A compound targeting Polo-like kinase 4 protein and its application in medicine

Abstract

The present invention belongs to the field of biomedicine and drug synthesis, and relates to a compound targeting Polo-like kinase 4 (PLK4) protein, and a pharmaceutically acceptable salt, hydrate, solvate or prodrug of the compound, and its application as a drug for treating malignant tumors, especially as a PLK4 degrader. The compound of general formula I and its geometric isomers or their pharmaceutically acceptable salts, hydrates, solvates, prodrugs and their preparation methods. The preferred compound has the activity of being a protein kinase degrader, especially a PLK4 kinase degrader.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and drug synthesis, and relates to a compound targeting Polo-like kinase 4 (PLK4) protein, as well as a pharmaceutically acceptable salt, hydrate, solvate or prodrug of the compound, and its application as a drug for treating malignant tumors, in particular as a PLK4 degrader. Background Art

[0002] 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.

[0003] PLK4 is a key protein for centrosome duplication in cells and plays an important role in centrosome duplication and mitosis. Polo-like kinase 4 is a member of the Polo-like protein kinase family and 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 duplication in the cell cycle, and tumor cells have the characteristics of unlimited proliferation. Therefore, PLK4 plays an irreplaceable role in various biological functions of tumor cells, such as proliferation, migration, invasion, and apoptosis. Many studies have shown that PLK4 protein interacts with other proteins, or that knocking out PLK4 protein is closely related to tumor cell growth, migration, and invasion. At the same time, 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.

[0004] The Proteolysis targeting chimera (PROTAC) molecule is a heterobifunctional molecule designed based on the ubiquitin proteasome system (UPS). One end of the molecule is used to connect to the ligand of the target protein, and the other end is used to connect to the ligand of the E3 ligase. The molecule is connected in the middle by a suitable linker. Today, with the vigorous development of the Proteolysis targeting chimera (PROTAC) technology, the use of PROTAC technology to degrade PLK4 may be a very promising therapeutic strategy for tumor treatment. Summary of the invention

[0005] The object of the present invention is to provide a compound targeting Polo-like kinase 4 (PLK4) protein, a preparation method thereof and an application of the compound as a drug for treating malignant tumors, in particular as a PLK4 degrader.

[0006] To achieve the above purpose, the present invention adopts the following technical solution:

[0007] A compound targeting Polo-like kinase 4 protein, the compound is

[0008] The compound represented by the general formula I, and its geometric isomers, enantiomers or pharmaceutically acceptable salts, hydrates, solvates or prodrugs; the compound represented by the general formula I is as follows:

[0009]

[0010] Wherein, the A ring is selected from an aliphatic heterocyclic ring consisting of 4 to 12 atoms;

[0011] Ring B is selected from

[0012] o is 0 or 1; p is 0 or 1; q is 0, 1 or 2;

[0013] The L part of the ring is selected from aliphatic heterocyclic rings containing 4 to 12 atoms,

[0014] n is an integer between 0 and 20; r is an integer between 0 and 4; s is an integer between 0 and 6; t is an integer between 0 and 6; u is an integer between 0 and 20; v is an integer between 0 and 4; w is an integer between 0 and 4;

[0015] The aliphatic heterocycle mentioned above may contain 1-3 heteroatoms of N, O or S; when the heteroatom is S, the sulfur atom may be further oxidized to sulfoxide or sulfone.

[0016] Preferably, the compound is a compound represented by general formula I, and its geometric isomers, enantiomers or pharmaceutically acceptable salts, hydrates, solvates or prodrugs;

[0017] wherein the A ring is selected from a piperidine ring;

[0018] Ring B is selected from

[0019] o is 0 or 1; p is 0 or 1; q is 0, 1 or 2;

[0020] The L ring is selected from piperazine ring, piperidine ring, pyrrole ring, azetidine ring, 2-azaspiro[3.3]heptyl ring,

[0021]

[0022] n is an integer between 0 and 20; r is an integer between 0 and 4; s is an integer between 0 and 6; t is an integer between 0 and 6; u is an integer between 0 and 20; v is an integer between 0 and 4; and w is an integer between 0 and 4.

[0023] Further preferably, the compound is a compound represented by general formula I, and its geometric isomers, enantiomers or pharmaceutically acceptable salts, hydrates, solvates or prodrugs;

[0024] wherein the A ring is selected from a piperidine ring;

[0025] Ring B is selected from

[0026] o is 0 or 1; p is 0 or 1; q is 0 or 1;

[0027] The L ring is selected from piperazine ring, piperidine ring, pyrrole ring, azetidine ring, 2-azaspiro[3.3]heptyl ring,

[0028]

[0029] n is 0, 2, 4, 6, 8, 10, 12, 14 or 16; r is 0 or 1; s is 0 or 1; t is 0 or 1; u is 4, 6 or 8; v is 1; and w is 0 or 1.

[0030] Specific compounds,

[0031] 5-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-(4-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-oxobutoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0032] 5-((6-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-6-oxohexyl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0033] 5-((8-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-8-oxooctyl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 4)

[0034] 5-((10-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-10-oxodecanyl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0035] 5-((12-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-12-oxododecanyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0036] 5-((14-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-14-oxotetradecyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0037] 5-((16-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-16-oxohexadecyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0038] 5-((18-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-18-oxooctadecyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0039] 5-(2-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0040] 5-(2-(3-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3-oxopropoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0041] 5-(2-(2-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3, 4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1, 3-dione

[0042] 5-(3-(2-(3-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3, 4-d]pyrimidin-1-yl)piperidin-1-yl)-3-oxopropoxy)ethoxy)propoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0043] 5-(4-(4-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3, 4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethyl)phenyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0044] 5-(4-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0045] 5-(3-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0046] 5-(3-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethyl)azetidin-1-yl)-2-(2,6-dioxapiperidin-3-yl)isoindoline-1,3-dione

[0047] 5-(6-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-azaspiro[3.3]hept-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0048] 5-(3-((4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0049] 5-(3-((4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)methyl)azetidin-1-yl)-2-(2,6-dioxapiperidin-3-yl)isoindoline-1,3-dione

[0050] 5-(4-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0051] 4-(4-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0052] (2S,4R)-1-((S)-2-(6-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-6-oxohexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0053] (2S,4R)-1-((S)-2-(8-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-8-oxooctylamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide

[0054] (2S,4R)-1-((S)-2-(10-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-10-oxodecanoylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxylic

[0055] (2S,4R)-1-((S)-2-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3-oxopropoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine

[0056] (2S,4R)-1-((S)-2-(2-(3-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3-oxopropoxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl).

[0057] A pharmaceutical composition comprises the compound represented by the general formula I and its geometric isomers or pharmaceutically acceptable salts, hydrates, solvates or prodrugs as active ingredients and pharmaceutically acceptable excipients.

[0058] Use of the compound of general formula I and its geometric isomers or pharmaceutically acceptable salts, hydrates, solvates or prodrugs or the composition according to claim 5 in the preparation of preventive or anti-tumor drugs.

[0059] Use of the compound of general formula I and its geometric isomers or pharmaceutically acceptable salts, hydrates, solvates or prodrugs or the composition of claim 5 in the preparation of drugs for preventing or treating diseases related to the expression or activity of PLK4 kinase.

[0060] The use of the compound of the general formula of the composition and its geometric isomers or pharmaceutically acceptable salts, hydrates, solvates or prodrugs or the composition of claim 5 in the preparation of a PLK4 degrader.

[0061] The advantages of the present invention are:

[0062] 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 degradation activity.

[0063] The compounds of the structure shown in the general formula I of the present invention are not limited to their specific isomers, and all show good inhibitory and degradation activities on PLK4, and are used to treat other diseases related to abnormal PLK4 expression. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 The results of the in vitro PLK4 protein degradation experiment of some products provided in the examples of the present invention are shown in the figure.

[0065] Figure 2 The results of the in vitro PLK4 protein degradation experiment of some products provided in the examples of the present invention are shown in the figure.

[0066] Figure 3 The results of the in vitro PLK4 protein degradation experiment of some products provided in the examples of the present invention are shown in the figure.

[0067] Figure 4 The results of the in vitro PLK4 protein degradation experiment of the compound of Example 21 provided in the embodiments of the present invention are shown; wherein the left is the in vitro PLK4 protein degradation experiment result of Example 21, and the right is the degradation activity DC of Example 21 50 value. DETAILED DESCRIPTION

[0068] The specific implementation modes of the present invention are further described below in conjunction with examples. It should be noted that the specific implementation modes described here are only for illustrating and explaining the present invention, and are not limited to the present invention.

[0069] The preparation methods of some compounds are as follows:

[0070] 1) When ring A is a piperidine ring, the preparation route of intermediate 6 is as follows:

[0071]

[0072] The specific synthesis steps are as follows:

[0073] Synthesis of 2,4,6-Trichloropyrimidine-5-carboxaldehyde (2)

[0074] Add 9.2 mL of phosphorus oxychloride to a 50 mL dry reaction bottle, place at -40 °C, then drop 1.2 mL of dry N,N-dimethylformamide, and then add barbituric acid (2.00 g, 15.60 mmol) in batches. After the addition is complete, move to 120 °C and reflux for 15 h. TLC monitoring (PE: EA = 4: 1) shows that the raw material reacts completely. After the reaction is cooled to room temperature, the reaction solution is slowly poured into ice water, and a yellow solid precipitates. A brown solid is obtained by suction filtration. The filtrate is extracted with ethyl acetate three times, concentrated, and combined and purified by column chromatography (PE: EA = 40: 1) to obtain a white solid with a yield of 82%.

[0075] Synthesis of tert-butyl 4-(4,6-dichloro-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate (3)

[0076] Dissolve tert-butyl 4-hydrazinopiperidine-1-carboxylate (1.00 g, 4.65 mmol) in 15.0 mL of ethanol, place at -78°C, slowly dropwise add 15.0 mL of ethanol solution of intermediate 2 (0.98 g, 4.65 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%.

[0077] Synthesis of tert-butyl 4-(6-chloro-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate (4)

[0078] The intermediate 3 (0.70 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%.

