4-aryl-2-[1-pyrazol-4-yl] aminopyrimidine derivative as well as preparation method and application thereof

Abstract

The invention discloses a 4-aryl-2-[1-pyrazol-4-yl] aminopyrimidine derivative as well as a preparation method and application thereof. The 4-aryl-2-[1-pyrazol-4-yl] aminopyrimidine compound and the pharmaceutical salt or isomer thereof are shown in the following structural formula. The micromolecule and the derivative thereof have excellent water solubility, can improve the bioavailability of the micromolecule in a living body, can be used as an active component, and can be developed into various dosage forms of treatment drugs of related targets, especially injections. The small molecules and derivatives and pharmaceutical compositions thereof are wide in application and can be prepared into drugs for treating tumors. The drug effect can be achieved at the molecular level and the cellular level, the treatment effect is more excellent, and the optimal IC50 value can reach the nanomolar concentration level.

Description

4-Aryl-2-[1-pyrazol-4-yl]aminopyrimidine derivatives, their preparation methods and applications Technical Field

[0001] This invention belongs to the field of CDK9 inhibitor preparation technology, specifically relating to a 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine derivative and its preparation method and application. Background Technology

[0002] Cyclin-dependent kinases (CDKs) belong to the serine / threonine protein kinase family and play a crucial role in controlling the cell cycle. As a member of the CDK family, CDK9 binds to cyclins to form positive elongation factor b (P-TEFb), which phosphorylates Ser2 within the C-terminal domain (CTD) of RNA polymerase II (RNAPII), resulting in gene transcription elongation. CDK9 is an important regulator that stimulates the transcription of anti-apoptotic proteins. Inhibition of CDK9-mediated RNAPII phosphorylation rapidly reduces anti-apoptotic protein levels, thereby promoting apoptosis and inhibiting tumor cell proliferation. Dysregulation of CDK9-related pathways is frequently observed in many human malignancies; therefore, CDK9 inhibitors are an attractive class of compounds for cancer therapy.

[0003] First-generation CDK9 inhibitor clinical candidates, such as Flavoiridol, possess excellent pharmacodynamic properties and in vivo anti-tumor efficacy. However, due to their lack of selectivity for the CDK family, they suffered from toxicity and adverse reactions, ultimately preventing their clinical application. Currently, selective CDK9 inhibitors have become a research hotspot. Among selective CDK9 inhibitors, three have entered clinical trials: BAY-1251152, AZD-4573, and KB-0742, all in Phase I clinical trials. An increasing number of researchers are focusing on designing drugs and inhibitors that target CDK9. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine derivative with excellent antitumor activity and its preparation method, wherein the derivative is an isomer, diastereomer, enantiomer, tautomer, solvate, salt of solvate, pharmaceutically acceptable salt, or mixture thereof.

[0005] Another object of the present invention is to provide a method for preparing such derivatives and their application in the treatment of tumors.

[0006] To address the problems of the prior art, the technical solution adopted by this invention is: 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine compounds of formula (I) and their pharmaceutical salts or isomers. (I) Where X represents -N- and Y represents -CR 4 -; or X represents -CH-, Y represents -N-; or X represents -CH-, Y represents -CR 4 -;R 1 The group to be represented is selected from the following groups: , , or And G is -NH- or -O-; R 2 Represents morpholinyl, morpholinylalkyl, alkyl-substituted piperazine, acyl-substituted piperazine, sulfonyl-substituted piperazine, piperazinealkyl, piperazinealkoxy, piperazinealkylamino, homopiperazine, homopiperazinealkyl, homopiperazinealkoxy, homopiperazinealkylamino, piperidinyl, piperidinylalkyl, piperidinylalkoxy, piperidinylalkylamino, tetrahydropyrrolyl, tetrahydropyrrolylalkyl, tetrahydropyrrolylalkylamino, tetrahydrofuranyl, tetrahydrofuranalkyl, tetrahydrofuranalkoxy, tetrahydrofuranalkylamino, tetrahydropyranyl, tetrahydropyranalkyl, tetrahydropyranalkoxy, or tetrahydropyranalkylamino; R 3 Represents an aromatic ring substituted or unsubstituted by a halogen, nitro, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, or phenoxy group, wherein the C1-C6 alkyl, C1-C6 alkoxy, or C1-C6 alkylamino group may be substituted or unsubstituted by any 1-3 substituents, and the substituents are halogens, C1-C6 alkyl, C3-C7 cycloalkyl, heterocyclic, C1-C6 alkenyl, C1-C6 alkynyl, phenyl groups substituted or unsubstituted by at least one halogen, heteroaryl groups substituted or unsubstituted by at least one halogen, or Het1 substituent groups substituted or unsubstituted by at least one halogen; R 3 R 4 They can be the same or different, each representing a hydrogen atom, halogen, C1-C6 alkyl, cyano, C1-C6 alkoxy, or C1-C6 alkylamino.

[0007] Preferably, X represents -CH-, and Y represents -CR. 4 - The general formula is as follows:

[0008] R 1 Represents 2-methoxyphenyl, 4-fluoro-2-methoxyphenyl, 2,4-dimethoxyphenyl, 4-cyano-2-methoxyphenyl, 2-ethoxyphenyl, 4-fluoro-2-ethoxyphenyl, benzofuranyl, or pyrazolyl; R 2Represents methyl, cyclopropyl, cyclobutyl, morpholino, piperazine, N-methylpiperazine, homopiperazine, or N-methylhomoperazine; R 5 It represents a hydrogen atom, a fluorine atom, or a chlorine atom.

[0009] Preferably, X represents -N-, and Y represents -CR. 4 - The general formula is as follows:

[0010] Among them, R 1 Represents 2-methoxyphenyl, 4-fluoro-2-methoxyphenyl, 2,4-dimethoxyphenyl, 4-cyano-2-methoxyphenyl, 2-ethoxyphenyl, 4-fluoro-2-ethoxyphenyl, benzofuranyl, or pyrazolyl; R 2 Represents methyl, cyclopropyl, cyclobutyl, morpholino, piperazine, N-methylpiperazine, homopiperazine, or N-methylhomoperazine; R 5 It represents a hydrogen atom, a fluorine atom, or a chlorine atom.

[0011] Preferably, the above-mentioned 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine derivatives and their pharmaceutical salts or isomers are: 5-chloro-4-(1-cyclopropylpyrazol-4-yl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridine (I-1), 5-chloro-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}-4-(1-methylpyrazol-4-yl)pyridine (I-2), 5-fluoro-4-( 4-Fluoro-2-methoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridine (I-3), 5-chloro-4-(4-fluoro-2-methoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridine (I-4), 4-(5-fluoro-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridin-4-yl)-3-methoxybenzonitrile (I-5), 5-fluoro-4-( 2,4-Dimethoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazole-4-yl]amino}pyridine (I-6), 4-[2-(benzyloxy)-4-fluorophenyl]-5-fluoro-2-{[1-(piperidin-4-yl)pyrazole-4-yl]amino}pyridine (I-7), 5-fluoro-4-[4-fluoro-2-(isopropoxy)phenyl]-2-{[1-(piperidin-4-yl)pyrazole-4-yl]amino}pyridine (I-8), 5-fluoro -2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}-4-[2-(trifluoromethoxy)phenyl]pyridine (I-9), 5-chloro-4-(4-fluoro-1-methoxyphenyl-2-yl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridine (I-10), 5-(5-chloro-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridin-4-yl)-1-(propyl-2-yl)-1,2-Dihydropyridin-2-one (I-11), 4-(1-benzofuran-7-yl)-5-chloro-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridine (I-12), 4-(4-fluoro-2-methoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyrimidine (II-1), 6-(4-fluoro-2-methoxyphenyl)-4-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyrimidine (II-2), 5-fluoro-4-(4-fluoro-2-methoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyrimidine (II-3), 5-Chloro-4-(4-fluoro-2-methoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyrimidine (II-4), 5-fluoro-4-(4-fluoro-2-methoxyphenyl)-2-{[1-(piperidin-3-yl)pyrazol-4-yl]amino}pyrimidine (II-5), 2-{[1-(azacyclobut-3-yl)pyrazol-4-yl]amino}-5-fluoro-4-(4-fluoro-2-methoxyphenyl)pyrimidine (II-6), 2-({1-[1-(1-(ethylsulfonyl)azacyclobut-3-yl]pyrazol-4-yl}amino)-4-(4-fluoro-2-methoxyphenyl)pyrimidine (II-7).

