3-(1H-pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivatives and their preparation and application

Abstract

The invention discloses a 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative and a preparation and application thereof, and relates to the technical field of pharmaceutical chemistry. The invention designs and synthesizes a 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative with a novel structure. Through in vitro CDK8 kinase activity and in vitro cell activity screening, it is found that some compounds thereof exhibit strong inhibitory activity on CDK8 kinase and tumor cells, and have low toxicity, and can be applied to the research and development of CDK8 inhibitors and anti-tumor drugs. In addition, the preparation method provided by the invention is simple and easy to operate, has good repeatability, and can obtain high-purity 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivatives for further research.

Description

Technical field:

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative and a preparation method and application thereof. Background technology:

[0002] Currently, the main treatments for cancer are chemotherapy, CAR-T cell immunotherapy, and the use of targeted inhibitors. Chemotherapy causes patients to have reduced immunity, are susceptible to infection, and have serious side effects. CAR-T therapy has many adverse reactions, such as cytokine release syndrome CRS, off-target effects, neurotoxicity, allergic reactions, graft-versus-host disease, tumor lysis syndrome, etc. The most serious of these is CRS, a fatal uncontrolled systemic inflammatory response. The increased risk of TRM often makes these patients unable to receive optimal chemotherapy or stem cell transplantation. Therefore, new targeted therapies offer the hope of effective anti-tumor activity with reduced toxicity from off-target effects.

[0003] Cyclin-dependent kinase 8 (CDK8) was originally called protein K35 and was discovered as a putative kinase partner of cyclin C. The CDK8 gene is located on human chromosome 13q12.13 and is transcribed into a 53kDa protein containing 464 amino acids. Its kinase activity is regulated by association with Cyc-C. The 13q12.13 chromosome region is amplified in most colon cancers. Among the many cellular functions of CDK8, the most noteworthy is its involvement in transcription. CDK8, MED12, MED13, and Cys-C form the mediator complex, a large multi-subunit protein complex that is central to regulating transcription in eukaryotes.

[0004] In 2008, Hahn and his collaborators at the Dana-Farber Cancer Institute in the United States first proposed that CDK8 is an oncogene for colorectal cancer by regulating β-catenin. Subsequent studies have shown that CDK8 is overexpressed in melanoma, breast cancer, acute myeloid leukemia, pancreatic cancer, prostate cancer and other cancers. Studies have shown that CDK8 kinase activity weakens the defense of natural killer cells against malignant cells and inhibits tumor surveillance of progenitor cells. Gene knockout has been used to verify that CDK8 plays an important role in the survival of these cancers. These evidences indicate the carcinogenic role of CDK8 in these cancers and that inhibiting CDK8 protein activity can inhibit tumorigenesis. Therefore, the discovery of effective and selective small molecule CDK8 inhibitors for the treatment of cancer can be used as a new strategy for cancer treatment. Summary of the invention:

[0005] The technical problem to be solved by the present invention is to design and synthesize a structural skeleton with 3-(1H-pyrrolo[2,3-b]pyridin-5-yl)benzoyl as the core to develop a "CDK8 inhibitor" based on drug chemical structure modification with the help of computer-aided drug design technology, and to discover highly active CDK8 inhibitors through pharmacological evaluation, thereby enriching the small molecule library targeting CDK8.

[0006] The first object of the present invention is to provide a 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative, the structure of which is shown in Formula I:

[0007]

[0008] Wherein, R1 is selected from any one of hydrogen, amino, alkylamino, alkyl, alkoxy and other substituents;

[0009] R2 is any one of substituents selected from hydrogen, halogen, phenyl, substituted phenyl, pyrazolyl, substituted pyrazolyl, furyl, substituted furyl, thienyl, substituted thienyl, pyridyl, substituted pyridyl and the like.

[0010] The 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivatives of the present invention include compounds 1-12 of the following structural formulas:

[0011]

[0012] The second object of the present invention is to provide a method for preparing the 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative, comprising the following steps:

[0013] (1) Suzuki-Miyaura reaction with 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine to give compound 1-3;

[0014] (2) Compound 1-3 undergoes chlorination reaction with N-chlorosuccinimide to obtain compound 4;

[0015] The reaction equation is as follows:

[0016]

[0017] (3) 5-bromo-7-azaindole undergoes amino protection reaction to obtain intermediate M1;

[0018] (4) Intermediate M1 and Suzuki reaction occurs to obtain intermediate M2;

[0019] (5) Intermediate M2 undergoes iodination reaction with N-iodosuccinimide to obtain intermediate M3;

[0020] (6) Intermediate M3 undergoes Suzuki reaction with R2B(OH)2 to obtain intermediate M4;

[0021] (7) Intermediate M4 undergoes amino deprotection reaction to obtain compound 5-12.

