CN116768889A - A new type of CDK9 kinase inhibitor and its preparation method and application - Google Patents

Abstract

A novel CDK9 kinase inhibitor, a preparation method and application thereof. The invention discloses a compound with a general formula (I) or a stereoisomer, a solvate, a deuterated compound, a prodrug, a metabolite and an intermediate thereof, and pharmaceutically acceptable salts or eutectic crystals thereof, and application of a pharmaceutical composition containing the compound as a CDK9 inhibitor for preventing or treating CDK9 related diseases, in particular application of the CDK9 inhibitor for preparing medicines for treating diseases such as tumors and the like.

Description

A novel CDK9 kinase inhibitor and its preparation method and application

Technical Field

The present invention relates to the technical field of chemical drugs, and in particular to a novel CDK9 kinase inhibitor and a preparation method and application thereof.

Background Art

CDK9 plays an important role in transcriptional regulation, such as the transcription of apoptosis regulator myeloid cell leukemia 1 (Mcl-1) and downstream proto-oncogene MYC, controlling the proliferation and survival of tumor cells. Therefore, the dysregulation of CDK9 signaling is significant in a variety of cancer cells. Mechanistically, CDK9 inhibition blocks the phosphorylation of RNAPII CTD and induces downregulation of MYC and Mcl-1 protein levels, which has been confirmed in various hematological malignancies. Studies have shown that miR-613, as a tumor suppressor gene, inhibits the migration and invasion of gastric cancer by targeting CDK9; inhibiting CDK9 can interfere with the proliferation of esophageal cancer cells and inhibit tumor formation in animal models; CDK9 inhibitors have also been shown to inhibit the growth of breast cancer cells and tumors. Targeting CDK9 can also provide some ideas for the treatment of triple-negative breast cancer; intervening in CDK9 will affect the biological functions of glioma cells, which also indirectly confirms that CDK9 may become a potential therapeutic target for brain glioma. All of the above indicate that CDK9 is an important target for the development of cancer therapeutics.

Summary of the invention

The purpose of the present invention is to solve the above problems in the prior art and to provide a novel CDK9 kinase inhibitor and a preparation method and application thereof.

A novel CDK9 kinase inhibitor, the general formula of which is a 3H-imidazole [4,5-b] pyridine compound derivative shown in the structure (I) or a tautomer, stereoisomer, N-oxide, solvate, hydrate, metabolite, pharmaceutically acceptable salt or prodrug thereof of the compound shown in the general formula (I), and the planar structure of the general formula (I) is as follows:

Wherein, X is selected from hydrogen or halogen, preferably H, Cl; L is selected from -CH ₂ -, -C ₂ H ₄ - or L is a chemical bond, preferably -CH ₂ -; R ₁ is selected from N-isopropylpiperazine, 1-tert-butoxycarbonyl-piperazine, piperazine, morpholine, trans-1,4-cyclohexanediamine, 1-tert-butoxycarbonyl-1,4-cyclohexanediamine, 4-methylpiperidine, 4-aminopiperidine, 4-hydroxypiperidine, 3-aminopiperidine, 4-(methylamino)cyclohexane-1-ol, and R ₁ is preferably trans-1,4-cyclohexanediamine; R ₂ is selected from mono-substituted aryl or poly-substituted aryl or 3-methylphenyl, and R ₂ is preferably 3,5-difluorophenyl.

The above-mentioned 3H-imidazole [4,5-b] pyridine compounds have good CDK9 inhibitory activity and selectivity.

A pharmaceutical combination comprises the above-mentioned compound or its tautomer, stereoisomer, N-oxide, hydrate, solvate, deuterated substance, prodrug, metabolite, intermediate, pharmaceutically acceptable salt or cocrystal, and a pharmaceutically acceptable carrier.

The preparation method of the compound is to condense the aromatic ring of bromine-substituted o-phenylenediamine with substituted phenylacetic acid, phenylpropionic acid, etc. at high temperature to obtain an intermediate (2), and then react (2) with the synthesized borate intermediate (1) through Suzuki coupling reaction or acid hydrolysis or condensation to obtain the compound of formula (I). The synthesis route is as follows:

The compound of the present invention is used in the preparation of a drug for treating a disease related to the activity or expression of cell cycle dependent kinase CDK9. Further, the compound of the present invention is used in the preparation of a drug for treating a disease related to the activity or expression of CDK9. The disease includes breast cancer, liver cancer, cervical cancer, brain glioma, etc.

Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

The CDK9 inhibitor disclosed in the present invention selectively inhibits the activity of CDK9 kinase, blocks the phosphorylation of RNA polymerase II CTD, and then regulates the related signal pathways to inhibit the proliferation of tumor cells in vivo and in vitro. Therefore, the CDK9 inhibitor disclosed in the present invention can be used for the treatment of cancer or related diseases.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a graph showing the crystal violet staining experimental results of compound A32 in Example 7 on U87 and U251 cells.

