A novel CDK9 kinase inhibitor and preparation method and application thereof

Abstract

A novel CDK9 kinase inhibitor and a preparation method and application thereof. The compound disclosed in the application has a general formula (I) or a stereoisomer, a solvate, a deuterated compound, a prodrug, a metabolite, an intermediate thereof, and a pharmaceutically acceptable salt or a co-crystal thereof, and a pharmaceutical combination containing the compound, and the compound is used as a CDK9 inhibitor for preventing or treating CDK9 related diseases, and is particularly used as a CDK9 inhibitor for preparing a drug for treating diseases such as tumors.

Description

Technical Field

[0001] This invention relates to the field of chemical drug technology, and in particular to a novel CDK9 kinase inhibitor, its preparation method, and its application. Background Technology

[0002] CDK9 plays a crucial role in transcriptional regulation, such as the transcription of the apoptosis regulator myeloid leukemia 1 (Mcl-1) and its downstream proto-oncogene MYC, controlling tumor cell proliferation and survival. Therefore, dysregulation of CDK9 signaling is significant in various cancer cells. Mechanistically, CDK9 inhibition blocks the phosphorylation of RNAPII CTD and induces downregulation of MYC and Mcl-1 protein levels, as confirmed in various hematologic malignancies. Studies have shown that miR-613, as a tumor suppressor gene, inhibits gastric cancer migration and invasion by targeting CDK9; CDK9 inhibition can interfere with esophageal cancer cell proliferation and inhibit tumor formation in animal models; CDK9 inhibitors have also been shown to inhibit breast cancer cell and tumor growth. Targeting CDK9 can also provide insights for the treatment of triple-negative breast cancer; intervention with CDK9 affects the biological function of glioma cells, which also indirectly confirms that CDK9 may be a potential therapeutic target for glioma. All of the above indicate that CDK9 is an important target for cancer therapeutic drug development. Summary of the Invention

[0003] The purpose of this invention is to solve the above-mentioned problems in the prior art and to provide a novel CDK9 kinase inhibitor, its preparation method, and its application.

[0004] A novel CDK9 kinase inhibitor, having the general formula (I) as shown in the structure of a 3H-imidazolium[4,5-b]pyridine derivative or a tautomer, stereoisomer, N-oxide, solvate, hydrate, metabolite, pharmaceutically acceptable salt or prodrug thereof, wherein the planar structure of general formula (I) is as follows:

[0005]

[0006] Wherein, X is selected from hydrogen or halogen, preferably H or Cl; L is selected from -CH2-, -C2H4- or L is a chemical bond, preferably -CH2-; R1 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, R1 is preferably trans-1,4-cyclohexanediamine; R2 is selected from monosubstituted aryl or polysubstituted aryl or 3-methylphenyl, R2 is preferably 3,5-difluorophenyl.

[0007] The above-mentioned 3H-imidazolium[4,5-b]pyridine compounds exhibit good CDK9 inhibitory activity and selectivity.

[0008] A pharmaceutical combination comprising the above-mentioned compound or its tautomers, stereoisomers, N-oxides, hydrates, solvates, deuterates, prodrugs, metabolites, intermediates, pharmaceutically acceptable salts or cocrystals, and pharmaceutically acceptable carriers.

[0009] The preparation method of the compound is as follows: the aromatic ring of the bromine-substituted o-phenylenediamine is condensed with substituted phenylacetic acid, phenylpropionic acid, etc. at high temperature to obtain intermediate (2), and (2) is then combined with the synthesized borate ester intermediate (1) through Suzuki coupling reaction or through acid hydrolysis or condensation to obtain the compound of formula (I) involved. The synthetic route is as follows:

[0010]

[0011] The invention relates to the use of the compounds in the preparation of medicaments for treating diseases related to the activity or expression level of the cell cycle-dependent kinase CDK9. Further, it relates to their use in the preparation of medicaments for treating diseases related to CDK9 activity or expression level. These diseases include breast cancer, liver cancer, cervical cancer, and glioma.

