2-amino-pyrimidines containing 2-chloroacrylamide, process for their preparation and use

Abstract

The application provides a 2-amino pyrimidine compound containing 2-chloro acrylamide, a preparation method and application thereof, and particularly provides a compound shown in formula I, which has good CDK7 inhibiting activity, can be used as a small molecule selective covalent CDK7 inhibitor, and is used for treating tumors, for example, colorectal cancer, breast cancer, human biphenotypic acute myeloid leukemia and human acute myeloid leukemia.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular, the present application relates to a new 2-amino pyrimidine compound containing 2-chloro acrylamide, its preparation method and application, and the application of the compound in the preparation of tumor prevention and treatment drugs. BACKGROUND

[0002] CDK7 is a kind of protein kinase with regulatory effect existing in vivo, and is polymerized into ternary complex CAK with Cyclin H and MAT1, and is involved in the regulation of cell cycle and transcription cycle by phosphorylation. In cell cycle regulation, CDK7 ensures that different CDKs have kinase activity at the right time through phosphorylation regulation, thereby promoting the progress of cell cycle; in the transcription cycle, CDK7 itself is an important subunit of transcription initiation complex TFIIH, and plays an important role in the initiation and proximal pause of transcription. At present, CDK7 has been found to be overexpressed in a variety of tumor cells, including leukemia, breast cancer, non-small cell lung cancer, melanoma, multiple myeloma, lung cancer, colorectal cancer, brain cancer, ovarian cancer, etc. CDK7 inhibitors aim to inhibit the overexpressed CDK7 in tumor cells, thereby inhibiting the cell cycle and transcription cycle of tumor cells, thereby inhibiting the proliferation and differentiation of tumor cells, and even inducing tumor cell apoptosis, to achieve the purpose of preventing and treating diseases.

[0003] In recent years, the research of CDK7 inhibitors is steadily advancing, and many potential compounds with development value have been found. However, the development of this field is still in its infancy. SUMMARY

[0004] In view of the deficiencies of the prior art, the technical problem to be solved by the present application is to develop 2-amino pyrimidine compounds containing 2-chloro acrylamide, test and screen their CDK7 inhibition ability. Research shows that the compounds have good inhibition activity on CDK7. The results of molecular level kinase inhibition activity test and cell proliferation inhibition test show that the compounds have good CDK7 inhibition activity and good inhibition effect on tumor cells. The molecules are expected to become an effective choice for inhibiting tumor cells by inhibiting CDK7.

[0005] Definitions of terms:

[0006] The term "aryl" used in the present application refers to a fully carbon monocyclic or fused polycyclic group of 5-12 carbon atoms, having a fully conjugated pi electron system. Non-limiting examples of aryl rings are benzene rings and naphthalene rings.

[0007] The term "heteroaromatic" as used herein refers to an unsaturated carbocyclic ring of 5-6 ring atoms, in which one or more carbons are replaced by heteroatoms such as oxygen, nitrogen, sulfur, etc. Specific heteroaromatic groups can be pyridyl, pyrimidyl, pyrazinyl, thiazolyl, oxazolyl, and isoxazolyl, etc.

[0008] The term "heterocyclyl" as used herein refers to a monocyclic group having 4-6 ring atoms in the ring, at least one or two of which are heteroatoms selected from nitrogen, oxygen, sulfur, and the remaining ring atoms are carbon atoms. Specific heterocyclyl groups can be piperidinyl, piperazinyl, pyrrolidinyl, tetrahydropyranyl, oxetanyl, etc.

[0009] The term "alkoxy" as used herein refers to an -O-alkyl group. Specific alkoxy groups can be methoxy, ethoxy, t-butoxy, etc.

[0010] The term "halogen" as used herein refers to fluorine, chlorine, bromine, or iodine, preferably fluorine, chlorine, or bromine.

[0011] The object of the present application is to provide 2-aminopyrimidine compounds containing 2-chloroacrylamide, which have the following general structure (I):

[0012]

[0013] wherein:

[0014] R is selected from H, C 1-6 alkyl, C 1-6 alkoxy, C 1-6 haloalkyl, amino, halogen, nitro, cyano, carboxyl;

[0015] A1 is selected from C 1-6 alkyl, heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaromatic, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocycloalkyl; the substituted aryl, substituted heteroaromatic, substituted cycloalkyl, substituted heterocycloalkyl are substituted by halogen, nitro, cyano, carbamoyl, C 1-4 alkyl, C 1-4 alkoxy optionally substituted;

[0016] A2 is selected from C 2-8 alkyl, heterocyclyl, unsubstituted or substituted aryl, unsubstituted or substituted heteroaromatic, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocycloalkyl; the substituted aryl, substituted heteroaromatic, substituted cycloalkyl, substituted heterocycloalkyl are substituted by halogen, nitro, cyano, carbamoyl, C 1-4 alkyl, C 1-4 alkoxy optionally substituted.

[0017] Another object of the present application is to provide a preparation method of the above-mentioned target compound, which is achieved by the following steps:

[0018] (1) Compound a reacts with compound b under the action of AlCl3 to obtain compound c, the reaction temperature is 60-90°C, the reaction time is 10-14 hours, the obtained crude product is separated by column chromatography to obtain the pure product;

[0019] (2) Compound c reacts with compound d under the action of hydrochloric acid to obtain compound e, the obtained crude product is separated by column chromatography to obtain the pure product, the reaction temperature is 100-130°C, the reaction time is 7-9 hours. If the obtained compound has a Boc protecting group, the Boc protecting group is removed under the action of TFA after the reaction is completed to obtain the corresponding compound e, the reaction temperature is 0°C, the reaction time is 1 hour, and the obtained crude product is separated by column chromatography to obtain the pure product;

[0020] (3) Compound e reacts with compound f under the action of a condensing agent to obtain compound g, the obtained crude product is separated by column chromatography to obtain the pure product, the selected condensing agent is 2-(7-azabenzotriazol)-N,N,N',N'-tetramethyluronium hexafluorophosphate, the reaction temperature is 0°C-rt, and the reaction time is 4-6 hours. If compound f is a benzene ring-substituted p-nitrobenzoic acid, compound f is first reacted with SOCl2 at 70-90°C to generate benzene ring-substituted p-nitrobenzoyl chloride, and then reacts with TFA, DMF and TEA to obtain an intermediate, the reaction temperature is rt, and the reaction is overnight, and the intermediate is reduced under the conditions of Fe, NH4Cl, EtOH, THF and H2O to obtain the corresponding compound g crude product, which is separated by column chromatography to obtain the pure product;

[0021] (4) Compound g is removed from the Boc protecting group under the action of TFA to obtain compound h, the reaction temperature is 0°C, the reaction time is 1-2 hours, and the obtained crude product is separated by column chromatography to obtain the pure product;

[0022] (5) Compound i is reacted under the action of oxalyl chloride to obtain a 2-chloropropenoyl chloride solution, the reaction temperature is 40-60°C, and the reaction time is 3-5 hours; compound h is reacted with the prepared 2-chloropropenoyl chloride solution under the action of TEA, the reaction temperature is rt, and the reaction time is 2-4 hours to obtain compound formula I;

[0023] The reaction formula of the above-mentioned main reaction process is as follows:

[0024]

[0025] Among them:

[0026] Another object of the present application is to provide a pharmaceutical composition comprising at least one of the compounds of any one of the above-mentioned forms, a stereoisomer or a pharmaceutically acceptable salt thereof as an active ingredient, and one or more pharmaceutically acceptable carriers or excipients.

[0027] The pharmaceutical composition of the present application can be prepared in various pharmaceutical dosage forms, for example, oral, injection, inhalation, implantation, etc. The injection administration and oral administration are preferred, for example, injection, lyophilized powder, needle, tablet, capsule or granule, etc.

[0028] The pharmaceutical composition of the present application and various preparations of the composition can be prepared using conventional pharmaceutical carriers.

[0029] Another object of the present application is to provide the pharmaceutical use of the compound of general formula (I) or the pharmaceutical composition containing the compound. That is, the present application provides the use of the compound of general formula (I) or the pharmaceutical composition containing the compound in the preparation of CDK7 inhibitor, and further provides the use thereof in the preparation of the drug for treating CDK7 related diseases.

[0030] Further, the disease is selected from cancer (including leukemia, human biphenotypic acute leukemia, human acute myeloid leukemia lymphoma, breast cancer, non-small cell lung cancer, melanoma, multiple myeloma, lung cancer, colorectal cancer, brain cancer, ovarian cancer, osteosarcoma), benign tumor, cardiovascular disease, inflammation and autoimmune disease.

[0031] Experiments prove that the 2-aminopyrimidine compound containing 2-chloroacrylamide of the present application has good CDK7 inhibitory activity. The raw materials required for the synthesis of the compound of the present application are easy to obtain, the route design is reasonable, the reaction conditions are mild, the yield of each step is high, the operation is simple, and it is suitable for industrial production. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1 Effects of compounds P-15 and P-19 on MV-4-11 tumor cell related proteins;

[0033] Figure 2 Effects of compounds P-15 and P-19 on MV-4-11 tumor cell cycle;

[0034] Figure 3 Effects of compounds P-15 and P-19 on MV-4-11 tumor cell cycle; Figure 2 Comparison of quantitative results in different groups;

[0035] Figure 4 Effects of compounds P-15 and P-19 on apoptosis related proteins. DETAILED DESCRIPTION

[0036] The application is further described in combination with the examples below, which are only used to illustrate the application and not to limit the application in any way.