FIG2 is a graph showing the inhibition results of compound A32 of Example 8 on the activity of CDK family kinases.

FIG3 is a graph showing the experimental results of some compounds of the A32 series in Example 9 that can inhibit the function of CDK9.

FIG4 is a graph showing the pharmacodynamics experimental results of compound A32 of Example 10 on a subcutaneous transplant tumor model of U87 cells in mice.

FIG5 is a graph showing the pharmacodynamics experimental results of Compound A32 of Example 11 on the U87-Luc cell orthotopic transplanted tumor model in mice.

DETAILED DESCRIPTION

Table 1 lists the structures of the compounds of the present invention and the mass spectrometry and nuclear magnetic resonance characterization data of the compounds.

Table 1 Structures of 3H-imidazole [4,5-b] pyridine compounds of the present invention and their high-resolution mass spectrometry and nuclear magnetic resonance characterization

Example 1: Synthesis of (1r,4r)-N1-(5-chloro-4-(2-(3,5-difluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)cyclohexane-1,4-diamine (A32)

(1) Synthesis of intermediate 6-bromo-2-(3,5-difluorobenzyl)-3H-imidazo[4,5-b]pyridine:

Under nitrogen protection, a mixture of 3,5-difluorophenylacetic acid (4.30 g, 25.0 mmol) and 2,3-diamino-5-bromopyridine (1.87 g, 10.0 mmol) was melted at 150 ° C and stirred for 3 h. The reaction mixture was treated with 3N aqueous hydrochloric acid solution and then made alkaline by adding ammonia water. The filter cake was filtered and washed and dried, and purified by silica gel column chromatography using a gradient elution method. The eluent was a mixture of ethyl acetate and methanol in a volume ratio of 1:0 to 10:1, and then the product was crystallized from ethyl acetate to obtain 2.75 g of 6-bromo-2-(3,5-difluorobenzyl)-3H-imidazo[4,5-b]pyridine with a yield of 90%. ¹ H NMR (600MHz, DMSO-d ₆ ) δ11.54-14.04(m,1H),7.88(br d,J＝8.25Hz,1H),7.45(ddd,J＝2.02,7.98,11.65Hz,1H),7.38-7.42(m,1H),7.37(d,J＝8.25Hz,1 H), 7.19 (ddd, J=2.11, 4.08, 6.28Hz, 1H), 4.24 (s, 2H), LRMS (ESI) m/z: 324.0 [M+H] ⁺ .

(2) Synthesis of the intermediate tert-butyl ((1r,4r)-4-((5-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxin-2-yl)pyridin-2-yl)amino)cyclohexyl)carbamate:

2-Fluoro-4-iodo-5-bromopyridine (2.57 g, 10 mmol) and N-Boc-1,4-cyclohexanediamine (2.14 g, 10 mmol) were dissolved in 25 mL of N,N-dimethylformamide, and N,N-diisopropylethylamine (3.87 g, 30 mmol) was added. After nitrogen was replaced in the reaction system, the temperature was raised to 120°C and the reaction was continued for 4 h. After TLC detected that the raw materials reacted completely, the heating was stopped, and the reaction solution was poured into 100 mL of ice water under stirring. Solids precipitated, and the filter cake was taken by suction filtration and dried to obtain 4.29 g of white solid tert-butyl ((1r, 4r)-4-((5-chloro-4-iodopyridin-2-yl)amino)cyclohexyl)carbamate, with a yield of 95%.

The tert-butyl ((1r, 4r)-4-((5-chloro-4-iodopyridin-2-yl)amino)cyclohexyl)carbamate (4.29 g, 9.5 mmol), boronic acid pinacol ester (3.05 g, 12 mmol), 1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (0.1 mmol), and dry anhydrous potassium acetate (0.93 g, 30 mmol) were added to 25 mL of dry N,N-dimethylformamide, and the nitrogen was replaced. The temperature was raised to 90°C under nitrogen protection for reaction for 3 h. The tert-butyl ((1r, 4r)-4-((5-chloro-4-iodopyridin-2-yl)amino)cyclohexyl)carbamate (4.29 g, 9.5 mmol) was detected by TLC. After the reaction of (1r,4r)-4-((5-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxin-2-yl)pyridin-2-yl)amino)cyclohexyl)carbamate is completed, the reaction solution is poured into 100 mL of ice water with stirring, extracted with (3*25 mL) of ethyl acetate, the organic phases are combined, and after back-extraction with saturated sodium chloride, the organic phase is added with anhydrous sodium sulfate to dry, the desiccant is filtered to remove the organic phase, and the organic phase is concentrated and silica gel is added to mix the sample, and the mixture is separated and purified by column chromatography on silica gel with the developing solvent of petroleum ether:ethyl acetate=5:1 to obtain 3.43 g of white powder intermediate tert-butyl ((1r,4r)-4-((5-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxin-2-yl)pyridin-2-yl)amino)cyclohexyl)carbamate, with a yield of 80%.