[0079] Preparation of tert-butyl 4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carboxylate (5)

[0080] The intermediate 4 (0.11 g, 0.24 mmol) was dissolved in 1.5 mL of isopropanol, and then 2,4-difluorobenzylamine (0.041 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. TLC monitoring (DCM: MeOH = 10:1) showed that the raw material reaction was complete, 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 46%.

[0081] N 6 -(2,4-difluorobenzyl)-N 4 Preparation of -(5-methyl-1H-pyrazol-3-yl)-1-(piperidin-4-yl)-1H-pyrazolo[3,4-d]pyrimidine-4,6-diamine (Intermediate 6)

[0082] Intermediate 5 (0.07 g, 0.13 mmol) was dissolved in 1 mL of saturated hydrogen chloride solution in ethyl acetate and stirred at room temperature for 4 h. The reaction was complete under TLC monitoring (DCM:MeOH=8:1), and the mixture was filtered under reduced pressure, and the filter cake was washed three times with ethyl acetate to obtain a light yellow solid with a yield of 92%. 1H NMR(600MHz,DMSO-d6)δ9.04(s,1H),8.79(s,1H),8.25(s,1H), 7.61(s,1H),7.26(t,J=8.8Hz,1H),7.06(s,1H),6.30(s,1H),4.80(t,J=10.9Hz,1H),4.61 (s,2H),3.42(d,J=11.8Hz,2H),3.16(d,J=6.4Hz,2H),2.33–2.21(m,5H),2.04(d,J=12.7Hz,2H). 13 C NMR (151MHz, DMSO-d6) δ160.8 (dd, J=243.1Hz, 7.6Hz), 159.8 (dd, J=252.1Hz,10.6Hz),158.9,156.0,149.8,136.1,132.6,131.2,129.0,124.2,110.5(d,J= 18.1Hz),103.0(t,J=25.7Hz),96.5,94.7,60.5,49.8,41.6(2C),26.9(2C),10.2.HRMS (ESI,m / z)calcd for C 21 H2F2N9[M+H] + ,440.2117; found 440.2129.

[0083] 2) The preparation routes of Examples 1-9 are as follows:

[0084]

[0085] Example 1

[0086] The specific synthesis steps are as follows:

[0087] Synthesis of tert-butyl 3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)propanoate (9a)

[0088] Compound 7 (0.15 g, 0.55 mmol) was dissolved in 3 mL DMF, and compound 8a (0.11 g, 0.55 mmol) and sodium carbonate (0.12 g, 1.09 mmol) were added in sequence, and then reacted at 40°C for 8 h. TLC detection (DCM: MeOH = 30: 1) showed that the raw materials reacted completely. After the reaction was cooled to room temperature, 30 mL of water was added, and ethyl acetate (10 mL × 3) was used for extraction three times. The organic layer was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. No further purification was required to obtain a colorless oily liquid with a yield of 92%.

[0089] Synthesis of 3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)propionic acid (10a)

[0090] Dissolve tert-butyl 3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)propanoate (0.20 g, 0.50 mmol) in 1 mL of dichloromethane, add 2 mL of trifluoroacetic acid, and react at room temperature for 4 h. TLC monitoring (DCM:MeOH=15:1) shows that the raw material reacts completely and is spin-dried to obtain a white solid with a yield of 95%.

[0091] Preparation of 5-(3-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3-oxopropoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 1)

[0092] The intermediate 10a (0.03 g, 0.06 mmol) was dissolved in 2 mL DMF, followed by the intermediate 6 (0.02 g, 0.06 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.02 g, 0.06 mmol), and sodium bicarbonate (0.02 g, 0.23 mmol), and the reaction was carried out at room temperature for 3 h. TLC detection (DCM:MeOH=10:1) showed that the raw materials reacted completely, 20 mL of water was added, and ethyl acetate (10 mL×3) was used for extraction three times. The organic layer was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. A white solid was obtained by thick preparative silica gel plate (DCM:MeOH=12:1) with a yield of 46%. 1 H NMR (600MHz, DMSO-d6) δ12.05(s,1H),11.12(s,1H),10.25 (s,1H),8.06(s,1H),7.84(d,J=6.2Hz,1H),7.57–7.45(m,4H),7.37(d,J=7.9Hz,1H), 7.20(s,1H),7.01(s,1H),5.25–5.17(m,3H),5.14(s,1H),4.70(s,1H),4.53–4.41(m,4H), 3.97(d,J=9.0Hz,1H),2.89(d,J=10.1Hz,2H),2.63–2.58(m,2H),2.21–2.01(m,5H), 1.90(s,4H). 13C NMR(151MHz,DMSO-d6)δ173.3,170.4,167.4,167.2,165.4,164.2, 161.6(dd,J=244.0,3.8Hz),160.7,160.6(dd,J=246.4,5.6Hz),155.3,153.8,134.1(2C), 132.3,131.9,129.9,125.6,124.1,123.7,121.6,111.4(d,J=20.7Hz),109.5,103.8(t,J= 25.8Hz),96.4(2C),66.7,53.5,49.4(2C),43.6,41.0,31.4(2C),30.8,22.5,11.4.

[0093] Preparation of 5-(4-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-oxobutoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 2)

[0094] Referring to the method for preparing Example 1, the raw material 8a in step a was replaced with compound 8b in equal proportion to obtain Example 2. 1 H NMR (600MHz, CDCl3) δ7.80(t,J=8.2Hz, 1H),7.64–7.60(m,1H),7.38(d,J=7.0Hz,1H),7.37–7.31(m,1H),7.21(t,J=6.1Hz, 1H),6.81–6.77(m,2H),6.46(s,1H),5.55(s,1H),5.07–5.00(m,1H),4.72–4.60(m,4H), 4.20–4.13(m,2H),4.03(t,J=14.3Hz,1H),3.29–3.19(m,2H),2.98–2.86(m,4H),2.79 –2.73(m,2H),2.44–2.37(m,2H),2.25(s,3H),2.20–2.15(m,2H),2.01–1.92(m,2H). 13C NMR (151MHz, CDCl3) δ172.2,172.0,170.5,167.7,167.5,164.6,164.5,162.0(dd,J= 248.4,11.3Hz),160.9,160.7(dd,J=246.4,7.7Hz),154.8,153.7,134.1(d,J=20.0Hz), 131.7,130.3,125.7,123.5,122.6(t,J=13.4Hz),119.9,111.0(dd,J=20.9,2.9Hz),110.3,10 3.5(td,J=25.1,3.5Hz),97.0,96.5,68.0,51.9,49.7(2C),45.4,39.0,31.7,27.9,26.9(2C), 24.5,22.5,11.4.

[0095] Preparation of 5-((6-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-6-oxohexyl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 3)

[0096] Referring to the method for preparing Example 1, the raw material 8a in step a was replaced with compound 8c in equal proportion to obtain Example 3. 1 H NMR(600MHz, CDCl3)δ9.32(s,1H),7.75(t,J=9.1Hz,1H),7.51(s,1H),7.44–7.40(m,1H),7.3 7(dd,J=15.5,8.0Hz,1H),7.19(d,J=8.1Hz,1H),6.78(dd,J=15.7,7.7Hz,2H),6.50(s,1H),5. 43(s,1H),5.03(dt,J=13.0,4.6Hz,1H),4.82(t,J=12.2Hz,1H),4.71–4.64(m,3H),4.17–4.14 (m,1H),4.09(d,J=5.4Hz,2H),3.27–3.22(m,1H),2.97–2.88(m,2H),2.79–2.68(m,3H),2.32– 2.29(m,1H),2.24(s,3H),2.21–2.15(m,2H),1.93–1.86(m,5H),1.71(d,J=5.4Hz,2H), 1.63–1.52(m,2H). 13C NMR (151MHz, CDCl3) δ172.4,171.5,167.8,167.6,164.8,162.0 (dd,J=247.5,11.7Hz),160.8,160.7(dd,J=248.3,11.8Hz),154.9,153.8,134.2,131.5, 130.3,125.5,125.5,123.3,123.2,122.7(dd,J=14.8,3.4Hz),120.8,120.2,111.0(dd,J= 20.7, 3.1Hz), 109.1, 103.5 (t, J = 25.4Hz), 96.9, 96.8, 68.8, 52.6, 49.8 (2C), 45.6, 41.1, 39.0, 32.4, 31.7, 28.9, 28.7, 25.8 (2C), 22.6, 11.3.

[0097] Preparation of 5-((8-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-8-oxooctyl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 4)

[0098] Referring to the method for preparing Example 1, the raw material 8a in step a was replaced with compound 8d in equal proportion to obtain Example 4. 1H NMR (600MHz, CDCl3) δ8.97(s,1H), 7.73(d,J=6.7Hz,1H),7.67(s,1H),7.35(d,J=6.1Hz,2H),7.16(d,J=8.1Hz,1H),6.80 –6.76(m,2H),6.33(s,1H),5.70(s,1H),5.03–4.98(m,1H),4.74(d,J=11.9Hz,1H),4.69 –4.65(m,1H),4.59(s,2H),4.09(t,J=5.7Hz,2H),4.00(d,J=11.4Hz,1H),3.20(t,J= 12.8Hz,1H),2.87(dd,J=12.3,4.3Hz,2H),2.78–2.72(m,2H),2.49(d,J=6.0Hz,1H), 2.30(dd,J=7.4,4.0Hz,1H),2.24(s,3H),2.18–2.11(m,2H),1.95(d,J=11.9Hz,3H), 1.84–1.80(m,2H),1.70–1.63(m,2H),1.44–1.39(m,6H). 13 C NMR (151MHz, CDCl3) δ 172.5, 171.6, 170.3, 167.4, 167.2, 164.7, 161.9 (dd, J = 247.5, 11.8Hz), 160.7 (dd, J = 248.0, 11.8Hz),160.6,155.1,154.0,134.2,131.4,130.3,129.8(dd,J=23.3,18.7Hz),125.5,123.1, 122.6(dd,J=14.9,3.2Hz),120.7,111.0(dd,J=20.9,3.3Hz),108.9,103.4(t,J=25.4Hz), 96.8,96.3,68.9,53.5,49.4(2C),45.1,40.9,38.8,33.3,31.6,29.6,28.9,28.6,25.5(2C),25.3, 22.7,11.6.

