2-aminopyrimidine compound containing 2-chloroacrylamide as well as preparation method and application of 2-aminopyrimidine compound

Abstract

The invention provides a 2-aminopyrimidine compound containing 2-chloroacrylamide as well as a preparation method and application thereof, and particularly provides a compound as shown in a formula I. The compound has relatively good CDK7 inhibitory activity, can be used as a small-molecule selective covalent CDK7 inhibitor and is used for treating tumors, such as tumor cells, tumor cells and the like. For example, colorectal cancer, breast cancer, human biphenotypic myelomonocytic leukemia, and human acute myeloid leukemia.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to a novel 2-aminopyrimidine compound containing 2-chloroacrylamide, a preparation method and application thereof, and application of the compound in preparing tumor prevention and treatment drugs. Background Art

[0002] CDK7 is a regulatory protein kinase in the body. It aggregates with Cyclin H and MAT1 to form a ternary complex CAK, which participates in the regulation of the cell cycle and transcription cycle through phosphorylation. In cell cycle regulation, CDK7 ensures that different CDKs have kinase activity at the right time through phosphorylation regulation, thereby promoting the cell cycle process; in the transcription cycle, CDK7 itself is an important component subunit of the 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 many 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 are designed to inhibit the overexpression of 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, so as to achieve the purpose of preventing and treating diseases.

[0003] In recent years, the research on CDK7 inhibitors has been progressing steadily, and many potential compounds with development value have been discovered, but the development of this field is still in its early stages. Summary of the invention

[0004] In view of the deficiencies of the prior art, the technical problem to be solved by the present invention is to develop 2-aminopyrimidine compounds containing 2-chloroacrylamide, and test and screen their CDK7 inhibitory ability. Studies have shown that such compounds have good inhibitory activity against CDK7. The results of molecular level kinase inhibitory activity tests and cell proliferation inhibition tests show that such compounds have good CDK7 inhibitory activity and have good inhibitory effects on tumor cells. Such molecules are expected to become an effective choice for inhibiting tumor cells by inhibiting CDK7.

[0005] Definition of terms: The term "aryl" as used herein refers to an all-carbon monocyclic or condensed polycyclic group of 5 to 12 carbon atoms having a completely conjugated π electron system. Non-limiting examples of aromatic rings include benzene ring and naphthalene ring.

[0006] The term "heterocyclic aryl" used in the present invention refers to an unsaturated carbon ring of 5-6 ring atoms, wherein one or more carbon atoms are replaced by heteroatoms such as oxygen, nitrogen, sulfur, etc. Specific heterocyclic aryl groups can be: pyridyl, pyrimidinyl, pyrazinyl, thiazolyl, oxazolyl and isoxazolyl, etc.

[0007] The term "heterocyclic group" used in the present invention refers to a monocyclic group having 4-6 ring atoms in the ring, wherein at least one or two ring atoms are selected from nitrogen, oxygen, sulfur heteroatoms, and the remaining ring atoms are carbon atoms. Specific heterocyclic groups can be piperidinyl, piperazinyl, pyrrolidinyl, tetrahydropyranyl, oxetanyl, etc.

[0008] The term "alkoxy" used in the present invention refers to an -O-alkyl group. Specific alkoxy groups include methoxy, ethoxy, tert-butoxy, and the like.

[0009] The term "halogen" as used herein refers to fluorine, chlorine, bromine or iodine, preferably fluorine, chlorine or bromine.

[0010] The object of the present invention is to provide a 2-aminopyrimidine compound containing 2-chloroacrylamide, wherein the compound has the following general formula (I):

[0011] in: R is selected from H, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, amino, halogen, nitro, cyano, carboxyl; A1 is selected from C 1-6 alkyl, heterocyclic group, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic aryl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclic alkyl; the substituted aryl, substituted heterocyclic aryl, substituted cycloalkyl, substituted heterocyclic alkyl is substituted by halogen, nitro, cyano, carbamoyl, C 1-4 Alkyl, C 1-4 Alkoxy groups are optionally substituted; A2 is selected from C 2-8 alkyl, heterocyclic group, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic aryl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclic alkyl; the substituted aryl, substituted heterocyclic aryl, substituted cycloalkyl, substituted heterocyclic alkyl is substituted by halogen, nitro, cyano, carbamoyl, C 1-4 Alkyl, C 1-4 The alkoxy group is optionally substituted.