[0012] The preparation method of the above-mentioned 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine compounds and their pharmaceutical salts or isomers includes the following steps: under nitrogen protection, using key intermediates A1~A13 and B1 as raw materials, Xantphos (4,5-bis(diphenylphosphino)-9,9-dimethyloxanthracene) as a ligand, sodium tert-butoxide as a base, and Pd2(dba)3tris(dibenzylideneacetone)dipalladium) as a catalyst, a Buchwald-Hartwig coupling reaction is carried out in anhydrous toluene, followed by removal of the tert-butyloxycarbonyl group via trifluoroacetic acid, finally yielding the target compound.

[0013] The preparation method of the above-mentioned 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine compounds and their pharmaceutical salts or isomers includes the following steps: under nitrogen protection, using key intermediates A14-A15 and B1 as raw materials, Xantphos (4,5-bis(diphenylphosphino)-9,9-dimethyloxanthracene) as a ligand, sodium tert-butoxide as a base, and Pd2(dba)3 (tris[dibenzylideneacetone]dipalladium) as a catalyst, a Buchwald-Hartwig coupling reaction is carried out in anhydrous toluene, followed by removal of the tert-butyloxycarbonyl group via trifluoroacetic acid to obtain the target compounds II-1 and II-2.

[0014] The preparation method of the above-mentioned 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine compounds and their pharmaceutical salts or isomers includes the following steps: using key intermediates A18 and B7 as raw materials, t-BuBrettphos (2-di-tert-butylphosphine-2′,4′,6′-triisopropyl-3,6-dimethoxy-1,1′-biphenyl) as a ligand, sodium tert-butoxide as a base, and Pd2(dba)3 as a catalyst, a Buchwald-Hartwig coupling reaction is carried out in anhydrous toluene. Subsequently, the tert-butyloxycarbonyl group is removed by trifluoroacetic acid to obtain the target compound II-3.

[0015] The preparation method of the above-mentioned 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine compounds and their pharmaceutical salts or isomers includes the following steps: using key intermediates A17 and B1 as raw materials, t-BuBrettphos as a ligand, sodium tert-butoxide as a base, and Pd2(dba)3 as a catalyst, a Buchwald-Hartwig coupling reaction is carried out in anhydrous toluene. Subsequently, the tert-butyloxycarbonyl group is removed by trifluoroacetic acid to obtain the target compound II-4.

[0016] The preparation method of the above-mentioned 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine derivatives and their pharmaceutical salts or isomers includes the following steps: using key intermediates A16 and B9-B10 as raw materials, under sealed tube conditions, Cs2CO3 is used as a base to carry out a nucleophilic substitution reaction in tert-butanol to obtain target compounds II-5 and II-6.

[0017] The preparation method of the above-mentioned 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine derivatives and their pharmaceutical salts or isomers includes the following steps: using key intermediates A14 and B4 as raw materials, under sealed tube conditions, Cs2CO3 is used as a base to carry out a nucleophilic substitution reaction in tert-butanol to obtain the target compound II-7.

[0018] A pharmaceutical composition comprising any one of the above-mentioned 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine derivatives and their pharmaceutical salts or isomers, and a pharmaceutically acceptable carrier.

[0019] Specifically, the protein kinase inhibitory small molecules and their derivatives can be added to pharmaceutically acceptable carriers to form common pharmaceutical preparations, such as sterile preparations like injections and lyophilized preparations. Common pharmaceutical excipients include solubilizers, stabilizers, osmotic pressure regulators, pH regulators, etc., and corresponding drug delivery devices can also be included.

[0020] The use of any of the above-mentioned 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine derivatives and their pharmaceutical salts or isomers, or the above-mentioned pharmaceutical compositions, in the preparation of cell cycle-dependent kinase inhibitor drugs.

[0021] The use of any of the above-mentioned 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine derivatives and their pharmaceutical salts or isomers, or the above-mentioned pharmaceutical compositions, in the preparation of a medicament for treating tumors.

[0022] Preferably, the tumors include, but are not limited to, lung cancer, prostate cancer, liver cancer, stomach cancer, cervical cancer, colorectal cancer, melanoma, ovarian cancer, breast cancer, kidney cancer, nervous system tumors, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, Burkitt's lymphoma, and follicular lymphoma. Beneficial effects

[0023] (1) These small molecules and their derivatives have highly efficient inhibitory activity. At the molecular level, CDK9 kinase inhibits IC50. 50 The optimal concentration is less than 10 nM, reaching the nanomolar concentration level; at the cellular level, MV4-11 inhibits tumor cell proliferation with an IC50 value. 50 The optimal concentration is less than 50 nM, reaching the nanomolar concentration level; and it can effectively inhibit the proliferation of various tumor cells, reaching the nanomolar concentration level, with the optimal concentration being less than 50 nM; (2) This type of small molecule and its derivatives have excellent water solubility, which can improve their bioavailability in vivo, and are conducive to the development of various dosage forms of therapeutic drugs for related targets, especially injections; (3) This type of small molecule and its derivatives and drug compositions have wide applications and can be prepared as drugs for the treatment and / or prevention of protein kinase-related hyperproliferative diseases, virus-induced infectious diseases, and cardiovascular diseases; they can exert their effects at both the molecular and cellular levels, and the therapeutic effect is even better, IC50 50 The optimal value can reach the nanomolar concentration level; (4) The compound preparation method is easy to operate and the reaction substrate is widely applicable. Detailed Implementation

[0024] The chemical reagents used in the preparation of the reagents and materials compounds were sourced from Shanghai Bid Pharmaceutical Technology Co., Ltd., Shanghai Haohong Biomedical Technology Co., Ltd., and Saen Chemical Technology Co., Ltd.

[0025] Proteins and Cells: Human DMEM medium (Gibco); Fetal Bovine Serum (FBS) (BI); 0.25% Trypsin-EDTA (Kaiji Biotechnology); DMEM medium (Gibco); 0.25% Trypsin-EDTA (Kaiji Biotechnology); 5000 U / mL Penicillin and Streptomycin solution (Shanghai Yisheng); MV4-11 cells (Shanghai Cell Bank); DMSO (Shanghai Yisheng); Ultrapure water was used for the experiments.

