WO2024114574A1

WO2024114574A1 - High-selectivity plk4 inhibitor, preparation method therefor and use thereof

Info

Publication number: WO2024114574A1
Application number: PCT/CN2023/134323
Authority: WO
Inventors: 赵冬梅; 孙印; 程卯生; 薛艳丽; 孙宇; 王林; 王靖凯; 孙鹏昆; 牟书毅
Original assignee: 沈阳药科大学
Priority date: 2022-12-02
Filing date: 2023-11-27
Publication date: 2024-06-06
Also published as: CN118126043A; CN118126043B

Abstract

The present invention belongs to the field of drug synthesis; and relates to a high-selectivity PLK4 inhibitor, a preparation method therefor, and the use thereof as a PLK4 inhibitor. The inhibitor is a pyrazolopyrimidine derivative as shown in formula I, and a geometric isomer, an enantiomer or a pharmaceutically acceptable salt thereof; preferably, the compound has the activity of acting as a protein kinase inhibitor, especially as a PLK4 kinase inhibitor.

Description

A highly selective PLK4 inhibitor and its preparation method and application

Technical Field

The invention belongs to the field of drug synthesis and relates to a highly selective PLK4 inhibitor and a preparation method thereof and an application thereof as a PLK4 inhibitor.

Background technique

Malignant tumors are one of the leading causes of death worldwide. Chemical drugs for malignant tumors are undergoing a transformation from early non-selective drugs to small molecule drugs with specific targets. With the introduction of the concept of precision medicine, targeting protein kinases abnormally expressed in tumor cells should be the main research direction in the future anti-tumor field. In recent years, the treatment method of inhibiting tumor cell proliferation by controlling centrosome amplification has become an emerging solution for tumor chemotherapy.

Polo-like kinase 4 (PLK4) is a key protein for centrosome duplication in cells and plays an important role in centrosome duplication and mitosis. Many studies have shown that overexpression of PLK4 can lead to centrosome amplification and induce cancer. Currently, the design of PLK4 small molecule ATP-competitive inhibitors has become an important means of treating tumors induced by incorrect centrosome duplication, and PLK4 inhibitors can achieve precise treatment of tumors containing TRIM37 gene amplification. This study has been confirmed in breast cancer and glioblastoma.

Polo-like kinase 4 is a member of the Polo-like protein kinase family. It is an evolutionarily highly conserved serine/threonine protein kinase with five subtypes, namely PLK1-5. PLK4 is mainly expressed in active dividing tissues and cells. A series of biological studies have shown that PLK4 is closely related to centrosome replication in the cell cycle, and tumor cells have the characteristics of unlimited proliferation. Therefore, PLK4 plays an irreplaceable role in various biological functions such as tumor cell proliferation, migration, invasion, and apoptosis. A large number of studies have found that PLK4 is abnormally expressed in most human tumors, such as liver cancer, gastric cancer, lung cancer, breast cancer, melanoma, and malignant blood diseases. At present, no PLK4 inhibitor has been marketed, and only two types of parent nuclear structures have been reported so far. One type of representative inhibitor, LCR-263, has nanomolar kinase activity and high selectivity for PLK4, but it has not yet entered preclinical research; the other type of representative inhibitor, CFI-400945, has entered Phase II clinical research, but it lacks selectivity for TRKA and Aurora A/B. Therefore, finding new PLK4 inhibitors that are highly efficient, highly selective and have good in vivo properties has great scientific value and research significance.

Summary of the invention

The purpose of the present invention is to provide a highly selective PLK4 inhibitor and a preparation method thereof and an application thereof as a PLK4 inhibitor.

To achieve the above purpose, the present invention adopts the following technical solution:

A highly selective PLK4 inhibitor, the inhibitor is a pyrazolopyrimidine derivative represented by general formula I, and its geometric isomers, enantiomers or pharmaceutically acceptable salts thereof;

wherein X is selected from C or N;

Y is selected from C or N;

Z is selected from hydrogen, halogen;

R ₁ is selected from hydrogen, C1-C4 alkyl;

_R2 is selected from hydrogen, C1-C4 alkyl;

_R3 is selected from hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl;

_R4 is selected from hydrogen, C1-C6 alkyl, C4-C7 aliphatic ring containing up to 2 heteroatoms, wherein the heteroatom is N, O or S; when the heteroatom is S, the sulfur atom can be further oxidized to sulfoxide or sulfone; when the heteroatom is N, the nitrogen atom can be further substituted by C1-C4 alkyl, C1-C4 alkanoyl or C1-C4 alkanesulfonyl;

Ring A is selected from aryl which is unsubstituted or substituted with 1 to 3 R _a which may be the same or different;

_Ra is selected from halogen, C1-C4 alkyl, C1-C6 alkoxy.

Preferably, the compound represented by the general formula I, and its geometric isomers, enantiomers or pharmaceutically acceptable salts thereof;

wherein X is selected from C or N;

Y is selected from C or N;

Z is selected from hydrogen, halogen;

_R1 is selected from hydrogen;

_R2 is selected from hydrogen, methyl;

_R3 is selected from hydrogen, methyl, ethyl, cyclopropyl;

_R4 is selected from hydrogen, methyl, isopropyl, a C4-C7 aliphatic ring containing up to 2 heteroatoms, wherein the heteroatom is N, O or S; when the heteroatom is S, the sulfur atom may be further oxidized to sulfoxide or sulfone; when the heteroatom is N, the nitrogen atom may be further substituted by a C1-C4 alkyl group, a C1-C4 alkanoyl group or a C1-C4 alkanesulfonyl group;

Ring A is selected from phenyl, pyridyl, pyrimidinyl, which may or may not contain 1-3 R _a substituted groups which may be the same or different;

_Ra is selected from fluorine, chlorine, bromine, methyl, methoxy.

Further preferably, the compound represented by the general formula I, and its geometric isomers, enantiomers or pharmaceutically acceptable salts thereof;

wherein X is selected from C or N;

Y is selected from C or N;

Z is selected from chlorine;

_R1 is selected from hydrogen;

_R2 is selected from hydrogen, methyl;

_R3 is selected from hydrogen, methyl, ethyl, cyclopropyl;

_R4 is selected from hydrogen, methyl, isopropyl, a C4-C7 aliphatic ring containing up to 2 heteroatoms, wherein the heteroatom is N, O or S; when the heteroatom is S, the sulfur atom can be further oxidized to sulfone; when the heteroatom is N, the nitrogen atom can be further substituted by acetyl or methylsulfonyl;

Ring A is selected from phenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 2,5-difluorophenyl, 3,5-difluorophenyl, 3,4,5-trifluorophenyl, 3-methoxyphenyl, and 3-methylphenyl.