[0012] Another object of the present invention is to provide a method for preparing the above-mentioned target compound, which is achieved by the following steps: (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, and the obtained crude product is separated by column chromatography to obtain a pure product; (2) Compound c reacts with compound d under the action of hydrochloric acid to obtain compound e, and the obtained crude product is separated by column chromatography to obtain a pure product. The reaction temperature is 100-130°C 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 first removed under the action of TFA after the reaction to obtain the corresponding compound e. The reaction temperature is 0°C and the reaction time is 1 hour. The obtained crude product is separated by column chromatography to obtain a pure product; (3) Compound e reacts with compound f under the action of a condensing agent to obtain compound g, and the obtained crude product is separated by column chromatography to obtain a pure product. The selected condensing agent is 2-(7-azabenzotriazole)-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-substituted p-nitrobenzoic acid, compound f is first reacted with SOCl2 at 70-90°C to generate a benzene-substituted p-nitrobenzoyl chloride, which is then reacted with TFA, DMF and TEA to obtain an intermediate, the reaction temperature is rt, the reaction is carried out 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 a pure product; (4) Compound g is subjected to the removal of the Boc protecting group under the action of TFA to obtain compound h. The reaction temperature is 0°C and the reaction time is 1-2 hours. The obtained crude product is separated by column chromatography to obtain a pure product. (5) Compound i is reacted with oxalyl chloride to obtain a 2-chloroacryloyl chloride solution at a temperature of 40-60° C. for a reaction time of 3-5 hours; Compound h is reacted with the obtained 2-chloroacryloyl chloride solution under the action of TEA at a reaction temperature of rt for a reaction time of 2-4 hours to obtain a compound of formula I; The reaction formula of the above main reaction process is:

[0013] in: Another object of the present invention is to provide a pharmaceutical composition comprising at least one compound in any form as described above, a stereoisomer or a pharmaceutically acceptable salt of the compound as an active ingredient, and one or more pharmaceutically acceptable carriers or excipients.

[0014] The pharmaceutical composition of the present invention can be prepared into various pharmaceutical dosage forms, such as oral administration, injection, inhalation, implantation, etc. Injection and oral administration are preferred, such as injection, lyophilized powder, injection, tablet, capsule or granule.

[0015] The pharmaceutical composition of the present invention and various preparations of the composition can be prepared using conventional pharmaceutical carriers.

[0016] Another object of the present invention is to provide the pharmaceutical use of the compound of formula (I) or a pharmaceutical composition containing the compound. That is, the present invention provides the use of the compound of formula (I) or a pharmaceutical composition containing the compound in the preparation of CDK7 inhibitors, and further provides its use in the preparation of drugs for treating CDK7-related diseases.

[0017] Furthermore, the disease is selected from cancer (including leukemia, human biphenotypic myelomonocytic 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 tumors, cardiovascular diseases, inflammation and autoimmune diseases.

[0018] Experiments have shown that the 2-aminopyrimidine compounds containing 2-chloroacrylamide of the present invention have good CDK7 inhibitory activity. The raw materials required for the synthesis of the compounds of the present invention are easily available, 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 THE DRAWINGS

[0019] Figure 1 The effects of compounds P-15 and P-19 on MV-4-11 tumor cell-related proteins; Figure 2 The effects of compounds P-15 and P-19 on the cell cycle of MV-4-11 tumor cells; Figure 3 for Figure 2 Comparison of quantitative results; Figure 4 This is the effect of compounds P-15 and P-19 on cell apoptosis-related proteins. DETAILED DESCRIPTION

[0020] The present invention is further described in conjunction with the examples. The following examples are only intended to illustrate the present invention, but are not intended to limit the present invention in any way.