[0037] Example 1 Preparation of 3-(2,5-dichloro-4-pyrimidinyl)-1H-indole

[0038]

[0039] A magnetic stirrer, 2,4,5-trichloropyrimidine (1.17 g, 10 mmol), AlCl3(1.83 g, 10 mmol), 30 mL of DCE were sequentially added into a 100 mL single-necked flask, and stirred at room temperature for 10 min. Then indole (1.17 g, 10 mmol) was added, and heated to reflux at 80 ℃ for 4 h. After the reaction was completed, the reaction system was poured into water, and a large amount of yellow solid was precipitated. The filtrate was removed by suction filtration, and the dried solid was obtained as a crude product. The crude product was separated and purified by silica gel column chromatography to obtain 2.4 g of yellow solid compound 5, with a yield of 91%; 1 H NMR (400 MHz, DMSO-d6) δ 12.21 (s, 1H), 8.71 – 8.65 (m, 2H), 8.47 (dd, J = 6.8, 2.0 Hz, 1H), 7.51 (dd, J = 6.4, 2.0 Hz, 1H), 7.23 (pd, J = 7.0, 3.6 Hz,2H).

[0040]

[0041] Synthesis of compound 6: Compound 1 and 3 were used as raw materials, and the synthesis and post-treatment were the same as in Example 1 to obtain yellow solid 6 with a yield of 83%; 1 H NMR (400 MHz, DMSO-d6) δ 12.26 (s, 1H), 8.69 (d, J = 3.6 Hz, 1H),8.54 (dd, J = 6.4, 2.8 Hz, 1H), 8.34 (d, J = 3.2 Hz, 1H), 7.58 (dd, J = 6.4, 2.6Hz, 1H), 7.30 (qt, J = 7.2, 3.6 Hz, 2H).

[0042]

[0043] Synthesis of compound 7: Compound 7 was synthesized from compound 1 and 4, and the synthesis and work-up were the same as example 1, to give yellow solid 7 in 78% yield; 1 H NMR (400 MHz, DMSO-d6) δ 12.22 (d, J = 26.8 Hz, 1H), 9.05 (d, J = 27.6Hz, 1H), 8.43 (ddd, J = 26.0, 6.3, 2.3 Hz, 1H), 8.11 (d, J = 2.4 Hz, 1H), 7.57(ddd, J = 8.8, 5.6, 1.7 Hz, 1H), 7.33 – 7.24 (m, 2H).

[0044] Example 2 N 1 Preparation of (5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)benzene-1,3-diamine

[0045]

[0046] A magnetic stirrer, compound 5 (3.00 g, 11.4 mmol), 1,3-phenylenediamine (2.50 g, 22.8 mmol), 30 mL of n-butanol were sequentially added into a 100 mL single-necked flask, heated to 110°C, and 0.5 mL of concentrated hydrochloric acid solution was added dropwise, and the reaction was refluxed overnight. After the reaction was completed, it was cooled to room temperature, the reaction solution was concentrated, and column chromatography on silica gel was used for separation and purification to obtain 1.53 g of yellow solid in a yield of 40%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.87 (s, 1H), 9.31 (s, 1H), 8.65 (d, J =8.0 Hz, 1H), 8.49 (s, 1H), 8.41 (s, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.22 (t, J =7.6 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.00 (s, 1H), 6.97 – 6.90 (m, 2H), 6.25(d, J = 6.8 Hz, 1H), 4.91 (s, 2H).

[0047]

[0048] Synthesis of compound 17: Compound 6 and 8 were used as raw materials, synthesis and post-processing were the same as example 2, 17 was obtained as a yellow solid with a yield of 41%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.94 (s, 1H), 9.20 (s, 1H), 8.82 (d, J =8.0 Hz, 1H), 8.39 (d, J = 4.0 Hz, 1H), 8.19 (d, J = 2.0 Hz, 1H), 7.53 (d, J = 8.0Hz, 1H), 7.26 (t, J = 7.6 Hz, 1H), 7.20 (t, J = 7.6 Hz, 1H), 7.07 (s, 1H), 6.96(d, J = 4.8 Hz, 2H), 6.26 (d, J = 3.6 Hz, 1H), 4.94 (s, 2H).

[0049]

[0050] Synthesis of compound 18: Compound 7 and 8 were used as raw materials, synthesis and post-processing were the same as example 2, 18 was obtained as a yellow solid with a yield of 39%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.79 (s, 1H), 9.99 (s, 1H), 8.66 (d, J =31.2 Hz, 1H), 8.46 – 8.33 (m, 1H), 8.28 (s, 1H), 7.85 (d, J = 16.4 Hz, 1H),7.41 (dd, J = 20.4, 8.0 Hz, 2H), 7.23 (dd, J = 16.4, 8.0 Hz, 1H), 7.10 (s, 1H),6.99 (s, 1H), 3.18 (d, J = 4.4 Hz, 2H).

[0051]

[0052] Synthesis of compound 19: Compound 19 was synthesized from compound 5 and 9 following the procedure of example 2. The product was obtained as a yellow solid in 52% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 12.25 (s, 2H), 8.73 (s, 2H), 8.72 (d, J= 3.0 Hz, 2H), 8.53 (d, J = 2.4 Hz, 1H), 8.51 (d, J = 2.0 Hz, 1H), 7.56 (d, J= 1.6 Hz, 1H), 7.55 (d, J = 2.4 Hz, 1H), 7.29 – 7.25 (m, 4H).

[0053]

[0054] Synthesis of compound 20: Compound 20 was synthesized from compound 5 and 10 following the procedure of example 2. The product was obtained as a yellow solid in 27% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 8.63 (s, 1H), 8.46 (d, J = 2.8 Hz, 1H),8.24 (d, J = 4.0 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.25 (d, J = 8.0 Hz, 1H), 7.21(t, J = 7.6 Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 7.09 (d, J = 8.0 Hz, 1H), 3.72 (d, J =77.6 Hz, 5H), 3.25 (s, 1H), 2.81 (t, J = 10.8 Hz, 1H), 2.15 (d, J = 11.0 Hz, 1H),1.97 (s, 1H), 1.79 (dd, J = 24.4, 10.4 Hz, 2H), 1.68 – 1.59 (m, 1H).

[0055]

[0056] Synthesis of compound 21 : starting from compound 5 and 11, synthesis and workup as in example 2, 21 was obtained as a yellow solid in 33% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 8.63 (s, 1H), 8.46 (s, 1H), 8.24 (d, J = 4.8 Hz, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.20 (dd, J = 13.2, 5.7 Hz, 1H), 7.14(dd, J = 14.0, 7.2 Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 3.82 (d, J = 54.4 Hz, 1H),3.27 (s, 3H), 2.97 (d, J = 4.0 Hz, 1H), 2.68 (s, 1H), 2.01 (s, 1H), 1.84 (dt, J =13.2, 6.8 Hz, 2H), 1.68 (s, 1H), 1.60 (d, J = 5.6 Hz, 3H).

[0057] Example 3 N 1 Preparation of (5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)benzene-1,3-diamine

[0058]

[0059] Synthesis and workup as in example 2, except that 1,3-phenylenediamine was replaced by 1-Boc-4-aminopiperidine and the intermediate was stirred with TFA in DCM at 0°C for 1.5 h. The solvent and TFA were removed under reduced pressure to give the target compound 22 as a yellow solid in 56% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.90 (s, 1H), 8.71 (s, 1H), 8.56 (d, J =26.0 Hz, 2H), 8.48 (d, J = 2.8 Hz, 1H), 8.28 (s, 1H), 7.49 (dd, J = 10.0, 8.0 Hz,2H), 7.20 (dt, J= 14.8, 7.2 Hz, 2H), 4.05 (s, 1H), 3.38 (s, 1H), 3.06 (s, 2H),2.13 (d, J = 12.0 Hz, 2H), 1.75 (q, J = 10.4 Hz, 2H).

[0060]

[0061] Compound 23 was synthesized from compound 5 and 12, synthesis and workup as in example 3, to give 23 as a yellow solid in 43% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.94 (s, 1H), 8.89 (d, J = 25.2 Hz,1H), 8.48 (d, J = 3.0 Hz, 1H), 8.31 (s, 1H), 8.15 (s, 1H), 7.51 (t, J = 6.4 Hz,1H), 7.41 (d, J = 7.6 Hz, 1H), 7.22 (dd, J = 10.8, 4.0 Hz, 1H), 7.20 – 7.14 (m,1H), 4.20 (s, 1H), 3.44 – 3.34 (m, 1H), 3.22 (d, J = 12.4 Hz, 1H), 2.89 (d, J =8.4 Hz, 2H), 2.07 (s, 1H), 1.97 – 1.88 (m, 1H), 1.80 – 1.70 (m, 1H), 1.65 –1.54 (m, 1H).

[0062]

[0063] Compound 24 was synthesized from compound 5 and 13, synthesis and workup as in example 3, to give 24 as a yellow solid in 36% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.96 (s, 1H), 9.06 (s, 2H), 8.49 (d, J = 2.8 Hz, 1H), 8.33 (s, 1H), 7.59 (d, J= 6.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 6.8 Hz, 1H), 7.17 (t, J = 7.6 Hz, 1H), 4.56 (s, 1H), 3.40 (dd, J =12.0, 6.4 Hz, 2H), 3.33 – 3.23 (m, 2H), 2.31 – 2.19 (m, 1H), 2.07 (td, J =12.8, 6.0 Hz, 1H).