[0022] The reaction equation is as follows:

[0023]

[0024] The third object of the present invention is to provide a pharmaceutical composition containing the 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative or a pharmaceutically acceptable salt thereof.

[0025] The fourth object of the present invention is to provide a pharmaceutical preparation comprising an active ingredient and a pharmaceutically acceptable excipient and / or carrier, wherein the active ingredient contains the 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative or a pharmaceutically acceptable salt thereof.

[0026] The fifth object of the present invention is to provide the use of the 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative or a pharmaceutically acceptable salt thereof in the preparation of a CDK8 inhibitor.

[0027] The sixth object of the present invention is to provide the use of the 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative or a pharmaceutically acceptable salt thereof in the preparation of anti-tumor drugs.

[0028] The tumors are acute myeloid leukemia, gastric cancer, breast cancer, malignant melanoma, non-small cell lung cancer, colorectal cancer and the like.

[0029] The beneficial effects of the present invention are as follows: the present invention designs and synthesizes a 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative with a novel structure, and through in vitro CDK8 kinase activity and in vitro cell activity screening, it is found that some of the compounds exhibit strong inhibitory activity on CDK8 kinase and tumor cells, and have low toxicity, and can be applied to the research and development of CDK8 inhibitors and anti-tumor drugs; and the preparation method provided by the present invention is simple and easy, has good repeatability, and can obtain high-purity 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivatives for further research. Description of the drawings:

[0030] Figure 1 To study the in vitro mechanism of action of compound 2 of the present invention;

[0031] Figure 2 This is an acute myeloid leukemia cell apoptosis test of compound 2 of the present invention;

[0032] Figure 3 This is an acute toxicity test of compound 2 of the present invention. Specific implementation method:

[0033] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments and diagrams.

[0034] Example 1

[0035] Synthesis of 3-(1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (Compound 1):

[0036]

[0037] 3-Bromo-benzaldehyde (0.74g, 4mmol), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine (1.2g, 5mmol), potassium carbonate (2.2g, 16mmol), dichloro[1,1'-bis(diphenylphosphino)ferrocene]palladium (0.15g, 0.02mmol), 1,4-dioxane / water (30mL / 5mL) were added to the reaction bottle, replaced with nitrogen, and heated to 80°C for 12h. After the reaction was completed, water and ethyl acetate were added for stirring, diatomaceous earth was used for filtering, and the filter cake was washed with ethyl acetate; the filtrate was allowed to stand for separation, the aqueous phase was extracted with ethyl acetate, the organic phase was combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and dried to obtain a black oil; column chromatography was performed to obtain a yellow solid with a yield of 75.6% and a purity of 97.60%.

[0038] 1 H NMR (400MHz, DMSO-d6) δ11.81(s,1H),10.11(s,1H),8.60(d,J=2.2Hz,1H),8.33-8.24(m,2H),8.07(d,J= 7.8Hz,1H),7.88(d,J=7.6Hz,1H),7.70(t,J=7.7Hz,1H),7.58-7.53(m,1H),6.54(dd,J=3.3,1.8Hz,1H). 13CNMR(101MHz,DMSO-d6)δ193.74,148.77,141.93,140.47,137.37,133.24,130. 32,128.81,127.70(2C),127.35,126.80,120.21,100.78.HRMS(ESI):m / z[M+H] + Calculate for C 14 H 10 N2O:239.223.0866; found:223.0866.

[0039] Example 2

[0040] Synthesis of 3-(1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (Compound 2):

[0041]

[0042] The synthesis steps are the same as those of Example 1, except that 3-bromobenzaldehyde is replaced by 3-bromobenzamide.