[0021] Example 1 Preparation of 3-(2,5-dichloro-4-pyrimidinyl)-1H-indole

[0022] A magnetic stirrer, 2,4,5-trichloropyrimidine (1.17 g, 10 mmol), AlCl3 (1.83 g, 10 mmol), and 30 mL of DCE were added to a 100 mL single-necked flask in sequence and stirred at room temperature for 10 min. Indole (1.17 g, 10 mmol) was then added and heated to reflux at 80 °C for 4 h. After the reaction was completed, the reaction system was poured into water to precipitate a large amount of yellow solid. The filtrate was removed by suction and the solid was dried to obtain 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).

[0023] Synthesis of compound 6: Compounds 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).

[0024] Synthesis of compound 7: Compounds 1 and 4 were used as raw materials, and the synthesis and post-treatment were the same as in Example 1 to obtain yellow solid 7 with a yield of 78%; 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). Example 2 N 1 Preparation of -(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)benzene-1,3-diamine

[0025] A magnetic stirrer, compound 5 (3.00 g, 11.4 mmol), 1,3-phenylenediamine (2.50 g, 22.8 mmol), and 30 mL of n-butanol were added to a 100 mL single-necked flask in sequence, heated to 110°C, and 0.5 mL of concentrated hydrochloric acid solution was added dropwise. The mixture was refluxed overnight. After the reaction was completed, the mixture was cooled to room temperature, concentrated, and purified by silica gel column chromatography to obtain 1.53 g of a yellow solid with 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).

[0026] Synthesis of compound 17: Compounds 6 and 8 were used as raw materials, and the synthesis and post-treatment were the same as in Example 2 to obtain yellow solid 17 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).

[0027] Synthesis of compound 18: Compounds 7 and 8 were used as raw materials, and the synthesis and post-treatment were the same as in Example 2 to obtain yellow solid 18 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).

[0028] Synthesis of compound 19: Compounds 5 and 9 were used as raw materials, and the synthesis and post-treatment were the same as in Example 2 to obtain yellow solid 19 with a yield of 52%; 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).

[0029] Synthesis of compound 20: Compounds 5 and 10 were used as raw materials, and the synthesis and post-treatment were the same as in Example 2 to obtain yellow solid 20 with a yield of 27%; 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).

[0030] Synthesis of compound 21: Compounds 5 and 11 were used as raw materials, and the synthesis and post-treatment were the same as in Example 2 to obtain yellow solid 21 with a yield of 33%; 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). Example 3 N 1 Preparation of -(5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)benzene-1,3-diamine

[0031] The synthesis and post-treatment of the intermediate were the same 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 obtain the target compound 22 as a yellow solid with a yield of 56%; 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).

[0032] Synthesis of compound 23: Compounds 5 and 12 were used as raw materials, and the synthesis and post-treatment were the same as in Example 3 to obtain yellow solid 23 with a yield of 43%; 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).

[0033] Synthesis of compound 24: Compounds 5 and 13 were used as raw materials, and the synthesis and post-treatment were the same as in Example 3 to obtain yellow solid 24 with a yield of 36%; 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).

[0034] Synthesis of compound 25: Compounds 5 and 14 were used as raw materials, and the synthesis and post-treatment were the same as in Example 3 to obtain yellow solid 25 with a yield of 32%; 1H 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). Example 4 Preparation of (1r, 4r)-4-amino-N-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)cyclohexanecarboxamide

[0035] A magnetic stirrer, trans-4-(Boc-amino)cyclohexanecarboxylic acid (0.83 g, 3.6 mmol), HATU (1.37 g, 3.6 mmol), 10 mL DMF, and DIPEA (0.78 g, 6.0 mmol) were added to a 25 mL single-necked flask in sequence. Stir at 0 °C for 1 h. Compound 16 (1.00 g, 3.0) was added to the reaction system and reacted at room temperature overnight. After the reaction was completed, the reaction system was poured into water, and a large amount of yellow solid precipitated. The filtrate was removed by suction, and the solid was dried to obtain a crude product. The obtained crude product was added to a 40% TFA in DCM solution, stirred at 0 °C for 1.5 h, and TFA and solvent were removed under reduced pressure. The product was separated and purified by silica gel column chromatography to obtain 1.03 g of a 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).