[0064]

[0065] Synthesis of compound 25: Using compounds 5 and 14 as raw materials, the synthesis and post-treatment were the same as in Example 3, yielding yellow solid 25 in a yield of 32%; 1 H NMR (400 MHz, DMSO- d 6) δ 12.01 (s, 1H), 9.19 (s, 2H), 8.49 (s,1H), 8.33 (s, 1H), 7.56 (d, J = 38.8 Hz, 2H), 7.20 (s, 2H), 4.57 (s, 1H), 3.45(d, J = 37.2 Hz, 2H), 3.29 (d, J = 20.4 Hz, 2H), 2.25 (s, 1H), 2.08 (s, 1H).

[0066] Example 4 Preparation of (1r,4r)-4-amino-N-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)cyclohexanecarboxamide

[0067]

[0068] A magnetic stir bar, trans-4-(Boc-amino)cyclohexanecarboxylic acid (0.83 g, 3.6 mmol), HATU (1.37 g, 3.6 mmol), 10 mL DMF, DIPEA (0.78 g, 6.0 mmol) were added into a 25 mL single neck flask in turn. Stirring at 0 °C for 1 h. Compound 16 (1.00 g, 3.0) was added into the reaction system, and the reaction was carried out at room temperature overnight. After the reaction was completed, the reaction system was poured into water, and a large amount of yellow solid was precipitated. The filtrate was removed by suction filtration, and the dried solid was obtained as a crude product. The obtained crude product was added into a 40% TFA solution in DCM, and stirred at 0 °C for 1.5 h. The TFA and solvent were removed under reduced pressure, and the product was separated and purified by silica gel column chromatography to obtain 1.03 g of yellow solid, with a yield of 56%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.95 (s, 1H), 9.84 (s, 1H), 9.62(s, 1H), 8.61 (d, J = 8.0 Hz, 1H), 8.53 (d, J = 3.0 Hz, 1H), 8.44 (s, 1H), 7.94(s, 1H), 7.84 (d, J = 3.6 Hz, 2H), 7.47 (dd, J = 18.4, 8.0 Hz, 2H), 7.27 (d, J =8.0 Hz, 1H), 7.23 – 7.17 (m, 2H), 7.10 (t, J = 7.6 Hz, 1H), 3.00 (d, J = 6.4 Hz,1H), 2.30 (t, J = 11.6 Hz, 1H), 1.99 (d, J = 10.4 Hz, 2H), 1.87 (d, J = 12.0 Hz,2H), 1.49 (q, J = 11.6 Hz, 2H), 1.33 (q, J = 11.6 Hz, 2H).

[0069]

[0070] Synthesis of compound 28: Compound 28 was synthesized using compounds 17 and 26 as raw materials, and the synthesis and post-treatment were the same as in Example 4, to obtain yellow solid 28 with a yield of 63%; 1 H NMR (400 MHz, DMSO-d 6) δ 12.02 (s, 1H), 9.94 – 9.80 (m, 1H),9.47 (s, 1H), 8.74 (d, J = 8.0 Hz, 1H), 8.39 (d, J = 3.6 Hz, 1H), 8.19 (s, 1H),7.98 (s, 4H), 7.51 (d, J = 8.0 Hz, 1H), 7.42 (t, J = 12.8 Hz, 1H), 7.21 (dd, J =17.2, 7.2 Hz, 2H), 7.15 – 7.07 (m, 1H), 3.01 (s, 1H), 2.32 (t, J = 11.2 Hz,1H), 2.00 (d, J = 10.0 Hz, 2H), 1.88 (d, J = 11.6 Hz, 2H), 1.50 (dd, J = 23.6, 11.6Hz, 2H), 1.36 (dd, J = 23.0, 11.2 Hz, 2H).

[0071]

[0072] Synthesis of compound 29: Compound 18 and 26 were used as raw materials, synthesis and post-processing were the same as example 4, 29 was obtained as a yellow solid with a yield of 49%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.81 (d, J = 52.4 Hz, 1H), 9.98 (d, J =17.6 Hz, 1H), 9.81 (s, 1H), 8.69 (t, J = 52.8 Hz, 1H), 8.22 (dd, J = 61.6, 4.8Hz, 1H), 8.00 – 7.84 (m, 4H), 7.46 (d, J = 8.0 Hz, 2H), 7.39 – 7.26 (m, 1H),7.16 (dd, J= 14.0, 6.4 Hz, 2H), 7.10 – 6.83 (m, 1H), 3.05 – 2.92 (m, 1H), 2.27(t, J = 11.6 Hz, 1H), 1.96 (d, J = 10.0 Hz, 2H), 1.89 – 1.80 (m, 2H), 1.46 (q, J =12.4 Hz, 2H), 1.31 (dd, J = 25.6, 13.2 Hz, 2H).

[0073]

[0074] Compound 30 was synthesized from compound 19 and 26, synthesis and workup as in example 4, to give 30 as a yellow solid in 52% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.93 (s, 1H), 9.81 (s, 1H), 9.55 (s,1H), 8.57 (d, J = 7.6 Hz, 1H), 8.50 (d, J = 3.0 Hz, 1H), 8.45 (s, 1H), 7.84 (s,3H), 7.69 (d, J = 9.0 Hz, 2H), 7.53 (d, J = 9.0 Hz, 2H), 7.24 (t, J = 7.6 Hz, 1H),7.13 (t, J = 7.6 Hz, 1H), 3.04 (s, 1H), 2.31 (t, J = 12.0 Hz, 1H), 2.05 – 1.97(m, 2H), 1.92 (d, J = 14.0 Hz, 2H), 1.54 (q, J = 11.2 Hz, 2H), 1.42 – 1.33 (m,2H).

[0075]

[0076] Compound 31 was synthesized from compound 20 and 26, synthesis and workup as in example 4, to give 31 as a yellow solid in 43% yield; 1 H NMR (400 MHz, DMSO-d 6) δ 11.88 (s, 1H), 8.62 (s, 1H), 8.46 (s,1H), 8.24 (s, 1H), 7.87 (s, 2H), 7.72 (t, J = 9.2 Hz, 1H), 7.49 (d, J = 8.4 Hz,1H), 7.20 (ddd, J = 20.8, 14.4, 8.0 Hz, 3H), 4.18 (s, 1H), 3.99 (s, 1H), 3.83(d, J = 13.0 Hz, 1H), 3.66 (s, 1H), 2.97 (d, J = 16.0 Hz, 1H), 2.14 (s, 1H), 1.94(d, J = 9.0 Hz, 3H), 1.75 (d, J = 10.8 Hz, 3H), 1.40 (dd, J = 22.4, 10.4 Hz, 4H),1.28 (dd, J = 26.4, 13.6 Hz, 4H).

[0077]

[0078] Synthesis of compound 32: Compound 21 and 26 were used as raw materials, synthesis and post-processing were the same as in Example 4, and yellow solid 32 was obtained with a yield of 49%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.87 (s, 1H), 8.62 (s, 1H), 8.47 (d, J = 2.8 Hz, 1H), 8.24 (d, J = 7.6 Hz, 1H), 7.85 (s, 4H), 7.64 (d, J = 6.8 Hz, 1H),7.49 (d, J = 8.0 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.15 (t, J = 7.2 Hz, 1H), 7.06(d, J= 6.4 Hz, 1H), 3.85 (s, 1H), 3.73 (s, 2H), 3.59 (s, 3H), 2.97 (s, 1H),2.16 (d, J = 11.6 Hz, 1H), 2.05 (t, J = 12.4 Hz, 1H), 1.95 (d, J = 11.6 Hz, 3H),1.76 (d, J = 10.4 Hz, 6H).

[0079]

[0080] Compound 33 was synthesized from compound 22 and 26, synthesis and workup as in example 4, to give 33 as a yellow solid in 41% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.88 (s, 1H), 8.61 (s, 1H), 8.47 (d, J = 2.8 Hz, 1H), 8.26 (s, 1H), 7.89 (s, 4H), 7.50 (d, J = 7.6 Hz, 1H), 7.27 (d, J = 7.6 Hz, 1H), 7.24 – 7.14 (m, 2H), 4.37 (d, J = 12.4 Hz, 1H), 4.01 (d, J = 12.4Hz, 2H), 3.43 (s, 4H), 2.99 (s, 1H), 2.75 (s, 1H), 2.60 (t, J = 10.8 Hz, 1H),2.05 (d, J = 13.6 Hz, 1H), 1.94 (d, J = 10.4 Hz, 4H), 1.74 (d, J = 12.0 Hz, 2H).

[0081]

[0082] Compound 34 was synthesized from compound 23 and 26, synthesis and workup as in example 4, to give 34 as a yellow solid in 41% yield; 1 H NMR (400 MHz, DMSO- d6) δ 11.99 - 11.87 (m, 1H), 8.46 (s, 1H), 8.34 (d, J = 6.4 Hz, 1H), 8.27 (s, 1H), 7.94 (s, 2H), 7.49 (d, J = 7.0 Hz, 1H),7.34 (dd, J = 8.4, 4.4 Hz, 1H), 7.20 (d, J = 7.2 Hz, 1H), 7.13 (d, J = 7.6 Hz, 1H),4.51 - 4.29 (m, 1H), 4.11 (s, 1H), 3.91 - 3.72 (m, 1H), 3.02 (dd, J = 25.0,15.6 Hz, 2H), 2.74 (s, 1H), 2.03 (s, 1H), 1.95 (d, J = 9.6 Hz, 2H), 1.88 - 1.74(m, 3H), 1.64 (d, J = 13.0 Hz, 1H), 1.53 - 1.32 (m, 6H).