[0099] Preparation of 5-((10-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-10-oxodecanyl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 5)

[0100] Referring to the method for preparing Example 1, the raw material 8a in step a was replaced with compound 8e in equal proportion to obtain Example 5. 1 H NMR(600MHz, DMSO-d6)δ 12.06(s,1H),11.11(s,1H),9.76(s,3H),8.07(s,1H),7.82(d,J=8.3Hz,1H),7.47(s,1H), 7.42(d,J=2.0Hz,1H),7.34(dd,J=8.3,2.1Hz,1H),7.19(s,1H),7.01(t,J=7.9Hz,1H), 5.12(dd,J=12.9,5.4Hz,1H),4.64(s,1H),4.50(d,J=10.4Hz,3H),4.16(t,J=6.5Hz, 2H),4.00(d,J=12.9Hz,1H),3.61–3.57(m,5H),3.11(td,J=7.2,3.0Hz,5H),2.92–2.86 (m,1H),2.74(d,J=12.7Hz,1H),2.61–2.58(m,1H),2.56–2.52(m,1H),2.39–2.30(m, 2H),2.18(s,3H),2.04–1.96(m,2H),1.84(d,J=11.5Hz,2H),1.77–1.73(m,2H),1.53–1.49(m,2H),1.46–1.40(m,2H). 13 C NMR (151MHz, DMSO-d6) δ173.3, 171.0, 170.4, 167.4, 167.2, 164.6, 161.5 (dd, J=242.5, 9.1Hz), 160.7 (dd, J=233.5, 9.7Hz), 160.4, 155.3, 153.8,134.4,132.5,132.0,130.1,129.1,125.7,124.2(d,J=11.7Hz),123.3,121.2,111.4 (dd,J=19.5,2.4Hz),109.3,103.7(t,J=24.8Hz),96.5,96.4,69.3,53.7,49.4( 2C),42.0,32.8,31.4,29.4,29.3(3C),29.1,28.8,25.8,25.5(2C),22.5,12.6.

[0101] Preparation of 5-((12-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-12-oxododecanyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 6)

[0102] Referring to the method for preparing Example 1, the raw material 8a in step a was replaced with compound 8f in equal proportion to obtain Example 6. 1 H NMR (600MHz, DMSO-d6) δ12.05(s,1H),11.11(s,1H),10.23(s,1H),8.05(s,1H),7.82(d,J=8.2Hz,1H),7.56–7.36 (m,3H),7.33(d,J=8.1Hz,1H),7.19(s,1H),7.01(t,J=7.5Hz,1H),5.12(dd,J=12.8,5.3Hz,1H),4.64(s,1H ),4.51(s,2H),4.15(t,J=6.1Hz,2H),3.99(d,J=11.6Hz,1H),3.24–3.14(m,2H),2.92–2.86(m,1H),2.72(d, J=11.2Hz,1H),2.60(d,J=17.9Hz,1H),2.57–2.53(m,1H),2.34(dd,J=15.6,7.6Hz,2H),2.19(s,3H),2.04( dd,J=15.4,8.9Hz,2H),1.91(s,1H),1.88–1.82(m,2H),1.76–1.72(m,2H),1.53–1.48(m,2H),1.44–1.40(m, 2H),1.27–1.21(m,12H). 13C NMR (151MHz, DMSO-d6) δ173.2,172.5,171.0,170.4, 167.3,167.2,164.5,161.5(dd,J=245.7,12.6Hz),160.6(dd,J=240.5,6.0Hz),155.3, 153.8,138.7,134.4,132.3,131.9,130.1,125.7,124.1,123.3,121.1,111.4(d,J=19.2Hz), 109.2,103.7(t,J=25.8Hz),96.4(2C),69.2,54.1,53.3,49.4(2C),46.7,44.5,32.7,31.4, 29.4(3C),29.3,29.2,29.1,28.8,25.8,25.4(2C),22.5,11.7.

[0103] Preparation of 5-((14-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-14-oxotetradecyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 7)

[0104] Referring to the method for preparing Example 1, the raw material 8a in step a was replaced with compound 8g in equal proportion to obtain Example 7. 1H NMR (600MHz, DMSO-d6) δ12.03(s,1H),11.11(s,1H),10.22(s,1H),8.09(s,1H),7.82(d,J=8. 2Hz,1H),7.54–7.39(m,3H),7.33(d,J=8.2Hz,1H),7.19(s,1H),7.01(t,J=7.3Hz,1H),5.12( dd,J=12.7,5.0Hz,1H),4.64(s,1H),4.51(s,2H),4.15(t,J=5.9Hz,2H),3.99(d,J=12.5Hz,1 H),3.22–3.18(m,1H),2.90(d,J=12.9Hz,1H),2.73(s,1H),2.61–2.58(m,1H),2.35–2.31(m, 2H),2.18(s,3H),2.06–1.99(m,4H),1.88–1.84(m,2H),1.75–1.72(m,2H),1.63(s,1H), 1.54–1.50(m,2H),1.41–1.36(m,J=6.3Hz,2H),1.28–1.24(m,16H). 13 C NMR (151 MHz, DMSO-d6) δ173.2, 171.0, 170.4, 167.3, 167.2, 164.5, 161.5 (dd, J = 245.5, 8.5Hz), 160.7,160.6(dd,J=236.1,8.2Hz),155.3,153.8,134.4,132.3,131.9,130.1,129.9,125.7, 124.1,123.3,121.1,111.4(d,J=21.2Hz),109.2,103.7(t,J=25.7Hz),96.7,96.4,69.2, 54.2,53.2,49.4(2C),44.5,32.7,31.4,29.5,29.4(5C),29.3,29.2,29.1,28.8,25.8,25.4(2C),22.5,11.4.

[0105] Preparation of 5-((16-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-16-oxohexadecyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 8)

[0106] Referring to the method for preparing Example 1, the raw material 8a in step a was replaced with compound 8h in equal proportion to obtain Example 8. 1 H NMR(600MHz, DMSO-d6)δ 12.00(s,1H),11.11(s,1H),10.21(s,1H),8.09(s,1H),7.82(d,J=8.2Hz,1H),7.57–7.40 (m,3H),7.32(s,1H),7.19(s,1H),7.01(s,1H),5.12(dd,J=12.7,5.1Hz,1H),4.64(s,1H),4.51(s,2H),4.15(t,J=5.9Hz,2H),3.99( d,J=11.4Hz,1H),3.25–3.10(m,2H),2.89(t,J=12.9Hz,1H),2.73(s,1H),2.62–2.57(m,1H),2.34(d,J=7.7Hz,2H),2.18(s,3H),2.08 –1.94(m,4H),1.84(s,2H),1.72(d,J=6.5Hz,2H),1.50(s,2H),1.42–1.35(m,2H),1.32 –1.22(m,20H). 13 C NMR(151MHz,DMSO-d6)δ173.2,171.0,170.4,167.4,167.3,164.5, 161.5(dd,J=247.1,3.5Hz),160.8,160.6(dd,J=237.1,7.6Hz),155.2,154.3,134.4,131.9 ,130.1,129.9,129.1,125.7,124.2,123.3,121.1,111.4(d,J=21.7Hz),109.2,103.7(t,J= 25.5Hz),96.4,96.3,69.3,54.1,53.0,49.4(2C),44.6,32.8,31.4,29.5(3C),29.4(4C),29.3, 29.2,29.1,28.8,28.5,25.8,25.4(2C),22.6,11.4.

[0107] Preparation of 5-((18-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-18-oxooctadecyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 9)

[0108] Referring to the method for preparing Example 1, the raw material 8a in step a was replaced with compound 8i in equal proportion to obtain Example 9. 1 H NMR(600MHz,DMSO-d6)δ 12.00(s,1H),11.11(s,1H),10.26(s,1H),8.09(s,1H),7.82(d,J=8.3Hz,1H),7.33(dd,J= 8.3,2.3Hz,1H),7.23(s,1H),7.20(s,2H),7.15(s,1H),7.06(s,1H),7.01(t,J=8.1Hz,1H), 5.11(dd,J=12.9,5.4Hz,1H),4.64(s,1H),4.51(s,2H),4.24(t,J=5.2Hz,2H),4.15(t,J= 6.5Hz,2H),3.50(s,2H),3.23–3.15(m,2H),2.91–2.85(m,1H),2.75–2.72(m,1H),2.61 (d,J=1.8Hz,1H),2.34(d,J=7.8Hz,2H),2.22–2.16(m,4H),2.03–2.00(m,2H),1.99– 1.96(m,2H),1.89–1.79(m,4H),1.75–1.72(m,2H),1.51–1.47(m,2H),1.42–1.39(m,2H),1.36–1.34(m,2H),1.32–1.27(m,16H). 13 C NMR(151MHz,DMSO-d6)δ174.7, 173.2,171.0,170.4,167.3,167.2,165.5,164.5,161.6(dd,J=236.0,3.5Hz),160.3(dd,J= 237.2,5.3Hz),157.5,134.4,134.2,132.0(dd,J=37.2,10.3Hz),130.1,129.9,129.3(t,J= 11.3Hz),125.7,121.1,111.4(dd,J=22.3,3.7Hz),109.2,103.8(t,J=24.2Hz),96.3,95.9, 69.3,53.2,52.2,49.4(2C),44.5,35.5,31.7,30.3,29.5,29.4(3C),29.3,29.2,29.1,29.0,28.8, 27.0,25.8,25.6,25.4(2C),23.8,22.8,22.5,11.4.

[0109] 3) The preparation routes of Examples 10-13 are as follows:

[0110]

[0111] Embodiment 10The specific synthesis steps are as follows:

[0112] Synthesis of tert-butyl 3-(2-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)propoxy)ethoxy)propanoate (12d)

[0113] Compound 7 (0.15 g, 0.55 mmol) was dissolved in 3 mL DMF, and compound 11a (0.17 g, 0.55 mmol) and sodium carbonate (0.12 g, 1.09 mmol) were added in sequence, and then reacted at 40°C for 8 h. TLC detection (DCM: MeOH = 30: 1) showed that the raw materials reacted completely. After the reaction was cooled to room temperature, 30 mL of water was added, and ethyl acetate (10 mL × 3) was used for extraction three times. The organic layer was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. No further purification was required to obtain a colorless oily liquid with a yield of 88%.