Still further preferably, the compound represented by the general formula I, and its geometric isomers, enantiomers or pharmaceutically acceptable salts thereof;

wherein X is selected from C or N;

Y is selected from C or N;

Z is selected from chlorine;

_R1 is selected from hydrogen;

_R2 is selected from hydrogen, methyl;

_R3 is selected from hydrogen, methyl, ethyl, cyclopropyl;

_R4 is selected from hydrogen, methyl, isopropyl,

Furthermore, the compound is:

2-chloro-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)-N-phenylbenzamide;

2-Chloro-N-(2-fluorophenyl)-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-Chloro-N-(2-chlorophenyl)-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-chloro-N-(2-bromophenyl)-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-Chloro-N-(2,5-difluorophenyl)-5-(((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-Chloro-N-(3,5-difluorophenyl)-5-(((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-chloro-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)-N-(3,4,5-trifluorophenyl)benzamide;

2-chloro-N-(3-methoxyphenyl)-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-chloro-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)-N-(m-tolyl)benzamide;

2-Chloro-N-(2,5-difluorophenyl)-5-(((4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-Chloro-N-(2,5-difluorophenyl)-5-((1-methyl-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-Chloro-N-(2,5-difluorophenyl)-5-((1-isopropyl-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

5-((1-(1-acetylpiperidin-4-yl)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)-2-chloro-N-(2,5-difluorophenyl)benzamide;

2-Chloro-N-(2,5-difluorophenyl)-5-(((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-Chloro-N-(2,5-difluorophenyl)-5-((1-(1,1-dioxotetrahydro-2H-thiopyran-4-yl)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-Chloro-N-(2,5-difluorophenyl)-5-(1-((1-isopropyl-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)ethyl)benzamide;

5-chloro-N-(2,5-difluorophenyl)-2-((1-isopropyl-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)isonicotinamide;

2-Chloro-N-(2,5-difluorophenyl)-5-((1-isopropyl-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)nicotinamide;

2-Chloro-N-(2,5-difluorophenyl)-5-(((4-((5-ethyl-1H-pyrazol-3-yl)amino)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-Chloro-5-((4-((5-cyclopropyl-1H-pyrazol-3-yl)amino)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)-N-(2,5-difluorophenyl)benzamide.

In the definition of compounds of general formula I given above, the terms used are generally defined as follows:

Halogen: refers to fluorine, chlorine, bromine or iodine. Alkyl: straight chain or branched chain alkyl, such as methyl, ethyl, propyl, isopropyl, n-butyl or tert-butyl. Cycloalkyl: substituted or unsubstituted cyclic alkyl, such as cyclopropyl, cyclopentyl or cyclohexyl; substituents such as methyl, halogen, etc. Alkoxy: straight chain or branched chain alkyl, the hydrogen atom of the hydroxyl group can be replaced by these straight chain or branched chain alkyl, such as methyloxy, ethyloxy, propyloxy, isopropyloxy, etc. Aliphatic ring containing 1-2 heteroatoms, such as cyclic alkyl of N, O, S, such as tetrahydrofuranyl, piperidine base,

The preparation method of the derivative represented by the above general formula I, the preparation reaction of the derivative represented by the general formula I is as follows:

The starting material 1 is condensed with the corresponding aromatic amine to obtain intermediate 2, and intermediate 2 is then subjected to a free radical reaction with NBS to obtain intermediate 3; intermediate 4 is subjected to a ring-locking reaction with hydrazine with different substituents to obtain intermediate 5, and then is substituted with aminopyrazole with different substituents to obtain intermediate 6. Intermediate 3 and intermediate 6 are subjected to a substitution reaction to obtain target compound 7. Target compounds with similar structures can also be prepared according to the above general method.

Further, compound 1 is used as a starting material, and chlorinated with dichlorothionyl at high temperature, and then reacted with the corresponding aromatic amine to obtain intermediate 2, the high temperature reaction temperature is 60-100°C, preferably 80°C, and the reaction solvent can be dimethyl sulfoxide, dichloromethane, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, preferably N,N-dimethylformamide; intermediate 2 is reacted with a free radical initiator and NBS at high temperature to obtain intermediate 3, the reaction solvent can be dichloromethane, chloroform, tetrachloromethane, benzene, 1,2-dichloromethane, preferably chloroform, and the reaction temperature is 50-90°C, preferably 60°C; intermediate 4 is reacted with hydrazine with different substituents under low temperature alkaline conditions to obtain intermediate 5, and the reaction solvent can be methanol, ethanol, isopropanol, n-propanol, tert-butanol, sec-butanol, n-butanol, dimethyl sulfoxide, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, preferably Ethanol, the reaction temperature is -78 to 0°C, preferably -78°C, the base in the reaction can be triethylamine, N,N-diisopropylethylamine, cesium carbonate, potassium carbonate, sodium carbonate, sodium bicarbonate, etc., preferably triethylamine; intermediate 5 is subjected to substitution reaction under alkaline conditions to obtain intermediate 6, and the reaction solvent can be methanol, ethanol, isopropanol, n-propanol, tert-butanol, sec-butanol, n-butanol, dimethyl sulfoxide, tetrahydrofuran, 1,4-dioxane, N,N-dimethyl Formamide, preferably N,N-dimethylformamide, the base in the reaction can be triethylamine, N,N-diisopropylethylamine, cesium carbonate, potassium carbonate, sodium carbonate, sodium bicarbonate, etc., preferably N,N-diisopropylethylamine, the reaction temperature is 40-80°C, preferably 65°C; intermediate 6 undergoes substitution reaction with intermediate 3 under high temperature alkaline conditions to obtain intermediate 7, the reaction solvent can be methanol, ethanol, isopropanol, n-propanol, tert-butanol, sec-butanol, n-butanol, Dimethyl sulfoxide, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, preferably isopropanol, the reaction temperature is 100-160°C, preferably 135°C, the base in the reaction can be triethylamine, N,N-diisopropylethylamine, cesium carbonate, potassium carbonate, sodium carbonate, sodium bicarbonate, etc., preferably triethylamine;

Application of pyrazolopyrimidine derivatives, the compound represented by general formula I, and its geometric isomers or pharmaceutically acceptable salts, hydrates, solvates or prodrugs in the preparation of drugs for preventing or treating diseases related to the expression or activity of PLK4 kinase.

The compound represented by the general formula I, and its geometric isomers or pharmaceutically acceptable salts, hydrates, solvates or prodrugs thereof are used in the preparation of preventive or anti-tumor drugs.

A pharmaceutical composition comprises a compound represented by general formula I and its geometric isomers or pharmaceutically acceptable salts, hydrates, solvates or prodrugs as active ingredients and a pharmaceutically acceptable excipient.

The composition is used in preparing medicines for preventing or treating diseases related to the expression or activity of PLK4 kinase.

The composition is used in preparing preventive or anti-tumor drugs.