[0026] Instruments and equipment 1 H-NMR was performed using a BRUKER AVANCE-300 NMR spectrometer (Brucker, Switzerland), with TMS as the internal standard. Shift values ​​(δ) were measured in ppm. Low-resolution mass spectrometry was performed using an expression compact Fourier transform mass spectrometer.

[0027]

[0028] Reagents and conditions: (a) Pd2(dba)3, Xantphos, t-BuONa, anhydroustoluene, 100 °C, 4 h; (b) TFA, DCM, rt 2 h.

[0029] Example 1: 5-Chloro-4-(1-Cyclopropylpyrazol-4-yl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridine (I-1) was reacted in a 25 mL double-necked flask with intermediates A2 (250 mg, 0.98 mmol), B1 (288 mg, 1.08 mmol), tris(dibenzylacetone)palladium (82 mg, 0.09 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (104 mg, 0.18 mmol), and sodium tert-butoxide (131 mg, 1.37 mmol). 10 mL of anhydrous toluene was added, and the mixture was heated to reflux at 100 °C for 4 h under nitrogen protection. The reaction solution was concentrated and purified by column chromatography (dichloromethanol:methane = 40:1) to obtain 190 mg of a brown oily substance. This oil was dissolved in 10 mL of dichloromethane, and 2 mg of the solution was added dropwise at room temperature. mL of trifluoroacetic acid was added, and the reaction was carried out for 2 h. Subsequently, saturated sodium carbonate solution was added, the organic phase was separated and concentrated, and purified by rapid preparative chromatography (methanol:water = 3:1) to obtain 81 mg (0.21 mmol) of white solid, namely compound I-1, with a yield of 21.6%. mp: 113-115 °C. 1H NMR (300 MHz, DMSO-d6) δ8.92 (s, 1H), 8.32 (d, J = 0.8 Hz, 1H), 8.14 (s, 1H), 7.96 -7.90 (m, 1H),7.85 (d, J = 0.8 Hz, 1H), 7.44 (d, J = 0.8 Hz, 1H), 6.80 (s, 1H), 4.13 (m, J= 11.5, 4.1 Hz, 1H), 3.84 (m, J = 7.4, 3.9 Hz, 1H), 3.45 (q, J = 7.2 Hz, 2H),3.02 (m, J = 12.8, 3.3 Hz, 2H), 2.58 (dd, J = 12.3, 2.5 Hz, 2H), 1.96 -1.85(m, 2H), 1.74 (m, J = 12.0, 4.1 Hz, 2H), 1.15-1.09 (m, 2H), 1.03 -0.96 (m,2H). MSI-MS m / z: 384.16 [M+H]+.

[0030] Example 2 5-Chloro-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}-4-(1-methylpyrazol-4-yl)pyridine (I-2) was synthesized from intermediates A1 (250 mg, 1.09 mmol), B1 (299 mg, 1.12 mmol), tris(dibenzylideneacetone)dipalladium (82 mg, 0.09 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (104 mg, 0.18 mmol), and sodium tert-butoxide (133 mg, 1.40 mmol) using the same method as compound I-1, yielding 76 mg (0.19 mmol) of pale yellow solid, namely compound I-2, with a yield of 20.4%. mp: 118 -119 °C. 1H NMR (300 MHz, Methanol-d4) δ8.21 (s, 1H), 8.10 (d, J = 12.8 Hz, 2H), 7.93 (s, 1H), 7.57 (s, 1H), 6.86 (s,1H), 4.53 (m, J = 10.3, 5.3 Hz, 1H), 4.00 (s, 3H), 3.60 (m, J = 13.0 Hz, 2H), 3.25 (m, J = 12.6, 3.9 Hz, 2H), 2.38-2.20 (m, 4H). MSI-MS m / z: 358.15 [M+H]+.

[0031] Example 3 5-Fluoro-4-(4-Fluoro-2-methoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridine (I-3) was synthesized from intermediates A3 (235 mg, 0.92 mmol), B1 (290 mg, 1.09 mmol), tris(dibenzylideneacetone)dipalladium (82 mg, 0.09 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (104 mg, 0.18 mmol), and sodium tert-butoxide (133 mg, 1.40 mmol) using the same method as compound I-1, yielding 56 mg (0.14 mmol) of white solid, namely compound I-3, with a yield of 15.2%. mp: 142 – 145 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.80(s, 1H), 8.07 (d, J = 1.9 Hz, 1H), 7.95 (s, 1H), 7.42 (d, J = 0.7 Hz, 1H),7.32 (dd, J = 8.4, 6.8 Hz, 1H), 7.08 (dd, J = 11.5, 2.5 Hz, 1H), 6.90 (m, J =8.4, 2.4 Hz, 1H), 6.58 (d, J = 5.0 Hz, 1H), 4.13 (m, J = 11.5, 8.0, 4.0 Hz,1H), 3.79 (s, 3H), 3.02 (m, J = 12.1 Hz, 2H), 2.58 (m, J = 12.3, 2.5 Hz, 2H), 1.91 (m, J = 12.0 Hz, 2H), 1.74 (m, J = 12.0, 4.1 Hz, 2H). ESI-MS m / z: 386.42[M+H]+.

[0032] Example 4 5-Chloro-4-(4-fluoro-2-methoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridine (I-4) was synthesized from intermediates A4 (250 mg, 0.91 mmol), B1 (290 mg, 1.09 mmol), tris(dibenzylideneacetone)dipalladium (82 mg, 0.09 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (104 mg, 0.18 mmol), and sodium tert-butoxide (131 mg, 1.37 mmol) using the same method as compound I-1, yielding 60 mg (0.15 mmol) of white solid, namely compound I-4, in a yield of 16.5%. mp: 116 – 118 °C. 1H NMR (300 MHz, DMSO-d6) δ 8.96(s, 1H), 8.14 (s, 1H), 7.94 (d, J = 0.7 Hz, 1H), 7.44 (d, J = 0.7 Hz, 1H),7.23 (dd, J = 8.4, 6.8 Hz, 1H), 7.06 (dd, J = 11.5, 2.4 Hz, 1H), 6.88 (m, J =8.4, 2.4 Hz, 1H), 6.55 (s, 1H), 4.14 (m, J = 11.6, 4.1 Hz, 1H), 3.77 (s, 3H),3.44 (m, J = 7.0, 4.7 Hz, 2H), 3.02 (m, J = 12.0 Hz, 2H), 2.58 (m, J = 12.3,2.5 Hz, 2H), 1.91 (d, J = 11.0 Hz, 2H), 1.74 (m, J = 12.0, 4.1 Hz, 2H). 13CNMR (126 MHz,Chloroform-d) δ 164.99, 157.78 (3JC-F = 10.1 Hz), 155.87,147.12, 146.29, 133.51, 131.11(3JC-F = 10.1 Hz), 129.08, 128.27, 122.47,121.31, 120.86, 109.40, 107.04(2JC-F = 21.42 Hz), 99.54(2JC-F = 26.46 Hz), 59.98, 55.90, 45.63, 33.77.ESI-MS m / z: 402.87 [M+H]+.