[0114] Synthesis of 3-(2-(3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)oxy)propoxy)ethoxy)propanoic acid (13d)

[0115] Compound 12a (0.24 g, 0.50 mmol) was dissolved in 1 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 4 h. TLC monitoring (DCM:MeOH=15:1) showed that the reaction of the raw material was complete, and the mixture was dried by spin drying to obtain a white solid with a yield of 92%.

[0116] Preparation of 5-(3-(2-(3-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3-oxopropoxy)ethoxy)propoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 13)

[0117] Intermediate 13d (0.03 g, 0.06 mmol) was dissolved in 2 mL DMF, followed by intermediate 6 (0.02 g, 0.06 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.02 g, 0.06 mmol), and sodium bicarbonate (0.02 g, 0.23 mmol), and reacted at room temperature for 3 h. TLC detection (DCM:MeOH=10:1) showed that the raw materials reacted completely, 20 mL of water was added, and ethyl acetate (10 mL×3) was extracted three times. The organic layer was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. A white solid was obtained by thick preparative silica gel plate (DCM:MeOH=10:1) with a yield of 46%. 1 H NMR (600MHz, DMSO-d6) δ12.00(s,1H),11.11(s,1H), 10.20(s,1H),8.09(s,1H),7.81(d,J=8.3Hz,1H),7.58–7.36(m,4H),7.33(dd,J=8.3, 1.9Hz,1H),7.19(s,1H),7.01(t,J=7.1Hz,1H),5.11(dd,J=12.9,5.4Hz,1H),4.63(s, 1H),4.50(s,2H),4.22(t,J=6.1Hz,2H),4.08–3.96(m,2H),3.64(d,J=6.5Hz,2H),3.55 (d,J=6.2Hz,2H),3.52(s,4H),3.23–3.15(m,2H),2.88(dd,J=14.5,11.2Hz,1H),2.74(s, 1H),2.65–2.57(m,2H),2.19(s,3H),2.05–2.01(m,2H),1.99–1.96(m,2H),1.84(s,4H). 13C NMR (151MHz, DMSO-d6) δ173.2,170.4,169.1,167.3,167.2,164.4,161.6(dd,J= 245.9, 6.2Hz), 160.7, 160.6 (dd, J = 237.0, 7.1Hz), 155.3, 153.6, 134.3, 132.4, 132.0, 130. 1,129.9,125.8,124.2,123.4,121.1,111.4(d,J=20.5Hz),109.3,103.8(t,J=26.0Hz),96.4 (2C),70.1,70.0,67.4,67.1,66.4,53.9,49.4(2C),44.6,38.7,33.2,31.7,31.4,29.3(2C),22.5, 11.4.

[0118] Preparation of 5-(2-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 10)

[0119] Referring to the method for preparing Example 13, the raw material 11d in step a was replaced with compound 11a in equal proportion to obtain Example 10. 1 H NMR (600MHz, DMSO-d6) δ11.99(s,1H),11.11(s,1H),10.20(s,1H),8.06(s,1H),7.82(d,J=7.5Hz,1H),7.53– 7.33(m,5H),7.19(s,1H),7.01(s,1H),5.11(d,J=8.2Hz,1H),4.65(s,1H),4.52–4.44(m, 2H),4.36(s,2H),4.34–4.24(m,2H),3.97–3.83(m,4H),3.17(s,2H),2.87(t,J=12.2Hz, 1H),2.80(s,1H),2.61–2.56(m,2H),2.19(s,3H),2.08–1.97(m,2H),1.86(s,2H). 13C NMR (151MHz, DMSO-d6) δ173.2,170.4,167.5,167.3,167.2,164.3,161.5(dd,J=243.3,12.8Hz),160.8,160.6(dd,J=238.8,10.1Hz), 155.3,153.8,140.6,138.3,134.4,132.4,130.2,125.7,124.3,123.6,121.4,111.4(dd,J=22.2,3.3Hz),103.8(t,J=25.7Hz),96.5, 96.4,69.9,69.2,68.7,53.5,49.4(2C),43.9,40.8,31.4(2C),30.9,22.5,11.2.

[0120] Preparation of 5-(2-(3-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3-oxopropoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 11)

[0121] Referring to the method for preparing Example 13, the raw material 11d in step a was replaced with compound 11b in equal proportion to obtain Example 11. 1 H NMR (600MHz, DMSO-d6) δ12.02(s,1H),11.12(s,1H),10.26(s,1H),8.04(s,1H),7.82(d,J=8.3Hz,1H),7.49(s,1H ),7.44(d,J=1.8Hz,1H),7.36(dd,J=8.3,1.9Hz,1H),7.20(s,2H),7.01(t,J=7.6Hz,1H),5.11(dd,J=12.9,5.4Hz, 1H),4.64(s,1H),4.50(s,2H),4.33–4.31(m,2H),4.04(d,J=12.9Hz,1H),3.79(d,J=4.3Hz,2H),3.74(d,J=6.6Hz, 2H),3.22–3.12(m,2H),2.91–2.86(m,1H),2.76(t,J=10.7Hz,1H),2.70–2.63(m,2H),2.61–2.55(mi,1H),2.20(s, 3H),2.06–2.02(m,2H),1.89–1.74(m,4H). 13C NMR (151MHz, DMSO-d6) δ173.2, 170.4, 169.1, 167.3, 167.2, 164.4, 161.6 (dd, J=240.5, 4.9Hz), 161.2, 160.7, 160.1 (dd, J= 242.0,5.2Hz),155.1,134.3,132.4,131.9,130.1,128.7,125.7,123.9,123.5,121.3,111.4(d, J=21.1Hz),109.3,103.8(t,J=25.9Hz),96.4(2C),69.0,68.9,67.5,53.9,49.4(2C),44.6, 40.6,33.2,31.4(2C),26.8,22.5,11.3.

[0122] Preparation of 5-(2-(2-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 12)

[0123] Referring to the method for preparing Example 13, the raw material 11d in step a was replaced with compound 11c in equal proportion to obtain Example 12. 1 H NMR (600MHz, DMSO-d6) δ11.98(s,1H),11.09(s,1H),10.18(s,1H),8.06(s,1H),7.80(d,J=8.3Hz,1H),7.57– 7.43(m,3H),7.34(dd,J=8.3,2.1Hz,1H),7.18(s,1H),7.00(t,J=7.7Hz,1H),5.10(dd,J =12.9,5.4Hz,1H),4.63(s,1H),4.50(s,2H),4.32–4.30(m,2H),4.27–4.23(m 1H),4.19 –4.15(m,1H),3.93(d,J=12.7Hz,1H),3.81(t,J=4.2Hz,2H),3.64(s,2H),3.62–3.58 (m,2H),3.18–3.14(m,2H),2.90–2.85(m,1H),2.81–2.75(m,1H),2.60–2.56(m,1H), 2.18(s,3H),2.05–2.01(m,2H),1.89–1.79(m,4H). 13C NMR (151MHz, DMSO-d6) δ 173.2, 170.4, 167.5, 167.3, 167.2, 164.4, 161.5 (dd, J=244.3, 12.1Hz), 161.4, 160.4 (dd, J= 245.8,6.5Hz),159.1,155.3,138.4,134.3,132.4,131.8,129.7,125.7,124.3,123.5,121. 3,111.4(d,J=20.6Hz),109.3,103.8(t,J=25.8Hz),96.4(2C),70.3,70.2,69.9,69.0,68.9, 53.0,49.4(2C),43.9,40.8,31.4(2C),26.8,22.5,11.2.

[0124] 4) The preparation routes of Examples 14-22 are as follows:

[0125]

[0126] Embodiment 14 The specific synthesis steps are as follows:

[0127] Synthesis of tert-butyl 4-(4-(2-(4-(4-((1H-pyrazol-3-yl)amino)-6-((2,4-difluorobenzyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethyl)phenyl)piperazine-1-carboxylate (15a)

[0128] The intermediate 14a (0.10 g, 0.30 mmol) was dissolved in 2 mL of DMF, and then the intermediate 6 (0.13 g, 0.30 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.10 g, 0.30 mmol), and sodium bicarbonate (0.10 g, 1.20 mmol) were added in sequence, and the reaction was carried out at room temperature for 3 h. TLC detection (DCM:MeOH=15:1) showed that the raw materials reacted completely, 20 mL of water was added, and ethyl acetate (10 mL×3) was extracted three times. The organic layer was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The solid was obtained by thick preparative silica gel plate (DCM:MeOH=15:1) with a yield of 78%.

[0129] Synthesis of 1-(4-(4-((1H-pyrazol-3-yl)amino)-6-((2,4-difluorobenzyl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-(4-(piperazin-1-yl)phenyl)ethan-1-one (16a)

[0130] Compound 15a (0.15 g, 0.20 mmol) was dissolved in 1 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 4 h. TLC monitoring (DCM:MeOH=10:1) showed that the reaction of the raw material was complete, and the mixture was dried by spin drying to obtain a white solid with a yield of 90%.

[0131] Preparation of 5-(4-(4-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethyl)phenyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 14)

[0132] The intermediate 16a (0.04 g, 0.06 mmol) was dissolved in 2 mL DMSO, followed by 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (0.02 g, 0.07 mmol) and DIEA (0.05 g, 0.30 mmol), and the reaction was carried out at 100°C for 4 h. TLC detection (DCM: MeOH = 10: 1) showed that the raw material was completely reacted, 20 mL of water was added, and ethyl acetate (10 mL × 3) was used for extraction three times. The organic layer was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The solid was obtained by thick silica gel preparation (DCM: MeOH = 10: 1) with a yield of 58%. 1 H NMR (600 MHz, DMSO-d6) δ12.01(s,1H),11.09(s,1H),10.22(s,1H),8.02(s,1H),7.71(d,J=8.5 Hz,1H),7.47(s,2H),7.41(d,J=1.6Hz,1H),7.32(d,J=2.0Hz,1H),7.23–7.18(m,2H), 7.15–7.13(m,2H),7.00(t,J=7.9Hz,1H),6.95(d,J=8.4Hz,2H),5.08(dd,J=12.8,5.4 Hz,1H),4.62(s,1H),4.50(d,J=10.5Hz,2H),4.10(d,J=12.6Hz,1H),3.67(s,2H),3.62 –3.60(m,4H),3.29–3.26(m,4H),3.21–3.17(m,1H),2.92–2.87(m,1H),2.76(d,J= 11.3Hz,1H),2.61–2.54(m,2H),2.18(s,3H),2.05–1.96(m,4H),1.90–1.84(m,2H). 13C NMR (151MHz, DMSO-d6) δ174.8,173.3,170.6,169.6,168.0,167.5,161.6(dd,J=238.2, 12.0Hz),161.3,160.7(dd,J=243.7,3.0Hz),155.6,155.2,149.6,134.3,132.3,132.0,130.1 (2C),129.9(2C),129.1,127.0,125.4,124.6,119.0,118.4,116.2,111.4(d,J=20.5Hz), 108.5,103.8(t,J=26.1Hz),96.4(2C),63.3,53.9,49.3(2C),48.5(2C),47.2(2C),45.1,35.5, 31.4(2C),27.0,22.6,11.3.