(3) Synthesis of (1r,4r)-N1-(5-chloro-4-(2-(3,5-difluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)cyclohexane-1,4-diamine (A32):

Take a thick-walled pressure bottle, weigh the intermediate 6-bromo-2-(3,5-difluorobenzyl)-3H-imidazo[4,5-b]pyridine (324 mg, 1 mmol) and the intermediate tert-butyl ((1r,4r)-4-((5-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxin-2-yl)pyridin-2-yl)amino)cyclohexyl)carbamate (497 mg, 1.1 mmol) and potassium carbonate (414 mg, 3 mmol) and 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (30 mg) and dissolve them in 5 mL of solvent (ethylene glycol dimethyl ether: water = 4:1). The mixture was replaced with nitrogen three times, and the temperature was raised to 90°C for overnight reaction. After the reaction was completed by TLC detection, the reaction solution was cooled to room temperature and filtered, and the filter cake was washed with a small amount of ethylene glycol dimethyl ether and dried to obtain a crude product. The crude product was dissolved in a dichloromethane:methanol system of 10:1, and silica gel was added to mix the sample. The crude product was separated and purified by column chromatography, and the developing solvent was petroleum ether:ethyl acetate = 1:1 to obtain the compound tert-butyl ((1r,4r)-4-((5-chloro-4-(2-(3,5-difluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)amino)cyclohexyl)carbamate (A26), a white powder, with a yield of 50%. ¹ H NMR (600MHz, CHLOROFORM-d) δ13.04-13.31(m,1H),8.17-8.28(m,1H),8.12(br d,J＝6.97Hz,2H),6.90(br d,J＝6.05Hz,2H),6.68(br d,J＝6.24Hz,1H),6.30-6 .37(m,1H),4.64(br s,1H),4.44-4.52(m,1H),4.36(s,1H),4.34(s,1H),3.54-3.63(m,1H),3.42-3.51(m,1H),2.11-2.19(m,2H),2.03-2.09(m,2H) ,1.45(br s,9H),1.26-1.35(m,4H); ¹³ C NMR(151MHz,CHLOROFORM-d)δ163.3(br dd,J=12.7,248.1Hz,2C),156.9,148.8(br s,1C),148.0(br s,1C),146.0,143.0(br s,1 C),142.6(d,J＝4.4Hz,1C),139.7(br d,J＝8.8Hz,1C),135.4(br s,1C),128.5(br s,1C),127.8(br s,1C),118.6-117.7(m,1C),112.2-111.8(m,1C),109 .0(br s,1C),103.3-102.8(m,1C),79.4(br s,1C),69.7,49.9(br s,1C),49.2(br s,1C),36.1(br s,1C),32.1(2C),31.9(2C),28.5(3C).

The obtained A26 was dissolved in a system of dichloromethane:trifluoroacetic acid in a volume ratio of 2:1, and stirred at room temperature for 2 hours. The dichloromethane and trifluoroacetic acid were evaporated under reduced pressure, and the residual solid was dispersed with dichloromethane. The product was treated with triethylamine under stirring until it became alkaline (pH=8). After the organic phase was concentrated, silica gel was added and the sample was mixed. The product was separated and purified by column chromatography on silica gel with a developing solvent of dichloromethane:methanol:ammonia=10:1:0 to dichloromethane:methanol:ammonia=10:1:0.1 to obtain (1r,4r)-N1-(5-chloro-4-(2-(3,5-difluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)cyclohexane-1,4-diamine (A32) as a yellow solid. ¹ H NMR (600 MHz, METHANOL-d ₄ )δ8.40(d,J＝1.8Hz,1H),8.05(s,1H),8.03(d,J＝1.8Hz,1H),6.95(br d,J＝6.2Hz,2H),6.75(br t,J＝9.0Hz,1H),6.48(d,J＝2.0Hz,1H),4.34-4.27(m,2H), 3.60 (br t, J＝9.9Hz, 1H), 2.87-2.76 (m, 1H), 2.14 (br d, J＝12.1Hz, 2H), 2.02-1.94 (m, 2H), 1.42-1.26 (m, 5H); ¹³ C NMR (151MHz, METHANOL-d ₄ )δ163.2(dd,J＝12.7,249.2Hz,2C),157.0,156.0(br s,1C),147.0,146.1,143.7,140.2-139.2(m,1C),131.7(brd,J＝8.8Hz,1C),128.3,124.6(br s,1C) ,117.5,112.3-111.4(m,1C),110.0(br s,1C),102.7-102.3(t,2C),49.6,49.4,35.1,33.2(2C),31.2(2C).