The advantages of the present invention are:

The present invention focuses on tumors caused by centrosome abnormalities, designs compounds with a structure shown in general formula I, and finds that compounds with such a structure exhibit good PLK4 inhibitory activity and also exhibit good kinase selectivity.

The compounds of the structure represented by the general formula I of the present invention are not limited to their specific isomers, and all exhibit good inhibitory activity against PLK4, and are used to treat other diseases related to abnormal PLK4 expression.

Detailed ways:

The examples are intended to illustrate rather than limit the scope of the invention. The H NMR spectra of the compounds were measured using a Bruker ARX-400; all reagents used were analytically pure or chemically pure.

The specific embodiment structure is as follows:

The preparation route of Example 1 is as follows:

The specific synthesis steps are as follows:

Synthesis of 2-chloro-5-methyl-N-benzamide (2)

The raw material 1 (3.00 g, 17.65 mmol) was dissolved in 40 mL of thionyl chloride and refluxed at 80 ° C for 4 h. The excess thionyl chloride was then removed by vortexing, dissolved in DMF, and slowly added dropwise to a DMF solution of aniline (1.65 g, 17.65 mmol) to react at room temperature for 5 h. TLC monitoring (PE: EA = 2: 1) showed that the raw material had reacted completely, 100 mL of water was added, and then extracted three times with ethyl acetate (30 mL each time), the organic layers were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and sodium sulfate was filtered out, the solvent was removed by vortexing, and purified by column chromatography (PE: EA = 4: 1) to obtain a white solid with a yield of 77%. Synthesis of 5-(bromomethyl)-2-chloro-N-phenylbenzamide (3)

Intermediate 2 (2.00 g, 8.16 mmol) was dissolved in 40 mL of chloroform, and AIBN (0.82 mmol) and NBS (1.45 g, 8.16 mmol) were added at 60°C. TLC monitoring (PE:EA=2:1) showed that the raw material reacted completely, the solvent was removed by rotary evaporation, and column chromatography purification (PE:EA=4:1) was performed to obtain a white solid with a yield of 63%.

Synthesis of 4,6-dichloro-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidine (5)

Dissolve (tetrahydro-2H-pyran-4-yl)hydrazine hydrochloride (1.00 g, 6.58 mmol) in 15.0 mL of ethanol, place at -78°C, slowly dropwise add 15.0 mL of ethanol solution of intermediate 2 (1.38 g, 6.58 mmol), then add 4.58 mL of triethylamine, continue to react at -78°C for 30 min, then move to room temperature for 1 h. TLC monitoring (PE:EA=4:1) shows that the raw material reacts completely, the solvent is removed, and column chromatography purification (PE:EA=10:1) is performed to obtain a white solid with a yield of 85%.

Synthesis of 6-chloro-N-(5-methyl-1H-pyrazol-3-yl)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine (6)

The intermediate 5 (0.50 g, 1.84 mmol) was dissolved in 5 mL DMF, followed by 5-methyl-1H-pyrazole-3-amine (0.18 g, 1.84 mmol), N,N-diisopropylethylamine (0.425 mL, 2.40 mmol), potassium iodide (0.37 g, 2.21 mmol), and then reacted at 65 ° C for 1 h. TLC detection (DCM: MeOH = 15: 1) showed that the raw materials reacted completely. After the reaction was cooled to room temperature, 50 mL of water was added to precipitate a white precipitate, which was filtered to obtain a white solid with a yield of 88%.

Preparation of 2-chloro-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)-N-phenylbenzamide (Example 1) Intermediate 6 (0.08 g, 0.24 mmol) was dissolved in 1.5 mL of isopropanol, and then intermediate 3 (0.094 g, 0.29 mmol) and 0.10 mL of triethylamine were added, and then heated to 135° C. for 15 h under a sealed tube. The reaction of the raw materials was completed by TLC monitoring (DCM:MeOH=10:1), and the reaction was cooled to room temperature, the solvent was dried, and a white solid was obtained by column chromatography (DCM:MeOH=40:1) with a yield of 51%. ¹ H NMR (600 MHz, DMSO-d ₆ )δ11.94(s,1H),10.41(s,1H),10.12(s,1H),7.97(s,1H),7.66–7.51(m,4H),7.43(t,J＝10.2Hz,2H),7.27(t,J＝7.8Hz,2H),7.03(t,J＝7.3Hz,1H),4.57(t,J＝11.4Hz,1H),4.45(s,2H),3.80(s,2H),3.36(t,J＝11.3Hz,2H),2.13(s,3H),2.01(d,J＝9.3Hz,2H),1.68(d,J＝9.6Hz,2H). ¹³ C NMR (151 MHz, DMSO-d ₆ )δ165.5,161.0,155.2,153.8,140.7,139.3,137.0,131.9,131.6,131.1,130.2,129.7,129.3,129.2(2C),128.2,124.2,119.9(2C),97.4,96.4,66.7(2C),52.6,44.1,32.2(2C),11.7.

Example 2 2-Chloro-N-(2-fluorophenyl)-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetramethyl Preparation of 2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide

Referring to the method of Preparation Example 1, the aniline raw material in step a was replaced by 2-fluoroaniline in equal proportion to obtain Example 2. ¹ H NMR (600 MHz, CD ₃ OD) δ7.92 (t, J = 7.7 Hz, 1H), 7.83 (s, 1H), 7.70 (s, 1H), 7.51 (d, J = 8.3 Hz, 1H), 7.43 (d, J = 8.3 Hz, 1H), 7.23-7.16 (m, 3H), 6.14 (s, 1H), 4.71 (q, J = 9.1, 6.7 Hz, 1H), 4.64 (s, 2H), 4.03-3.97 (m, 2H), 3.55 (t, J = 11.9 Hz, 2H), 2.27-2.18 (m, 5H), 1.80 (d, J = 12.8 Hz, 2H). ¹³ C NMR (151 MHz, CD ₃ OD) δ 167.1, 160.8, 155.0, 154.9 (d, J = 241.6 Hz), 140.1, 135.8, 135.7, 131.5, 130.2, 129.6, 128.8, 128.0, 126.4, 126.3, 125.2 (d, J = 12.1 Hz), 124.8, 124.0, 123.8, 115.3 (d, J = 19.8 Hz), 101.1, 96.0, 66.8 (2C), 53.0, 43.9, 31.5 (2C), 10.4.

