×10 ⁷ /mL, and place the cell suspension in an ice water bath; on the day of inoculation, wipe the axillary skin of the mouse with alcohol for disinfection, and draw 0.1mL/mouse of the above tumor cell suspension with a sterile syringe and inoculate it subcutaneously in the axillary area of the mouse, containing about 5×10 ⁶ tumor cells. When the tumor volume grows to about 231mm ³ , select mice with good tumor growth, divide the animals into balanced groups according to the tumor volume, and start to administer the drugs. The dosage and administration method are shown in the table below. The tumor volume is measured 2-3 times a week, and the volume is measured in cubic millimeters. When the average tumor volume of the solvent group grows to more than 800 cubic millimeters, the administration is terminated to compare the difference in average tumor volume between the test compound group and the solvent group. The anti-tumor efficacy of the compound is evaluated by TGI (%). TGI (%) reflects the tumor growth inhibition rate.

Calculation of 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)/(average tumor volume of the solvent control group at the end of drug administration-average tumor volume of the solvent control group at the beginning)] × 100%.

The results are shown in Table 8.

Table 8 In vivo tumor inhibition test data

Group Number of animals Dosage Dosage Dosing days TGI (%) Solvent Group 8 qd,p.o. -- 12 -- Compound 45 8 qd,p.o. 5mg/kg 12 45.6 Compound 45 8 qd,p.o. 7.5 mg/kg 12 76.7 Lenvatinib 8 qd,p.o. 10mg/kg 12 44.1

Solvent: an aqueous solution containing 2% DMSO and 20% 0.5 g/mL HP-β-CD (the ratio of DMSO: HP-β-CD (0.5 g/mL): water is 2%: 20%: 78%, v/v/v).

Lenvatinib preparation: Weigh an appropriate amount of Lenvatinib (FDA-approved for first-line treatment of patients with unresectable hepatocellular carcinoma), add it to a mixture of castor oil and anhydrous ethanol (volume ratio of 1:1), vortex and sonicate until a uniform solution is obtained, then add ultrapure water, and vortex and sonicate until a uniform solution is obtained. (The volume ratio of castor oil: anhydrous ethanol: ultrapure water is 1:1:6).

Dosing volume: 10mL/kg

Test conclusion:

The compounds of the present application showed good in vivo efficacy and tolerability in a subcutaneous xenograft tumor model of human liver cancer HepG2-Luc cells; at a dosage of 5 mg/kg, the tumor inhibition degree of compound 45 was equivalent to that of Lenvatinib (dosage: 10 mg/kg); at a dosage of 7.5 mg/kg, the tumor inhibition degree of compound 45 was significantly better than that of Lenvatinib; during the experiment, the mice tolerated it well.

Experimental Example 7: Investigation of the in vivo anti-tumor activity of the drug - In vivo efficacy of the compound of the present application in a subcutaneous xenograft tumor model of human liver cancer Hep3B cells.

BALB/c Nude mice, weighing approximately 17-20 g.

Resuscitate the in vitro passaged cells to the required cell number, dilute the cells with serum-free medium, count them under a microscope, adjust the number of tumor cells, add matrix gel and mix well, so that the final cell number is about 5×10 ⁷ /mL, and place the cell suspension in an ice water bath; on the day of inoculation, wipe the axillary skin of the mice with alcohol for disinfection, and draw 0.1mL/mouse of the above tumor cell suspension with a sterile syringe and inoculate it subcutaneously in the axillary area of the mice, containing about 5×10 ⁶ tumor cells. When the tumor volume grows to about 230mm ³ , select mice with good tumor growth, divide the animals into balanced groups according to the tumor volume, and start to administer the drugs. The dosage and administration method are shown in the table below. The tumor volume is measured 2-3 times a week, and the volume is measured in cubic millimeters. When the average tumor volume of the solvent group grows to more than 800 cubic millimeters, the administration is terminated to compare the difference in average tumor volume between the test compound group and the solvent group. The anti-tumor efficacy of the compound is evaluated by TGI (%). TGI (%) reflects the tumor growth inhibition rate.