Example 2: Synthesis of 4-(5-chloro-4-(2-(4-fluorophenylethyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)morpholine (A22)

(1) Synthesis of intermediate 6-bromo-2-(4-fluorophenethyl)-3H-imidazole[4,5-b]pyridine:

Under nitrogen protection, a mixture of 4-fluorophenylpropionic acid (4.20 g, 25.0 mmol) and 2,3-diamino-66-bromopyridine (1.87 g 10.0 mmol) was melted at 150 ° C and stirred for 2 h. The reaction mixture was treated with 3N aqueous hydrochloric acid solution and then made alkaline by adding ammonia water. The filter cake was filtered and washed and dried, and purified by silica gel column chromatography using a gradient elution method. The eluent was a mixture of ethyl acetate and methanol in a volume ratio of 1:0 to 10:1. The product was then crystallized from ethyl acetate to obtain 2.72 g of 6-bromo-2-(4-fluorophenethyl)-3H-imidazole [4,5-b] pyridine with a yield of 85%. ¹ H NMR (600MHz, DMSO-d ₆ ) δ8.35 (d, J = 2.02Hz, 1H), 8.16 (d, J = 1.83Hz, 1H), 7.28-7.31 (m, 2H), 7.08-7.12 (m, 2H), 3.16-3.20 (m, 2H), 3.12-3.16 (m, 2H); ³ C NMR (151MHz, DMSO-d ₆ ) δ 174.1, 161.2 (d, J = 241.0Hz, 1C), 158.6 (br s,1C),143.4,137.2(d,J＝3.3Hz,1C),130.9-129.9(m,1C),115.5(d,J＝22.0Hz,1C),112.7,32.4,31.2; LRMS(ESI)m/z:320.0[M+H] ⁺ .

(2) Synthesis of 4-(5-chloro-4-(2-(4-fluorophenylethyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)morpholine (A22):

2-Fluoro-4-iodo-5-bromopyridine (2.57 g, 10 mmol) and morpholine (0.87 g, 10 mmol) were dissolved in 25 mL of N,N-dimethylformamide, and N,N-diisopropylethylamine (3.87 g, 30 mmol) was added. After nitrogen was replaced in the reaction system, the temperature was raised to 120°C and the reaction was continued for 4 h. After TLC detected that the raw materials had reacted completely, the heating was stopped, and the reaction solution was poured into 100 mL of ice water with stirring. Solids precipitated, and the filter cake was taken by suction filtration and dried to obtain 2.92 g of white solid 4-(5-chloro-4-iodopyridin-2-yl)morpholine, with a yield of 90%.

The 4-(5-chloro-4-iodopyridin-2-yl)morpholine (2.92 g, 9.0 mmol) obtained in the previous step, pinacol boronic acid ester (3.05 g, 12 mmol), 1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (0.1 mmol), and dry anhydrous potassium acetate (0.93 g, 30 mmol) were added to 25 mL of dry N,N-dimethylformamide, and the nitrogen was replaced. The temperature was raised to 90°C under nitrogen protection for reaction for 3 h. 4-(5-chloro-4-iodopyridin-2-yl)morpholine was detected by TLC. After the reaction is complete, the reaction solution is poured into 100 mL of ice water with stirring, extracted with (3*25 mL) ethyl acetate, the organic phases are combined, and after back extraction with saturated sodium chloride, the organic phase is added with anhydrous sodium sulfate to dry, and the desiccant is filtered to remove the organic phase. After the organic phase is concentrated, silica gel is added to mix the sample, and the mixture is separated and purified by column chromatography on silica gel with the developing agent of petroleum ether: ethyl acetate = 5:1 to obtain 1.95 g of a white powder intermediate 4-(5-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxin-2-yl)pyridin-2-yl)morpholine, with a yield of 67%.

Take a thick-walled pressure bottle, weigh the intermediate 6-bromo-2-(4-fluorophenethyl)-3H-imidazole [4,5-b] pyridine (320 mg, 1 mmol) and the intermediate 4-(5-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxin-2-yl) pyridin-2-yl) morpholine (357 mg, 1.1 mmol) and potassium carbonate (414 mg, 3 mmol) and 1,1'-bis (diphenylphosphino) ferrocene] dichloropalladium (30 mg) and dissolve them in 5 mL of solvent (ethylene glycol dimethyl ether: water = 4:1) The mixture was replaced with nitrogen three times, and the temperature was raised to 90°C for overnight reaction. After the reaction was completed by TLC detection, the reaction solution was cooled to room temperature and filtered, and the filter cake was washed with a small amount of ethylene glycol dimethyl ether and dried to obtain a crude product. The crude product was dissolved in a dichloromethane:methanol system of 10:1, and silica gel was added to mix the sample. The product was separated and purified by column chromatography, and the developing solvent was petroleum ether:ethyl acetate = 1:1 to obtain compound 4-(5-chloro-4-(2-(4-fluorophenylethyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)morpholine (A22) as a white powder. ¹ H NMR (600MHz, DMSO-d ₆ ) δ8.42 (d, J = 2.02Hz, 1H), 8.28 (s, 1H), 8.08 (d, J = 2.02Hz, 1H), 7.38 (dd, J = 5.69, 8.44Hz, 2H), 7.18 (t, J = 8.89Hz, 2H), 7.00 (s, 1H), 4 .31(br d,J＝13.02Hz,2H),3.21-3.28(m,4H),2.98-3.05(m,2H),2.87-2.95(m,1H),1.83(br d,J＝10.09Hz,2H),1.26-1.34(m,2H); ¹³ C NMR (151MHz, DMSO-d ₆ )δ161.2(d,J＝242.1Hz,1C),158.2,156.2,153.4,147.5,146.5,143.4,137.3(d,J＝3.3Hz,1C),130.5(d,J＝7.7Hz,2C),130.3,127.3,117.4,115.5(d ,J＝20.9Hz,2C),109.5,48.8(2C),44.2(2C),34.6,32.5,31.2.