[0043] 1 H NMR (400MHz, DMSO-d6) δ11.78(s,1H),8.60(d,J=2.1Hz,1H),8.30(d,J=2.0Hz,1H),8.23(t,J=1.6Hz,1H),8.15( s,1H),7.90-7.84(m,2H),7.57(d,J=7.7Hz,1H),7.54(t,J=2.9Hz,1H),7.47(s,1H),6.53(dd,J=3.4,1.8Hz,1H). 13 C NMR(101MHz,DMSO-d6)δ168.31,148.64,142.05,139.52,135.42,130.01,129.46 ,128.02,127.57,126.74,126.52,126.21,120.16,100.69.HRMS(ESI):m / z[M+H] + Calculate for C 14 H 11 N3O:238.0975; found:238.0975.

[0044] Example 3

[0045] Synthesis of N-methyl-3-(1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (Compound 3):

[0046]

[0047] The synthesis steps are the same as those of Example 1, except that 3-bromo-N-methylbenzamide is used instead of 3-bromobenzaldehyde.

[0048] 1 H NMR (400MHz, DMSO-d6) δ11.81(s,1H),8.62(d,J=2.2Hz,2H),8.29(d,J=2.1Hz,1H),8.21(s,1H ),7.86(t,J=7.6Hz,2H),7.62-7.47(m,2H),6.53(dd,J=3.3,1.8Hz,1H),2.85(d,J=4.5Hz,3H). 13 C NMR(101MHz,DMSO-d6)δ167.05,148.64,142.03,139.55,135.64,129.82,129.53,12 8.03,127.59,126.75,126.20,125.76,120.19,100.70,26.76.HRMS(ESI):m / z[M+H] + Calculate for C 15 H 13 N3O:252.1131; found:252.1130.

[0049] Example 4

[0050] Synthesis of 3-(3-chloro-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (Compound 4):

[0051]

[0052] Compound 2 (0.24 g, 1 mmol), N-chlorosuccinimide (0.27 g, 2 mmol) and N,N-dimethylformamide (10 mL) were added to the reaction bottle, and the temperature was raised to 50°C for 4 h. After the reaction was completed, sodium thiosulfate aqueous solution was added to quench; ethyl acetate was stirred, diatomaceous earth was used for filtering, and the filter cake was washed with ethyl acetate; the liquid was allowed to stand for separation, the aqueous phase was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and spin-dried to obtain a black oil; column chromatography was performed to obtain a yellow solid with a yield of 23.5% and a purity of 97.21%.

[0053] 1H NMR(500MHz,DMSO-d6)δ12.24(s,1H),8.73(d,J=2.1Hz,1H),8.55(d,J=2.1Hz,1H), 8.23(s,1H),8.15(s,1H),7.93(t,J=7.3Hz,2H),7.59(t,J=7.7Hz,1H),7.49(s,2H). 13 C NMR(126MHz,DMSO-d6)δ169.37,168.04,153.38,149.72,136.46,135.62,132.0 2,131.90,129.74,129.65,127.80,125.89,124.15,74.50.HRMS(ESI):m / z[M+H] + Calculate for C 14 H 10 N3OCl:272.0585; found:272.0580.

[0054] Example 5

[0055] 5.1 Synthesis of 5-bromo-1-methylphenyl-1H-pyrrolo[2,3-b]pyridine (Intermediate M1):

[0056] 5-Bromo-7-azaindole (20 g, 100 mmol), p-toluenesulfonyl chloride (21 g, 110 mmol), sodium hydroxide (6 g, 150 mmol) and tetrahydrofuran / water (150 mL / 30 mL) were added to the reaction bottle, and the temperature was raised to 40°C for 6 h. After the reaction was completed, the mixture was concentrated, ethyl acetate and water were added and stirred, and the mixture was allowed to stand for separation. The aqueous phase was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and subjected to column chromatography to obtain a white solid with a yield of 85.2%.

[0057] 5.2 Synthesis of 3-(1-methylphenyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (Intermediate M2):

[0058] The synthesis steps were the same as those in Example 1, except that the intermediate M1 and 3-carboxamidophenylboronic acid were used to replace 3-bromo-benzaldehyde and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine.

[0059] 5.3 Synthesis of 3-(3-iodo-1-methylphenyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (Intermediate M3):

[0060] The synthesis steps are the same as those of Example 4, except that intermediate M2 and N-iodosuccinimide are used to replace compound 3 and N-chlorosuccinimide.