[0012] Compared with the prior art, the beneficial effects achieved by the technical solution of this invention are:

[0013] The CDK9 inhibitor disclosed in this invention selectively inhibits CDK9 kinase activity, blocks the phosphorylation of RNA polymerase II CTD, and thereby regulates related signaling pathways, inhibiting the proliferation of tumor cells in vitro and in vivo. Therefore, the CDK9 inhibitor disclosed in this invention can be used for the treatment of cancer or related diseases. Attached Figure Description

[0014] Figure 1 The image shows the results of crystal violet staining experiments on U87 and U251 cells using compound A32 from Example 7.

[0015] Figure 2 The graph shows the inhibition results of compound A32 in Example 8 on CDK family kinase activity.

[0016] Figure 3 The figure shows the experimental results of how some compounds in the A32 series can inhibit CDK9 function in Example 9.

[0017] Figure 4 The figure shows the pharmacodynamic results of compound A32 in Example 10 on a mouse subcutaneous xenograft model of U87 cells.

[0018] Figure 5The figure shows the pharmacodynamic results of compound A32 in Example 11 on a mouse orthotopic xenograft model of U87-Luc cells. Detailed Implementation

[0019] Table 1 lists the structures of the compounds of the present invention and their mass spectrometry and nuclear magnetic resonance characterization data.

[0020] Table 1. Structures and high-resolution mass spectrometry and NMR characterization of the 3H-imidazolium[4,5-b]pyridine compounds of the present invention.

[0021]

[0022]

[0023]

[0024]

[0025]

[0026]

[0027]

[0028]

[0029]

[0030]

[0031]

[0032]

[0033]

[0034]

[0035]

[0036] 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)

[0037] (1) Synthesis of intermediate 6-bromo-2-(3,5-difluorobenzyl)-3H-imidazo[4,5-b]pyridine:

[0038]

[0039] 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 under nitrogen protection. The reaction mixture was treated with 3N hydrochloric acid aqueous solution and then made alkaline by adding ammonia. The mixture was filtered, the filter cake was washed and dried, and purified by silica gel column chromatography using a gradient elution method with 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 give 2.75 g of 6-bromo-2-(3,5-difluorobenzyl)-3H-imidazo[4,5-b]pyridine in 90% yield. 1 H NMR(600MHz,DMSO-d6)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,1H),7.19(ddd,J=2.11,4.08,6.28Hz,1H),4.24(s,2H), LRMS(ESI)m / z:324.0[M+H] + .

[0040] (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:

[0041]

[0042] 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. N,N-diisopropylethylamine (3.87 g, 30 mmol) was added, and the reaction mixture was purged with nitrogen. The temperature was raised to 120 °C and reacted for 4 h. After TLC analysis showed complete reaction of the starting materials, heating was stopped, and the reaction mixture was poured into 100 mL of ice water with stirring. A solid precipitated out. The solid was filtered, and the filter cake was dried to obtain 4.29 g of a white solid, tert-butyl((1r,4r)-4-((5-chloro-4-iodopyridin-2-yl)amino)cyclohexyl)carbamate, with a yield of 95%.

[0043] The tert-butyl((1r,4r)-4-((5-chloro-4-iodopyridin-2-yl)amino)cyclohexyl)carbamate (4.29 g, 9.5 mmol), pinacol terbinate (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) obtained in the previous step were added to 25 mL of dry N,N-dimethylformamide. Nitrogen gas was purged, and the mixture was heated to 90 °C for 3 h under nitrogen protection. The tert-butyl((1r,4r)-4-((5-chloro-4-iodopyridin-2-yl))carbamate was detected by TLC. After the reaction of amino)cyclohexyl)carbamate was complete, the reaction solution was poured into 100 mL of ice water with stirring, extracted with (3*25 mL) ethyl acetate, the organic phases were combined, back-extracted with saturated sodium chloride, dried with anhydrous sodium sulfate, filtered to remove the drying agent, concentrated the organic phase, added silica gel and mixed, and separated and purified by column chromatography with silica gel. The developing solvent was petroleum ether:ethyl acetate = 5:1, yielding 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, yield 80%.

[0044] (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):

[0045]

[0046] Take a thick-walled pressure-resistant bottle and weigh the intermediate 6-bromo-2-(3,5-difluorobenzyl)-3H-imidazo[4,5-b]pyridine (324 mg, 1 mmol), 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), potassium carbonate (414 mg, 3 mmol), and 1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (30 mg). Dissolve them in 5 mL of solvent (ethylene glycol dimethyl ether:water = 4:1). The reaction mixture was purged with nitrogen three times, heated to 90°C, and reacted overnight. After the reaction was completed, it 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 the crude product. The crude product was dissolved in a 10:1 dichloromethane:methanol system, mixed with silica gel, and purified by column chromatography using petroleum ether:ethyl acetate in a 1:1 ratio 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); 13C 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,1C),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).