Example 3 Preparation of 2-chloro-N-(2-chlorophenyl)-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide

Referring to the method of preparing Example 1, the aniline raw material in step a was replaced with 2-chloroaniline in equal proportion to obtain Example 3. ¹ H NMR (600MHz, DMSO-d ₆ )δ11.99(s,1H),10.16(s,2H),8.05(s,1H),7.65(d,J＝6.7Hz,2H),7.60–7.48(m,4H),7.38(t,J＝7.4Hz,1H),7.27(t,J＝7.3Hz,1H),4.63(t,J＝11.2Hz,1H),4.56(s,2H),3.99–3.94(m,2H),3.47(t,J＝11.5Hz,2H),2.23(s,3H),2.10(dt,J＝11.6,8.3Hz,2H),1.77(d,J＝10.9Hz,2H). ¹³ C NMR(151MHz,DMSO-d ₆ )δ165.8,161.0,155.3,153.9,148.3,140.8,138.4,136.4,135.9,134.9,132.3,131.1,130.1,129.8,128.8,128.4,128.1,127.9,127.8,97.3,96.4,66.7(2C),52.8,44.1,32.2(2C),11.3.

Example 4 Preparation of 2-chloro-N-(2-bromophenyl)-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide

Referring to the method of Preparation Example 1, the aniline raw material in step a was replaced with 2-bromoaniline in equal proportion to obtain Example 4. ¹ H NMR (600 MHz, CD ₃ OD) δ7.84-7.73 (m, 3H), 7.64 (d, J = 7.9 Hz, 1H), 7.53 (d, J = 7.8 Hz, 1H), 7.47 (d, J = 8.2 Hz, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.18 (t, J = 7.5 Hz, 1H), 6.14 (s, 1H), 4.74-4.66 (m, 3H), 4.03 (d, J = 8.2 Hz, 2H), 3.56 (t, J = 11.8 Hz, 2H), 2.29-2.20 (m, 5H), 1.81 (d, J = 12.3 Hz, 2H). ¹³ C NMR (101 MHz, CD ₃ OD) δ 163.7, 160.5, 160.1, 157.3, 141.4, 140.1, 136.7, 136.4, 135.5, 133.7, 132.6, 131.6, 130.8, 129.7, 128.8, 128.2, 127.7, 127.5, 120.0, 92.7, 89.8, 66.8 (2C), 53.1, 43.9, 31.6 (2C), 11.4.

Example 5 2-Chloro-N-(2,5-difluorophenyl)-5-(((4-((5-methyl-1H-pyrazol-3-yl)amino

Preparation of 1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide

Referring to the method of preparing Example 1, the aniline raw material in step a was replaced with 2,5-difluoroaniline in equal proportion to obtain Example 5. ¹ H NMR (600MHz, CD ₃ OD) δ7.92 (s, 1H), 7.84 (s, 1H), 7.70 (s, 1H), 7.54 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 8.3 Hz, 1H), 7.19 (td, J = 9.7, 4.8 Hz, 1H), 6.93 (ddd, J = 11.7, 8.4, 3.2 Hz, 1H), 6.12 (s, 1H), 4.75–4.71 (m, 1H), 4.66 (s, 2H), 4.03 (d, J = 8.2 Hz, 2H), 3.57 (t, J = 11.4 Hz, 2H), 2.28–2.22 (m, 5H), 1.82 (d, J = 10.9 Hz, 2H). ¹³ C NMR (151 MHz, CD ₃ OD)δ166.9,159.6,158.3(d,J＝240.9Hz),154.6,151.4,150.3(d,J＝243.9Hz),139.8,139.7,135.7,135.6,131.8,130.4,129.6,128.9,128.0,126.6(dd,J＝15.2,10.1Hz),115.9(dd,J＝22.5,9.9Hz),111.6(dd,J＝24.6,7.4Hz),110.5(d,J＝28.8Hz),95.7(2C),66.7(2C),53.2,43.9,31.5(2C),10.2.

Example 6 Preparation of 2-chloro-N-(3,5-difluorophenyl)-5-(((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide

Referring to the method of Preparation Example 1, the aniline raw material in step a was replaced with 3,5-difluoroaniline in equal proportion to obtain Example 6. ¹ H NMR (600 MHz, CD ₃ OD) δ7.79 (s, 1H), 7.66 (s, 1H), 7.53 (d, J = 8.2 Hz, 1H), 7.44 (d, J = 8.3 Hz, 1H), 7.34-7.31 (m, 2H), 6.73-6.69 (m, 1H), 6.15 (s, 1H), 4.71 (dd, J = 10.0, 5.8 Hz, 1H), 4.64 (s, 2H), 4.02 (d, J = 8.5 Hz, 2H), 3.56 (t, J = 11.4 Hz, 2H), 2.27-2.21 (m, 5H), 1.80 (d, J = 11.2 Hz, 2H). ¹³ C NMR (151 MHz, CD ₃ OD) δ 166.9, 163.26 (d, J = 244.5 Hz, 2C), 160.2, 154.9, 141.0, 140.9, 140.8, 140.0, 135.9, 131.6, 130.4, 129.5, 128.6, 127.8, 124.9, 102.5 (d, J = 30.2 Hz, 2C), 98.8 (t, J = 26.2 Hz), 95.9, 95.7, 66.8 (2C), 53.1, 43.9, 31.5 (2C), 10.4.

Example 7 Preparation of 2-chloro-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)-N-(3,4,5-trifluorophenyl)benzamide

Referring to the method of Preparation Example 1, the aniline raw material in step a was replaced with 3,4,5-trifluoroaniline in equal proportion to obtain Example 7. ¹ H NMR (600 MHz, DMSO-d ₆ )δ12.02(s,1H),10.87(s,1H),10.20(s,1H),8.04(s,1H),7.61-7.56(m,4H),7.52(d,J＝7.8Hz,1H),4.65-4.60(m,1H),4.53(s,2H),3.91(s,2H),3.45(t,J＝11.5Hz,2H),2.20(s,3H),2.09(dt,J＝11.9,8.0Hz,2H),1.75(d,J＝10.1Hz,2H). ¹³ C NMR (151 MHz, DMSO-d ₆ )δ166.0,160.9,155.2,153.8,150.57(ddd,J＝244.5,10.0,5.4Hz,2C),141.0,136.2,135.5(td,J＝11.2,2.4Hz),135.4(ddd,J＝245.8,31.6,15.9Hz),134.7,132.3,131.6,130.1,129.9,128.9,128.1,104.2(d,J＝25.2Hz,2C),96.4(2C),66.7(2C),52.7,44.1,32.2(2C),11.3.