[0133] Preparation of 5-(4-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 15)

[0134] Referring to the method for preparing Example 14, the raw material 14a in step a was replaced with compound 14b in equal proportion to obtain Example 15. 1 H NMR (600MHz, DMSO-d6) δ12.02(s,1H),11.08(s,1H),10.29(s,1H),8.09(s,1H),7.65(d,J=8.5Hz,1H),7.53(s,1H),7.3 1(d,J=1.6Hz,1H),7.26–7.18(m,3H),7.02(t,J=7.6Hz,1H),5.07(dd,J=12.8,5.4Hz,1H),4.66(s,1H),4.54(s,2H),4.0 3(s,2H),3.22(d,J=12.6Hz,1H),2.99(t,J=12.0Hz,2H),2.89–2.87(m,1H),2.77(d,J=13.7Hz,1H),2.62–2.54(m,2H), 2.34(d,J=6.7Hz,2H),2.20(s,3H),2.04–1.96(m,4H),1.85(d,J=7.5Hz,2H),1.79(d,J=11.6Hz,2H),1.30–1.24(m,4H).13 C NMR(151MHz,DMSO-d6)δ173.3,170.6,169.8,168.1,167.4,162.8, 161.6(dd,J=239.1,10.5Hz),160.7,160.6(dd,J=244.4,6.4Hz),155.4,155.1,134.5, 132.4,131.9,130.1,128.8,125.4,123.9,118.0,117.8,111.5(d,J=21.1Hz),108.2,103.8(t, J=25.8Hz),96.4(2C),54.0,49.2(2C),47.8(2C),44.7,40.6,38.9,33.0,31.7,31.3(2C),27.3(2C),22.7,11.4.

[0135] Preparation of 5-(3-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 16)

[0136] Referring to the method for preparing Example 14, the raw material 14a in step a was replaced with compound 14c in equal proportion to obtain Example 16. 1 H NMR(600MHz,DMSO-d6)δ 12.00(s,1H),11.06(s,1H),10.19(s,1H),8.05(s,1H),7.63(t,J=7.0Hz,1H),7.47(s,2H),7.21–7.14(m,3H),7 .01(s,1H),5.05(dd,J=12.7,5.5Hz,1H),4.65(s,1H),4.52(s,2H),4.11(q,J=5.2Hz,2H),4.04(d,J=13.6Hz,1H) ,3.67(d,J=9.8Hz,1H),3.52(d,J=8.4Hz,1H),3.42(t,J=8.2Hz,1H),3.26–3.20(m,2H),3.17(d,J=5.0Hz,4H),3. 09(d,J=10.5Hz,1H),2.86(d,J=4.6Hz,1H),2.79(t,J=12.0Hz,1H),2.18(s,3H),1.87(s,4H),1.47–1.44(m,2H). 13C NMR(151MHz,DMSO-d6)δ174.8,173.3,170.6,169.8,168.1, 167.7,166.0,161.5(dd,J=240.0,6.9Hz),160.7,160.6(dd,J=239.5,5.9Hz),152.3,134.4 ,131.9,130.1,128.8,128.2,125.4,124.6,115.9,115.6(d,J=3.4Hz),111.4(d,J=18.9Hz), 105.8,103.8(t,J=26.4Hz),96.5(2C),59.4,53.7,53.2,49.1,49.0(2C),47.7,44.5,35.6, 31.7(2C),27.0,25.6,22.6,11.3.

[0137] Preparation of 5-(3-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethyl)azetidin-1-yl)-2-(2,6-dioxapiperidin-3-yl)isoindoline-1,3-dione (Example 17)

[0138] Referring to the method for preparing Example 14, the raw material 14a in step a was replaced with compound 14d in equal proportion to obtain Example 17. 1 H NMR (600MHz, DMSO-d6) δ11.99 (s, 1H), 11.07 (s, 1H), 10.25 (s, 1H), 8.11–8.05 (m, 2H), 7.62 (d, J = 8.3Hz, 1H), 7.27 (s, 1H), 7.20(s,2H),7.19(s,1H),7.10(s,1H),7.01(t,J=8.2Hz,1H),5.05(dd,J=12.8,5.4Hz, 1H),4.66(s,1H),4.51(s,2H),4.23–4.18(m,2H),4.04–3.94(m,2H),3.70(dd,J=13.9, 7.8Hz,2H),3.17(s,2H),3.13(dd,J=7.4,4.3Hz,1H),2.87(d,J=8.2Hz,2H),2.62–2.59 (m,2H),2.39(dt,J=3.4,1.7Hz,1H),2.19(s,3H),1.89–1.83(m,5H).

[0139] Embodiment 18

[0140] Preparation of 5-(6-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-azaspiro[3.3]hept-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 18)

[0141] Referring to the method for preparing Example 14, the raw material 14a in step a was replaced with compound 14e in equal proportions to obtain Example 18. 1 H NMR (600MHz, DMSO-d6) δ12.02(s,1H),11.07(s,1H),10.28(s,1H),8.06(s,1H),7.63(d,J=8.1Hz ,1H),7.51(s,2H),7.31–7.12(m,3H),7.02(s,1H),6.75(s,1H),6.62(d,J=7.8Hz,1H),5.05(dd, J=12.4,5.1Hz,1H),4.65(s,1H),4.50(s,2H),4.07(s,2H),4.00(d,J=11.6Hz,1H),3.94(s,2H), 3.23–3.17(m,2H),2.87(t,J=13.1Hz,1H),2.75–2.72(m,1H),2.60(s,2H),2.38(d,J=5.5Hz,2H), 2.20(s,3H),2.07–1.94(m,6H),1.90–1.77(m,4H). 13 C NMR (151MHz, DMSO-d6) δ 173.3, 170.5, 169.9, 167.9, 167.6, 166.0, 161.6 (dd, J=240.1, 13.3Hz), 160.9, 160.7 (dd, J= 245.7,12.7Hz),155.5,155.2,134.2,132.3,131.9,130.1,128.8,125.2,124.1,117.2,114.7,111.4(d,J=20.9Hz),105.0,10 3.8(t,J=25.3Hz),96.4(2C),64.6,63.3,59.3,54.1,49.1(2C),44.6,39.0,35.6,31.7,31.4(2C),30.8,27.0,26.7(2C),11.3.

[0142] Preparation of 5-(3-((4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 19)

[0143] Referring to the method for preparing Example 14, the raw material 14a in step a was replaced with compound 14f in equal proportion to obtain Example 19. 1 H NMR(600MHz,DMSO-d6)δ 12.01(s,1H),11.07(s,1H),10.21(s,1H),8.09(s,1H),7.65(s,1H),7.55–7.39(m,2H), 7.21(s,1H),7.01(s,1H),6.93(s,1H),6.83(d,J=7.6Hz,1H),5.06(dd,J=12.7,5.4Hz, 1H),4.54(s,2H),4.40(s,1H),3.81–3.61(m,2H),3.53(s,1H),3.42–3.38(m,2H),3.17(d, J=5.1Hz,2H),2.89(ddd,J=16.6,13.8,5.2Hz,2H),2.63–2.52(m,4H),2.18(s,5H),2.04–1.99(m,2H),1.82(s,2H),1.29–1.23(m,2H). 13 C NMR(151MHz,DMSO-d6)δ173.3, 170.6,168.1,167.7,161.5(dd,J=244.7,5.4Hz),160.7,160.6(dd,J=241.3,5.3Hz),158.5,1 55.5,152.2,138.3,134.4,132.1,131.9,129.9,125.4,124.4,116.1,115.7,111.4(d,J=22.4 Hz), 105.9, 103.8 (t, J = 25.8Hz), 96.3 (2C), 65.4, 52.6 (2C), 51.7, 49.2 (2C), 47.6, 40.5, 38.2, 31.5 (2C), 29.8, 28.8, 22.7, 11.4.

[0144] Preparation of 5-(3-((4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)methyl)azetidin-1-yl)-2-(2,6-dioxapiperidin-3-yl)isoindoline-1,3-dione (Example 20)

[0145] Referring to the method for preparing Example 14, the raw material 14a in step a was replaced with compound 14g in equal proportion to obtain Example 20. 1 H NMR(600MHz, DMSO-d6)δ12.02 (s,1H),11.08(s,1H),10.21(s,1H),8.09(s,1H),7.66(d,J=7.0Hz,1H),7.55–7.42(m, 2H),7.22(s,1H),7.02(s,1H),6.79(s,1H),6.66(d,J=7.3Hz,1H),5.06(dd,J=12.1,4.3 Hz,1H),4.63–4.52(m,3H),4.22(s,2H),3.84(s,2H),2.93–2.84(m,2H),2.63–2.57(m, 2H),2.52(s,2H),2.42–2.31(m,2H),2.26–2.13(m,6H),2.07–1.93(m,4H). 13 C NMR (151MHz, DMSO-d6)δ173.3,170.6,167.9,167.6,161.6(dd,J=245.9,6.6Hz),160.8, 160.4(dd,J=240.6,7.4Hz),155.5,153.9,148.3,134.2,132.6,131.9,130.1,128.8,125.3, 124.2,117.5,114.7,111.5(d,J=21.2Hz),104.9,103.8(t,J=25.6Hz),96.4(2C) ,59.4,56.0(2C),51.7,49.2(2C),46.6,38.2,31.5(2C),29.5,27.0,22.7,11.4.