[0047] The obtained A26 was dissolved in a system with a volume ratio of dichloromethane:trifluoroacetic acid = 2:1 and stirred at room temperature for 2 h. Dichloromethane and trifluoroacetic acid were removed by vacuum distillation. The residual solid was further dispersed in dichloromethane and treated with triethylamine under stirring until alkaline (pH = 8). After the organic phase was concentrated, silica gel was added and the sample was mixed. The sample was then purified by column chromatography with silica gel. The solvent ratio of dichloromethane:methanol:ammonia was changed from 10:1:0 to 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), a yellow solid. 1 H NMR (600MHz, METHANOL-d4) 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); 13C NMR(151MHz,METHANOL-d4)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).

[0048] Example 2: Synthesis of 4-(5-chloro-4-(2-(4-fluorophenylethyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)morpholine (A22)

[0049] (1) Synthesis of intermediate 6-bromo-2-(4-fluorophenylethyl)-3H-imidazolium[4,5-b]pyridine:

[0050]

[0051] A mixture of 4-fluorophenylpropionic acid (4.20 g, 25.0 mmol) and 2,3-diamino-6,6-bromopyridine (1.87 g, 10.0 mmol) was melted at 150 °C and stirred for 2 h under nitrogen protection. The reaction mixture was treated with 3N hydrochloric acid aqueous solution and then made alkaline by adding ammonia. The mixture was filtered, the filter cake was washed and dried, and purified by silica gel column chromatography using a gradient elution method with 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 give 2.72 g of 6-bromo-2-(4-fluorophenylethyl)-3H-imidazolium[4,5-b]pyridine, in 85% yield. 1 H NMR (600MHz, DMSO-d6) 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); 13C NMR(151MHz,DMSO-d6)δ174.1,161.2(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] + .

[0052] (2) Synthesis of 4-(5-chloro-4-(2-(4-fluorophenylethyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)morpholine (A22):

[0053]

[0054] 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. N,N-diisopropylethylamine (3.87 g, 30 mmol) was added, and the reaction mixture was purged with nitrogen. The temperature was then raised to 120 °C and reacted for 4 h. After TLC analysis confirmed the reaction was complete, heating was stopped, and the reaction mixture was poured into 100 mL of ice water with stirring. A solid precipitated out. The precipitate was filtered, and the filter cake was dried to obtain 2.92 g of a white solid, 4-(5-chloro-4-iodopyridin-2-yl)morpholine, with a yield of 90%.

[0055] The 4-(5-chloro-4-iodopyridin-2-yl)morpholine (2.92 g, 9.0 mmol), pinacol ester borate (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) obtained in the previous step were added to 25 mL of dry N,N-dimethylformamide. Nitrogen gas was purged, and the mixture was heated to 90 °C for 3 h under nitrogen protection. 4-(5-chloro-4-iodopyridin-2-yl)morpholine was detected by TLC. After the reaction was complete, the reaction solution was poured into 100 mL of ice water with stirring, and extracted with (3*25 mL) ethyl acetate. The organic phases were combined, back-extracted with saturated sodium chloride, and dried with anhydrous sodium sulfate. The desiccant was removed by filtration, and the organic phase was concentrated and mixed with silica gel. The mixture was then purified by column chromatography with silica gel. The developing solvent was petroleum ether:ethyl acetate = 5:1. 1.95 g of 4-(5-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxin-2-yl)pyridin-2-yl)morpholine was obtained as a white powder intermediate, with a yield of 67%.