Example 8 Preparation of 2-chloro-N-(3-methoxyphenyl)-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide

Referring to the method of preparing Example 1, the aniline raw material in step a was replaced with 3-methoxyaniline in equal proportion to obtain Example 8. ¹ H NMR (600MHz, DMSO-d ₆ )δ12.05(s,1H),10.47(s,1H),10.34(s,1H),8.06(s,1H),7.88–7.65(m,2H),7.53–7.48(m,2H),7.39(s,1H),7.24(d,J＝4.8Hz,2H),6.69(dd,J＝6.9,4.3Hz,1H),4.64(t,J＝11.4Hz,1H),4.53(s,2H),3.87(s,2H),3.73(s,3H),3.44(t,J＝11.7Hz,2H),2.21(s,3H),2.07(d,J＝9.5Hz,2H),1.76(d,J＝10.0Hz,2H). ¹³ C NMR (151MHz,DMSO-d ₆ )δ165.5,160.7,159.9,154.9,147.7,141.2,140.5,139.7,138.4,137.0,132.4,130.0,129.7,129.2,128.4,128.2,112.2,109.6,105.7,97.0,96.2,66.6(2C),55.4,52.6,44.1,32.2(2C),11.7.

Example 9 Preparation of 2-chloro-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)-N-(m-tolyl)benzamide

Referring to the method of preparing Example 1, the aniline raw material in step a was replaced by 3-methylaniline in equal proportion to obtain Example 9. ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 12.05 (s, 1H), 10.41–10.27 (m, 2H), 8.07 (s, 1H), 7.64 (s, 2H), 7.55–7.49 (m, 3H), 7.45 (d, J=7.5 Hz, 1H), 7.21 (t, J=7.4 Hz, 1H), 6.92 (d, J=6.9 Hz, 1H), 4.64 (s, 1H), 4.52 (s, 2H), 3.87 (s, 2H), 3.44 (s, 2H), 2.29 (s, 3H), 2.20 (s, 3H), 2.08 (d, J=9.6 Hz, 2H), 1.76 (s, 2H). ¹³ C NMR (151 MHz, DMSO-d ₆ ) δ 165.5, 160.9, 155.1, 153.7, 143.7, 140.6, 139.3, 138.4, 137.1, 136.2, 132.4, 131.1, 129.9, 129.3, 128.9, 128.2, 124.9, 120.4, 117.1, 96.9, 96.3, 66.7 (2C), 52.6, 44.0, 32.2 (2C), 21.7, 11.3.

Example 10 Preparation of 2-chloro-N-(2,5-difluorophenyl)-5-(((4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide

Referring to the method for preparing Example 5, the (tetrahydro-2H-pyran-4-yl)hydrazine hydrochloride raw material in step c was replaced with hydrazine hydrate in equal proportion to obtain Example 10. ¹ H NMR (600 MHz, DMSO-d ₆ )δ10.55 (s, 1H), 10.21 (s, 1H), 8.05 (s, 1H), 7.77 (s, 1H), 7.56 (s, 1H), 7.48 (d, J＝5.8 Hz, 2H), 7.36–7.32 (m, 1H), 7.10–7.05 (m, 1H), 4.57 (s, 2H), 2.21 (s, 3H). ¹³ C NMR (151 MHz, DMSO-d ₆ )δ166.0,161.3,158.8,158.0 (d, J = 239.9 Hz), 153.7,150.8 (d, J = 242.6 Hz), 140.7,136.2,133.3,131.1,130.5,130.2,130.0,128.3,127.9,127.16 (dd, J = 13.8,11.8 Hz), 117.2 (dd, J = 22.2,9.8 Hz), 112.6 (dd, J = 24.5,7.5 Hz), 111.6 (d, J = 27.3 Hz), 95.8,85.8,43.7,11.4. Example 11 Preparation of 2-chloro-N-(2,5-difluorophenyl)-5-((1-methyl-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide

Referring to the method for preparing Example 5, the (tetrahydro-2H-pyran-4-yl)hydrazine hydrochloride raw material in step c was replaced with methylhydrazine sulfate in equal proportion to obtain Example 11. ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 11.99 (s, 1H), 10.54 (s, 1H), 10.14 (s, 1H), 8.03 (s, 1H), 7.76 (s, 1H), 7.61 (s, 1H), 7.55 (s, 1H), 7.50 (s, 2H), 7.34 (dd, J = 14.0, 9.3 Hz, 1H), 7.08 (d, J = 8.0 Hz, 1H), 4.58 (s, 2H), 3.72 (s, 3H), 2.20 (s, 3H). ¹³ C NMR (151 MHz, DMSO-d ₆ )δ166.1,161.4,158.0(d,J＝239.0Hz),155.9,153.9,150.8(d,J＝243.6Hz),148.3,140.8,138.4,136.1,132.3,130.9,129.8,128.8,128.3,127.1(dd,J＝14.2,11.6Hz),117.2(dd,J＝22.2,9.7Hz),112.6(dd,J＝24.0,7.5Hz),111.7(d,J＝27.9Hz),97.5,96.1,43.9,33.3,11.2.

Example 12 Preparation of 2-chloro-N-(2,5-difluorophenyl)-5-((1-isopropyl-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide

Referring to the method of Preparation Example 5, the (tetrahydro-2H-pyran-4-yl)hydrazine hydrochloride raw material in step c was replaced with isopropylhydrazine hydrochloride in equal proportions. Example 12 was obtained. ¹ H NMR (600 MHz, DMSO-d ₆ )δ11.98(s,1H),10.53(s,1H),10.08(s,1H),8.03(s,1H),7.76(s,1H),7.63(s,1H),7.49(s,3H),7.34(dd,J＝14.3,9.3Hz,1H),7.07(t,J＝8.2Hz,1H),4.82-4.76(m,1H),4.55(s,2H),2.20(s,3H),1.37(d,J＝6.3Hz,6H). ¹³ C NMR (151 MHz, DMSO-d ₆ )δ166.0,161.1,158.0(d,J＝239.1Hz),154.9,150.8(d,J＝242.5Hz),148.4,140.7,138.3,136.1,135.8,132.2,131.0,129.7,128.9,128.3,127.1(dd,J＝13.9,11.9Hz),117.2(dd,J＝22.2,9.7Hz),112.5(dd,J＝24.1,7.0Hz),111.6(d,J＝28.5Hz),97.6,96.4,47.8,43.9,22.1(2C),11.2.

Example 13 Preparation of 5-((1-(1-acetylpiperidin-4-yl)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)-2-chloro-N-(2,5-difluorophenyl)benzamide

Referring to the method of preparing Example 5, the (tetrahydro-2H-pyran-4-yl)hydrazine hydrochloride raw material in step c was replaced with 1-(4-hydrazinopiperidin-1-yl)ethanone dihydrochloride in equal proportion to obtain Example 13. ¹ H NMR (600 MHz, CD ₃ OD) δ7.92 (s, 1H), 7.83 (s, 1H), 7.70 (s, 1H), 7.52 (d, J = 8.0 Hz, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.21-7.17 (m, 1H), 6.93 (t, J = 8.3 Hz, 1H), 6.15 (s, 1H), 4.78-4.73 (m, 1H), 4.65-4.60 (m, 2H), 4.00 (d, J = 12.3 Hz, 1H), 3.30-3.20 (m, 2H), 2.82 (t, J = 11.9 Hz, 1H), 2.26 (s, 3H), 2.12-2.03 (m, 5H), 1.97-1.90 (m, 2H). ¹³ C NMR (101 MHz, CD ₃ OD)δ170.1,166.9,164.9,158.3(d,J＝240.1Hz),155.1,153.4,150.3(d,J＝242.7Hz),142.7,140.0,137.4,135.5,131.7,131.6,130.4,129.5,128.8,128.1(dd,J＝18.4,9.0Hz),115.9(dd,J＝22.5,9.6Hz),111.5(dd,J＝24.4,8.2Hz),110.5(d,J＝27.5Hz),96.3,95.9,53.5,45.3,43.9,40.6,31.0,30.2,19.8,10.4.