Calculation of 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)/(average tumor volume of the solvent control group at the end of drug administration-average tumor volume of the solvent control group at the beginning)] × 100%.

The results are shown in Table 9.

Table 9 In vivo tumor inhibition test data

Group Number of animals Dosage Dosage Dosing days TGI (%) Solvent Group 8 qd,p.o. -- 12 -- Compound 45 8 qd,p.o. 5mg/kg 12 43.9 Compound 45 8 qd,p.o. 7.5 mg/kg 12 64.6 Lenvatinib 8 qd,p.o. 10mg/kg 12 66.6

Solvent: an aqueous solution containing 2% DMSO and 20% 0.5 g/mL HP-β-CD (the ratio of DMSO: HP-β-CD (0.5 g/mL): water is 2%: 20%: 78%, v/v/v).

Preparation of Lenvatinib: Weigh an appropriate amount of Lenvatinib, add it to a mixture of castor oil and anhydrous ethanol (volume ratio is 1:1), vortex and sonicate until a uniform solution is obtained, then add ultrapure water, vortex and sonicate until a uniform solution is obtained. (The volume ratio of castor oil: anhydrous ethanol: ultrapure water is 1:1:6).

Dosing volume: 10mL/kg

Test conclusion:

The compounds of the present application showed good in vivo efficacy and tolerability in a subcutaneous xenograft tumor model of human liver cancer Hep3B cells; at a dosage of 7.5 mg/kg, the tumor inhibition degree of compound 45 was comparable to that of Lenvatinib (dosage: 10 mg/kg); during the experiment, the mice tolerated it well.

Experimental Example 8: In vitro hERG inhibitory activity investigation

1. Purpose of the test:

The rapidly activated human delayed rectifier outward potassium current (IKr) is mainly mediated by the hERG ion channel and is involved in the repolarization of human cardiomyocytes. Drug blockade of this current is the main cause of clinical QT interval prolongation syndrome, acute arrhythmia and even sudden death. The whole-cell patch clamp technique was used to detect the blocking effect of the compound on the hERG channel in the CHO-K1 cell line stably expressing the hERG channel and the half-maximal inhibitory concentration IC ₅₀ of the compound was determined. As part of the comprehensive cardiac safety assessment, it was preliminarily evaluated in the in vitro screening of the safety of cardiotoxicity.

2. Test methods:

This test includes the following aspects:

hERG currents were recorded using manual patch clamp technique in CHO-K1 cells stably expressing hERG channels;

· Calculate the inhibition rate of each concentration based on hERG tail current;

Each compound was tested at 5 concentrations and the _IC50 value was calculated;

·Three cells were tested at each concentration;

A positive control drug.

The hERG current was recorded by whole-cell patch clamp technique. The cell suspension was added to the cell tank and placed on the upright microscope stage. After the cells adhered to the wall, they were perfused with extracellular fluid at a flow rate of 1-2 mL/min. The glass microelectrode was pulled in two steps by a microelectrode puller, and its water resistance was 2-5 MΩ. After the whole-cell recording was established, the clamping potential was maintained at -80 mV. When voltage stimulation was given, the hERG tail current was elicited by depolarization to +60 mV and then repolarization to -50 mV. All recordings were performed after the current stabilized. Extracellular perfusion administration started with a low concentration, and each concentration was given for 5-10 minutes until the current stabilized before the next concentration was given. The half-maximal inhibitory concentration (IC ₅₀ ) of the test compound was obtained by the best fit of the Logistic equation.

Amitripyline is one of the most widely used drugs for blocking hERG current and was therefore used as a positive control drug in this study.