[0056] Take a thick-walled, pressure-resistant bottle and weigh the intermediate 6-bromo-2-(4-fluorophenylethyl)-3H-imidazol[4,5-b]pyridine (320 mg, 1 mmol), 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), potassium carbonate (414 mg, 3 mmol), and 1,1'-bis(diphenylphosphine)ferrocene]palladium dichloride (30 mg). Dissolve them in 5 mL of solvent (ethylene glycol dimethyl ether:water = 4:1). The reaction mixture was purged with nitrogen three times, heated to 90°C, and reacted overnight. After the reaction was completed by TLC, the mixture 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 the crude product. The crude product was dissolved in a 10:1 dichloromethane:methanol system, mixed with silica gel, and purified by column chromatography using petroleum ether:ethyl acetate as the developing solvent. The resulting compound was 4-(5-chloro-4-(2-(4-fluorophenylethyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)morpholine (A22), a white powder. 1 H NMR(600MHz,DMSO-d6)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-d6)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.

[0057] 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)

[0058] (1) Synthesis of intermediate 6-bromo-2-(3,5-difluorobenzyl)-3H-imidazo[4,5-b]pyridine:

[0059]

[0060] The method and post-processing are described in Example 1. Spectral data: 1 H NMR (600MHz, DMSO-d6) δ8.35(d,J=1.83Hz,1H),8.17(d,J=1.83Hz,1H),7.32-7.39(m,1H),7.20(br d,J=10.09Hz,1H),7.18(d,J=7.70Hz,1H),7.07(dt,J=2.57,8.62Hz,1H),4.25(s,2H); 13 C NMR(151MHz,DMSO-d6)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),1 25.53(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] + .

[0061] (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):

[0062]

[0063] 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. N,N-diisopropylethylamine (3.87 g, 30 mmol) was added, and the reaction mixture was purged with nitrogen. The temperature was then raised to 120 °C and reacted for 4 h. After the reaction proceeded completely as determined by TLC, heating was stopped, and the reaction mixture was poured into 100 mL of ice water with stirring. A solid precipitated out. The precipitate was filtered, and the filter cake was dried to obtain 3.05 g of a white solid, 1-(5-chloro-4-iodopyridin-2-yl)piperidin-4-ol, with a yield of 90%.

[0064] The 1-(5-chloro-4-iodopyridin-2-yl)piperidin-4-ol (3.05 g, 9.0 mmol), pinacol ester of borate (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) obtained in the previous step were added to 25 mL of dry N,N-dimethylformamide. Nitrogen gas was purged, and the mixture was reacted at 90 °C for 3 h under nitrogen protection. TLC was used to detect the 1-(5-chloro-4-iodopyridin-2-yl)piperidin-4-ol. After the alcohol reaction was complete, the reaction solution was poured into 100 mL of ice water with stirring, and extracted with (3*25 mL) ethyl acetate. The organic phases were combined, back-extracted with saturated sodium chloride, and dried with anhydrous sodium sulfate. The desiccant was removed by filtration, and the organic phase was concentrated and mixed with silica gel. The mixture was then purified by column chromatography with silica gel as the developing solvent (petroleum ether:ethyl acetate = 5:1) to obtain 2.19 g of 1-(5-chloro-4-(4,4,5,5-tetramethyl-1,3,2-dioxin-2-yl)pyridin-2-yl)piperidin-4-ol, a white powder intermediate, with a yield of 72%.

[0065] Take a thick-walled, pressure-resistant bottle and weigh the intermediate 6-bromo-2-(3,5-difluorobenzyl)-3H-imidazo[4,5-b]pyridine (306 mg, 1 mmol), 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), potassium carbonate (414 mg, 3 mmol), and 1,1'-bis(diphenylphosphino)ferrocene]palladium dichloride (30 mg). Dissolve them in 5 mL of solvent (ethylene glycol dimethyl ether:water = 4... In step 1), nitrogen was purged three times, and the temperature was raised to 90°C and reacted overnight. After the reaction was completed by TLC, 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 the crude product. The crude product was dissolved in a 10:1 system of dichloromethane and methanol, and silica gel was added and stirred. The mixture was then purified by column chromatography with petroleum ether and ethyl acetate as the developing solvent 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), a white powder. 1H NMR(600MHz,METHANOL-d4)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-d4)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)。

[0066] Among them, the compounds listed in this 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- (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 (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)piperidine-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), 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[4,5-b]pyridin-6-yl)pyridin-2-yl)cyclohexane-1,4-diamine (A31), (1r,4r)-N1- The synthetic methods for (5-chloro-4-(2-(3,4-difluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)cyclohexane-1,4-diamine (A33) and (5-chloro-N-(piperidin-3-yl)-4-(2-(3-(trifluoromethyl)benzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-amine (A34) are similar to those for compound A32. Their mass spectra and NMR spectra are shown in Table 1.