Example 14 Preparation of 2-chloro-N-(2,5-difluorophenyl)-5-(((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide

Referring to the method of preparing Example 5, the (tetrahydro-2H-pyran-4-yl)hydrazine hydrochloride raw material in step c was replaced with 4-hydrazine-1-(methylsulfonyl)piperidine in equal proportion to obtain Example 14. ¹ H NMR (600 MHz, CD ₃ OD) δ7.93–7.90 (m, 1H), 7.79 (s, 1H), 7.70 (s, 1H), 7.53 (d, J = 7.9 Hz, 1H), 7.46 (d, J = 8.3 Hz, 1H), 7.19 (td, J = 9.7, 4.9 Hz, 1H), 6.95–6.91 (m, 1H), 6.17 (s, 1H), 4.62–4.56 (m, 3H), 3.81 (d, J = 11.3 Hz, 2H), 2.95 (td, J = 12.2, 10.3 Hz, 2H), 2.85 (s, 3H), 2.31–2.25 (m, 5H), 1.96 (d, J = 11.5 Hz, 2H). ¹³ C NMR (151 MHz, CD ₃ OD)δ167.0,164.2,161.0,158.3(d,J＝240.0Hz),155.2,154.5,150.3(d,J＝241.4Hz),140.2,140.0,135.5,131.6,130.5,129.5,128.7,128.1,126.6(dd,J＝12.7,11.8Hz),115.9(dd,J＝22.5,9.6Hz),111.5(dd,J＝24.2,6.9Hz),110.5(d,J＝29.0Hz),96.1(2C),53.3,45.1(2C),44.9,43.9,30.3(2C),10.4.

Example 15 Preparation of 2-chloro-N-(2,5-difluorophenyl)-5-((1-(1,1-dioxotetrahydro-2H-thiopyran-4-yl)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide

Referring to the method for preparing Example 5, the (tetrahydro-2H-pyran-4-yl)hydrazine hydrochloride raw material in step c was replaced with (1,1-dioxothio-4-yl)hydrazine hydrochloride in equal proportion to obtain Example 15. ¹ H NMR (600 MHz, DMSO-d ₆ )δ12.02(s,1H),10.54(s,1H),10.15(s,1H),8.09(s,1H),7.78(s,1H),7.69(s,1H),7.61(s,1H),7.49(d,J＝7.8Hz,1H),7.34(td,J＝9.6,5.1Hz,1H),7.08(t,J＝8.4Hz,1H),4.84(s,1H),4.53(s,2H),3.44(t,J＝11.5Hz,2H),3.22–3.14(m,2H),2.62–2.55(m,2H),2.22–2.16(m,5H). ¹³ C NMR(151MHz,DMSO-d ₆ )δ166.1,164.2,161.0,158.0(d,J＝239.7Hz),155.3,153.9,150.8(d,J＝242.3Hz),140.7,138.4,136.2,132.8,131.5,129.7,129.2,128.3,127.1(dd,J＝13.9,11.8Hz),117.2(dd,J＝22.2,9.7Hz),112.6(dd,J＝24.3,7.4Hz),111.5(d,J＝28.2Hz),97.4,96.4,51.4,49.4(2C),44.0,29.5(2C),11.2.

Example 16 Preparation of 2-chloro-N-(2,5-difluorophenyl)-5-(1-((1-isopropyl-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)ethyl)benzamide

Referring to the method for preparing Example 12, the raw material 1 in step a was replaced with 2-chloro-5-ethylbenzoic acid in equal proportion to obtain Example 16. ¹ H NMR (600 MHz, DMSO-d ₆ )δ12.00(s,1H),10.54(s,1H),10.14(s,1H),7.98(s,1H),7.77(s,1H),7.68(s,1H),7.58(s,1H),7.48(d,J＝8.2Hz,2H),7.34(dd,J＝14.2,9.4Hz,1H),7.08(d,J＝7.9Hz,1H),5.17(s,1H),4.78(s,1H),2.22(s,3H),1.51–1.31(m,9H). ¹³ C NMR (151 MHz, DMSO-d ₆ )δ166.1,160.1,158.0(d,J＝238.9Hz),154.8,153.8,150.8(d,J＝242.6Hz),145.8,138.6,136.1,131.8,129.9,129.6,128.2,127.7,127.2(dd,J＝14.0,11.9Hz),117.2(dd,J＝22.2,9.7Hz),112.6(dd,J＝24.0,7.1Hz),111.6(d,J＝27.8Hz),97.4,96.2,49.6,48.0,24.1,22.0,11.7.

Example 17 Preparation of 5-chloro-N-(2,5-difluorophenyl)-2-((1-isopropyl-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)isonicotinamide

Referring to the method for preparing Example 12, the raw material 1 in step a was replaced with 5-chloro-2-methylisonicotinic acid in equal proportion to obtain Example 17. ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 12.00 (s, 1H), 10.68 (s, 1H), 9.12 (s, 1H), 8.35 (s, 1H), 8.07 (s, 2H), 7.82–7.80 (m, 1H), 7.50 (s, 1H), 7.35–7.31 (m, 1H), 7.10–7.04 (m, 1H), 4.80 (s, 1H), 4.52 (s, 2H), 2.20 (s, 3H), 1.35 (d, J=6.6 Hz, 6H).

Example 18 Preparation of 2-chloro-N-(2,5-difluorophenyl)-5-((1-isopropyl-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)nicotinamide

Referring to the method for preparing Example 12, the raw material 1 in step a was replaced with 2-chloro-5-methylnicotinic acid in equal proportion to obtain Example 18. ¹ H NMR (600 MHz, DMSO-d ₆ )δ12.01(s,1H),10.72(s,1H),10.11(s,1H),8.55(s,1H),8.07(s,2H),7.84–7.81(m,1H),7.52(s,1H),7.37–7.33(m,1H),7.11–7.06(m,1H),4.81(s,1H),4.55(s,2H),2.21(s,3H),1.37(d,J＝6.6Hz,6H).