[0036] Synthesis of compound 28: Compounds 17 and 26 were used 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).

[0037] Synthesis of compound 29: Compounds 18 and 26 were used as raw materials, and the synthesis and post-treatment were the same as in Example 4 to obtain yellow solid 29 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).

[0038] Synthesis of compound 30: Compounds 19 and 26 were used as raw materials, and the synthesis and post-treatment were the same as in Example 4 to obtain yellow solid 30 with a yield of 52%; 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).

[0039] Synthesis of compound 31: Compounds 20 and 26 were used as raw materials, and the synthesis and post-treatment were the same as in Example 4 to obtain yellow solid 31 with a yield of 43%; 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).

[0040] Synthesis of compound 32: Compounds 21 and 26 were used as raw materials, and the synthesis and post-treatment were the same as in Example 4 to obtain yellow solid 32 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).

[0041] Synthesis of compound 33: Using compounds 22 and 26 as raw materials, the synthesis and post-treatment were the same as in Example 4 to obtain yellow solid 33 with a yield of 41%; 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).

[0042] Synthesis of compound 34: Compounds 23 and 26 were used as raw materials, and the synthesis and post-treatment were the same as in Example 4 to obtain yellow solid 34 with a yield of 41%; 1 H NMR (400 MHz, DMSO- d 6) 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).

[0043] Synthesis of compound 35: Compounds 24 and 26 were used as raw materials, and the synthesis and post-treatment were the same as in Example 4 to obtain yellow solid 35 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).

[0044] Synthesis of compound 36: Compounds 25 and 26 were used as raw materials, and the synthesis and post-treatment were the same as in Example 4 to obtain 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). Example 5 Preparation of 4-amino-N-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)benzamide

[0045] 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, and TEA (0.30 g, 3.0 mmol) were added to a 50 mL single-necked flask in sequence and reacted at room temperature overnight. The reaction solution was concentrated under reduced pressure, and 10 mL saturated NaOH aqueous solution was added to quench. The reaction system was poured into water, and a large amount of yellow solid precipitated. The filtrate was removed by suction 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, and 20 mL THF were added to a 100 mL single-necked flask in sequence, and the mixture was heated to reflux at 80 °C and reacted overnight. After the reaction was completed, the solid was removed by suction while hot, and the filtrate was concentrated under reduced pressure. The filtrate 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). Example 6 Preparation of 4-amino-N-(3-((5-chloro-4-(1H-indol-3-yl)pyrimidin-2-yl)amino)phenyl)-2-fluorobenzamide

[0046] A magnetic stirrer, 2-fluoro-4-nitrobenzoic acid (2 g, 10.8 mmol), and 15 mL SOCl2 were added to a 50 mL single-necked flask in sequence, and the mixture was heated to reflux at 80°C for overnight reaction. After the reaction, the reaction solution was concentrated under reduced pressure to obtain a white solid 2-fluoro-4-nitrobenzoyl chloride. The synthesis and post-treatment of compound 42 were the same as those in 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), 9.66 (s, 2H), 8.65 (d, J = 6.8 Hz, 1H), 8.54 (s, 1H), 8.45 (s, 1H), 8.06 (s,1H), 7.49 (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).

[0047] Synthesis of compound 43: Compounds 16 and 39 were used as raw materials, and the synthesis and post-treatment were the same as in 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).

[0048] Synthesis of compound 44: Compounds 16 and 40 were used as raw materials, and the synthesis and post-treatment were the same as in Example 5 to obtain yellow solid 44 with a yield of 38%; 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).

[0049] Synthesis of compound 53: Compounds 16 and 45 were used as raw materials, and the synthesis and post-treatment were the same as in Example 5 to obtain yellow solid 53 with a yield of 52%; 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).