[0033] Example 5 4-(5-fluoro-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridin-4-yl)-3-methoxybenzonitrile (I-5) was synthesized from intermediates A7 (355 mg, 1.35 mmol), B1 (300 mg, 1.12 mmol), tris(dibenzylideneacetone)dipalladium (100 mg, 0.11 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (127 mg, 0.22 mmol), and sodium tert-butoxide (194 mg, 2.02 mmol) using the same method as compound I-1, yielding 93 mg (0.24 mmol) of pale yellow solid, namely compound I-5, with a yield of 21.1%. mp: 205 – 208 °C. 1H NMR (300 MHz, DMSO-d6) δ8.87 (s, 1H), 8.12 (d, J = 1.8 Hz, 1H), 7.95 (d, J = 0.8 Hz, 1H), 7.67 (d, J= 1.4 Hz, 1H), 7.64 – 7.46 (m, 2H), 7.43 (d, J = 0.8 Hz, 1H), 6.60 (d, J =4.9 Hz, 1H), 4.13 (m, J = 11.3, 7.9, 4.0 Hz, 1H), 3.85 (s, 3H), 3.03 (d, J =12.3 Hz, 2H), 2.63 – 2.53 (m, 2H), 1.91 (d, J = 11.7 Hz, 2H), 1.75 (m, J =12.0, 4.1 Hz, 2H). MSI-MS m / z: 393.44 [M+H]+.

[0034] Example 6. 5-Fluoro-4-(2,4-dimethoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridine (I-6) was synthesized from intermediates A8 (337 mg, 1.26 mmol), B1 (280 mg, 1.05 mmol), tris(dibenzylideneacetone)dipalladium (91 mg, 0.10 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (115 mg, 0.20 mmol), and sodium tert-butoxide (181 mg, 1.89 mmol) using the same method as compound I-1, yielding 84 mg (0.21 mmol) of a pale yellow solid, namely compound I-6, in a yield of 20.1%. mp: 120 – 123 °C. 1H NMR (300 MHz, DMSO-d6) δ8.76 (s, 1H), 8.04 (d, J = 2.0 Hz, 1H), 7.95 (s, 1H), 7.42 (s, 1H), 7.19 (d,J = 8.4 Hz, 1H), 6.73 – 6.54 (m, 3H), 4.16 (m, J = 11.4, 8.2, 4.0 Hz, 1H), 3.80 (d, J = 15.9 Hz, 6H), 3.07 (d, J = 12.3 Hz, 2H), 2.62 (m, J = 12.4, 2.6Hz, 2H), 1.94 (d, J = 12.0 Hz, 2H), 1.78 (m, J = 12.0, 4.0 Hz, 2H). MSI-MS m / z: 398.45 [M+H]+.

[0035] Example 7 4-[2-(benzyloxy)-4-fluorophenyl]-5-fluoro-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridine (I-7) was synthesized from intermediates A6 (268 mg, 0.81 mmol), B1 (260 mg, 0.97 mmol), tris(dibenzylideneacetone)dipalladium (73.28 mg, 0.08 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (92 mg, 0.16 mmol), and sodium tert-butoxide (116 mg, 1.21 mmol) using the same method as compound I-1, yielding 78 mg (0.17 mmol) of white solid, namely compound I-7, in a yield of 20.9%.mp: 101 – 103 °C. 1H NMR (300 MHz, DMSO-d6) δ8.81 (s, 1H), 8.09 (d, J = 1.9 Hz, 1H), 7.98 – 7.92 (m, 1H), 7.42 (d, J = 0.7Hz, 1H), 7.40 – 7.25 (m, 6H), 7.15 (dd, J = 11.4, 2.4 Hz, 1H), 6.92 (m, J =8.4, 2.4 Hz, 1H), 6.62 (d, J = 4.9 Hz, 1H), 5.18 (s, 2H), 4.13 (m, J = 11.5,4.1 Hz, 1H), 3.08 – 2.97 (d, 2H), 2.63 – 2.53 (m, 2H), 1.96 – 1.85 (d, 2H),1.74 (m, J = 12.0, 4.1 Hz, 2H). 13C NMR (126 MHz, Chloroform-d) δ 165.01,163.04, 157.10(3JC-F = 10.08 Hz), 153.70, 152.76, 150.81, 136.15, 135.78(3JC-F = 15.12 Hz), 134.82,(2JC-F = 27.72 Hz), 133.47, 131.74(3JC-F = 10.08 Hz),128.65, 128.06, 126.93, 123.24, 120.61, 119.53, 108.96, 107.69(2JC-F = 21.42Hz), 101.12(2JC-F = 26.46 Hz), 70.70, 58.85, 58.44, 44.86, 32.60, 18.49. MSI-MS m / z: 462.52 [M+H]+。

[0036] Example 8. 5-Fluoro-4-[4-fluoro-2-(isopropoxy)phenyl]-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridine (I-8) was synthesized from intermediates A5 (300 mg, 1.00 mmol), B1 (319 mg, 1.20 mmol), tris(dibenzylideneacetone)dipalladium (91 mg, 0.10 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (115 mg, 0.20 mmol), and sodium tert-butoxide (144 mg, 1.50 mmol) using the same method as compound I-1, yielding 82 mg (0.20 mmol) of brown solid, namely compound I-8, in a yield of 20.0%. mp: 193 – 195 °C. 1H NMR (400 MHz, DMSO-d6) δ8.94 (s, 1H), 8.08 (d, J = 1.9 Hz, 1H), 8.02 (s, 1H), 7.47 (s, 1H), 7.31 (dd,J = 8.5, 6.9 Hz, 1H), 7.09 (dd, J = 11.7, 2.5 Hz, 1H), 6.86 (m, J = 8.4, 2.4Hz, 1H), 6.61 (d, J = 5.0 Hz, 1H), 4.70 (h, J = 6.0 Hz, 1H), 4.46 (m, J =9.9, 4.7 Hz, 1H), 3.42 – 3.37 (m, 2H), 3.05 (d, J = 11.7 Hz, 2H), 2.20 – 2.03(m, 4H), 1.20 (d, J = 6.0 Hz, 6H). ESI-MS m / z: 414.47 [M+H]+.

[0037] Example 9 5-Fluoro-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}-4-[2-(trifluoromethoxy)phenyl]pyridine (I-9) was synthesized from intermediates A9 (394 mg, 1.35 mmol), B1 (300 mg, 1.13 mmol), tris(dibenzylideneacetone)dipalladium (100 mg, 0.11 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (127 mg, 0.22 mmol), and sodium tert-butoxide (194 mg, 2.02 mmol) using the same method as compound I-1, yielding 89 mg (0.21 mmol) of light brown solid, compound I-9, in 18.7% yield. mp: 193 – 195 °C. 1H NMR (300 MHz, DMSO-d6) δ8.95 (s, 1H), 8.18 (d, J = 1.8 Hz, 1H), 7.98 (d, J = 0.7 Hz, 1H), 7.65 (m, J= 8.2, 6.6, 2.8 Hz, 1H), 7.62 – 7.49 (m, 3H), 7.44 (d, J = 0.8 Hz, 1H), 6.64(d, J = 4.9 Hz, 1H), 4.14 (m, J = 11.5, 4.2 Hz, 1H), 3.03 (d, J = 12.6, 3.4Hz, 2H), 2.64 – 2.53 (m, 2H), 2.02 – 1.86 (d, 2H), 1.75 (m, J = 12.0, 4.1 Hz, 2H). MSI-MS m / z: 422.40 [M+H]+.