Example 3: Synthesis of 1-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)piperidin-4-ol (Compound A18)

(1) Synthesis of intermediate 6-bromo-2-(3,5-difluorobenzyl)-3H-imidazo[4,5-b]pyridine:

The method and post-treatment are shown in Example 1. Spectral data: ¹ H NMR (600 MHz, DMSO-d ₆ ) δ 8.35 (d, J = 1.83 Hz, 1H), 8.17 (d, J = 1.83 Hz, 1H), 7.32-7.39 (m, 1H), 7.20 (br d, J = 10.09 Hz, 1H), 7.18 (d, J = 7.70 Hz, 1H), 7.07 (dt, J = 2.57, 8.62 Hz, 1H), 4.25 (s, 2H); ¹³ C NMR (151 MHz, DMSO-d ₆ )δ162.61(d,J＝243.2Hz,1C),157.22,143.83,139.87(d,J＝7.7Hz,1C),130.87(d,J＝8.8Hz,1C),125.53(d,J＝3.3Hz,1C),116.24(d,J＝22.0Hz,1C),114 .05(d,J＝20.9Hz,1C),112.94,35.18; LRMS(ESI)m/z:307.1[M+H] ⁺ .

(2) Synthesis of 1-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)piperidin-4-ol (A18):

2-Fluoro-4-iodo-5-bromopyridine (2.57 g, 10 mmol) and 4-hydroxypiperidine (1.01 g, 10 mmol) were dissolved in 25 mL of N,N-dimethylformamide, and N,N-diisopropylethylamine (3.87 g, 30 mmol) was added. After nitrogen was replaced in the reaction system, the temperature was raised to 120°C and the reaction was continued for 4 h. After TLC detected that the raw materials reacted completely, the heating was stopped, and the reaction solution was poured into 100 mL of ice water with stirring. Solids precipitated, and the filter cake was taken by suction filtration and dried to obtain 3.05 g of 1-(5-chloro-4-iodopyridin-2-yl)piperidin-4-ol as a white solid with a yield of 90%.

The 1-(5-chloro-4-iodopyridin-2-yl)piperidin-4-ol (3.05 g, 9.0 mmol) obtained in the previous step, pinacol borates (3.05 g, 12 mmol), 1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (0.1 mmol), and dry anhydrous potassium acetate (0.93 g, 30 mmol) were added to 25 mL of dry N,N-dimethylformamide, and the nitrogen was replaced. The temperature was raised to 90 ° C under nitrogen protection for 3 h. The 1-(5-chloro-4-iodopyridin-2-yl)piperidin-4-ol was detected by TLC. After the reaction of the -alcohol is complete, the reaction solution is poured into 100 mL of ice water with stirring, extracted with (3*25 mL) ethyl acetate, the organic phases are combined, and after saturated sodium chloride back extraction, the organic phase is added with anhydrous sodium sulfate to dry, and the desiccant is filtered to remove the organic phase. After the organic phase is concentrated, silica gel is added to mix the sample, and the sample is separated and purified by column chromatography on silica gel, and the developing agent is petroleum ether: ethyl acetate = 5:1 to obtain 2.19 g of a white powder intermediate 1-(5-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxin-2-yl)pyridin-2-yl)piperidin-4-ol, with a yield of 72%.