Example 19 Preparation of 2-chloro-N-(2,5-difluorophenyl)-5-(((4-((5-ethyl-1H-pyrazol-3-yl)amino)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide Referring to the method for preparing Example 12, the raw material 5-methyl-1H-pyrazole-3-amine in step d was replaced with 5-ethyl-1H-pyrazole-3-amine in equal proportion to obtain Example 19. ¹ H NMR (600 MHz, DMSO-d ₆ )δ12.05(s,1H),10.54(s,1H),10.22(s,1H),8.04(s,1H),7.77(s,1H),7.64(s,1H),7.50(t,J＝9.6Hz,3H),7.36–7.32(m,1H),7.07(t,J＝8.4Hz,1H),4.81(dt,J＝13.2,6.6Hz,1H),4.57(s,2H),2.67–2.57(m,2H),1.38(d,J＝6.6Hz,6H),1.22(t,J＝7.6Hz,3H). ¹³ C NMR (151MHz,DMSO-d ₆ )δ166.0,161.1,158.0(d,J＝238.6Hz),154.9,153.8,150.8(d,J＝242.5Hz),145.3,140.6,136.1,132.7,132.1,131.1,129.8,128.9,128.4,127.2(dd,J＝14.3,11.6Hz),117.2(dd,J＝22.3,9.9Hz),112.5(dd,J＝24.1,7.4Hz),111.6(d,J＝27.9Hz),96.4,95.9,47.8,44.0,22.3,22.1(2C),11.7.

Example 20 Preparation of 2-chloro-5-((4-((5-cyclopropyl-1H-pyrazol-3-yl)amino)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)-N-(2,5-difluorophenyl)benzamide

Referring to the method for preparing Example 12, the raw material 5-methyl-1H-pyrazole-3-amine in step d was replaced with 5-cyclopropyl-1H-pyrazole-3-amine in equal proportion to obtain Example 20. ¹ H NMR (600 MHz, DMSO-d ₆ δ12.07 (s, 1H), 10.54 (s, 1H), 10.20 (s, 1H), 8.03 (s, 1H), 7.77 (s, 1H), 7.65 (s, 1H), 7.50 (d, J = 7.4 Hz, 3H), 7.37-7.32 (m, 1H), 7.08 (t, J = 8.4 Hz, 1H), 6.50 (s, 1H), 4.81 (dt, J = 13.1, 6.4 Hz, 1H), 4.58 (s, 2H), 1.99-1.92 (m, 1H), 1.38 (d, J = 6.6 Hz, 6H), 1.01-0.92 (m, 4H). ¹³ C NMR (151 MHz, DMSO-d ₆ )δ164.9,156.9(d,J＝239.8Hz),156.2,152.8,152.2,149.7(d,J＝243.2Hz),146.2,139.6,135.1,131.7,131.0,130.1,128.7,127.8,127.4,126.1(dd,J＝14.1,11.8Hz),116.1(dd,J＝22.4,9.9Hz),111.5(dd,J＝24.1,7.6Hz),110.5(d,J＝28.1Hz),95.3,93.7,46.7,42.8,21.2(2C),7.1(2C),6.2.

Example 21: Study on in vitro enzyme inhibition activity of some products of the present invention

Experimental Materials:

Tecan ELISA reader.

Kinase Binding Assay Kit (including Kinase Tracer 236, Eu-Anti-GST Antibody, kinase buffer solution (1X Kinase Buffer A)), 384 shallow well plates, recombinant human PLK4 protein (aa 1-836, containing a GST tag).

Recombinant human PLK4 protein kinase concentration 50 ng/μL (Thermo Scientific: PV6395), distilled water, DMSO.

experimental method:

First, the compound samples prepared in the above examples were prepared into a 20 mM solution with DMSO, and then diluted with kinase buffer solution (1X Kinase Buffer A)) to 200 μM, 40 μM, 10 μM, 1.6 μM, 0.32 μM, 0.064 μM, 0.0128 μM, 0.00256 μM, 0.000512, and 0.0001024 μM according to test requirements; then the compound samples (4 μL) were added to the 384-well plate, followed by 8 μL of kinase buffer solution containing recombinant human PLK4 kinase (at a concentration of 50 ng/μL) and Eu-Anti-GST Antibody, and 4 μL of kinase buffer solution containing Tracer 236, and then incubated at room temperature for 60 minutes and the plate was read.

Method for evaluating results: Kinase "tracer" binding is detected by adding Eu-labeled antibodies. Binding of the tracer and antibody to the kinase results in a high degree of FRET, whereas the use of a kinase inhibitor in place of the tracer results in a loss of FRET. The europium donor is excited by a 340nm excitation filter with a 30nm grating, using a filter centered at 665nm with a 10nm bandpass to detect the energy transferred to the Alexa Fluor 647 tracer. This signal is then replaced by the peak excitation of europium, which is accomplished using a 615nm, 10nm grating filter. The "emission ratio" is calculated by dividing the 665nm signal by the 615nm signal. Therefore, the ratio of the HTRF signal (665/615) is calculated for each well plate reaction. The results are characterized as Delta F (DF%):

Calculation of inhibition rate (% activity): In the absence of compound sample, the DF% of kinase activity is defined as 100%. When compound sample is added, the kinase activity rate is:

Calculation of _IC50 : DF% of kinase activity in the presence of added compound is plotted on the Y-axis and the logarithm of the compound concentration is plotted on the X-axis. The _IC50 value is obtained by fitting the data to a S-shaped stoichiometric response curve. Centrinone was used as a positive control in the experiment and the _IC50 value of centrinone was tested to be 3.0 nM.

Table 1 IC ₅₀ values of some of the compounds in the examples

As shown in Table 1, the compounds in the table all showed strong inhibitory activity against PLK4. Among them, the inhibitory activity of compounds 5, 10, 11, 12, 13, 14, and 15 was better than that of the positive compounds. Compound 12 was further selected for kinase selectivity test.

Example 22: Kinase selectivity of Example 12 of the present invention

Table 2 Kinase selectivity of Example 12 at 0.5 μM concentration

As shown in Table 2, at a concentration of 0.5 μM, the inhibition rate of compound 12 on the above 68 kinases was less than 50%, which indicates that compound 12 has a certain in vitro kinase selectivity.