[0038] Example 10 5-Chloro-4-(4-fluoro-1-methoxyphenyl-2-yl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridine (I-10) was synthesized from intermediates A10 (230 mg, 0.85 mmol), B1 (266 mg, 1.01 mmol), tris(dibenzylideneacetone)dipalladium (82 mg, 0.09 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (104 mg, 0.18 mmol), and sodium tert-butoxide (131 mg, 1.37 mmol) using the same method as compound I-1, yielding 52 mg (0.13 mmol) of white solid, namely compound I-10, in a yield of 15.2%. mp: 133 – 135 °C. 1H NMR (300 MHz, DMSO-d6) δ8.99 (s, 1H), 8.16 (s, 1H), 7.95 (s, 1H), 7.45 (d, J = 0.7 Hz, 1H), 7.28 (m,J = 8.7, 3.1 Hz, 1H), 7.21 – 7.09 (m, 2H), 6.57 (s, 1H), 4.20 – 4.12 (m, 1H), 3.74 (s, 3H), 3.03 (d, J = 12.1 Hz, 2H), 2.59 (dd, J = 12.3, 2.5 Hz, 2H),1.91 (t, J = 7.7 Hz, 2H), 1.75 (m, J = 12.0, 4.1 Hz, 2H). MSI-MS m / z: 402.87[M+H]+.

[0039] Example 11 5-(5-chloro-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridin-4-yl)-1-(propyl-2-yl)-1,2-dihydropyridin-2-one (I-11) was synthesized from intermediates A11 (250 mg, 1.09 mmol), B1 (349 mg, 1.31 mmol), tris(dibenzylideneacetone)dipalladium (99 mg, 0.11 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (127 mg, 0.22 mmol), and sodium tert-butoxide (156 mg, 1.64 mmol) using the same method as compound I-1, yielding 52 mg (0.15 mmol) of a light brown solid, namely compound I-11, in a yield of 13.8%. mp: 155 – 158 °C. 1H NMR (300 MHz, DMSO-d6) δ8.75 (s, 1H), 8.12 (d, J = 2.5 Hz, 1H), 8.05 (d, J = 2.6 Hz, 1H), 7.61 (d, J= 9.5, 2.2 Hz, 1H), 7.38 (d, J = 1.8 Hz, 1H), 6.82 (d, J = 5.6 Hz, 1H), 6.51(d, J = 9.5 Hz, 1H), 6.20 (d, J = 1.9 Hz, 1H), 5.09 (p, J = 6.7 Hz, 1H), 4.20(s, 1H), 3.35 (d, J = ESI-MS m / z:397.47 [M+H]+.

[0040] Example 12 4-(1-benzofuran-7-yl)-5-chloro-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridine (I-12) was synthesized from intermediates A13 (300 mg, 1.14 mmol), B1 (362 mg, 1.36 mmol), tris(dibenzylideneacetone)dipalladium (104 mg, 0.11 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (127 mg, 0.22 mmol), and sodium tert-butoxide (164 mg, 1.71 mmol) using the same method as compound I-1. 72 mg (0.18 mmol) of light brown solid was obtained, yielding compound I-12, with a yield of 15.7%. mp: 218 – 220 °C. 1H NMR (300 MHz, DMSO-d6) δ 9.10 (s, 1H), 8.27 (s, 1H), 8.08 – 7.96 (m, 2H), 7.78 (dd, J = 7.4, 1.6Hz, 1H), 7.49 – 7.46 (s, 1H), 7.44 – 7.29 (m, 2H), 7.07 (d, J = 2.2 Hz, 1H), 6.78 (s, 1H), 3.09 (d, J = 12.2 Hz, 2H), 2.65 (t, J = 11.3 Hz, 2H), 1.96 (d,J = 12.1 Hz, 2H), 1.89 – 1.72 (m, 2H). MSI-MS m / z: 394.88 [M+H]+.

[0041] Example 13 4-(4-fluoro-2-methoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyrimidine (II-1)

[0042] Reagents and conditions: (a) Pd2(dba)3, Xantphos, t-BuONa, anhydroustoluene, 100 °C, 4 h; (b) TFA, DCM, rt, 2 h. Using intermediates A14 (250 mg, 1.04 mmol), B1 (334 mg, 1.26 mmol), tris(dibenzylacetone)dipalladium (91 mg, 0.10 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (115 mg, 0.20 mmol), and sodium tert-butoxide (149 mg, 1.56 mmol) as starting materials, compound II-1 was synthesized using the same method as compound I-1, yielding 74 mg (0.20 mmol) of pale yellow solid, with a yield of 19.2%. mp: 156 – 158 °C. 1 H NMR (300 MHz, DMSO-d6)δ 9.46 (s, 1H), 8.42 (d,J= 5.2 Hz, 1H), 7.92 (s, 1H), 7.56 (s, 1H), 7.20 (d,J= 5.2 Hz, 1H), 7.11 (dd,J= 11.5, 2.4 Hz, 1H), 7.04 – 6.93 (m, 1H), 4.25 – 4.09 (m, 1H), 3.90 (s, 3H), 3.08 (d,J= 12.6 Hz, 2H), 2.72 – 2.56 (m,2H), 1.96 (d,J= 12.3 Hz, 2H), 1.78 (m,J= 12.1, 6.0 Hz, 2H). MSI-MSm / z:369.42 [M+H] + .

[0043] Example 14 6-(4-fluoro-2-methoxyphenyl)-4-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyrimidine (II-2)

[0044] Reagents and conditions: (a) Pd2(dba)3, t-BuBrettphos, t-BuONa, anhydrous toluene, 100 °C, 4 h; (b) TFA, DCM, rt, 2 h. In a 25 mL double-necked flask, add intermediates A15 (310 mg, 1.40 mmol), B1 (389 mg, 1.18 mmol), tris(dibenzylacetone)palladium (109 mg, 0.12 mmol), 2-di-tert-butylphosphine-2′,4′,6′-triisopropyl-3,6-dimethoxy-1,1′-biphenyl (116 mg, 0.24 mmol), sodium tert-butoxide (226 mg, 2.36 mmol), and 10 mL of anhydrous toluene. The reaction mixture is heated to reflux at 100 °C under nitrogen protection for 3 hours. The reaction solution was concentrated and purified by column chromatography (dichloromethanol:methane = 40:1) to obtain 170 mg of a brown oily substance. This oil was dissolved in 10 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise at room temperature. The reaction was allowed to proceed for 2 h. Subsequently, saturated sodium carbonate solution was added for extraction, and the organic phase was concentrated. After rapid preparative chromatography purification, 43 mg (0.12 mmol) of a white solid, compound II-2, was obtained, with a yield of 10.2%. mp: 171 – 173 °C. 1 H NMR (300MHz, DMSO-d6) δ 8.62 (d,J= 1.2 Hz, 1H), 8.09 – 7.98 (m, 2H), 7.52 (s, 1H),7.29 (s, 1H), 7.09 (dd,J= 11.5, 2.5 Hz, 1H), 6.91 (m,J= 8.4, 2.5 Hz, 1H),4.24 (m,J= 11.3, 8.2, 4.1 Hz, 1H), 3.91 (s, 3H), 3.18 – 2.93 (m, 2H), 2.68(m,J= 12.3, 2.7 Hz, 2H), 1.98 – 1.74 (m, 4H). MSI-MSm / z: 369.42 [M+H] + .