[0050] Synthesis of compound 54: Compounds 16 and 46 were used as raw materials, and the synthesis and post-treatment were the same as in Example 5 to obtain yellow solid 54 with a yield of 64%; 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).

[0051] Synthesis of compound 55: Compounds 16 and 47 were used as raw materials, and the synthesis and post-treatment were the same as in Example 5 to obtain yellow solid 55 with a yield of 59%; 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).

[0052] Synthesis of compound 56: Compounds 16 and 48 were used as raw materials, and the synthesis and post-treatment were the same as in Example 5 to obtain yellow solid 56 with a yield of 55%; 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).

[0053] Synthesis of compound 57: Compounds 16 and 49 were used as raw materials, and the synthesis and post-treatment were the same as in Example 5 to obtain yellow solid 57 with a yield of 66%; 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).

[0054] Synthesis of compound 58: Compounds 16 and 50 were used as raw materials, and the synthesis and post-treatment were the same as in Example 5 to obtain yellow solid 58 with a yield of 64%; 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).

[0055] Synthesis of compound 59: Compounds 16 and 51 were used as raw materials, and the synthesis and post-treatment were the same as in Example 5 to obtain yellow solid 59 with a yield of 64%; 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).

[0056] Synthesis of compound 60: Compounds 16 and 52 were used as raw materials, and the synthesis and post-treatment were the same as in Example 5 to obtain yellow solid 60 with a yield of 64%; 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). Example 7 Preparation of Compound P-1

[0057] A magnetic stirrer and 2-chloroacrylic acid (233 mg, 2.2 mmol) were added to a two-necked flask. Under the protection of N2, 4 mL of anhydrous DCM, 0.1 mL of anhydrous DMF, and oxalyl chloride (270 mg, 2.2 mmol) were added in sequence. The mixture was heated to reflux at 50°C for 4 h. After the reaction was completed, a colorless 2-chloroacryloyl chloride solution was obtained. Compound 27 (100 mg, 0.22 mmol), 3 mL of anhydrous DCM, 2 mL of anhydrous DMF, TEA (258 mg, 2.6 mmol), and 2-chloroacryloyl chloride were added to a 25 mL single-necked flask in sequence and reacted at room temperature overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the system was poured into water to precipitate a large amount of yellow solid. The filtrate was removed by suction and filtered. After drying, it was 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] + .

[0058] Synthesis of compound P-2: Compounds 28 and 61 were used as raw materials, and the synthesis and post-treatment 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] + .

[0059] Synthesis of compound P-3: Compounds 29 and 61 were used as raw materials, and the synthesis and post-treatment 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] + .

[0060] Synthesis of compound P-4: Compounds 30 and 61 were used as raw materials, and the synthesis and post-treatment 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] + .

[0061] Synthesis of compound P-5: Compounds 31 and 61 were used as raw materials, and the synthesis and post-treatment 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 11.81 (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 = 10.4 Hz, 6H); HRMS: C 28 H 33 Cl2N6O2[M+H] + .

[0062] Synthesis of compound P-6: Compounds 32 and 61 were used as raw materials, and the synthesis and post-treatment 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 11.82 (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] + .

[0063] Synthesis of compound P-7: Compounds 33 and 61 were used as raw materials, and the synthesis and post-treatment 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 11.87 (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] + .

[0064] Synthesis of compound P-8: Compounds 34 and 61 were used as raw materials, and the synthesis and post-treatment 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 11.83 (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: C27 H 31 Cl2N6O2[M+H] + .

[0065] Synthesis of compound P-9: Compounds 35 and 61 were used as raw materials, and the synthesis and post-treatment were the same as in Example 7 to obtain a yellow solid with a yield of 19%; 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] + .

[0066] Synthesis of compound P-10: Compounds 36 and 61 were used as raw materials, and the synthesis and post-treatment were the same as in Example 7 to obtain a yellow solid with a yield of 19%;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 HRMS: C 26 H 29 Cl2N6O2[M+H] + .