[0083]

[0084] Synthesis of compound 35: using compound 24 and 26 as raw materials, synthesis and post-processing were the same as in Example 4, and yellow solid 35 was obtained with a yield of 29%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.94 (s, 1H), 8.48 (d, J = 2.8 Hz,1H), 8.30 (d, J = 6.4 Hz, 1H), 7.92 (s, 2H), 7.88 (s, 1H), 7.57 (dd, J = 18.0,6.0 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.15 (t, J = 7.6Hz, 1H), 4.47 (d, J= 38.0 Hz, 1H), 3.89 (s, 1H), 3.76 – 3.68 (m, 1H), 3.64(dd, J = 11.6, 6.0 Hz, 1H), 3.50 (d, J = 4.4 Hz, 1H), 2.98 (d, J = 9.2 Hz, 2H), 2.31 (dd, J = 31.6, 13.2 Hz, 1H), 2.14 (t, J = 10.4 Hz, 1H), 1.94 (d, J = 10.0 Hz, 6H), 1.77 (t, J = 14.0 Hz, 2H).

[0085]

[0086] Synthesis of compound 36: Using compounds 25 and 26 as raw materials, the synthesis and post-treatment were the same as in Example 4, yielding a yellow solid 36 with a yield of 24%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.95 (s, 1H), 8.48 (d, J = 3.0 Hz, 1H), 8.30 (d, J = 6.4 Hz, 1H), 7.95 (s, 2H), 7.91 (s, 1H), 7.58 (dd, J = 17.6, 6.4 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.21 (t, J = 7.6 Hz, 1H), 7.15 (t, J = 7.2Hz, 1H), 4.47 (d, J = 37.2 Hz, 1H), 3.89 (s, 1H), 3.77 – 3.69 (m, 1H), 3.64(dd, J = 12.4, 6.0 Hz, 1H), 3.40 – 3.36 (m, 1H), 2.98 (d, J = 10.4 Hz, 2H), 2.31(dd, J = 30.0, 14.0 Hz, 1H), 2.14 (t, J= 10.4 Hz, 1H), 1.94 (d, J = 10.0 Hz, 6H),1.77 (t, J = 15.2 Hz, 2H).

[0087] Example 5 Preparation of 4-amino-N-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2- yl)amino)phenyl)benzamide

[0088]

[0089] A magnetic stirrer, compound 16 (0.5 g, 1.5 mmol), 4-nitrobenzoyl chloride (0.42 g, 2.3 mmol), 10 mL TFA, 10 mL DMF, TEA (0.30 g, 3.0 mmol) were sequentially added into a 50 mL single-necked flask, and the reaction was carried out at room temperature overnight. The reaction solution was concentrated under reduced pressure, 10 mL saturated NaOH aqueous solution was added to quench, and the reaction system was poured into water to precipitate a large amount of yellow solid. The filtrate was removed by suction filtration, and the solid was dried to obtain a crude product. The crude product, a magnetic stirrer, reduced iron powder (0.42 g, 7.5 mmol), ammonium chloride (0.24 g, 4.5 mmol), 15 mL EtOH, 5 mL H2O, 20 mL THF were sequentially added into a 100 mL single-necked flask, and the reaction was carried out at 80 °C under reflux overnight. After the reaction was completed, the solid was removed by suction filtration while hot, the filtrate was concentrated under reduced pressure, and was separated and purified by silica gel column chromatography to obtain 76 mg of a yellow solid, with a yield of 11%; 1 H NMR (400 MHz, DMSO- d 6) δ11.92 (s, 1H), 9.72 (s, 1H), 9.63 (s, 1H), 8.65 (d, J = 7.8 Hz, 1H), 8.53 (d, J =2.4 Hz, 1H), 8.45 (s, 1H), 8.11 (s, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.49 (d, J =8.0 Hz, 2H), 7.37 (d, J = 7.6 Hz, 1H), 7.24 (d, J = 8.0 Hz, 1H), 7.21 – 7.16 (m,1H), 7.10 (t, J = 7.2 Hz, 1H), 6.59 (d, J= 8.4 Hz, 2H), 5.74 (s, 2H).

[0090] Example 6 Preparation of 4-amino-N-(3-((5-chloro-4-(lH-indol-3- yl)pyrimidin-2-yl)amino)phenyl)-2-fluorobenzamide

[0091]

[0092] A magnetic stir bar, 2-fluoro-4-nitrobenzoic acid (2 g, 10.8 mmol), 15 mL SOCl2 were added into a 50 mL single necked flask successively, and the reaction was heated to reflux at 80 °C overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain white solid 2-fluoro-4-nitrobenzoyl chloride. The synthesis and work-up of compound 42 were the same as example 5, except that 4-nitrobenzoyl chloride was replaced by 2-fluoro-4-nitrobenzoyl chloride. Yellow solid, yield 28%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.93 (s, 1H), 10.21 (s, 1H), 9.69 (s, 1H), 8.68 (d, J = 7.2 Hz, 1H), 8.54 (s, 1H), 8.45 (s, 1H), 8.06 (s, 1H), 7.58 (d, J = 8.0 Hz, 3H), 7.45 - 7.38 (m, 1H), 7.31 (s, 1H), 7.21 (d, J = 11.2 Hz, 3H), 7.11 (s, 1H), 6.39 (dd, J = 36.0, 10.8 Hz, 1H), 6.00 (s, 1H).

[0093]

[0094] Synthesis of compound 43: Compound 43 was synthesized using compound 16 and 39 as starting materials, and the synthesis and work-up were the same as example 5 to obtain yellow solid 43 with a yield of 34%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.93 (s, 1H), 10.21 (s, 1H), 9.69 (s, 1H), 8.68 (d, J = 7.2 Hz, 1H), 8.54 (s, 1H), 8.45 (s, 1H), 8.06 (s, 1H), 7.58 (d, J = 7.2 Hz, 1H), 7.50 (d,J = 7.6 Hz, 1H), 7.31 (s, 2H), 7.28 – 7.18 (m,2H), 7.13 (t, J = 6.8 Hz, 1H), 6.95 (s, 1H), 6.80 (d, J = 7.6 Hz, 1H), 5.93 (s,2H).

[0095]

[0096] Compound 44 was synthesized from compound 16 and 40, synthesis and workup as in example 5, to give 44 as a yellow solid in 38% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.89 (s, 1H), 9.70 (s, 1H), 9.60 (s,1H), 8.65 (d, J = 7.0 Hz, 1H), 8.49 (d, J = 31.6 Hz, 2H), 8.11 (s, 1H), 7.61 (d, J = 12.4 Hz, 2H), 7.49 (d, J = 6.8 Hz, 2H), 7.38 (d, J = 7.2 Hz, 1H), 7.27 – 7.15(m, 2H), 7.11 (d, J = 6.8 Hz, 1H), 6.87 (s, 1H), 6.65 (d, J = 8.0 Hz, 1H), 5.47(s, 2H), 2.12 (s, 3H).

[0097]

[0098] Compound 53 was synthesized from compound 16 and 45, synthesis and workup as in example 5, to give 53 as a yellow solid in 52% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.97 (s, 1H), 9.98 (s, 1H), 9.62 (s,1H), 8.80 (s, 1H), 8.62 (d, J = 8.0 Hz, 1H), 8.53 (d, J = 3.0 Hz, 1H), 8.46 (d,J = 15.2 Hz, 2H), 7.96 (s, 1H), 7.49 (t, J = 8.0 Hz, 2H), 7.27 (d, J = 8.0 Hz, 1H),7.21 (t, J = 7.0 Hz, 2H), 7.10 (t, J = 7.6 Hz, 1H), 3.33 (s, 1H), 2.92 (d, J = 8.4Hz, 2H), 2.64 (t, J = 10.8 Hz, 1H), 1.93 (d, J = 11.6 Hz, 2H), 1.80 (td, J = 14.8,3.6 Hz, 2H).

[0099]

[0100] Compound 54 was synthesized using compound 16 and 46 as starting materials, synthesis and workup as in example 5 to give 54 as a yellow solid in 64% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.95 (s, 1H), 10.11 (s, 1H), 9.63(s, 1H), 8.68 (s, 2H), 8.61 (d, J = 8.0 Hz, 1H), 8.53 (d, J = 3.0 Hz, 1H), 8.44(s, 1H), 7.96 (s, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 8.0 Hz, 1H), 7.28(d, J = 8.0 Hz, 1H), 7.22 (t, J = 8.0 Hz, 2H), 7.10 (t, J = 7.6 Hz, 1H), 3.30 (d, J =11.6 Hz, 1H), 3.19 (d, J = 13.6 Hz, 1H), 3.04 (dd, J = 11.0, 5.2 Hz, 1H), 2.93(s, 1H), 2.82 (s, 1H), 2.01 (d,J = 8.8 Hz, 1H), 1.83 (d, J = 11.2 Hz, 1H), 1.64(d, J = 8.8 Hz, 1H).

[0101]

[0102] Compound 55 was synthesized using compound 16 and 47 as starting material, synthesis and workup as in example 5 to give 55 as a yellow solid in 59% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 12.00 (s, 1H), 10.14 (s, 1H), 9.63(s, 1H), 8.85 (s, 2H), 8.61 (d, J = 7.2 Hz, 1H), 8.52 (s, 1H), 8.44 (s, 1H),7.96 (d, J = 5.6 Hz, 2H), 7.52 – 7.46 (m, 2H), 7.28 (d, J = 7.2 Hz, 1H), 7.12 –7.07 (m, 1H), 3.30 (d, J = 12.4Hz, 2H), 3.22 – 3.17 (m, 1H), 3.03 (s, 1H), 2.84(s, 1H), 2.01 (d, J = 9.2 Hz, 1H), 1.83 (d, J = 12.0 Hz, 1H), 1.64 (dd, J = 22.0,12.0 Hz, 2H).