[0061] 5.4 Synthesis of 3-(3-phenyl-1-tolyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (Intermediate M4):

[0062] The synthesis steps were the same as those in Example 1, except that the intermediate M3 and phenylboronic acid were used to replace 3-bromo-benzaldehyde and 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrrolo[2,3-b]pyridine.

[0063] 5.5 Synthesis of 3-(3-phenyl-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (Compound 5):

[0064]

[0065] Add intermediate M4 (0.46 g, 1 mmol), sodium hydroxide (0.08 g, 2 mmol) and ethanol / water (8 mL / 1 mL) to the reaction flask, and heat to 80°C for 1 h. After the reaction is completed, column chromatography is performed to obtain a white solid with a yield of 64.5% and a purity of 99.01%.

[0066] 1 H NMR (400MHz, DMSO-d6) δ12.08(s,1H),8.65(d,J=1.5Hz,1H),8.53(s,1H),8.26(s,1H),8.18(s,1H),7.95(d,J=6.4Hz, 2H),7.89(d,J=7.7Hz,1H),7.81(d,J=7.5Hz,2H),7.58(t,J=7.7Hz,1H),7.47(t,J=7.5Hz,3H),7.28(t,J=7.3Hz,1H). 13 C NMR(101MHz,DMSO-d6)δ168.29,149.24,142.61,139.49,135.42,135.37,130.39,129.48,129.42(2C) ,128.75,126.95(2C),126.72,126.38,126.25,126.09,125.24,117.78,115.26.HRMS(ESI):m / z[M+H] + Calculate for C 20 H 15 N3O:314.1288; found:314.1286.

[0067] Example 6

[0068] Synthesis of 3-(3-(1-methyl-1H-pyrazol-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (Compound 6):

[0069]

[0070] The synthesis steps were the same as those of Example 5, except that 1-methyl-1H-pyrazole-4-boronic acid was used instead of phenylboronic acid.

[0071] 1 H NMR (400MHz, DMSO-d6) δ11.83(s,1H),8.62(d,J=1.9Hz,1H),8.45(d,J=1.7Hz,1H),8.27(d,J=3.4Hz,2H),8.20(s,1H),7.96 (d,J=7.8Hz,1H),7.92(s,1H),7.89(d,J=7.7Hz,1H),7.77(d,J=2.3Hz,1H),7.59(t,J=7.7Hz,1H),7.51(s,1H),3.91(s,3H). 13 C NMR(101MHz,DMSO-d6)δ168.35,148.81,142.40,139.52,136.65,135.41,130.41,129.41,128.2 9,127.56,126.63,126.40,125.99,123.26,117.85,115.62,107.36,60.23.HRMS(ESI):m / z[M+H] + Calculate for C 18 H 15 N5O:318.1349; found:318.1346.

[0072] Example 7

[0073] Synthesis of 3-(3-(4-fluorophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (Compound 7):

[0074]

[0075] The synthesis steps are the same as those of Example 5, except that 4-fluorophenylboronic acid is used instead of phenylboronic acid.

[0076] 1H NMR (400MHz, DMSO-d6) δ12.11(s,1H),8.66(d,J=1.7Hz,1H),8.50(d,J=1.5Hz,1H),8.23(d,J=24.2 Hz,2H),7.88(ddd,J=15.1,14.0,6.7Hz,5H),7.57(dd,J=22.0,14.3Hz,2H),7.30(t,J=8.8Hz,2H). 13 C NMR(101MHz,DMSO-d6)δ168.29,162.32,159.91,149.14,142.66,139.46,135.40,131.83,130.41,129.47 ,128.77,128.70,126.73,126.40,125.98,125.23,117.68,116.28,116.07,114.27.HRMS(ESI):m / z[M+H] + Calculate for C 20 H 14 N3OF:332.1194; found:332.1198.

[0077] Example 8

[0078] Synthesis of 3-(3-(4-chlorophenyl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (Compound 8):

[0079]

[0080] The synthesis steps are the same as those of Example 5, except that 4-chlorophenylboronic acid is used instead of phenylboronic acid.