Take a thick-walled pressure bottle, weigh the intermediate 6-bromo-2-(3,5-difluorobenzyl)-3H-imidazo[4,5-b]pyridine (306 mg, 1 mmol) and the intermediate 1-(5-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxin-2-yl)pyridin-2-yl)piperidin-4-ol (372 mg, 1.1 mmol) and potassium carbonate (414 mg, 3 mmol) and 1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium (30 mg) and dissolve them in 5 mL of solvent (ethylene glycol dimethyl ether: water = 4 :1), replaced with nitrogen three times, heated to 90°C and reacted overnight. After the reaction was completed by TLC detection, the reaction solution was cooled to room temperature and filtered, the filter cake was washed with a small amount of ethylene glycol dimethyl ether, and dried to obtain a crude product; the crude product was dissolved in a dichloromethane:methanol system of 10:1, silica gel was added to mix the sample, and column chromatography was used for separation and purification, and petroleum ether:ethyl acetate = 1:1 was used as the developing solvent for separation to obtain compound 1-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)piperidin-4-ol (A18) as a white powder, ¹ H NMR (600MHz, METHANOL-d ₄ ) δ8.39 (brs, 1H), 8.18 (s, 1H), 7.93-8.12 (m, 1H), 7.32 (dt, J = 6.05, 7.98Hz, 1H), 7.13-7.20 (m, 1H), 7.10 (br d,J＝9.54Hz,1H),6.97(dt,J＝2.29,8.39Hz,1H),6.71(s,1H),4.31(s,2H),4.09-4.18(m,1H),4.06(td,J＝4.31,13.39Hz,2H),3.84-3.92(m,1H),3. 18(ddd,J＝3.03,10.13,13.25Hz,2H),1.93-2.01(m,2H),1.59(dtd,J＝3.85,9.45,13.02Hz,2H); ¹³ C NMR (151MHz, METHANOL-d ₄ )δ163.0(d,J＝246.5Hz,1C),158.0,147.3(2C),146.2,143.5(br s,1C),138.3(d,J＝7.7Hz,2C),130.3(d,J＝7.7Hz,1C),128.5(br s,1C),124.5(d,J＝3. 3Hz, 1C), 118.3, 115.7 (d, J = 22.0Hz, 1C), 114.0 (d, J = 20.9Hz, 1C), 109.3 (2C), 67.2, 43.3 (2C), 35.3, 33.3 (2C).

Among them, the compounds listed in the present invention are: (1r,4r)-N1-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-5-yl)pyridin-2-yl)cyclohexane-1,4-diamine (A1), (1r,4r)-N1-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)cyclohexane-1,4-diamine (A2), 5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-5-yl)-N-(piperidin-3 -yl)pyridin-2-amine (A5), 5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)-N-(piperidin-3-yl)pyridin-2-amine (A6), (1r,4r)-N1-(4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)cyclohexane-1,4-diamine (A7), (1r,4r)-N1-(5-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)cyclohexane-1,4-diamine Amine (A8), (1r,4r)-N1-(6-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)cyclohexane-1,4-diamine (A9), (1r,4r)-N1-(3-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)cyclohexane-1,4-diamine (A10), (1r,4r)-N1-(5-chloro-4-(2-(3-fluorophenyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)cyclohexane- 1,4-diamine (A11), (1r,4r)-N1-(5-chloro-4-(2-(3-fluorophenylethyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)cyclohexane-1,4-diamine (A12), 1-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)piperidin-3-amine (A13), 1-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)piperidin-4-amine (A14), 4), 6-(5-chloro-2-(piperazin-1-yl)pyridin-4-yl)-2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridine (A15), 5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)-N-(piperidin-4-yl)pyridin-2-amine (A16), (1r,4r)-N1-(5-chloro-4-(2-(3-(trifluoromethyl)benzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)cyclohexane-1,4-diamine (A23), 4-(( 6-(2-(((1r,4r)-4-aminocyclohexyl)amino)-5-chloropyridin-4-yl)-3H-imidazo[4,5-b]pyridin-2-yl)methyl)-2-fluorophenol (A24), (1r,4r)-N1-(5-chloro-4-(2-(3-chlorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)cyclohexane-1,4-diamine (A27), (1r,4r)-N1-(5-chloro-4-(2-(3-methylbenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl) )cyclohexane-1,4-diamine (A28), (1r,4r)-N1-(5-chloro-4-(2-(2-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)cyclohexane-1,4-diamine (A29), (1r,4r)-N1-(5-chloro-4-(2-(4-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)cyclohexane-1,4-diamine (A30), (1r,4r)-N1-(5-chloro-4-(2-(2,3-difluorobenzyl)-3H-imidazo[4 The synthesis methods of compound A32 are similar to those of compound A32. The attribution of mass spectra and NMR spectra are shown in Table 1.

Among them, the compounds listed in the present invention are: tert-butyl ((1r, 4r)-4-((3-(2-(3-fluorobenzyl)-3H-imidazo [4,5-b] pyridin-6-yl) pyridin-2-yl) amino) cyclohexyl) carbamate (A10-1), tert-butyl ((1r, 4r)-4-((5-chloro-4-(2-(3-fluorophenyl)-3H-imidazo [4,5-b] pyridin-6-yl) pyridin-2-yl) amino) cyclohexyl) carbamate (A11-1), tert-butyl ((1r, 4r)-4-((5-chloro-4-(2-(3-fluorophenylethyl)-3H-imidazo [4,5-b] pyridin-6-yl) pyridin-2-yl) amino) cyclohexyl) amino tert-butyl (1-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-5-yl)pyridin-2-yl)piperidin-3-yl)carbamate (A13-1), tert-butyl (1-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)piperidin-4-yl)carbamate (A14-1), tert-butyl 4-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-5-yl)pyridin-2-yl)piperazine-1-carboxylate (A15-1), tert-butyl 4-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-5-yl)pyridin-2-yl)piperazine-1-carboxylate (A15-1), tert-butyl 4-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)piperidin-4-yl)carbamate (A16-1), imidazo[4,5-b]pyridin-5-yl)pyridin-2-yl)amino)piperidine-1-carboxylic acid tert-butyl ester (A16-1), tert-butyl ((1r,4r)-4-((5-chloro-4-(2-(3-fluoro-4-hydroxybenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)amino)cyclohexyl)carbamate (A24-1), tert-butyl ((1r,4r)-4-((5-chloro-4-(2-(3-methylbenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)amino)cyclohexyl)carbamate (A28-1), tert-butyl ((1r,4r)-4-((5-chloro-4-(2-(2-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)amino)cyclohexyl)carbamate The synthesis methods of compound A26 are similar to those of compound A26. The attribution of mass spectra and NMR spectra are shown in Table 1.