[0103]

[0104] Compound 56 was synthesized using compound 16 and 48 as starting material, synthesis and workup as in example 5 to give 56 as a yellow solid in 55% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 12.00 (s, 1H), 10.14 (s, 1H), 9.63(s, 1H), 8.85 (s, 2H), 8.61 (d, J = 7.2 Hz, 1H), 8.52 (s, 1H), 8.44 (s, 1H),7.96 (d, J= 5.6 Hz, 2H), 7.52 – 7.46 (m, 2H), 7.28 (d, J = 7.2 Hz, 1H), 7.12 –7.07 (m, 1H), 3.30 (d, J = 12.4Hz, 2H), 3.22 – 3.17 (m, 1H), 3.03 (s, 1H), 2.84(s, 1H), 2.01 (d, J = 9.2 Hz, 1H), 1.83 (d, J = 12.0 Hz, 1H), 1.64 (dd, J = 22.0,12.0 Hz, 2H).

[0105]

[0106] Synthesis of compound 57: Compound 57 was synthesized from compound 16 and 49, and the work-up was the same as in example 5. Compound 57 was obtained as a yellow solid in 66% yield. 1 H NMR (400 MHz, DMSO- d 6) δ 12.02 (s, 1H), 9.92 (s, 1H), 9.64 (s,1H), 8.63 (d, J = 8.0 Hz, 1H), 8.55 (d, J = 2.8 Hz, 1H), 8.46 (s, 1H), 8.16 (s,2H), 7.98 (s, 1H), 7.50 (dd, J = 20.0, 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 1H),7.23 (t, J = 8.0 Hz, 2H), 7.11 (t, J = 7.6 Hz, 1H), 3.72 – 3.65 (m, 1H), 3.07 –2.99 (m, 1H), 2.42 (dd, J = 17.0, 9.0 Hz, 2H), 2.31 (dd, J = 20.4, 9.6 Hz, 2H).

[0107]

[0108] Synthesis of compound 58: compound 58 was synthesized from compound 16 and 50 following the procedure of example 5. The yellow solid 58 was obtained in 64% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.94 (s, 1H), 9.86 (s, 1H), 9.62 (s,1H), 8.63 (d, J = 8.0 Hz, 1H), 8.53 (d, J = 3.0 Hz, 1H), 8.44 (s, 1H), 7.93 (s,1H), 7.73 (s, 2H), 7.50 (d, J = 8.0 Hz, 1H), 7.47 (d, J = 8.4 Hz, 1H), 7.29 (d, J =8.0 Hz, 1H), 7.24 – 7.17 (m, 2H), 7.11 (t, J = 7.6 Hz, 1H), 2.81 (dd, J = 12.8,6.4 Hz, 2H), 2.34 (t, J = 6.8 Hz, 2H), 1.69 – 1.53 (m, 4H).

[0109]

[0110] Synthesis of compound 59: compound 59 was synthesized from compound 16 and 51 following the procedure of example 5. The yellow solid 59 was obtained in 64% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.95 (s, 1H), 9.84 (s, 1H), 9.60 (s,1H), 8.62 (d, J = 8.0 Hz, 1H), 8.52 (d, J = 2.8 Hz, 1H), 8.44 (s, 1H), 7.93 (s,1H), 7.76 (s, 2H), 7.50 (d, J = 8.0 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.28 (d, J =8.0 Hz, 1H), 7.24 – 7.16 (m, 2H), 7.10 (t, J= 7.6 Hz, 1H), 2.79 (d, J = 5.6 Hz,2H), 2.30 (t, J = 7.2 Hz, 2H), 1.65 – 1.52 (m, 4H), 1.39 – 1.29 (m, 2H).

[0111]

[0112] Synthesis of compound 60: Compound 60 was synthesized using compound 16 and 52 as starting materials, and the synthesis and workup were the same as in Example 5 to give 60 as a yellow solid in 64% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.95 (s, 1H), 9.81 (s, 1H), 9.59 (s,1H), 8.63 (d, J = 8.0 Hz, 1H), 8.53 (s, 1H), 8.44 (s, 1H), 7.93 (s, 1H), 7.75(s, 2H), 7.48 (dd, J = 13.6, 8.0 Hz, 2H), 7.27 (d, J = 7.6 Hz, 1H), 7.24 – 7.16(m, 2H), 7.10 (t, J = 7.2 Hz, 1H), 2.77 (d, J = 5.0 Hz, 2H), 2.29 (t, J = 7.2 Hz,2H), 1.58 (s, 2H), 1.53 (s, 2H), 1.30 (s, 7H).

[0113] Example 7 Preparation of compound P-1

[0114]

[0115] A magnetic stirrer, 2-chloropropenoic acid (233 mg, 2.2 mmol) were added into a two-necked flask, 4 mL of anhydrous DCM, 0.1 mL of anhydrous DMF, oxalyl chloride (270 mg, 2.2 mmol) were added in turn under the protection of N2, heated to reflux at 50°C for 4h, and a colorless 2-chloropropenoic acid chloride solution was obtained after the reaction was completed. Compound 27 (100 mg, 0.22 mmol), 3 mL of anhydrous DCM, 2 mL of anhydrous DMF, TEA (258 mg, 2.6 mmol), 2-chloropropenoic acid chloride were added into a 25 mL single-necked flask in turn, and the reaction was carried out at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and a large amount of yellow solid was precipitated by pouring the system into water. The filtrate was removed by suction filtration, dried, and then separated and purified by silica gel column chromatography to obtain a yellow solid with a yield of 32%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.91 (s,1H), 9.77 (s, 1H), 9.60 (s, 1H), 8.62 (d, J = 8.0 Hz, 1H), 8.53 (d, J = 3.0 Hz,1H), 8.44 (s, 1H), 8.16 (d, J = 8.0 Hz, 1H), 7.95 (s, 1H), 7.48 (dd, J = 17.0,8.0 Hz, 2H), 7.28 (d, J = 8.0 Hz, 1H), 7.20 (q, J = 7.6 Hz, 2H), 7.10 (t, J = 7.6Hz, 1H), 6.29 (d, J = 2.0 Hz, 1H), 5.90 (d, J = 2.0 Hz, 1H), 3.68 – 3.57 (m, 1H),2.29 (t, J = 11.6 Hz, 1H), 1.84 (s, 4H), 1.45 (tt, J = 23.0, 11.6 Hz, 4H) ; HRMS:C 28 H 27 Cl2N6O2[M+H] + .

[0116]

[0117] Synthesis of compound P-2: using compound 28 and 61 as raw materials, synthesis and post-processing were the same as in Example 7, to obtain a yellow solid with a yield of 38%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.82 (d, J = 44.4 Hz, 1H), 10.11 –9.78 (m, 1H), 8.75 (t, J = 58.4 Hz, 1H), 8.33 (s, 1H), 8.19 (s, 1H), 7.96 (dd, J = 32.4, 22.8 Hz, 2H), 7.50 (d, J = 7.2 Hz, 2H), 7.43 – 7.29 (m, 2H), 7.19 (d, J =7.2 Hz, 2H), 7.11 (s, 1H), 6.29 (s, 1H), 5.91 (s, 1H), 3.62 (s, 1H), 2.28 (s,1H), 1.83 (s, 4H), 1.46 (dd, J = 24.0, 11.6 Hz, 4H) ; HRMS: C 28 H 27 ClFN6O2[M+H] + .

[0118]

[0119] Synthesis of compound P-3: using compound 29 and 61 as raw materials, synthesis and post-processing were the same as in Example 7, to obtain a yellow solid with a yield of 33%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.97 (s, 1H), 9.82 (s, 1H), 9.51 (s,1H), 8.76 (d, J = 7.6 Hz, 1H), 8.41 (d, J = 2.8 Hz, 1H), 8.20 (s, 2H), 8.00 (s,1H), 7.51 (d, J = 7.6 Hz, 1H), 7.44 (d, J = 7.6 Hz, 1H), 7.22 (dd, J = 11.6, 7.2Hz, 3H), 7.14 (t, J= 7.2 Hz, 1H), 6.29 (s, 1H), 5.91 (s, 1H), 3.63 (d, J = 7.6Hz, 1H), 2.30 (t, J = 10.8 Hz, 1H), 1.85 (s, 4H), 1.54 – 1.37 (m, 4H) ; HRMS:C 29 H 27 ClF3N6O2[M+H] + .

[0120]

[0121] Synthesis of compound P-4: using compound 30 and 61 as raw materials, synthesis and post-processing were the same as in Example 7, to obtain a yellow solid with a yield of 36%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.89 (s, 1H), 9.74 (s, 1H), 9.53 (s,1H), 8.56 (d, J = 8.0 Hz, 1H), 8.48 (d, J = 2.8 Hz, 1H), 8.43 (s, 1H), 8.15 (d, J =8.0 Hz, 1H), 7.67 (d, J = 8.8 Hz, 2H), 7.55 – 7.48 (m, 3H), 7.23 (t, J = 7.2 Hz,1H), 7.12 (t, J = 7.2 Hz, 1H), 6.29 (d, J = 1.6 Hz, 1H), 5.90 (d, J = 2.0 Hz, 1H),3.63 (d, J = 7.6 Hz, 1H), 2.28 (t, J = 11.6 Hz, 1H), 1.86 (t, J = 12.4 Hz, 4H),1.54 – 1.40 (m, 4H) ; HRMS: C 28 H 27 Cl2N6O2[M+H] + .