[0045] Example 15 5-Fluoro-4-(4-Fluoro-2-methoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyrimidine (II-3)

[0046] Reagents and conditions: (a) Pd2(dba)3, t-BuBrettphos, t-BuONa, anhydrous toluene, 100 °C, 4 h; (b) TFA, DCM, rt, 2 h. In a 25 mL double-necked flask, add intermediates A18 (250 mg, 1.05 mmol), B7 (291 mg, 1.26 mmol), tris(dibenzylacetone)dipalladium (91 mg, 0.10 mmol), 2-di-tert-butylphosphine-2′,4′,6′-triisopropyl-3,6-dimethoxy-1,1′-biphenyl (96 mg, 0.20 mmol), sodium tert-butoxide (151 mg, 1.58 mmol), and 10 mL of anhydrous toluene. The reaction is carried out under nitrogen protection at 100 °C under reflux for 4 h. The reaction solution was concentrated and purified by column chromatography (dichloromethanol:methane = 40:1) to obtain 201 mg of a brown oily substance. This oil was dissolved in 10 mL of dichloromethane, and 2 mL of trifluoroacetic acid was added dropwise at room temperature. The reaction was allowed to proceed for 2 h. Subsequently, saturated sodium carbonate solution was added for extraction, and the organic phase was concentrated. After rapid preparative chromatography purification (methanol:water = 3:1), 72 mg (0.19 mmol) of a white solid, compound II-3, was obtained, with a yield of 18.1%. mp: 196 – 199 °C 1 H NMR (300MHz, DMSO-d6) δ 9.59 (s, 1H), 8.48 (d,J= 2.1 Hz, 1H), 7.86 (s, 1H), 7.52(d,J= 3.8 Hz, 2H), 7.13 (dd,J= 11.5, 2.4 Hz, 1H), 6.96 (m,J= 8.4, 2.4Hz, 1H), 4.25 – 4.12 (m, 1H), 3.83 (s, 3H), 3.10 (d,J= 12.2 Hz, 2H), 2.66(m,J= 12.4, 2.7 Hz, 2H), 1.95 (d,J= 12.4 Hz, 2H), 1.78 (m, J = 12.0, 4.0 Hz, 2H). 13 C NMR (126 MHz, Chloroform-d) δ 166.07, 164.08, 158.92 ( 3 J C-F = 10.3Hz), 156.46, 151.53, 149.53, 145.55( 2 J C-F = 22.68 Hz), 131.85 (3 J C-F = 10.6 Hz),130.19, 122.81, 119.52, 117.62, 107.70( 2 J C-F = 21.42 Hz), 99.72 ( 2 J C-F = 22.68Hz), 59.85, 56.06, 45.69, 33.82, 18.48.MSI-MSm / z: 387.41 [M+H] + .

[0047]

[0048] Reagents and conditions: (a) Pd2(dba)3, t-BuBrettphos, t-BuONa, anhydrous toluene, 100 °C, 4 h. Example 16 5-Chloro-4-(4-fluoro-2-methoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyrimidine (II-4) was synthesized from intermediates A17 (400 mg, 1.46 mmol), B1 (325 mg, 1.22 mmol), tris(dibenzylideneacetone)dipalladium (110 mg, 0.12 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (139 mg, 0.24 mmol), and sodium tert-butoxide (210 mg, 2.19 mmol) using the same method as compound I-1, yielding 112 mg (0.28 mg) of the product. A pale yellow solid (mmol), namely compound II-4, was obtained in 22.7% yield. mp: 163–165 °C. 1 H NMR (300 MHz, DMSO-d6) δ9.64 (s, 1H), 8.49 (d,J= 2.1 Hz, 1H), 7.98 (s, 1H), 7.55 (d,J= 12.4 Hz,2H), 7.13 (dd,J= 11.5, 2.4 Hz, 1H), 6.96 (m,J= 8.4, 2.4 Hz, 1H), 5.12 (p,J= 7.4 Hz, 1H), 3.87 (d,J= 7.6 Hz, 2H), 3.84 (s, 3H), 3.70 (t,J= 7.8 Hz,2H). 13C NMR (126 MHz, Chloroform-d) δ 165.67, 163.69, 162.65, 158.26( 3 J C-F =10.08 Hz), 158.03, 157.13, 131.12 ( 3 J C-F = 10.08 Hz), 130.35, 122.27, 120.31,117.99, 107.44( 2 J C-F = 22.68 Hz), 99.62 ( 2 J C-F = 25.20 Hz), 59.72, 58.43, 55.89,45.56, 33.62, 18.48. MSI-MSm / z: [M+H] + .

[0049] Example 17 5-Fluoro-4-(4-fluoro-2-methoxyphenyl)-2-{[1-(piperidin-3-yl)pyrazol-4-yl]amino}pyrimidine (II-5) was synthesized from intermediates A16 (288 mg, 1.12 mmol), B9 (250 mg, 0.94 mmol), tris(dibenzylacetone)dipalladium (82 mg, 0.09 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (104 mg, 0.18 mmol), and sodium tert-butoxide (131 mg, 1.37 mmol). The synthesis method was the same as for compound I-1, yielding 85 mg (0.36 mmol) of white solid, compound II-5, in 38.3% yield. mp: 198 – 200 °C. 1H NMR (300 MHz, DMSO-d6) δ 9.56(s, 1H), 8.47 (s, 1H), 7.87 (s, 1H), 7.52 (d,J= 8.7 Hz, 2H), 7.13 (d,J=11.4 Hz, 1H), 6.96 (t,J= 8.5 Hz, 1H), 4.05 (s, 1H), 3.83 (s, 3H), 3.10 (d,J= 11.6 Hz, 1H), 2.85 (d,J= 12.1 Hz, 1H), 2.67 (t,J= 11.0 Hz, 1H), 2.41(d,J= 12.2 Hz, 1H), 2.03 (s, 1H), 1.82 (t,J= 11.9 Hz, 1H), 1.69 (d,J=14.1 Hz, 1H), 1.47 (d,J= 12.9 Hz, 1H). MSI-MSm / z: 387.41 [M+H] + .

[0050] Example 18 2-{[1-(azacyclobut-3-yl)pyrazol-4-yl]amino}-5-fluoro-4-(4-fluoro-2-methoxyphenyl)pyrimidine (II-6) was synthesized from intermediates A16 (317 mg, 1.24 mmol), B10 (355 mg, 1.49 mmol), tris(dibenzylideneacetone)dipalladium (110 mg, 0.12 mmol), 4,5-bis(diphenylphosphine)-9,9-dimethyloxanthracene (139 mg, 0.24 mmol), and sodium tert-butoxide (144 mg, 1.50 mmol) using the same method as compound I-1, yielding 93 mg (0.36 mmol) of a yellow solid, namely compound II-6, in a yield of 29.0%. mp: 118 – 120 °C. 1 H NMR (300 MHz, DMSO-d6) δ9.64 (s, 21H), 8.49 (d,J= 2.1 Hz, 1H), 7.98 (s, 1H), 7.55 (d,J= 12.4 Hz,2H), 7.13 (dd,J= 11.5, 2.4 Hz, 1H), 6.96 (m,J= 8.4, 2.4 Hz, 1H), 5.12 (p,J= 7.4 Hz, 1H), 3.87 (d,J= 7.6 Hz, 2H), 3.84 (s, 3H), 3.70 (t,J= 7.8 Hz,2H). MSI-MSm / z: 359.35 [M+H] + .