Claims

A highly selective PLK4 inhibitor, characterized in that the inhibitor is a pyrazolopyrimidine derivative represented by general formula I, and its geometric isomers, enantiomers or pharmaceutically acceptable salts thereof;

wherein X is selected from C or N;

Y is selected from C or N;

Z is selected from hydrogen, halogen;

R 1 is selected from hydrogen, C1-C4 alkyl;

R2 is selected from hydrogen, C1-C4 alkyl;

R3 is selected from hydrogen, C1-C4 alkyl, C3-C6 cycloalkyl;

R4 is selected from hydrogen, C1-C6 alkyl, C4-C7 aliphatic ring containing up to 2 heteroatoms, wherein the heteroatom is N, O or S; when the heteroatom is S, the sulfur atom can be further oxidized to sulfoxide or sulfone; when the heteroatom is N, the nitrogen atom can be further substituted by C1-C4 alkyl, C1-C4 alkanoyl or C1-C4 alkanesulfonyl;

Ring A is selected from aryl which is unsubstituted or substituted with 1 to 3 R a which may be the same or different;

Ra is selected from halogen, C1-C4 alkyl, C1-C6 alkoxy.
The highly selective PLK4 inhibitor according to claim 1, characterized in that: the compound represented by the general formula I, and its geometric isomers, enantiomers or pharmaceutically acceptable salts thereof;

wherein X is selected from C or N;

Y is selected from C or N;

Z is selected from hydrogen, halogen;

R1 is selected from hydrogen;

R2 is selected from hydrogen, methyl;

R3 is selected from hydrogen, methyl, ethyl, cyclopropyl;

R4 is selected from hydrogen, methyl, isopropyl, a C4-C7 aliphatic ring containing up to 2 heteroatoms, wherein the heteroatom is N, O or S; when the heteroatom is S, the sulfur atom may be further oxidized to sulfoxide or sulfone; when the heteroatom is N, the nitrogen atom may be further substituted by a C1-C4 alkyl group, a C1-C4 alkanoyl group or a C1-C4 alkanesulfonyl group;

Ring A is selected from phenyl, pyridyl, pyrimidinyl, which may or may not contain 1-3 R a substituted groups which may be the same or different;

Ra is selected from fluorine, chlorine, bromine, methyl, methoxy.
The highly selective PLK4 inhibitor according to claim 2, characterized in that: the compound represented by the general formula I, and its geometric isomers, enantiomers or pharmaceutically acceptable salts thereof;

wherein X is selected from C or N;

Y is selected from C or N;

Z is selected from chlorine;

R1 is selected from hydrogen;

R2 is selected from hydrogen, methyl;

R3 is selected from hydrogen, methyl, ethyl, cyclopropyl;

R4 is selected from hydrogen, methyl, isopropyl, a C4-C7 aliphatic ring containing up to 2 heteroatoms, wherein the heteroatom is N, O or S; when the heteroatom is S, the sulfur atom can be further oxidized to sulfone; when the heteroatom is N, the nitrogen atom can be further substituted by acetyl or methylsulfonyl;

Ring A is selected from phenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 2,5-difluorophenyl, 3,5-difluorophenyl, 3,4,5-trifluorophenyl, 3-methoxyphenyl, and 3-methylphenyl.
The highly selective PLK4 inhibitor according to claim 3, characterized in that: the compound represented by the general formula I, and its geometric isomers, enantiomers or pharmaceutically acceptable salts thereof;

wherein X is selected from C or N;

Y is selected from C or N;

Z is selected from chlorine;

R1 is selected from hydrogen;

R2 is selected from hydrogen, methyl;

R3 is selected from hydrogen, methyl, ethyl, cyclopropyl;

R4 is selected from hydrogen, methyl, isopropyl,

Ring A is selected from phenyl, 2-fluorophenyl, 2-chlorophenyl, 2-bromophenyl, 2,5-difluorophenyl, 3,5-difluorophenyl, 3,4,5-trifluorophenyl, 3-methoxyphenyl, and 3-methylphenyl.
The highly selective PLK4 inhibitor according to claim 4, characterized in that the derivative is:

2-chloro-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)-N-phenylbenzamide;

2-Chloro-N-(2-fluorophenyl)-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-Chloro-N-(2-chlorophenyl)-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl 1-(4-(2-amino-3-yl)benzamide;

2-chloro-N-(2-bromophenyl)-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-Chloro-N-(2,5-difluorophenyl)-5-(((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-Chloro-N-(3,5-difluorophenyl)-5-(((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-chloro-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)-N-(3,4,5-trifluorophenyl)benzamide;

2-chloro-N-(3-methoxyphenyl)-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-chloro-5-((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(tetrahydro-2H-pyran-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)-N-(m-tolyl)benzamide;

2-Chloro-N-(2,5-difluorophenyl)-5-(((4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-Chloro-N-(2,5-difluorophenyl)-5-((1-methyl-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-Chloro-N-(2,5-difluorophenyl)-5-((1-isopropyl-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

5-((1-(1-acetylpiperidin-4-yl)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)-2-chloro-N-(2,5-difluorophenyl)benzamide;

2-Chloro-N-(2,5-difluorophenyl)-5-(((4-((5-methyl-1H-pyrazol-3-yl)amino)-1-(1-(methylsulfonyl)piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-Chloro-N-(2,5-difluorophenyl)-5-((1-(1,1-dioxotetrahydro-2H-thiopyran-4-yl)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide;

2-Chloro-N-(2,5-difluorophenyl)-5-(1-((1-isopropyl-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)ethyl)benzamide;

5-Chloro-N-(2,5-difluorophenyl)-2-((1-isopropyl-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)isonicotinamide;

2-Chloro-N-(2,5-difluorophenyl)-5-((1-isopropyl-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)nicotinamide;

2-Chloro-N-(2,5-difluorophenyl)-5-(((4-((5-ethyl-1H-pyrazol-3-yl)amino)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)benzamide; 2-Chloro-5-((4-((5-cyclopropyl-1H-pyrazol-3-yl)amino)-1-isopropyl-1H-pyrazolo[3,4-d]pyrimidin-6-yl)amino)methyl)-N-(2,5-difluorophenyl)benzamide.
A pharmaceutical composition, characterized in that it comprises a compound represented by general formula I according to any one of claims 1 to 6 and its geometric isomers or pharmaceutically acceptable salts, hydrates, solvates or prodrugs as active ingredients and a pharmaceutically acceptable excipient.
The use according to claim 1 or 6 is characterized in that: the compound represented by general formula I, and its geometric isomers or pharmaceutically acceptable salts, hydrates, solvates or prodrugs, or the pharmaceutical composition according to claim 6 is used in the preparation of a PLK4 inhibitor.
The use according to claim 1 or 6 is characterized in that: the compound represented by general formula I, and its geometric isomers or pharmaceutically acceptable salts, hydrates, solvates or prodrugs, or the pharmaceutical composition according to claim 6 is used in the preparation of a drug for preventing or treating a disease related to the expression or activity of PLK4 kinase.
The use according to claim 1 or 6 is characterized in that: the compound represented by general formula I, and its geometric isomers or pharmaceutically acceptable salts, hydrates, solvates or prodrugs, or the pharmaceutical composition according to claim 6 is used in the preparation of preventive or anti-tumor drugs.