[0122]

[0123] Synthesis of compound P-5: Using compounds 31 and 61 as raw materials, the synthesis and post-treatment were the same as in Example 7, yielding a yellow solid with a yield of 33%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.81 (s, 1H), 8.63 (s, 1H), 8.46 (s, 1H), 8.24 (s, 1H), 8.10 (d, J = 7.6 Hz, 1H), 7.68 (d, J = 7.2 Hz, 1H), 7.49 (d, J =7.2 Hz, 1H), 7.18 (dt, J = 14.4, 7.0 Hz, 3H), 6.26 (s, 1H), 5.87 (s, 1H), 4.09(d, J = 74.0 Hz, 1H), 3.74 (d, J = 63.2 Hz, 1H), 3.56 (s, 1H), 2.12 (s, 1H), 2.00(d, J = 10.4 Hz, 1H), 1.75 (s, 8H), 1.57 (d, J = 14.4 Hz, 1H), 1.37 (d, J = 10.4Hz, 6H); HRMS: C 28 H 33 Cl2N6O2[M+H] + .

[0124]

[0125] Synthesis of compound P-6: Using compounds 32 and 61 as raw materials, the synthesis and post-treatment were the same as in Example 7, yielding a yellow solid with a yield of 33%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.82 (s, 1H), 8.59 (d, J = 39.0 Hz, 1H), 8.45 (d, J = 17.0 Hz, 1H), 8.35 – 8.21 (m, 1H), 8.12 (d, J = 7.6 Hz, 1H), 7.60 (t, J = 13.6 Hz, 1H), 7.46 (dd, J= 22.4, 7.6 Hz, 1H), 7.25 – 7.12 (m, 2H),7.04 (d, J = 6.0 Hz, 1H), 6.27 (s, 1H), 5.88 (s, 1H), 3.85 (s, 1H), 3.73 (s,1H), 3.58 (s, 1H), 2.16 (s, 1H), 2.03 (s, 1H), 1.77 (d, J = 26.0 Hz, 9H), 1.57(s, 2H), 1.45 – 1.35 (m, 4H) ; HRMS: C 28 H 33 Cl2N6O2[M+H] + .

[0126]

[0127] Compound P-7: Compound 33 and 61 as starting material, synthesis and work-up as in Example 7, yield 33% as yellow solid; 1 H NMR (400 MHz, DMSO- d 6) δ 11.87 (s, 1H), 8.46 (s, 1H), 8.29 (s, 1H), 8.18 (s, 1H), 7.55 – 7.38 (m, 2H), 7.17 (d, J = 27.0 Hz, 2H), 6.45 (d, J =8.4 Hz, 1H), 6.27 (s, 1H), 5.90 (s, 1H), 4.47 (s, 1H), 4.10 (s, 1H), 3.83 (s,1H), 3.58 (s, 1H), 3.01 (s, 1H), 2.70 (s, 1H), 2.03 (s, 2H), 1.78 (d, J = 17.2Hz, 4H), 1.59 (s, 2H), 1.43 (s, 6H) ; HRMS: C 27 H 31 Cl2N6O2[M+H] + .

[0128]

[0129] Compound P-8: Compound 34 and 61 as starting material, synthesis and work-up as in Example 7, yield 33% as yellow solid; 1H NMR (400 MHz, DMSO- d 6) δ 11.83 (s, 1H), 8.61 (s, 1H), 8.47 (s,1H), 8.26 (s, 1H), 8.13 (d, J = 6.4 Hz, 1H), 7.49 (d, J = 6.0 Hz, 1H), 7.23 (dd, J = 22.0, 6.8 Hz, 3H), 6.26 (s, 1H), 5.88 (s, 1H), 4.38 (s, 1H), 4.00 (s, 2H),3.59 (s, 1H), 3.17 (s, 1H), 2.74 (s, 1H), 2.56 (s, 1H), 1.99 (s, 2H), 1.82(s, 2H), 1.71 (s, 2H), 1.43 (s, 6H) ; HRMS: C 27 H 31 Cl2N6O2[M+H] + .

[0130]

[0131] Compound P-9 was synthesized using compound 35 and 61 as starting material, synthesis and work-up as in example 7, as a yellow solid in 19% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.85 (s, 1H), 8.55 (d, J = 30.0 Hz,1H), 8.47 (s, 1H), 8.30 (d, J = 6.4 Hz, 1H), 8.10 (dd, J = 19.2, 7.6 Hz, 1H),7.57 (d, J = 6.4 Hz, 1H), 7.51 (dd, J = 11.6, 7.2 Hz, 1H), 7.21 (t, J = 7.6 Hz,1H), 7.15 (t, J = 7.2 Hz, 1H), 6.25 (dd, J = 9.6, 1.6 Hz, 1H), 5.87 (dd, J = 8.4,1.6 Hz, 1H), 4.47 (d, J= 33.0 Hz, 1H), 3.57 (ddd, J = 27.0, 17.2, 7.6 Hz, 3H),3.40 (d, J = 6.4 Hz, 1H), 2.35 (s, 1H), 2.27 (s, 1H), 2.21 – 1.93 (m, 2H), 1.79(t, J = 14.0 Hz, 4H), 1.38 (dd, J = 17.6, 7.0 Hz, 4H) ; HRMS: C 26 H 29 Cl2N6O2[M+H] + .

[0132]

[0133] Compound P-10: Compound 36 and 61 as starting material, synthesis and work-up as in Example 7, yield 19% of yellow solid; 1 H NMR (400 MHz, DMSO- d 6) δ 11.86 (s, 1H), 8.65 (d, J = 40.4 Hz,1H), 8.48 (s, 1H), 8.30 (s, 1H), 8.12 (s, 1H), 7.69 (d, J = 18.4 Hz, 1H), 7.55(d, J = 35.0 Hz, 2H), 7.19 (d, J = 16.8 Hz, 1H), 6.26 (s, 1H), 5.88 (s, 1H), 4.47(d, J = 32.8 Hz, 1H), 3.60 (s, 3H), 3.52 (s, 1H), 2.35 (s, 1H), 2.27 (s, 1H),2.16 – 1.92 (m, 2H), 1.78 (s, 4H), 1.41 (s, 4H) ; HRMS: C 26 H 29 Cl2N6O2[M+H] + .

[0134]

[0135] Compound P-11: Compound 41 and 61 as starting material, synthesis and work-up as in Example 7, yield 8% of yellow solid.1 H NMR (400 MHz, DMSO- d 6) δ 11.91 (s, 1H), 10.45 (s, 1H), 10.13 (s, 1H), 9.65 (s, 1H), 8.63 (d, J = 8.0 Hz, 1H), 8.53 (d, J = 2.8 Hz, 1H), 8.46(s, 1H), 8.16 (s, 1H), 7.95 (d, J = 8.4 Hz, 2H), 7.81 (d, J = 8.4 Hz, 2H), 7.50 (dd, J = 12.4, 8.4 Hz, 2H), 7.40 (d, J = 7.6 Hz, 1H), 7.27 (t, J = 8.0 Hz, 1H), 7.17 (t, J = 7.2 Hz, 1H), 7.09 (t, J = 7.2 Hz, 1H), 6.47 (t, J = 8.8 Hz, 1H), 6.14(d, J = 2.4 Hz, 1H); HRMS: C 28 H 21 Cl2N6O2[M+H] + .

[0136]

[0137] Synthesis of compound P-12: Using compounds 42 and 61 as raw materials, the synthesis and post-treatment were the same as in Example 7, yielding a yellow solid with a yield of 9%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.90 (s, 1H), 10.57 (s, 1H), 10.23 (s, 1H), 9.67 (s, 1H), 8.64 (d, J = 8.0 Hz, 1H), 8.53 (d, J = 3.0 Hz, 1H), 8.45(s, 1H), 8.09 (s, 1H), 7.73 (d, J = 12.8 Hz, 1H), 7.65 (t, J = 8.4 Hz, 1H), 7.58 (dd,J = 13.2, 4.7 Hz, 2H), 7.49 (d, J = 8.0 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H),7.25 (dd, J = 14.4, 6.4 Hz, 1H), 7.20 (t, J = 7.6 Hz, 1H), 7.12 (t, J = 7.6 Hz,1H), 6.51 (d, J = 2.8 Hz, 1H), 6.17 (d, J = 2.8 Hz, 1H) ; HRMS: C 28 H 20 Cl2FN6O2[M+H] + .

[0138]

[0139] Synthesis of compound P-13: Compound 43 and 61 were used as raw materials, synthesis and post-processing were the same as in Example 7, a yellow solid was obtained with a yield of 9%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.90 (s, 1H), 10.63 (s, 1H), 10.47(s, 1H), 9.66 (d, J = 20.0 Hz, 1H), 8.66 (d, J = 8.0 Hz, 1H), 8.53 (d, J = 2.8 Hz,1H), 8.44 (d, J = 4.0 Hz, 1H), 8.18 (s, 1H), 8.06 (d, J = 7.6 Hz, 2H), 7.66 (d, J =8.4 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 7.33 (d, J = 8.4Hz, 1H), 7.29 – 7.24 (m, 1H), 7.22 (d, J = 7.6 Hz, 1H), 7.14 (t, J = 7.2 Hz, 1H),6.54 (d,J = 2.4 Hz, 1H), 6.19 (d, J = 2.4 Hz, 1H) ; HRMS: C 29 H 20 Cl2F3N6O2[M+H] + .