[0051] Example 19 2-({1-[1-(ethylsulfonyl)azacyclobut-3-yl]pyrazol-4-yl}amino)-4-(4-fluoro-2-methoxyphenyl)pyrimidine (II-7) was dissolved in 25 mL sealed tubes by adding A14 (250 mg, 1.04 mmol), B4 (200 mg, 0.86 mmol), and p-toluenesulfonic acid monohydrate (586 mg, 3.08 mmol). 8 mL of tert-butanol was added, and the mixture was reacted at 80 °C for 3 h. The reaction was then extracted with water, and the organic phase was concentrated. Rapid preparative chromatography (methanol:water = 3:1) yielded 95 mg (0.22 mmol) of a brown solid, compound II-7, in 25.6% yield. mp: 118–119 °C. 1 H NMR (300 MHz, DMSO-d6) δ 9.58 (s, 1H), 8.43 (d,J= 5.2 Hz, 1H), 8.08 (d,J= 0.7 Hz, 1H), 7.71 (s, 1H), 7.23 (d,J= 5.2 Hz, 1H), 7.11 (dd,J= 11.5, 2.5 Hz, 1H), 6.97 (m,J= 8.4, 2.5 Hz,1H), 5.29 (p,J= 7.1 Hz, 1H), 4.33 – 4.17 (m, 4H), 3.90 (s, 3H), 3.22 (q,J= 7.4 Hz, 2H), 1.27 (t,J= 7.4 Hz, 4H). MSI-MSm / z: 433.47 [M+H] + .

[0052] Example 20: The inhibitory activity of the compound against CDK9 kinase was tested using the HotSpotSM kinase method / fluorescence resonance energy transfer (FRET). Taking CDK9 / Cyclin T1 as an example, the specific procedure was as follows: CDK9 / Cyclin T1 was diluted to an appropriate concentration with kinase diluent and set aside. The kinase reaction mixture contained CDK9 / Cyclin T1, peptide substrate, HEPES (pH 7.5), BRIJ-35, MgCl2, and EDTA. The CDK9 phospho-peptide substrate was used as a 100% phosphorylation control, and 0% phosphorylation control was used without ATP. After reacting at room temperature for 1 hour, a moderately diluted Development Reagent A was added to the reaction system. The reaction was continued at room temperature for another 1 hour, and then Stop Reagent was added to stop the reaction. The excitation wavelength was set to 400 nm, and the fluorescence intensity at wavelengths of 445 nm (Coumarin) and 520 nm (Fluorescein) was detected simultaneously. The inhibition rate of the test compound (n=2) was calculated using the formula, IC50. 50 The value was obtained by plotting the percentage inhibition rate and the logarithmic concentration.

[0053] Example 21 Inhibitory Effect of Compounds on the Proliferation of Leukemia Cell Line MV4-11 Tumor Cells The MTT assay for in vitro testing of antitumor proliferative activity is a method for detecting cell viability and growth. Its detection principle is that NADP-associated dehydrogenase (succinate dehydrogenase) in the mitochondria of living cells can reduce exogenous MTT to insoluble blue-purple formazan crystals, which are deposited in the cells. Dead cells do not have this function. The purple formazan crystals in the cells are dissolved using dimethyl sulfoxide (DMSO) or a triple solution (10% SDS-5% isobutanol-0.01 mol / L HCl), and the absorbance (OD value) at 570 nm is detected using an enzyme-linked immunosorbent assay (ELISA) reader, which can indirectly reflect the number of viable cells.

[0054] The specific procedure involves seeding tumor cells in the logarithmic growth phase into 96-well culture plates at a certain cell quantity. After culturing for 24 hours, the test compound is added (for suspension cells, it can be added directly after inoculation). The cells are then cultured at 37°C and 5% CO2 for 48 or 72 hours. MTT is then added and the cells are cultured for another 4 hours. The cells are then dissolved and crystallized in DMSO. The OD value is measured at 570 nm using an enzyme-linked immunosorbent assay (ELISA) reader, and the inhibition rate and IC50 value of the compound are calculated.

[0055] The pharmacological results of some of the preferred compounds are as follows: Table 1 Series I Compounds Inhibitory activity against CDK and inhibitory activity against the proliferation of MV4-11 cells

[0056] Table 2 Series II Compounds Inhibitory activity against CDK9 and MV4-11 cells

[0057] Table 3 Selectivity test of preferred compounds for CDKs

[0058] Based on the above data, it can be seen that the small molecules and their derivatives of this invention have highly efficient inhibitory activity. At the molecular level, CDK9 kinase inhibits IC50. 50 The optimal concentration is less than 10 nM, reaching the nanomolar concentration level; at the cellular level, MV4-11 inhibits tumor cell proliferation with an IC50 value. 50 The optimal concentration is less than 50 nM, reaching the nanomolar concentration level; and it can effectively inhibit the proliferation of various tumor cells, reaching the nanomolar concentration level, with an optimal concentration less than 50 nM. The preparation method is easy to operate, and the reaction substrate has a wide applicability, which can be used to prepare anti-tumor drugs for the treatment and / or prevention of protein kinase-related diseases; it can exert its efficacy at both the molecular and cellular levels, and the therapeutic effect is even better, showing good application prospects.

Claims

1. The 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine compounds of formula (I) and their pharmaceutical salts or isomers, (I) Among them, X represents -N-, Y represents -CR 4 -; or X represents -CH-, Y represents -N-; or X represents -CH-, Y represents -CR 4 -;R 1 The group to be represented is selected from the following groups: 、 、 or And G is -NH- or -O-; R 2 Represents morpholinyl, morpholinylalkyl, alkyl-substituted piperazine, acyl-substituted piperazine, sulfonyl-substituted piperazine, piperazinealkyl, piperazinealkoxy, piperazinealkylamino, homopiperazine, homopiperazinealkyl, homopiperazinealkoxy, homopiperazinealkylamino, piperidinyl, piperidinylalkyl, piperidinylalkoxy, piperidinylalkylamino, tetrahydropyrrolyl, tetrahydropyrrolylalkyl, tetrahydropyrrolylalkylamino, tetrahydrofuranyl, tetrahydrofuranalkyl, tetrahydrofuranalkoxy, tetrahydrofuranalkylamino, tetrahydropyranyl, tetrahydropyranalkyl, tetrahydropyranalkoxy, or tetrahydropyranalkylamino; R 3 Represents an aromatic ring substituted or unsubstituted by a halogen, nitro, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 alkylamino, or phenoxy group, wherein the C1-C6 alkyl, C1-C6 alkoxy, or C1-C6 alkylamino group may be substituted or unsubstituted by any 1-3 substituents, and the substituents are halogens, C1-C6 alkyl, C3-C7 cycloalkyl, heterocyclic, C1-C6 alkenyl, C1-C6 alkynyl, phenyl groups substituted or unsubstituted by at least one halogen, heteroaryl groups substituted or unsubstituted by at least one halogen, or Het1 substituent groups substituted or unsubstituted by at least one halogen; R 3 R 4 They can be the same or different, each representing a hydrogen atom, halogen, C1-C6 alkyl, cyano, C1-C6 alkoxy, or C1-C6 alkylamino.

2. The 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine compound and its pharmaceutical salt or isomer according to claim 1, characterized in that, X represents -CH-, Y represents -CR 4 - The general formula (I) is as follows: , where R 1 Represents 2-methoxyphenyl, 4-fluoro-2-methoxyphenyl, 2,4-dimethoxyphenyl, 4-cyano-2-methoxyphenyl, 2-ethoxyphenyl, 4-fluoro-2-ethoxyphenyl, benzofuranyl, or pyrazolyl; R 2 Represents methyl, cyclopropyl, cyclobutyl, morpholino, piperazine, N-methylpiperazine, homopiperazine, or N-methylhomoperazine; R 5 It represents a hydrogen atom, a fluorine atom, or a chlorine atom.