[0067] Synthesis of compound P-11: Compounds 41 and 61 were used as raw materials, and the synthesis and post-treatment were the same as in Example 7 to obtain a yellow solid with a yield of 8%; 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] + .

[0068] Synthesis of compound P-12: Compounds 42 and 61 were used as raw materials, and the synthesis and post-treatment were the same as in Example 7 to obtain 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] + .

[0069] Synthesis of compound P-13: Compounds 43 and 61 were used as raw materials, and the synthesis and post-treatment were the same as in Example 7 to obtain a yellow solid 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] + .

[0070] Synthesis of compound P-14: Using compounds 44 and 61 as raw materials, the synthesis and post-treatment were the same as in Example 7 to obtain a yellow solid with a yield of 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] + .

[0071] Synthesis of compound P-15: Compounds 53 and 61 were used as raw materials, and the synthesis and post-treatment were the same as in Example 7 to obtain a yellow solid with a yield of 37%; 1 H NMR (400 MHz, DMSO- d 6) 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] + .

[0072] Synthesis of compound P-16: Compounds 54 and 61 were used as raw materials, and the synthesis and post-treatment 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] + .

[0073] Synthesis of compound P-17: Compounds 55 and 61 were used as raw materials, and the synthesis and post-treatment 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 11.93 (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] + .

[0074] Synthesis of compound P-18: Compounds 56 and 61 were used as raw materials, and the synthesis and post-treatment were the same as in Example 7 to obtain a yellow solid with a yield of 35%; 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] + .

[0075] Synthesis of compound P-19: Compounds 57 and 61 were used as raw materials, and the synthesis and post-treatment were the same as in Example 7 to obtain a yellow solid with a yield of 34%; 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] + .

[0076] Synthesis of compound P-20: Compounds 58 and 61 were used as raw materials, and the synthesis and post-treatment 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 11.90 (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] + .

[0077] Synthesis of compound P-21: Compounds 59 and 61 were used as raw materials, and the synthesis and post-treatment 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 11.92 (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] + .

[0078] Synthesis of compound P-22: Compounds 60 and 61 were used as raw materials, and the synthesis and post-treatment 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 11.93 (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] + . CDK7 kinase inhibitory activity test of 2-aminopyrimidine compounds containing 2-chloroacrylamide Experimental method: CDK7 activity was determined using Perkin Elmer's LANCE Ultra KinaSelect Ser / Thr kit detection reagent. The total reaction volume was 10 µL. After adding 20 µmol of the target compound to a 384-well plate, CDK7 protein (final concentration 2 ng / µL), specific ULight 4EBP1 peptide substrate and ATP were reacted in buffer (50 mM HEPES (pH=7.5), 10 mM MgCl2, 1 mM EDTA, 2 mM DTT). After 2 hours, a specific Eu-labeled anti-phosphorylation antibody was added and incubated for 30 min to detect phosphorylation. The fluorescence signal intensity at 665 nm and 615 nm was detected by the fluorescence detector Envision to reflect CDK7 activity. The positive compound is THZ1, IC 50 The values ​​were calculated by Graphpad Prism 5 software, and the results are shown in Table 1. 50 A total of 10 target compounds with concentrations less than 20 nM were tested for proliferation inhibition activity in different tumor cells.

[0079] Table 1 Kinase inhibitory activity of compounds against CDK7

[0080] Proliferation inhibition activity test of some 2-aminopyrimidine compounds containing 2-chloroacrylamide in different tumor cells Experimental method: The cell collections of human myelomonocytic leukemia cells MV4-11, human polar myeloid leukemia cells Molm-13, and human breast cancer cells MDA-MB-231 were centrifuged, the supernatant was removed, the obtained cells were resuspended in complete culture medium, and inoculated in 96-well plates (8000-10000 wells) at 80 µL / well. The target compound was diluted 5-fold with a maximum concentration of 50 μM (DMSO concentration was 100%) in DMSO, and then placed in the culture medium and mixed at a total dilution of 100 times (DMSO concentration was 1%).