[0146] Preparation of 5-(4-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 21)

[0147] Referring to the method for preparing Example 14, the raw material 14a in step a was replaced with compound 14h in equal proportion to obtain Example 21. 1 H NMR(600MHz,DMSO-d6)δ11.99(s,1H),11.08(s,1H),10.20(s,1H),8.06(s ,1H),7.67(d,J=8.5Hz,1H),7.47(s,2H),7.34(s,1H),7.24(d,J=8.4Hz,1H ),7.22–7.17(m,1H),7.06–7.01(m,1H),5.07(dd,J=12.8,5.4Hz,1H),4.6 7(s,1H),4.52(s,2H),4.19(d,J=12.3Hz,1H),4.07(d,J=12.2Hz,2H),3.13 –3.03(m,4H),2.91–2.86(m,1H),2.77(d,J=5.0Hz,1H),2.62–2.55(m,2H),2.18(s, 3H),2.07–1.91(m,4H),1.84(s,2H),1.74(d,J=11.9Hz,2H),1.67–1.60(m,2H). 13 C NMR (151MHz, DMSO-d6) δ173.3,172.6,170.5,168.0,167.4,161.5(dd,J=245.3,4.3 Hz),160.7,160.2(dd,J=239.8,2.7Hz),159.9,158.5,155.3,138.4,134.5,132.5,131.9, 130.3,129.7,125.5,124.3,118.0,111.5(dd,J=21.8,2.3Hz),108.3,103.8(t,J=24.2Hz), 96.4 (2C), 54.4, 49.2 (2C), 47.2, 44.4, 40.9, 37.3, 32.0, 31.4, 30.9, 29.4 (2C), 28.0, 22.6, 11.2. Preparation of 4-(4-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (Example 22)

[0148] Referring to the method for preparing Example 21, the 2-(2,6-dioxopiperidine-3-yl)-5-fluoroisoindoline-1,3-dione in step c is replaced with 2-(2,6-dioxopiperidine-3-yl)-4-fluoroisoindoline-1,3-dione in equal proportion to obtain Example 22. 1 H NMR(600MHz,DMSO-d6)δ12.01(s,1H),11.09(s,1H),10.22(s,1H),8.06(s,1H),7.70–7.65(m,1H),7.58–7.43(m,2H),7.3 3(t,J=6.9Hz,1H),7.30(t,J=7.0Hz,1H),7.21(s,1H),7.02(t,J=7.7Hz,1H),5.11(dd,J=12.7,5.5Hz,1H),4.68(s,1H),4. 55–4.48(m,3H),4.17(d,J=12.4Hz,1H),3.73(d,J=10.2Hz,2H),3.46(s,1H),3.27(t,J=12.6Hz,1H),3.00(t,J=12.7Hz,2 H),2.92–2.86(m,1H),2.77(t,J=10.9Hz,1H),2.62–2.53(m,1H),2.32–2.23(m,1H),2.19(s,3H),2.10–2.01(m,2H),1.96– 1.88(m,2H),1.85–1.75(m,5H). 13 C NMR (151MHz, DMSO-d6) δ173.3,172.8,170.5, 167.5,166.7,161.5(dd,J=241.3,10.9Hz),161.1,160.6(dd,J=239.4,15.4Hz),155.2, 153.8,150.4,136.2,134.1,132.4,131.9,130.1,124.3,123.8,117.8,116.8,114.8(d,J=25.7 Hz),111.4(d,J=20.2Hz),103.8(t,J=25.8Hz),96.9,96.4,53.9,51.7( 2C),49.3(2C),44.4,40.9,37.1,31.0(2C),28.9(2C),24.0,22.5,11.4.

[0149] 5) The preparation routes of Examples 23-25 ​​are as follows:

[0150]

[0151] The specific synthesis steps of Example 23 are as follows:

[0152] Synthesis of tert-butyl 6-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-6-oxohexanoate (18a)

[0153] Intermediate 17a (0.12 g, 0.60 mmol) was dissolved in 2 mL DMF, followed by intermediate 6 (0.20 g, 0.60 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.20 g, 0.60 mmol), and sodium bicarbonate (0.20 g, 2.40 mmol), and the reaction was carried out at room temperature for 3 h. TLC detection (DCM:MeOH=15:1) showed that the raw materials reacted completely, 20 mL of water was added, and ethyl acetate (10 mL×3) was extracted three times. The organic layer was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. Solid was obtained by thick preparative silica gel plate (DCM:MeOH=15:1) with a yield of 74%.

[0154] Synthesis of 6-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-6-oxohexanoic acid (19a)

[0155] Compound 18a (0.17 g, 0.30 mmol) was dissolved in 1 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 4 h. TLC monitoring (DCM:MeOH=10:1) showed that the reaction of the raw material was complete, and the mixture was dried by spin drying to obtain a white solid with a yield of 92%.

[0156] Preparation of (2S,4R)-1-((S)-2-(6-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-6-oxohexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Example 23) Intermediate 19a (0.03 g, 0.06 mmol) was dissolved in 2 mL DMF, followed by (2S, 4R)-1-((S)-2-amino-3, 3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (0.03 g, 0.06 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.02 g, 0.06 mmol), and sodium bicarbonate (0.02 g, 0.23 mmol), and the mixture was reacted at room temperature for 3 h. TLC detection (DCM:MeOH=10:1) showed that the reaction of the raw materials was complete, 20 mL of water was added, and the mixture was extracted three times with ethyl acetate (10 mL×3). The organic layer was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The solid was obtained by thick silica gel preparation (DCM:MeOH=10:1) with a yield of 69%. 1 H NMR (600MHz, DMSO-d6) δ12.01(s,1H),10.22(s,1H),8.98(s,1H),8.37(d,J=7.7Hz,1H),8.05(s,1H),7.81(d,J=9.2Hz,1H),7.52–7.36(m,6H) ,7.20(s,1H),7.01(t,J=7.4Hz,1H),5.10(s,1H),4.95–4.90(m,1H),4 .64(s,1H),4.53(d,J=9.3Hz,2H),4.50–4.48(m,1H),4.43(t,J=8.0Hz, 1H),4.28(s,1H),4.01(d,J=12.2Hz,1H),3.64–3.58(m,2H),3.20(t,J=12.3Hz,1H), 2.74(s,1H),2.46(s,2H),2.36(s,3H),2.21–2.15(m,3H),2.03–1.98(m,2H),1.89–1.78 (m,4H),1.57–1.47(m,4H),1.38(d,J=7.0Hz,3H),1.30–1.20(m,3H),0.94(s,9H). 13C NMR (151MHz, DMSO-d6) δ172.4, 171.1, 170.9, 170.1, 161.5 (dd, J = 245.1, 12.6Hz), 160.9,160.6(dd,J=240.5,15.9Hz),160.4,155.3,151.9,148.2,145.1,138.4,132.3,131.5, 130.1,130.0,129.2(2C),126.8(2C),126.7,124.2,111.4(d,J=20.6Hz),103.8(t,J=25.7 Hz),96.4(2C),69.2,59.0,56.8,56.7,53.9,48.1(2C),44.5,38.2,35.7,35.2,32.5,26.9(3C),26.8(2C),25.7,25.1,22.9,16.4,11.7.

[0157] Embodiment 24

[0158] Preparation of (2S,4R)-1-((S)-2-(8-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-8-oxooctylamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Example 24) Referring to the method for preparing Example 23, 17a in step a is replaced by 17b in equal proportion to obtain Example 24. 1H NMR(600MHz,DMSO-d6)δ11.98(s, 1H),10.20(s,1H),8.98(s,1H),8.37(d,J=7.8Hz,1H),8.05(s,1H),7.79(d,J=9.3Hz, 1H),7.50(s,2H),7.42(d,J=8.1Hz,2H),7.38(d,J=8.1Hz,2H),7.20(s,1H),7.02(t,J=7.5Hz,1H),5.10(d,J=3.4Hz,1H),4.93–4.90(m,1H),4.64(s,1H),4.52(d,J=9.4Hz,2H),4.48(d,J=2.9Hz,1H),4.43(t,J=8.0Hz,1H),4.28(d,J=2.2Hz,1H),3.99(d,J=11.8Hz,1H),3.61(s,2H),3.22–3.17(m,2H),2.73(s,1H),2.45(s,3H),2.34(s,2H),2.28–2.25(m,1H),2.18(s,3H),2.12(d,J=7.2Hz,1H),2.03–1.99(m,2H),1.81(d,J=4.6Hz,2H),1.53–1.46(m,6H),1.37(d,J=7.0Hz,3H),1.29–1.24(m,4H),0.94(s,9H). 13 C NMR(151MHz,DMSO-d6)δ172.6,171.1,171.0,170.1,161.7(dd,J=254.2,18.8Hz), 160.9,160.3(dd,J=252.0,22.4Hz),158.2,155.3,151.9,148.2,145.1,138.4,132.3,131.5,130.1,130.0,129.3(2C),126.8(2C),126.7,121.2,111.4(d,J=20.3Hz),103.8(t,J=28.0 Hz),96.4(2C),69.2,59.0,56.8,56.7,54.0,48.1(2C),44.6,38.2,35.6,35.3,32.7,29.0,28.9, 26.9(3C),26.8(2C),25.8,25.3,22.9,16.4,11.1.