3. The 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine compound and its pharmaceutical salt or isomer according to claim 1, characterized in that, X represents -N-, Y represents -CR 4 - The general formula is as follows: , where R 1 Represents 2-methoxyphenyl, 4-fluoro-2-methoxyphenyl, 2,4-dimethoxyphenyl, 4-cyano-2-methoxyphenyl, 2-ethoxyphenyl, 4-fluoro-2-ethoxyphenyl, benzofuranyl, or pyrazolyl; R 2 Represents methyl, cyclopropyl, cyclobutyl, morpholino, piperazine, N-methylpiperazine, homopiperazine, or N-methylhomoperazine; R 5 It represents a hydrogen atom, a fluorine atom, or a chlorine atom.

4. The 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine compound and its pharmaceutical salt or isomer according to claim 1, characterized in that, The 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine compounds and their pharmaceutical salts or isomers are specifically: 5-chloro-4-(1-cyclopropylpyrazol-4-yl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridine (I-1), 5-chloro-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}-4-(1-methylpyrazol-4-yl)pyridine (I-2), 5-fluoro-4-(4-fluoro)pyridine 5-chloro-4-(4-fluoro-2-methoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazole-4-yl]amino}pyridine (I-3), 5-chloro-4-(4-fluoro-2-methoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazole-4-yl]amino}pyridine (I-4), 4-(5-fluoro-2-{[1-(piperidin-4-yl)pyrazole-4-yl]amino}pyridine-4-yl)-3-methoxybenzonitrile (I-5), 5-fluoro-4-(2, 4-Dimethoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazole-4-yl]amino}pyridine (I-6), 4-[2-(benzyloxy)-4-fluorophenyl]-5-fluoro-2-{[1-(piperidin-4-yl)pyrazole-4-yl]amino}pyridine (I-7), 5-fluoro-4-[4-fluoro-2-(isopropoxy)phenyl]-2-{[1-(piperidin-4-yl)pyrazole-4-yl]amino}pyridine (I-8), 5-fluoro- 2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}-4-[2-(trifluoromethoxy)phenyl]pyridine (I-9), 5-chloro-4-(4-fluoro-1-methoxyphenyl-2-yl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridine (I-10), 5-(5-chloro-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridin-4-yl)-1-(propyl-2-yl)-1,2-Dihydropyridin-2-one (I-11), 4-(1-benzofuran-7-yl)-5-chloro-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyridine (I-12), 4-(4-fluoro-2-methoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyrimidine (II-1), 6-(4-fluoro-2-methoxyphenyl)-4-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyrimidine (II-2), 5-fluoro-4-(4-fluoro-2-methoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyrimidine (II-3), 5-Chloro-4-(4-fluoro-2-methoxyphenyl)-2-{[1-(piperidin-4-yl)pyrazol-4-yl]amino}pyrimidine (II-4), 5-fluoro-4-(4-fluoro-2-methoxyphenyl)-2-{[1-(piperidin-3-yl)pyrazol-4-yl]amino}pyrimidine (II-5), 2-{[1-(azacyclobut-3-yl)pyrazol-4-yl]amino}-5-fluoro-4-(4-fluoro-2-methoxyphenyl)pyrimidine (II-6), 2-({1-[1-(1-(ethylsulfonyl)azacyclobut-3-yl]pyrazol-4-yl}amino)-4-(4-fluoro-2-methoxyphenyl)pyrimidine (II-7).

5. A method for preparing the 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine compound and its pharmaceutical salt or isomer according to any one of claims 1-4, characterized in that, Includes the following steps: Under nitrogen protection, using key intermediates A1~A13 and B1 as raw materials, Xantphos (4,5-bis(diphenylphosphino)-9,9-dimethyloxanthracene) as ligand, sodium tert-butoxide as base, and Pd2(dba)3tris(dibenzylideneacetone)dipalladium as catalyst, a Buchwald-Hartwig coupling reaction was carried out in anhydrous toluene. Subsequently, the tert-butyloxycarbonyl group was removed by trifluoroacetic acid to finally obtain the target compound.

6. A method for preparing the 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine compound and its pharmaceutical salt or isomer according to any one of claims 1-4, characterized in that, Includes the following steps: Under nitrogen protection, using key intermediates A14 and B1 as starting materials, Xantphos (4,5-bis(diphenylphosphino)-9,9-dimethyloxanthracene) as a ligand, sodium tert-butoxide as a base, and Pd2(dba)3 (tris[dibenzylideneacetone]dipalladium) as a catalyst, a Buchwald-Hartwig coupling reaction was carried out in anhydrous toluene. Subsequently, the tert-butyloxycarbonyl group was removed via trifluoroacetic acid to obtain the target compound II-1. Using key intermediates A15 and B1 as starting materials, t-BuBrettphos (2-di-tert-butylphosphino-2′,4′,6′-triisopropyl-3,6-dimethoxy-1,1′-biphenyl) as a ligand, sodium tert-butoxide as a base, and Pd2(dba)3 as a catalyst, a Buchwald-Hartwig coupling reaction was carried out in anhydrous toluene. Subsequently, the tert-butyloxycarbonyl group was removed by trifluoroacetic acid to obtain target compound II-2. Using key intermediates A18 and B7 as starting materials, t-BuBrettphos as a ligand, sodium tert-butoxide as a base, and Pd2(dba)3 as a catalyst, a Buchwald-Hartwig coupling reaction was carried out in anhydrous toluene. Subsequently, the tert-butyloxycarbonyl group was removed by trifluoroacetic acid to obtain target compound II-3. Using key intermediates A17 and B1 as starting materials, under sealed tube conditions and with Cs2CO3 as a base, a nucleophilic substitution reaction was carried out in tert-butanol to obtain target compound II-4. Using key intermediates A16 and B9-B10 as starting materials, under sealed tube conditions and with Cs2CO3 as a base, a nucleophilic substitution reaction was carried out in tert-butanol to obtain target compounds II-5 and II-6. Using key intermediates A14 and B4 as starting materials, under sealed tube conditions and with Cs2CO3 as a base, a nucleophilic substitution reaction was carried out in tert-butanol to obtain target compound II-7.

7. A pharmaceutical composition comprising any one of the 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine compounds of claims 1-4 and their pharmaceutically acceptable salts or isomers, and a pharmaceutically acceptable carrier.

8. The use of any 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine compound of any one of claims 1-4 and its pharmaceutical salt or isomer, or the pharmaceutical composition of claim 7, in the preparation of a cell cycle-dependent kinase inhibitor drug.

9. The use of any one of the 4-aryl-2-[1-pyrazol-4-yl]aminopyrimidine compounds of claims 1-4 and their pharmaceutical salts or isomers, or the pharmaceutical composition of claim 7, in the preparation of a drug for treating tumors.

10. The application according to claim 9, characterized in that, The tumors mentioned include, but are not limited to, lung cancer, prostate cancer, liver cancer, stomach cancer, cervical cancer, colorectal cancer, melanoma, ovarian cancer, breast cancer, kidney cancer, nervous system tumors, acute myeloid leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, mantle cell lymphoma, Burkitt's lymphoma, and follicular lymphoma.

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