[0140]

[0141] Synthesis of compound P-14: Compound 44 and 61 were used as raw materials, synthesis and post-processing were the same as in Example 7, a yellow solid was obtained, the yield was 6%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.89 (d, J = 18.0 Hz, 1H), 10.17 (s,1H), 9.94 (s, 1H), 9.66 (s, 1H), 8.63 (d, J = 8.4 Hz, 1H), 8.58 – 8.52 (m, 1H),8.46 (s, 1H), 8.16 (s, 1H), 7.82 (s, 1H), 7.77 (d, J = 8.4 Hz, 1H), 7.50 – 7.47(m, 2H), 7.40 (d, J = 8.4 Hz, 1H), 7.27 (t, J = 8.0 Hz, 1H), 7.17 (t, J = 7.6 Hz,1H), 7.09 (t, J = 7.6 Hz, 1H), 6.51 (s, 1H), 6.45 (s, 1H), 6.11 (d, J = 2.0 Hz,1H), 2.28 (s, 3H) ; HRMS: C 29 H 23 Cl2N6O2[M+H] + .

[0142]

[0143] Synthesis of compound P-15: Compound 53 and 61 were used as raw materials, synthesis and post-processing were the same as in Example 7, a yellow solid was obtained, the yield was 37%; 1 H NMR (400 MHz, DMSO- d6) δ 11.90 (s, 1H), 9.87 (s, 1H), 9.61 (s,1H), 8.62 (d, J = 8.0 Hz, 1H), 8.52 (d, J = 2.8 Hz, 1H), 8.44 (s, 1H), 7.96 (s,1H), 7.52 – 7.45 (m, 2H), 7.28 (d, J = 8.0 Hz, 1H), 7.20 (dd, J = 10.0, 5.6 Hz,2H), 7.10 (t, J = 7.6 Hz, 1H), 5.74 (s, 2H), 4.30 (s, 1H), 3.89 (s, 1H), 3.17(s, 1H), 2.81 (s, 1H), 2.64 (t, J = 11.2 Hz, 1H), 1.85 (d, J = 12.0 Hz, 2H), 1.56(s, 2H) ; HRMS: C 27 H 25 Cl2N6O2[M+H] + .

[0144]

[0145] Synthesis of compound P-16: using compound 54 and 61 as raw materials, synthesis and post-processing were the same as in Example 7, to obtain a yellow solid with a yield of 32%; 1 H NMR (400 MHz, DMSO- d 6) δ 11.93 (s, 1H), 10.00 (d, J = 12.4 Hz,1H), 9.65 (s, 1H), 8.63 (d, J = 8.0 Hz, 1H), 8.54 (d, J = 3.0 Hz, 1H), 8.45 (s,1H), 7.95 (s, 1H), 7.49 (t, J = 7.2 Hz, 2H), 7.27 (d, J = 7.6 Hz, 1H), 7.21 (dt, J = 12.0, 4.0 Hz, 2H), 7.10 (t, J= 7.2 Hz, 1H), 5.76 – 5.71 (m, 2H), 4.26 (dd, J = 76.8, 11.6 Hz, 1H), 3.81 (t, J = 16.4 Hz, 1H), 3.48 – 3.39 (m, 1H), 3.14 (t, J = 14.4 Hz, 1H), 2.95 – 2.78 (m, 1H), 1.99 (s, 1H), 1.79 (d, J = 13.2 Hz, 1H),1.70 (d, J = 10.0 Hz, 1H), 1.41 (s, 1H) ; HRMS: C 27 H 25 Cl2N6O2[M+H] + .

[0146]

[0147] Compound P-17 was synthesized using compound 55 and 61 as starting material, synthesis and work-up as in example 7, as a yellow solid in 33% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.93 (s, 1H), 10.01 (s, 1H), 9.65(s, 1H), 8.63 (d, J = 7.2 Hz, 1H), 8.54 (s, 1H), 8.45 (s, 1H), 7.95 (s, 1H),7.50 (d, J = 7.6 Hz, 2H), 7.26 (s, 1H), 7.21 (d, J = 3.0 Hz, 2H), 7.10 (s, 1H),5.74 (d, J = 7.6 Hz, 2H), 4.25 (d, J = 75.6 Hz, 1H), 3.82 (t, J = 16.0 Hz, 1H),3.14 (t, J = 12.0 Hz, 1H), 2.88 (s, 1H), 2.73 (s, 1H), 1.99 (s, 1H), 1.79 (d, J = 13.0 Hz, 1H), 1.70 (d, J= 7.6 Hz, 1H), 1.41 (s, 1H); HRMS: C 27 H 25 Cl2N6O2[M+H] + .

[0148]

[0149] Compound P-18 was synthesized using compound 56 and 61 as starting material, synthesis and work-up as in example 7, to yield a yellow solid in 35% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.93 (s, 1H), 9.95 (d, J = 14.0 Hz,1H), 9.65 (s, 1H), 8.62 (d, J = 7.2 Hz, 1H), 8.55 (d, J = 20.4 Hz, 2H), 8.44 (s,1H), 7.95 (s, 1H), 7.48 (dd, J = 11.0, 7.0 Hz, 2H), 7.30 (d, J = 7.6 Hz, 1H),7.21 (t, J = 7.6 Hz, 2H), 6.49 – 6.44 (m, 1H), 6.32 (s, 1H), 5.91 (s, 1H), 4.19(d, J = 6.0 Hz, 1H), 2.92 (s, 1H), 2.14 – 2.04 (m, 1H), 1.93 – 1.75 (m, 4H),1.67 (s, 1H) ; HRMS: C 27 H 25 Cl2N6O2[M+H] + .

[0150]

[0151] Compound P-19 was synthesized using compound 57 and 61 as starting material, synthesis and work-up as in example 7, to yield a yellow solid in 34% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.93 (s, 1H), 9.82 (s, 1H), 9.65 (s,1H), 8.70 (d, J = 7.2 Hz, 1H), 8.63 (d,J = 7.6 Hz, 1H), 8.54 (s, 1H), 8.45 (s,1H), 7.95 (s, 1H), 7.47 (dd, J = 19.0, 7.6 Hz, 2H), 7.31 (d, J = 7.6 Hz, 1H),7.22 (d, J = 6.8 Hz, 2H), 7.09 (t, J = 7.2 Hz, 1H), 6.31 (s, 1H), 5.92 (s, 1H),4.22 (dd, J = 15.6, 8.0 Hz, 1H), 2.93 – 2.83 (m, 1H), 2.38 (d, J = 7.2 Hz, 2H),2.29 (d, J = 9.6 Hz, 2H) ; HRMS: C 26 H 23 Cl2N6O2[M+H] + .

[0152]

[0153] Compound P-20: synthesized using compound 58 and 61 as starting materials, synthesis and work-up as in example 7, yield 36% as yellow solid; 1 H NMR (400 MHz, DMSO- d 6) δ 11.90 (s, 1H), 9.81 (s, 1H), 9.60 (s,1H), 8.63 (d, J = 6.8 Hz, 1H), 8.52 (s, 1H), 8.42 (d, J = 14.4 Hz, 2H), 7.93 (s,1H), 7.49 (t, J = 8.4 Hz, 2H), 7.27 (d, J = 7.2 Hz, 1H), 7.21 (d, J = 7.6 Hz, 2H),7.11 (t, J = 7.2 Hz, 1H), 6.31 (s, 1H), 5.89 (s, 1H), 3.18 (d, J = 5.2 Hz, 2H),2.31 (s, 2H), 1.54 (dd, J= 23.2, 5.6 Hz, 4H) ; HRMS: C 26 H 25 Cl2N6O2[M+H] + .

[0154]

[0155] Compound P-21 was synthesized from compound 59 and 61, and the synthesis and workup were the same as in Example 7 to give a yellow solid in 36% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.92 (s, 1H), 9.81 (s, 1H), 9.62 (s,1H), 8.65 (d, J = 8.0 Hz, 1H), 8.55 (d, J = 2.4 Hz, 1H), 8.46 (s, 1H), 8.40 (s,1H), 7.95 (s, 1H), 7.51 (t, J = 9.2 Hz, 2H), 7.29 (d, J = 8.0 Hz, 1H), 7.23 (q, J =8.4 Hz, 2H), 7.13 (t, J = 7.6 Hz, 1H), 6.32 (s, 1H), 5.90 (s, 1H), 3.21 – 3.16(m, 2H), 2.31 (t, J = 7.2 Hz, 2H), 1.66 – 1.57 (m, 2H), 1.56 – 1.48 (m, 2H),1.35 – 1.28 (m, 2H) ; HRMS: C 27 H 27 Cl2N6O2[M+H] + .

[0156]

[0157] Compound P-22 was synthesized from compound 60 and 61, and the synthesis and workup were the same as in Example 7 to give a yellow solid in 38% yield; 1 H NMR (400 MHz, DMSO- d 6) δ 11.93 (s, 1H), 9.82 (s, 1H), 9.63 (s,1H), 8.64 (d, J= 7.2 Hz, 1H), 8.53 (s, 1H), 8.44 (s, 1H), 8.39 (s, 1H), 7.93(s, 1H), 7.49 (t, J = 8.4 Hz, 2H), 7.25 (s, 1H), 7.20 (d, J = 7.6 Hz, 2H), 7.11(d, J = 6.8 Hz, 1H), 6.29 (s, 1H), 5.88 (s, 1H), 3.14 (d, J = 5.6 Hz, 2H), 2.28(s, 2H), 1.57 (s, 2H), 1.45 (s, 2H), 1.35 (s, 1H), 1.28 (s, 5H) ; HRMS:C 29 H 31 Cl2N6O2[M+H] + .