[0159] Preparation of (2S,4R)-1-((S)-2-(10-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-10-oxodecanoylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxylic acid (Example 25) Referring to the method for preparing Example 23, 17a in step a is replaced with 17c in equal proportion to obtain Example 25. 1 H NMR (600MHz, DMSO-d6) δ11.99 (s, 1H), 10.17 (s, 1H), 8.98 (s, 1H), 8.37 (d, J = 7.8Hz, 1H), 8.06 (s, 1H), 7.78 (d, J = 9.3Hz, 1H),7.51–7.42(m,4H),7.38(d,J=8.1Hz,2H),7.19(s,1H),7.01(s,1H),5.10(d,J=3.4 Hz,1H),4.93–4.90(m,1H),4.64(s,1H),4.52(d,J=9.3Hz,2H),4.42(s,1H),4.28(s,1 H),3.99(d,J=12.6Hz,1H),3.61(d,J=4.8Hz,2H),3.23–3.17(m,2H),2.73(t,J=13.3Hz ,2H),2.45(s,3H),2.34(s,2H),2.27–2.22(m,1H),2.18(s,3H),2.12–2.10(m,1H),2.0 2–1.98(m,2H),1.85–1.78(m,4H),1.52–1.49(m,4H),1.37(d,J=7.0Hz,3H),1.27–1.24 (m,8H),0.93(s,9H). 13C NMR(151MHz,DMSO-d6)δ172.0,170.5,170.4,169.5,161.1, 161.0(dd,J=244.6,9.9Hz),160.0(dd,J=242.2,8.4Hz),154.8,151.4,147.6,144.6,140.2,1 31.8,131.4,131.0,129.6,129.5,128.7(2C),126.3(2C),126.2,123.6,110.9(d,J=20.6Hz), 103.2(t,J=25.2Hz),95.9(2C),68.6,58.4,56.2,56.1,52.7,47.6(2C),44.0,37.6,35.1,34.8, 32.2,30.5,28.7(2C),28.6,28.5,26.4(2C),26.3(3C),25.3,24.8,22.3,15.9,10.6.

[0160] 6) The preparation routes of Examples 26-27 are as follows:

[0161]

[0162] Example 26 The specific synthesis steps are as follows:

[0163] Synthesis of tert-butyl 2-(3-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3-oxopropoxy)acetate (21a)

[0164] The intermediate 20a (0.12 g, 0.60 mmol) was dissolved in 2 mL of DMF, followed by the intermediate 6 (0.20 g, 0.60 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.20 g, 0.60 mmol), and sodium bicarbonate (0.20 g, 2.40 mmol), and the reaction was carried out at room temperature for 3 h. TLC detection (DCM:MeOH=15:1) showed that the raw materials reacted completely, 20 mL of water was added, and ethyl acetate (10 mL×3) was extracted three times. The organic layer was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The solid was obtained by thick silica gel preparation (DCM:MeOH=15:1) with a yield of 80%.

[0165] Synthesis of 2-(3-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3-oxopropoxy)acetic acid (22a)

[0166] Compound 21a (0.30 mmol) was dissolved in 1 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added, and the mixture was reacted at room temperature for 4 h. TLC monitoring (DCM: MeOH = 10: 1) showed that the reaction of the raw materials was complete and the mixture was spin-dried to obtain a white solid with a yield of 90%. Preparation of (2S, 4R)-1-((S)-2-(6-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-6-oxohexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (Example 26) Intermediate 22a (0.03 g, 0.06 mmol) was dissolved in 2 mL of DMF, followed by (2S, 4R)-1-((S)-2-amino-3, 3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (0.03 g, 0.06 mmol), 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (0.02 g, 0.06 mmol), and sodium bicarbonate (0.02 g, 0.23 mmol), and the mixture was reacted at room temperature for 3 h. TLC was performed (DCM:MeOH=10:1). The reaction of the raw materials was complete, 20 mL of water was added, and the mixture was extracted three times with ethyl acetate (10 mL×3). The organic layer was collected, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated. The solid was obtained by thick silica gel preparation (DCM:MeOH=10:1) with a yield of 73%. 1H NMR(600MHz,DMSO-d6)δ12.00(s, 1H),10.17(s,1H),8.98(s,1H),8.43(d,J=7.5Hz,1H),8.07(s,1H),7.50–7.41(m,5H), 7.34(d,J=8.1Hz,2H),7.20(s,1H),7.00(s,1H),5.15(s,1H),4.90–4.85(m,1H),4.67(d, J=7.4Hz,1H),4.57–4.50(m,3H),4.45(t,J=8.2Hz,2H),4.27(d,J=13.1Hz,2H),3.99 (d,J=5.0Hz,2H),3.66(d,J=4.7Hz,4H),3.60(dd,J=11.1,8.1Hz,2H),3.22–3.17(m, 1H),2.81(s,1H),2.45(s,3H),2.18(s,3H),2.07–2.03(m,2H),1.88(s,2H),1.79–1.75(m, 1H),1.40–1.36(m,1H),1.33(d,J=6.8Hz,3H),0.94(s,9H). 13 C NMR(151MHz, DMSO-d6)δ170.9,169.4,168.9,167.6,161.5(dd,J=245.5,8.1Hz),161.4,160.4(dd,J=243.1,9.2Hz),155.3,153.8,151.9,148.2,145.1,138.2,132.4,131.5,130.1,129.3,129.2 (2C),126.8,126.7(2C),124.2,111.4(d,J=21.5Hz),103.8(t,J=26.0Hz),97.5,96.4,70.7, 70.1,69.8,69.2,59.0,57.0,56.2,53.7,48.2(2C),43.8,40.8,38.2,36.2,26.8(2C),26.7(3C), 22.9,16.4,11.3.

[0167] Preparation of (2S,4R)-1-((S)-2-(2-(3-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3-oxopropoxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl) (Example 27) Referring to the method for preparing Example 26, 20a in step a is replaced with 20b in equal proportion to obtain Example 27. 1 H NMR (600MHz, DMSO-d6) δ12.01(s,1H), 10.24(s,1H),8.98(s,1H),8.42(d,J=7.6Hz,1H),8.07(s,1H),7.56(d,J=9.3Hz,1H), 7.51(s,1H),7.45–7.40(m,3H),7.36(d,J=8.1Hz,2H),7.21–7.17(m,1H),7.01(s,1H), 5.14(s,1H),4.92–4.89(m,1H),4.71–4.66(m,1H),4.60–4.47(m,5H),4.45(dd,J=8.8, 4.0Hz,1H),4.29(s,1H),4.05(d,J=12.6Hz,1H),3.97(s,2H),3.79–3.74(m,2H),3.61(d, J=3.8Hz,2H),3.25(t,J=12.6Hz,1H),2.78(t,J=10.9Hz,1H),2.75–2.69(m,2H),2.45 (s,3H),2.20(s,3H),2.07–2.03(m,2H),1.93–1.78(m,5H),1.52–1.44(m,1H),1.39(s,1H),1.36(d,J=6.9Hz,3H),0.97(s,9H). 13C NMR (151MHz, DMSO-d6) δ171.0,169.5, 169.1,168.9,161.5(dd,J=243.0,11.3Hz),160.9,160.7(dd,J=243.7,9.4Hz),155.2, 153.8,151.9,148.2,145.2,138.4,132.4,131.5,130.1(2C),129.3,129.2(2C),126.8(2C) ,124.1,111.4(d,J=20.9Hz),103.8(t,J=25.9Hz),96.8,96.4,70.1,69.3,67.8(2C),59.0, 56.9,56.4,55.3,53.8,48.2(2C),44.5,40.7,38.2,36.0,32.9,26.8(2C),26.7(3C),22.9,16.4, 11.3.

[0168] Example 28: Study on in vitro enzyme inhibition activity of some products of the present invention

[0169] Experimental Materials:

[0170] Tecan ELISA reader.

[0171] 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).

[0172] Recombinant human PLK4 protein kinase concentration 50ng / μL (Thermo Scientific: PV6395), distilled water, DMSO.

[0173] Experimental methods:

[0174] 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 the test requirements; then the compound samples (4 μL) were added to the 384-well plate, and then 8 μL of kinase buffer solution containing recombinant human PLK4 kinase (concentration of 50 ng / μL) and Eu-Anti-GST Antibody, and 4 μL of kinase buffer solution containing Tracer 236 were added, and then incubated at room temperature for 60 minutes and the plate was read.

[0175] 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 replacing the tracer with a kinase inhibitor 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 filter with a 10nm bandpass. 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%):

[0176]

[0177] 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:

[0178]

[0179] Calculation IC 50 : DF% of kinase activity in the presence of added compounds is plotted on the Y-axis and the logarithmic concentration of the compound is plotted on the X-axis. IC 50 The value is obtained by fitting the data into an S-shaped stoichiometric response curve. Centrinone was used as a positive control to test the IC 50 The value is 0.003 μM.

[0180] Table 1 Example Compound IC 50 value

[0181] Example <![CDATA[IC 50 (μM)]]> Example <![CDATA[IC 50 (μM)]]> Example 1 0.0056 Example 2 0.0015 Example 3 0.0055 Example 4 0.0422 Example 5 1.0820 Example 6 0.5315 Example 7 0.1538 Example 8 0.1213 Example 9 0.0197 Example 10 0.0020 Embodiment 11 0.0036 Example 12 0.0094 Embodiment 13 0.0379 Embodiment 14 0.2156 Embodiment 15 0.0191 Example 16 0.0081 Embodiment 17 0.0128 Embodiment 18 0.0611 Embodiment 19 0.0136 Embodiment 20 0.0101 Embodiment 21 0.0084 Embodiment 22 0.0195 Embodiment 23 0.0141 Embodiment 24 0.0241 Embodiment 25 0.0207 Embodiment 26 0.0095 Embodiment 27 0.0158

[0182] Example 29: Study on the in vitro PLK4 protein degradation activity of some products of the present invention

[0183] Experimental methods:

[0184] Step 1: Cell sample processing

[0185] ⑴ Add 6x10^5 MCF-7 cells to each well of a large dish and culture for 24 hours. Add drugs and culture for 48 hours.

[0186] ⑵ Add 1 mL of trypsin to each well of cells, lay them flat, then gently shake to wash the cells, and add 2 mL of culture medium.

[0187] ⑶ Use a pipette to transfer the cells to a 15mL centrifuge tube at 1000r / min for 5min.

[0188] (4) Resuspend the cell cake in 1 mL PBS and transfer the cell suspension to a 1.5 mL EP tube at 3000 r / min for 5 min.

[0189] Step 2: Lyse cells

[0190] ⑴ Calculate the amount of lysis buffer, add 10 μL PMSF (100 mM) per mL of lysis buffer, shake well and place on ice.

[0191] ⑵ Aspirate the PBS in each EP tube, then add 100 μL of lysis buffer to each EP tube cell, ultrasonically disrupt the cells for 2 s × 3 times, and place on ice for 30 minutes.

[0192] ⑶ Centrifuge at 4°C, 12000r / min for 15min.

[0193] (4) The supernatant obtained after centrifugation is the desired protein sample, which can be divided into 1.5 mL centrifuge tubes and stored in a -80°C refrigerator for later use, or protein quantification can be performed directly.