[0067] Among them, the compounds listed in this 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)carbamate ester (A12-1), 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), 4-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-5-yl)pyridin-2-yl)piperazine-1-carboxylic acid tert-butyl ester (A15-1), 4-((5-chloro-4-(2-(3-fluorobenzyl)-3H-) Imidazolo[4,5-b]pyridin-5-yl)pyridin-2-yl)amino)piperidin-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)) The synthetic methods of compounds A29-1, A31-1, and A33-1 are similar to those of compound A26. Their mass spectrometry and NMR spectra are shown in Table 1.

[0068] Among them, the compounds listed in this 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 synthetic methods of compound A18 are similar to those of compound A20 (-fluorobenzyl)-3H-imidazo[4,5-b]pyridine (A20), 6-(5-chloro-2-fluoropyridin-4-yl)-2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridine (A21), and 4-((6-(5-chloro-2-(((1r,4r)-4-hydroxycyclohexyl)amino)pyridin-4-yl)-3H-imidazo[4,5-b]pyridin-2-yl)methyl)-2-fluorophenol (A25). The mass spectra and NMR spectra are assigned in Table 1.

[0069] Among them, the compounds listed in this invention: 4-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-5-yl)pyridin-2-yl)morpholine (A3) and 4-(5-chloro-4-(2-(3-fluorobenzyl)-3H-imidazo[4,5-b]pyridin-6-yl)pyridin-2-yl)morpholine (A4) are synthesized in a similar manner to compound A22, and their mass spectra and NMR spectra are shown in Table 1.

[0070] Example 4: Cytotoxicity test results of some 3H-imidazolium[4,5-b]pyridine compounds of the present invention

[0071] Antiproliferation experiments were conducted on seven tumor cell lines (B16F10, Eahy-926, OCM-1a, Ovcar-3, SW480, A498, and U87) at concentrations of 1 μM, 10 μM, and 20 μM. The cell viability results are shown in Table 2.

[0072] Table 2. Cytotoxicity test results of some 3H-imidazol[4,5-b]pyridine compounds of the present invention.

[0073]

[0074] Example 5: Inhibition of CDK9 kinase activity by some of the 3H-imidazolium[4,5-b]pyridine compounds of the present invention.

[0075] The inhibitory effects of some compounds of this invention on CDK9 kinase activity were investigated at concentrations of 1 μM / 50 nM. The results are shown in Table 3.

[0076] Table 3. Results of CDK9 kinase activity inhibition by some 3H-imidazol[4,5-b]pyridine derivatives of the present invention.

[0077]

[0078] Example 6: Anti-cell proliferation experiment of the 3H-imidazolium[4,5-b]pyridine compound A32 of the present invention.

[0079] Cell proliferation was measured using the MTT assay. Different tumor cells or normal cells were seeded in 96-well plates, with 3000 cells per well. After 12 hours, the target compound was added for treatment. The compound was diluted in culture medium to a specific concentration gradient. After 72 hours, the culture medium was discarded, and fresh culture medium and MTT were mixed at a ratio of 20:1. 100 μL of the mixture was added to each well and incubated at 37°C for 2 hours. The cells were then detected at a wavelength of 490 nm. The results are shown in Table 4.

[0080] Table 4A32 shows the IC50 values ​​for different cell types. 50 value

[0081]

[0082] Example 7: Experiment on the inhibition of glioma cell proliferation by the 3H-imidazolium[4,5-b]pyridine compound A32 of the present invention.

[0083] Two types of glioma cells were treated with A32 small molecules at specific concentration gradients, and the crystal violet staining results were as follows: Figure 1 As shown, compared with the control group, different concentrations of A32 inhibited the proliferation of both cell types to a certain extent, and this effect was concentration-dependent. In particular, treatment with high concentrations (2.5 μM and 5 μM) of the small molecule inhibited glioma cell colony formation and almost halted growth.