[0158] CDK7 kinase inhibition activity test of 2-amino pyrimidine compounds containing 2-chloroacrylamide

[0159] Experimental method: CDK7 activity was determined using Perkin Elmer LANCE Ultra KinaSelect Ser / Thr kit reagent. The total volume of the reaction was 10 μΐ, and after adding 20 μιηοΐ target compound in a 384-well plate, CDK7 protein (final concentration 2 ng / μΐ), specific ULight 4EBP1 polypeptide substrate and ATP in buffer (50 mM HEPES (pH=7.5), 10 mM MgCl2, 1 mM EDTA, 2 mM DTT) were added to the reaction. After 2 hours, specific Eu-labeled anti-phosphorylated antibody was added to incubate for 30 min and detect phosphorylation, and the fluorescence signal intensity at 665 nm and 615 nm wavelengths was detected by fluorescence detector Envision to determine CDK7 activity. The positive compound was THZ1, and the IC 50 value was calculated by software Graphpad Prism 5, and the results are shown in Table 1. Subsequently, 10 target compounds with IC 50 less than 20 nM were tested for proliferation inhibition activity in different tumor cells.

[0160] Table 1 Kinase inhibition activity of compounds on CDK7

[0161]

[0162] Test of the proliferation inhibition activity of 2-amino pyrimidine compounds containing 2-chloro acrylamide on different tumor cells

[0163] Experimental method: the cell collection of human myelomonocytic leukemia cells MV4-11, human polar myeloid leukemia cells Molm-13, and human breast cancer cells MDA-MB-231 were centrifuged respectively, and the supernatant was removed. The obtained cells were resuspended with complete culture medium, and inoculated in a 96-well plate at 80 μL / well (8000-10000 well). The target compound was diluted in DMSO at 5 concentration gradients with the highest concentration of 50 μM (DMSO concentration of 100%), and the whole dilution was 100 times (DMSO concentration of 1%) in the culture medium and mixed.

[0164] The prepared target compound was added to the 96-well plate at 20 μL / well, and the initial highest concentration was 100 nM (DMSO was 0.2%). Then the cell plate was placed in a 37°C, 5% CO2 incubator for 3 days. After the culture was completed, the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Kit (MTS) was used for detection, 10 μL of MTS reagent was added to each well, and then mixed and placed in the incubator for about 1.5 h. Then the absorbance was measured by a microplate reader at 490 nm (background at 690 nm), until the DMSO treatment group reached 0.6-0.8. The formula cell survival rate (%) = (compound well value-pure culture medium well value) / (DMSO well value-pure culture medium well value)*100% was used to calculate and use Graphpad Prism 9.5 software to fit the growth curve and calculate the IC 50 value of the proliferation inhibition effect of the compound on different cells, and the results are shown in Table 2.

[0165] Table 2 Proliferation inhibition activity of compounds on three tumor cells

[0166]

[0167] Test of the CDK family selectivity of 2-amino pyrimidine compounds containing 2-chloro acrylamide

[0168] The test method of the kinase inhibition activity of CDK2, CDK9 and CDK12 is the same as that of CDK7, except that the CDK7 protein is replaced by CDK2, CDK9 and CDK12 protein, and the results are shown in Table 3.

[0169] Table 3 Proliferation inhibition activity of compounds on CDK2, CDK9 and CDK12 in CDK family

[0170]

[0171] Inhibition experiment of 2-amino pyrimidine compounds containing 2-chloro acrylamide on downstream proteins and cell cycle

[0172] The suspended cells 60w were plated into 12-well plates, the cells were treated after adding the compound at a specified concentration, and then the cells were collected, centrifuged at 5000 rpm for 3 min at 4 DEG C, the supernatant was discarded, 1 mL of PBS solution was added to the collection tube, the cells were resuspended, and the supernatant was discarded after centrifugation in the same way to obtain the target protein, and then a corresponding volume of 1xLoading Buffer was added, and the mixture was incubated in a 100 DEG C metal bath for 20 min, and then the mixture was used immediately or stored at -20 DEG C.

[0173] Western Blot protein level detection was performed on the collected target protein. First, 10% ordinary gel was prepared using Yezeng, then the gel holder was assembled, the protein samples were sequentially added to the gel holes, and the protein marker was added to the two side gel holes, and then electrophoresis was started. The electrophoresis conditions are as follows: 20 V sample collection, 60-80 V gel running for 1.5 h-2 h, and the electrophoresis is stopped when the bromophenol blue moves to the edge of the gel. Take the appropriate size NC membrane and two clean sponge pads, wet the NC membrane with the equilibration solution, assemble according to the order of “sponge pad-separation gel-NC membrane-sponge pad”, place the NC membrane close to the anode into the transmembrane instrument, and set the transmembrane time according to the size of the target protein, and transfer the protein from the separation gel to the NC membrane. Prepare 5% skim milk or 5% BSA blocking solution with TBST buffer, cut the membrane and place it in the blocking solution, and incubate at room temperature on a shaker for 1-2 h. After blocking, wash the membrane with TBST buffer for 3 times, each time for 5-10 min, add the specific antibody of the target protein, and incubate overnight at 4 DEG C on a shaker. After incubation of the primary antibody, wash the membrane with TBST buffer for 3 times, each time for 5-10 min, add the corresponding antibody of the primary antibody, and incubate at room temperature on a shaker for 1-2 h. After incubation of the secondary antibody, wash the membrane with TBST buffer for 3 times, each time for 5-10 min, rinse the membrane with developing solution, and then expose it to an imager to obtain a protein band image. After processing with Image Lab software, the results are analyzed. See Figure 1 、 Figure 2 、 Figure 3 and Figure 4 It can be seen that compounds P-15 and P-19 can inhibit the phosphorylation of downstream proteins in MV-4-11 tumor cells at a lower concentration, and can effectively inhibit the cell cycle process. The compounds of the present application have good kinase inhibition activity on CDK7, especially compounds P-1, P-2, P-5, P-6, P-7, P-8, P-9, P-15, P-19, P-20, which exhibit excellent CDK7 inhibition activity, IC 50All less than 20 nM; in the proliferation inhibition activity test of different tumors, it can be seen that compounds P-1, P-2, P-8, P-15, P-19, P-20 all have good tumor cell inhibition activity, especially compounds P-15, P-19, P-20 have excellent cell proliferation inhibition activity on both hematological tumors and solid tumors. As a CDK7 inhibitor with a new covalent warhead, this kind of molecule has good CDK7 kinase inhibition activity and cell proliferation inhibition activity, and is expected to become an effective choice for inhibiting tumors and diseases through the CDK7 pathway.

Claims

1. A compound having the general formula (Ⅰ): The compound is selected from: 。 2. A pharmaceutical composition comprising at least one compound as claimed in claim 1, wherein a pharmaceutically acceptable salt of the compound is an active ingredient, and comprising one or more pharmaceutically acceptable carriers or excipients.

3. The pharmaceutical composition according to claim 2, characterized in that: The administration route of the pharmaceutical composition is selected from oral, injection, inhalation, and implantation.

4. The pharmaceutical composition according to claim 2, characterized in that: The dosage form of the pharmaceutical composition is injection, tablet, capsule or granule.

5. Use of the compound of claim 1, a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 2 in the preparation of a tumor cell proliferation inhibitory drug.

6. A tumor treatment drug, characterized in that, The tumor contains any one or more of the compounds described in claim 1, wherein the tumor is colorectal cancer, breast cancer, human biphenotype myeloid monocytic leukemia, or human acute myeloid leukemia.

7. The method for preparing the compound according to claim 1, characterized in that, Including the following: (1) Compound a reacts with compound b under the action of AlCl3 to obtain compound c. The reaction temperature is 60-90℃ and the reaction time is 10-14 hours. The crude product obtained is purified by column chromatography. (2) Compound c reacts with compound d under the action of hydrochloric acid to obtain compound e. The crude product is purified by column chromatography. The reaction temperature is 100-130℃ and the reaction time is 7-9 hours. If the compound obtained by the reaction has a Boc protecting group, the Boc protecting group is removed under the action of TFA after the reaction to obtain the corresponding compound e. The reaction temperature is 0℃ and the reaction time is 1 hour. The crude product is purified by column chromatography. (3) Compound e reacts with compound f under the action of a condensing agent to obtain compound g. The crude product obtained is purified by column chromatography. The condensing agent used is 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate. The reaction temperature is 0℃-rt and the reaction time is 4-6 hours. If compound f is benzene ring-substituted p-nitrobenzoic acid, then compound f is first reacted with SOCl2 at 70-90℃ to generate benzene ring-substituted p-nitrobenzoyl chloride. Then it is reacted with TFA, DMF and TEA to obtain an intermediate. The reaction temperature is rt and the reaction is carried out overnight. The intermediate is reduced under the conditions of Fe, NH4Cl, EtOH, THF and H2O to obtain the crude product of compound g, which is purified by column chromatography. (4) Compound g was deprotected by Boc protecting group under the action of TFA to obtain compound h. The reaction temperature was 0℃ and the reaction time was 1-2 hours. The crude product was separated by column chromatography to obtain pure product. (5) Compound i reacts with oxalyl chloride to obtain a 2-chloroacryloyl chloride solution at a reaction temperature of 40-60℃ and a reaction time of 3-5 hours; Compound h reacts with the prepared 2-chloroacryloyl chloride solution under the action of TEA at a reaction temperature of rt and a reaction time of 2-4 hours to obtain compound I. The reaction equation for the main reaction process is as follows: Wherein: R, A1, and A2 are all as described in any compound of claim 1.

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