[0194] Step 3: BCA protein quantification

[0195] ⑴ Take 1.2mL of protein standard solution and add it to 1 tube of protein standard (30mg BSA), and prepare 25mg / mL protein standard solution after fully dissolving. Use immediately after preparation or store at -20℃ for a long time after aliquoting.

[0196] ⑵ Take an appropriate amount of 25 mg / mL protein standard and dilute it to a final concentration of 0.5 mg / mL.

[0197] (3) Prepare 0.5, 0.25, 0.1, 0.05, 0.025, and 0.0125 mg / mL BSA standard solutions by gradient dilution method.

[0198] (4) After diluting the protein supernatant of each group 50 times with TBS, take 20 μL of the diluted supernatant into a 96-well plate.

[0199] ⑸ According to the number of samples, prepare an appropriate amount of BCA working solution with a volume ratio of BCA reagent A to reagent B of 50:1. Prepare it before use and mix it thoroughly.

[0200] ⑹ Add 200 μL BCA working solution to each well and incubate at 37°C for 30 min.

[0201] ⑺The absorbance of the samples was detected by ELISA at a wavelength of 562 nm, and the protein concentration of each group was calculated based on the standard curve.

[0202] Step 4: Protein sample preparation

[0203] ⑴ Quantify the protein supernatant of each group to 2μg / μL using TBS and 5x loading buffer.

[0204] (2) Heat at 100℃ for 15 min to denature the protein and place in a -80℃ refrigerator for later use.

[0205] Step 5: VSDS-PAGE electrophoresis

[0206] ⑴ Before making glue, check the glass plate and clean it carefully to avoid affecting the quality of the glue.

[0207] ⑵ Prepare separation gel according to the formula (see Table 1 for the formula) as needed, mix thoroughly and quickly add the gel to the glass plate. The height of the gel added should be about 1.5 cm from the upper edge of the glass plate. Then immediately add 200 μL of isopropanol to seal the gel. After 1 hour, wait for the lower layer of gel to solidify and wash off the isopropanol with distilled water.

[0208] Table 1 SDS-PAGE separation gel formula (8%)

[0209]

[0210] ⑶ Prepare the laminating glue according to the formula as needed (see Table 2 for the formula), quickly add it to the glass plate, then insert the comb teeth (pore former), wait for 45 minutes for the upper layer of glue to solidify, and store it in a sealed container at 4°C for later use.

[0211] Table 2 SDS-PAGE stacking gel formula (5%)

[0212]

[0213] (4) Dilute 10× electrophoresis buffer to 1× working concentration.

[0214] ⑸ Assemble the electrophoresis device: Take out the glass plate with glue, carefully pull out the comb to avoid shaking. Assemble with the electrophoresis device that matches the glass plate. After assembly, pour the electrophoresis buffer into the inner tank until the liquid level is flush with the glass plate. After confirming that there is no leakage, continue to add electrophoresis buffer to the outer tank until the platinum wire is immersed.

[0215] ⑹ Sample loading / electrophoresis: Pipette the protein marker and quantitative protein sample into the sample well. Turn on the power supply, the voltage is 70V for the stacking gel, wait for the front edge of the sample to electrophoresed to the boundary between the two gels, the markers are separated, and change the voltage to 120V for the separation gel. When the bromophenol blue band at the front edge of the electrophoresis moves to the bottom of the glass plate, it indicates that the electrophoresis is complete, and the power supply can be turned off.

[0216] ⑺Prepare 1x transfer buffer, blocking solution (5% skim milk powder / TBS-T), filter paper and PVDF membrane.

[0217] ⑻ Transfer: Soak the prepared transfer clip, liner, PVDF membrane cut to specifications (pre-activated in methanol for 30 s) and 2 layers of filter paper in the transfer solution to remove bubbles. Place the liner, 1 layer of filter paper, gel, PVDF membrane, 1 layer of filter paper, and liner on the black side of the clip in order to avoid bubbles. Pay strict attention to the order of placing each item and carefully close the clip. Place it in the transfer tank. Fill it with transfer solution and place an ice box. Turn on the power and place the transfer tank in a foam box filled with ice. Transfer at a constant current of 200mA for 1-3h (depending on the molecular weight of the protein).

[0218] ⑼ Blocking: After the transfer time is up, soak the PVDF membrane in the blocking solution, mark the marker position, place it on a shaker, and block it at room temperature for 2 hours.

[0219] ⑽Prepare primary antibodies: dilute PLK4 antibody at 1:1000 in blocking solution, and dilute β-actin antibody at 1:2000 in blocking solution. Incubate at 4°C overnight.

[0220] ⑾Wash the membrane with TBS-T for 15 min each time, for 3 times.

[0221] ⑿ Prepare secondary antibody, dilute the secondary antibody with blocking solution at 1:10000, and incubate at room temperature for 2 hours.

[0222] ⒀Wash the membrane with TBS-T for 15 min each time, for 3 times.

[0223] Step 6: Development

[0224] ⑴ Prepare an appropriate amount of ECL luminescence working solution according to the ratio of A solution: B solution = 1:1.

[0225] ⑵ Develop with a developer. Scan the film and analyze the optical density of the target band using a gel image processing system (Gel-Pro-Analyzer software) (see Figure 1-4 ).

[0226] Depend on Figure 1-3 It can be seen that compounds 2, 3, and 4 have certain in vitro PLK4 degradation activity; Figure 4 It can be seen that Example 21 exhibits potent in vitro PLK4 degradation activity, DC 50 Reached 19.5±8.5nM.

Claims

1. A compound of formula I and a pharmaceutically acceptable salt thereof, characterized in that: The compound shown in general formula I is as follows: Wherein, ring A is an aliphatic heterocyclic ring containing 4 to 12 atoms; Ring B is , ; o is 0 or 1; p is 0 or 1; q is 0, 1 or 2; The L part of the ring is an aliphatic heterocyclic ring containing 4-12 atoms, , , , ; n is an integer between 0 and 20; r is an integer between 0 and 4; s is an integer between 0 and 6; t is an integer between 0 and 6; u is an integer between 0 and 20; v is an integer between 0 and 4; w is an integer between 0 and 4; The aliphatic heterocycle contains 1-3 heteroatoms of N, O or S. When the heteroatom is S, the sulfur atom can be further oxidized to sulfoxide or sulfone.

2. The compound of general formula I and its pharmaceutically acceptable salt according to claim 1, characterized in that: in, Ring A is a piperidine ring; Ring B is , ; o is 0 or 1; p is 0 or 1; q is 0, 1 or 2; The L ring is a piperazine ring, a piperidine ring, a pyrrole ring, an azetidine ring, a 2-azaspiro[3.3]heptyl ring, , , , ; n is an integer between 0 and 20; r is an integer between 0 and 4; s is an integer between 0 and 6; t is an integer between 0 and 6; u is an integer between 0 and 20; v is an integer between 0 and 4; and w is an integer between 0 and 4.

3. The compound of general formula I and its pharmaceutically acceptable salt according to claim 2, characterized in that: in, Ring A is a piperidine ring; Ring B is , ; o is 0 or 1; p is 0 or 1; q is 0 or 1; The L ring is a piperazine ring, a piperidine ring, a pyrrole ring, an azetidine ring, a 2-azaspiro[3.3]heptyl ring, , , , ; n is 0, 2, 4, 6, 8, 10, 12, 14 or 16; r is 0 or 1; s is 0 or 1; t is 0 or 1; u is 4, 6 or 8; v is 1; and w is 0 or 1.

4. The compound of general formula I and its pharmaceutically acceptable salt according to claim 3, characterized in that: The compound is: 5-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-(4-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-4-oxobutoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-((6-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-6-oxohexyl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-((8-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-8-oxooctyl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-((10-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-10-oxodecanyl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-((12-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-12-oxododecanyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-((14-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-14-oxotetradecyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-((16-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-16-oxohexadecyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-((18-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-18-oxooctadecyl)oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-(2-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-(2-(3-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3-oxopropoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-(2-(2-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethoxy)ethoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-(3-(2-(3-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3-oxopropoxy)ethoxy)propoxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-(4-(4-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethyl)phenyl)piperazin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-(4-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-(3-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-(3-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-oxoethyl)azetidin-1-yl)-2-(2,6-dioxapiperidin-3-yl)isoindoline-1,3-dione 5-(6-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-2-azaspiro[3.3]hept-2-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-(3-((4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)methyl)pyrrolidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 5-(3-((4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)methyl)azetidin-1-yl)-2-(2,6-dioxapiperidin-3-yl)isoindoline-1,3-dione 5-(4-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 4-(4-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidine-1-carbonyl)piperidin-1-yl)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (2S,4R)-1-((S)-2-(6-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-6-oxohexanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (2S,4R)-1-((S)-2-(8-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-8-oxooctylamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxamide (2S,4R)-1-((S)-2-(10-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-10-oxodecanoylamino)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxylic acid (2S,4R)-1-((S)-2-(2-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3-oxopropoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine (2S,4R)-1-((S)-2-(2-(3-(4-(6-((2,4-difluorobenzyl)amino)-4-((5-methyl-1H-pyrazol-3-yl)amino)-1H-pyrazolo[3,4-d]pyrimidin-1-yl)piperidin-1-yl)-3-oxopropoxy)ethoxy)acetamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl).

5. A pharmaceutical composition, characterized in that: The invention comprises the compound of general formula I as claimed in claim 1 and a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutically acceptable excipient.

6. The pharmaceutical use of the compound of general formula I according to claim 1 and its pharmaceutically acceptable salt or the pharmaceutical composition according to claim 5, characterized in that: Use of the compound of general formula I and its pharmaceutically acceptable salt or the pharmaceutical composition according to claim 5 in the preparation of drugs for preventing or treating diseases related to PLK4 kinase inhibition.

7. The pharmaceutical use according to claim 6, characterized in that: Use of the compound of general formula I and its pharmaceutically acceptable salt or the pharmaceutical composition according to claim 5 in the preparation of preventive or anti-tumor drugs.

8. The pharmaceutical use according to claim 7, characterized in that: Use of the compound of general formula I and its pharmaceutically acceptable salt or the pharmaceutical composition according to claim 5 in the preparation of a PLK4 degrader.

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