Among them, the compounds listed in the present invention are: 6-(5-chloro-2-(4-methylpiperidin-1-yl)pyridin-4-yl)-2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridine (A17), (1r,4r)-4-((5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)amino)cyclohexane-1-ol (A19), 6-(5-chloro-2-(4-isopropylpiperazin-1-yl)pyridin-4-yl)-2-(3 The synthesis method of compound A18 is similar to that of compound A18, and the attribution of mass spectra and NMR spectra are shown in Table 1.

Among them, the compounds listed in the present invention: 4-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-5-yl)pyridin-2-yl)morpholine (A3), 4-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)morpholine (A4) are synthesized similarly to compound A22, and the mass spectra and NMR spectra are shown in Table 1.

Example 4: Cytotoxicity test results of some 3H-imidazole [4,5-b] pyridine compounds of the present invention

The anti-proliferation experiments of the series of compounds of the present invention on seven tumor cells, B16F10, Eahy-926, OCM-1a, Ovcar-3, SW480, A498 and U87, were carried out at the concentrations of 1 μM/10 μM/20 μM. The cell survival rate results are shown in Table 2.

Table 2 Cytotoxicity test results of some 3H-imidazole [4,5-b] pyridine compounds of the present invention

Example 5: Inhibition results of some 3H-imidazole [4,5-b] pyridine compounds of the present invention on CDK9 kinase activity

The CDK9 kinase activity inhibition experiment was carried out on some compounds of the present invention at a concentration of 1 μM/50 nM. The experimental results are shown in Table 3.

Table 3 Inhibition results of some 3H-imidazole [4,5-b] pyridines of the present invention on CDK9 kinase activity

Example 6: Anti-cell proliferation experiment of 3H-imidazole [4,5-b] pyridine compound A32 of the present invention

MTT method to measure cell proliferation. Different tumor cells or normal cells were plated in 96-well plates, with 3000 cells per well. After 12 hours, the target compound was added for treatment. The compound was diluted in the culture medium to a certain concentration gradient. After 72 hours, the culture medium was discarded, and the fresh culture medium and MTT were mixed at a ratio of 20:1. 100 μL of the mixture was added to each well, incubated at 37°C for 2 hours, and then detected at a wavelength of 490 nm. The results are shown in Table 4.

Table 4 IC ₅₀ values of A32 for different cells

Example 7: Experiment on the inhibition of glioma cell proliferation by the 3H-imidazole [4,5-b] pyridine compound A32 of the present invention

The two types of brain glioma cells were treated with A32 small molecules at a certain concentration gradient. The crystal violet staining results are shown in Figure 1. Compared with the control group, different concentrations of A32 had a certain inhibitory effect on the proliferation of both cells, and showed a certain concentration dependence. In particular, after treatment with high concentrations (2.5μM and 5μM) of small molecules, the glioma cell clone formation was inhibited and the growth was almost stagnant.

Example 8: Kinase activity inhibition experiment of the 3H-imidazole [4,5-b] pyridine compound A32 of the present invention

This example tests the IC ₅₀ of compound A32 in Example 1 against CDK9 and the ability of A32 to inhibit the activity of other kinases in the same family at a concentration of 1 μM. The results show that at a concentration of 10 μM ATP, the IC ₅₀ of A32 against CDK9 is 38 nM; at a concentration of 45 μM ATP, the IC ₅₀ of A32 against CDK9 is 27 nM; at a concentration of 90 μM ATP, the IC ₅₀ of A32 against CDK7 is 326 nM, indicating that A32 has a 10-fold selectivity in inhibiting CDK7 and CDK9. At the same time, the results of the kinase selectivity experiment show that A32 has obvious differences in the inhibition of enzyme activity of each member of the CDKs family. After treatment with 1 μM A32, the kinase activity of CDK9 was almost completely inhibited, while the activity of other members was more than 50%, which also shows to a certain extent that A32 has CDKs family selectivity, as shown in Figure 2.