[0081] The prepared target compound was added to a 96-well plate at 20 µL / well, with the initial maximum concentration of 100 nM (DMSO was 0.2%), and the cell plate was then placed in an incubator at 37°C and 5% CO2 for 3 days. After the culture was completed, the CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Kit (MTS) was used for detection. 10 µL MTS reagent was added to each well, mixed and placed in an incubator for about 1.5 hours, and then the absorbance was measured at 490 nm (background was subtracted at 690 nm) by an ELISA reader until the DMSO treatment group reached 0.6-0.8. The formula cell viability (%) = (compound well value - pure culture well value) / (DMSO well value - pure culture well value) * 100% was used to calculate and use Graphpad Prism 9.5 software to fit the growth curve and calculate the IC of the compound's inhibitory effect on the proliferation of different cells. 50 The results are shown in Table 2.

[0082] Table 2 Proliferation inhibition activity of compounds on three types of tumor cells

[0083] CDK family selectivity test of 2-aminopyrimidines containing 2-chloroacrylamide The kinase inhibitory activity test method of CDK2, CDK9 and CDK12 is the same as that of CDK7, except that the CDK7 protein is replaced by CDK2, CDK9 and CDK12 proteins. The results are shown in Table 3.

[0084] Table 3 Proliferation inhibitory activity of compounds against CDK2, CDK9 and CDK12 in the CDK family

[0085] Inhibition experiment of 2-aminopyrimidine compounds containing 2-chloroacrylamide on downstream proteins and cell cycle 60w of suspended cells were plated into a 12-well plate, and the cells were treated by adding compounds of specified concentrations. After the treatment time was over, the cells were collected, centrifuged at 4°C, 5000 rpm, 3 min, and the supernatant was discarded. 1 mL of PBS solution was added to the collection tube, and the cells were resuspended. After the same centrifugation, the supernatant was discarded to obtain the target protein, and the corresponding volume of 1× Loading Buffer was added. The cells were placed in a 100°C metal bath for 20 min, and then cooled for use or stored at -20°C.

[0086] Western Blot protein level detection was performed on the collected target proteins. First, 10% ordinary gel was used for gel preparation, and then the gel rack was assembled. The protein samples were added to the gel wells in sequence, and the protein markers were added to the gel wells on both sides before electrophoresis was started. The electrophoresis conditions were 20 V for sample collection and 60-80 V for gel running for a total of 1.5 h~2 h. The electrophoresis was stopped when bromophenol blue moved to the edge of the gel. Take an NC membrane of appropriate size and two clean sponge pads, moisten the NC membrane with the balance solution, and assemble in the order of "sponge pad-separation gel-NC membrane-sponge pad". The NC membrane side was placed close to the positive electrode in the transfer instrument, and the transfer time was set according to the size of the target protein to transfer the protein from the separation gel to the NC membrane. TBST buffer was used to prepare 5% skim milk or 5% BSA blocking solution, the membrane was cut and placed in the blocking solution, and it was placed on a shaker at room temperature for 1~2 h. After the blocking was completed, the membrane was washed 3 times with TBST buffer, each time for 5~10 min, and the target protein-specific antibody was added, and incubated overnight on a shaker at 4 ℃. After the primary antibody incubation, the membrane was washed 3 times with TBST buffer, each time for 5-10 min, and the corresponding antibody of the primary antibody was added, and incubated on a shaker at room temperature for 1-2 h. After the secondary antibody incubation, the membrane was washed 3 times with TBST buffer, each time for 5-10 min, and the membrane was rinsed with developer and exposed in an imager to obtain protein band images, which were processed and analyzed using Image Lab software. 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 at lower concentrations in MV-4-11 tumor cells and can effectively inhibit the cell cycle process. The compounds of the present invention have good kinase inhibitory activity against CDK7, especially compounds P-1, P-2, P-5, P-6, P-7, P-8, P-9, P-15, P-19, and P-20 show excellent CDK7 inhibitory activity, IC 50 All of them are 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, and P-20 all have good tumor cell inhibition activity, especially compounds P-15, P-19, and P-20 have excellent cell proliferation inhibition activity on hematological tumors and solid tumors. As a CDK7 inhibitor with a new covalent warhead, this type 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 their diseases through the CDK7 pathway.