[0084] Example 8: Kinase activity inhibition experiment of the 3H-imidazolium[4,5-b]pyridine compound A32 of the present invention

[0085] This embodiment tested the IC of compound A32 in Example 1 on CDK9. 50 The study also investigated the inhibitory activity of A32 against other kinases in the same family at a concentration of 1 μM. The results showed that at a 10 μM ATP concentration, A32 had an IC50 inhibitory effect on CDK9. 50 The IC50 of A32 against CDK9 was 38 nM; at a ATP concentration of 45 μM, the IC50 of A32 against CDK9 was... 50 The IC50 of A32 against CDK7 was 27 nM; at a ATP concentration of 90 μM, the IC50 of A32 against CDK7 was... 50The concentration of A32 was 326 nM, indicating that A32 exhibits approximately 10-fold selectivity in inhibiting CDK7 and CDK9. Simultaneously, kinase selectivity experiments showed that A32 significantly differentiated the inhibitory activities of different members of the CDK family. Treatment with 1 μM A32 almost completely inhibited the kinase activity of CDK9, while the activities of other members remained above 50%, further demonstrating that A32 possesses CDK family selectivity. Figure 2 As shown.

[0086] Example 9: Study on the CDK9 inhibitory function of the 3H-imidazolium[4,5-b]pyridine compound A32 of the present invention.

[0087] CDK9 / cyclin T participates in the formation of the transcription elongation factor P-TEFb complex. CDK9 inhibitors can block the phosphorylation of serine 2 at the C-terminal domain of RNA polymerase II by CDK9 / cyclin T1. Therefore, this example uses the effect of the A32 series compounds on the phosphorylation level of serine 2 at the C-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 tested the phosphorylation level of serine 2 at the carboxyl terminus of RNA polymerase II in U87 cells treated with the A32 series of compounds. It was found that compounds A2, A12, A23, A27, A28, A29, A30, A31, A32, and A33 all inhibited the phosphorylation level of serine 2 at the carboxyl terminus of RNA polymerase II.

[0088] Example 10: Inhibition experiment of the 3H-imidazolium[4,5-b]pyridine compound A32 of the present invention on a U87 cell mouse subcutaneous xenograft model.

[0089] After constructing a mouse tumor model using ordinary U87 cells, wait until the initial tumor volume reaches 100 mmHg. 3 Afterwards, administration began, and the pharmacodynamic test results were as follows: Figure 4 As shown, after 14 days of intraperitoneal injection of 25 mg / kg, the tumor volume in mice increased slowly compared to the control group, and the final tumor weight after removal was also statistically different. During the experiment, the body weight of the mice in the treatment group did not change significantly, and HE staining of the organs of the mice in the treatment group showed no obvious damage, indicating that compound A32 is relatively safe for mice at this dose.

[0090] Example 11: Inhibition experiment of the 3H-imidazolium[4,5-b]pyridine compound A32 of the present invention on the U87-Luc cell mouse orthotopic xenograft model.

[0091] A U87-Luc cell orthotopic xenograft model was constructed, and the relevant experimental results are as follows: Figure 5 As shown in the figure. On the 3rd day after tumor grafting, mice were randomly divided into two groups and administered either the corresponding solvent or A32 solution (25 mg / kg). In vivo imaging was performed on days 7, 14, and 21 to detect fluorescence intensity. It can be seen that compared with the control group, the increase in intracranial fluorescence intensity in the drug-treated group was slow and significantly different. After day 21, drug administration was stopped. Mice in the control group died shortly afterward, while the survival time of the drug-treated mice was significantly prolonged. Protein extraction from the intracranial tumor tissue of mice and detection of apoptosis-related proteins revealed a decrease in the expression levels of MCL-1 and BCL-2, indicating that A32 promotes tumor cell apoptosis.

Claims

1. A CDK9 kinase inhibitor, characterized in that... Including compounds with the structure shown in formula (I): （I） Where X is Cl; L is -CH2-; R1 is trans-1,4-cyclohexanediamine; and R2 is 3,5-difluorophenyl.

2. The method for preparing a CDK9 kinase inhibitor according to claim 1, characterized in that, The synthesis route is as follows: 。 3. A drug combination, characterized in that: A compound containing the structure shown in formula (I) as described in claim 1.

4. The application of the CDK9 kinase inhibitor of claim 1 or the drug combination of claim 3, characterized in that: Application in the preparation of drugs for treating diseases related to CDK9 activity or expression levels.

5. The application as described in claim 4, characterized in that: The diseases mentioned are selected from breast cancer, stomach cancer, liver cancer, cervical cancer, and glioma.

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