Example 9: Study on the inhibitory effect of 3H-imidazole [4,5-b] pyridine compound A32 of the present invention on CDK9 function

CDK9/cyclinT participates in the formation of the transcription elongation factor P-TEFb complex, and CDK9 inhibitors can block the phosphorylation of CDK9/cyclinT1 on the serine 2 position of the carboxyl terminal domain of RNA polymerase II. Therefore, this embodiment uses the effect of the A32 series of compounds on the phosphorylation level of serine 2 of the carboxyl terminal domain of RNA polymerase II as an indicator to evaluate the inhibitory effect of this series of compounds on CDK9 (Figure 3). This embodiment tests the phosphorylation level of serine 2 of the carboxyl terminal domain of RNA polymerase II in U87 cells after treatment with the A32 series of compounds, and finds that compounds A2, A12, A23, A27, A28, A29, A30, A31, A32, A33, etc. all inhibit the phosphorylation level of serine 2 of the carboxyl terminal domain of RNA polymerase II.

Example 10: Inhibitory experiment of the 3H-imidazole [4,5-b] pyridine compound A32 of the present invention on the subcutaneous transplanted tumor model of U87 cells in mice

After constructing a common U87 cell mouse tumor model, administration began after the initial tumor volume reached 100 mm ^3. The pharmacodynamic experimental results are shown in Figure 4. It can be seen that after 14 days of intraperitoneal injection of 25 mg/kg, the tumor volume of mice increased slowly compared with the control group, and the final peeled tumor weight also had a statistical difference. During the experiment, there was no significant change in the body weight of mice in the administration group, and the organs of the mice in the administration group showed no obvious damage after HE staining, indicating that at this dose, compound A32 is relatively safe for mice.

Example 11: Inhibitory experiment of the 3H-imidazole [4,5-b] pyridine compound A32 of the present invention on the U87-Luc cell mouse orthotopic transplant tumor model

A U87-Luc cell orthotopic transplant tumor model was constructed, and the relevant experimental results are shown in Figure 5. On the third day after tumor implantation, the mice were randomly divided into two groups and given the corresponding solvent or A32 solution (25 mg/kg), and then the mice were imaged in vivo on the 7th, 14th, and 21st days to detect the fluorescence intensity. It can be seen that compared with the control group, the intracranial fluorescence intensity of the mice in the drug-treated group increased slowly and had significant differences. After the 21st day, the drug administration was stopped, the mice in the control group died in a short period of time, and the survival time of the mice given the drug was significantly prolonged. Protein extraction of intracranial tumor tissues of mice and detection of apoptosis-related proteins revealed that the expression levels of MCL-1 and BCL-2 decreased, which also shows that A32 promotes apoptosis of tumor cells.

Claims (8)

1. A novel CDK9 kinase inhibitor comprising a structural compound of formula (I):

wherein X is selected from hydrogen or halogen; l is selected from lower alkyl chains; r is R ₁ Selected from piperazinyl, morpholinyl, piperidinyl, 1, 4-cyclohexanediamino, and halogen atoms; r is R ₂ Selected from a single or multiple substituted aryl or 3-methylphenyl.

2. A novel CDK9 kinase inhibitor according to claim 1, wherein: x is selected from H, cl; l is selected from-CH ₂ -、-C ₂ H ₄ -；R ₁ Selected from N-isopropylpiperazine, 1-t-butoxycarbonyl-piperazine, morpholine, trans-1, 4-cyclohexanediamine, 1-t-butoxycarbonyl-1, 4-cyclohexanediamine, 4-methylpiperidine, 4-aminopiperidine, 4-hydroxypiperidine, 3-aminopiperidine, 4- (methylamino) cyclohexane-1-ol.

3. A novel CDK9 kinase inhibitor according to claim 1, wherein: l is-CH ₂ -，R ₁ Is trans-1, 4-cyclohexanediamine, R ₂ Is 3, 5-difluorophenyl.

4. A process for the preparation of a novel CDK9 kinase inhibitor according to any one of claims 1 to 3, which is characterized by the synthetic route:

5. a pharmaceutical combination, characterized in that: a pharmaceutical composition comprising a structural compound of formula (I) as defined in any one of claims 1 to 3 or a stereoisomer thereof, a hydrate thereof, a solvate thereof, a deuterate thereof, a prodrug thereof, a metabolite thereof, an intermediate thereof, a pharmaceutically acceptable salt or co-crystal thereof, and a pharmaceutically acceptable carrier.

6. Use of a CDK9 kinase inhibitor according to any one of claims 1 to 3 in combination with a medicament according to claim 5, wherein: use in the manufacture of a medicament for the treatment of a disease associated with the activity or expression of the cell cycle dependent kinase CDK 9.

7. Use of a CDK9 kinase inhibitor according to any one of claims 1 to 3 in combination with a medicament according to claim 5, wherein: use in the manufacture of a medicament for the treatment of a disorder associated with CDK9 activity or expression level.

8. Use according to claim 6 or 7, characterized in that: the diseases include breast cancer, gastric cancer, liver cancer, cervical cancer and brain glioma.