Claims

1. A compound having the general formula (I): ， in: R is selected from H, C 1-6 Alkyl, C 1-6 Alkoxy, C 1-6 Haloalkyl, amino, halogen, nitro, cyano, carboxyl; A1 is selected from C 1-6 alkyl, heterocyclic group, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic aryl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclic alkyl; the substituted aryl, substituted heterocyclic aryl, substituted cycloalkyl, substituted heterocyclic alkyl is substituted by halogen, nitro, cyano, carbamoyl, C 1-4 Alkyl, C 1-4 Alkoxy groups are optionally substituted; A2 is selected from C 2-8 alkyl, heterocyclic group, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic aryl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted heterocyclic alkyl; the substituted aryl, substituted heterocyclic aryl, substituted cycloalkyl, substituted heterocyclic alkyl is substituted by halogen, nitro, cyano, carbamoyl, C 1-4 Alkyl, C 1-4 The alkoxy group is optionally substituted.

2. The compound according to claim 1, characterized in that The compound is selected from: 。 3. A pharmaceutical composition comprising at least one compound according to any one of claims 1 to 2, a stereoisomer or a pharmaceutically acceptable salt of the compound as an active ingredient, and one or more pharmaceutically acceptable carriers or excipients.

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

5. The pharmaceutical composition according to claim 3, characterized in that: The dosage form of the pharmaceutical composition is injection, lyophilized powder, injection, tablet, capsule or granule.

6. Use of the compound according to any one of claims 1 to 2, its pharmaceutically acceptable salt, solvate, or the pharmaceutical composition according to claim 3 in the preparation of a drug for treating protein toxic diseases.

7. The use according to claim 6, characterized in that The drug for treating protein toxicity diseases is a CDK7 inhibitor.

8. A tumor treatment drug, characterized in that: Containing any one or more of the compounds according to any one of claims 1 to 2, wherein the tumor is colorectal cancer, breast cancer, human biphenotypic myelomonocytic leukemia or human acute myeloid leukemia.

9. The method for preparing the compound according to claim 1, characterized in that: These include: (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, and the obtained crude product is separated by column chromatography to obtain a pure product; (2) Compound c reacts with compound d under the action of hydrochloric acid to obtain compound e, and the obtained crude product is separated by column chromatography to obtain a pure product, the reaction temperature is 100-130°C, 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 first removed under the action of TFA after the reaction 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 a pure product; (3) Compound e reacts with compound f under the action of a condensing agent to obtain compound g, and the obtained crude product is separated by column chromatography to obtain a pure product. The selected condensing agent is 2-(7-azabenzotriazole)-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-substituted p-nitrobenzoic acid, compound f is first reacted with SOCl2 at 70-90°C to generate a benzene-substituted p-nitrobenzoyl chloride, which is then reacted with TFA, DMF and TEA to obtain an intermediate, the reaction temperature is rt, the reaction is carried out overnight, and the intermediate is reduced under the conditions of Fe, NH4Cl, EtOH, THF, and H2O to obtain a crude compound g, which is separated by column chromatography to obtain a pure product; (4) Compound g is subjected to the removal of the Boc protecting group under the action of TFA to obtain compound h. The reaction temperature is 0°C and the reaction time is 1-2 hours. The obtained crude product is separated by column chromatography to obtain a pure product. (5) Compound i is reacted with oxalyl chloride to obtain a 2-chloroacryloyl chloride solution at a temperature of 40-60° C. for a reaction time of 3-5 hours; Compound h is reacted with the obtained 2-chloroacryloyl chloride solution under the action of TEA at a reaction temperature of rt for a reaction time of 2-4 hours to obtain a compound of formula I; Among them, the reaction formula of the main reaction process is: ， Wherein: R, A1, A2 are as described in claim 1.

Images