3. The results are shown in Table 10:

Table 10 IC ₅₀ values of compounds on hERG current recorded in CHO-K1 stable cell line

In the above test, the IC ₅₀ result of the positive control drug Amitriptyline on hERG current inhibition is consistent with the historical results of the test party, and is also consistent with the results reported in the literature, indicating that the results of this test are credible. The above test results show that the tested compound cannot achieve half inhibition of hERG current at the highest test concentration, so IC ₅₀ cannot be measured, indicating that the compound of the present application has no obvious inhibitory effect on the hERG channel within the detection concentration range of this test, which can reflect to a certain extent that the compound of the present application has low or no cardiotoxicity, which has positive significance for drug safety evaluation.

Test Example 9: Investigation of drug toxicity to mice

Experimental animals: ICR mice (5 weeks old), half male and half female

Solvent: DMSO: HP-β-CD (0.5 g/mL): water in a ratio of 2%: 20%: 78% (v/v/v)

Test method: ICR mice were divided into 7 groups according to their weight, with 5 males and 5 females in each group. The drug was administered by gavage once a day for 7 consecutive days. The dosage and results are shown in the table below.

Test results:

Test conclusion: With increasing doses, the control drug BAY1251152 showed dose-dependent toxicity, and the number of animal deaths increased; at the same dose as the control drug, no test animal deaths occurred in the compound 45 of the present application. It can be seen that the tolerability of the compound 45 of the present application in female and male animals is significantly better than that of the control drug BAY1251152.

Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be apparent to one skilled in the art from the teachings of this application that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims (14)

1. Use of a compound as shown in Formula 45 or 68 or a pharmaceutically acceptable salt of the compound in the preparation of a drug for treating solid tumors, Wherein, the solid tumor is liver cancer, breast cancer or prostate cancer. 2. The use according to claim 1, characterized in that the liver cancer, breast cancer or prostate cancer is CDK9-related liver cancer, breast cancer or prostate cancer. 3. The use according to claim 1, characterized in that the liver cancer is hepatocellular carcinoma, and the breast cancer is triple-negative breast cancer. 4. The use according to claim 3, characterized in that the hepatocellular carcinoma is advanced hepatocellular carcinoma. 5. The use according to claim 3, characterized in that the hepatocellular carcinoma is CDK9-related advanced hepatocellular carcinoma, and the breast cancer is CDK9-related triple-negative breast cancer. 6. The use according to any one of claims 1 to 5, characterized in that the compound or the pharmaceutically acceptable salt of the compound is administered in a daily dosage of 0.1 mg to 100 mg. 7. The use according to claim 6, characterized in that the compound or the pharmaceutically acceptable salt of the compound is administered in a daily dosage of 2 mg to 50 mg. 8. The use according to claim 7, characterized in that the compound or a pharmaceutically acceptable salt of the compound is administered in a daily dose of 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 8 mg, 10 mg, 12 mg, 16 mg, 18 mg, 20 mg, 24 mg, 25 mg, 30 mg, 32 mg, 36 mg, 40 mg, 42 mg, 45 mg or 50 mg. 9. The use according to any one of claims 1 to 5, characterized in that a single dose of the drug contains 1 to 50 mg of the compound or a pharmaceutically acceptable salt of the compound. 10. The use according to claim 9, characterized in that a single dose of the drug contains 2-20 mg of the compound or a pharmaceutically acceptable salt of the compound. 11. The use according to any one of claims 1 to 5, characterized in that the compound or the pharmaceutically acceptable salt of the compound is the only active ingredient in the medicament. 12. The use according to any one of claims 1 to 5, characterized in that the compound or a pharmaceutically acceptable salt of the compound is used in combination with one or more targeted drugs or chemotherapeutic drugs to prepare the drug. 13. The use according to any one of claims 1 to 5, characterized in that the drug is prepared into a clinically accepted formulation. 14. The use according to claim 13, characterized in that the preparation is an oral preparation or an injection preparation.