[0081] 1 H NMR (400MHz, DMSO-d6) δ12.17(s,1H),8.66(d,J=1.4Hz,1H),8.53(s,1H),8.26(s,1H),8.18(s,1H),8.01(d,J=1.8Hz, 1H),7.95(d,J=7.6Hz,1H),7.89(d,J=7.7Hz,1H),7.85(d,J=8.4Hz,2H),7.59(t,J=7.7Hz,1H),7.51(d,J=8.3Hz,3H). 13C NMR(101MHz,DMSO-d6)δ168.27,149.21,142.76,139.41,135.41,134.30,130.59,130.44,129.47,129 .32(2C),128.90,128.52(2C),126.76,126.42,126.09,125.74,117.58,113.97.HRMS(ESI):m / z[M+H] + Calculate for C 20 H 14 N3OCl:348.0898; found:348.0893.

[0082] Example 9

[0083] Synthesis of 3-(3-(furan-3-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (Compound 9):

[0084]

[0085] The synthesis steps were the same as in Example 5, except that 3-furanboronic acid was used instead of phenylboronic acid.

[0086] 1 H NMR (400MHz, DMSO-d6-d6) δ11.91(s,1H),8.64(d,J=2.0Hz,1H),8.47(d,J=1.9Hz,1H),8.38(s,1H),8.26(s,1H),8.16(s,1H ),7.98(d,J=7.8Hz,1H),7.90–7.85(m,2H),7.75(t,J=1.6Hz,1H),7.59(t,J=7.7Hz,1H),7.49(s,1H),7.02(d,J=1.0Hz,1H). 13 CNMR(101MHz,DMSO-d6-d6)δ168.35,148.97,143.80,142.53,139.33,138.25,135.42,130.44,12 9.42,128.45,126.69,126.37(2C),124.80,119.74,117.62,109.81,107.05.HRMS(ESI):m / z[M+H] + Calculate for C 18 H 13 N3O2:239.0927; found:304.1084.

[0087] Example 10

[0088] Synthesis of 3-(3-(thiophene-3-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzamide (Compound 10):

[0089]

[0090] The synthesis steps are the same as those of Example 5, except that 3-thiopheneboronic acid is used instead of phenylboronic acid.

[0091] 1 H NMR (400MHz, DMSO-d6) δ11.98(s,1H),8.62(d,J=22.3Hz,2H),8.22(d,J=36.9Hz,2H),7.97 (t,J=10.3Hz,3H),7.88(d,J=7.5Hz,1H),7.65(s,2H),7.59(t,J=7.6Hz,1H),7.50(s,1H). 13 C NMR(101MHz,DMSO-d6)δ168.31,148.98,142.59,139.41,135.70,135.42,130.43,129.44,128.66 ,127.26,126.71,126.61,126.38,126.27,125.24,118.71,117.69,111.16.HRMS(ESI):m / z[M+H] + Calculate for C 18 H 13 N3OS : 320.0852; found:320.0853.

[0092] Embodiment 11

[0093] Synthesis of 3-(3-(pyridin-4-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (Compound 11):

[0094]

[0095] The synthesis steps are the same as those of Example 5, except that 4-pyridineboronic acid and 3-formylphenylboronic acid are used to replace phenylboronic acid and 3-carbamoylphenylboronic acid.

[0096] 1H NMR (400MHz, DMSO) δ12.43(s,1H),10.14(s,1H),8.69(s,2H),8.58(d,J=5.1Hz,2H),8.36(s,1H), 8.32(d,J=2.1Hz,1H),8.19(d,J=7.5Hz,1H),7.90(dd,J=14.1,6.5Hz,3H),7.74(t,J=7.6Hz,1H). 13 C NMR (101MHz, DMSO) δ193.77,150.38,149.55,142.94,142.77,140.16,137.35,133.78,130. 30,129.49,128.68,127.99,127.82,126.59,121.00,117.52,112.39.HRMS(ESI):m / z[M+H] + Calculate for C 19 H 13 N3O : 300.1131; found:300.1128.

[0097] Example 12

[0098] Synthesis of 3-(3-(furan-3-yl)-1H-pyrrolo[2,3-b]pyridin-5-yl)benzaldehyde (Compound 12):

[0099]

[0100] The synthesis steps are the same as those of Example 5, except that 3-furanboronic acid and 3-formylphenylboronic acid are used to replace phenylboronic acid and 3-carbamoylphenylboronic acid.

[0101] 1 H NMR (400MHz, DMSO) δ11.96(s,1H),10.15(s,1H),8.65(d,J=1.9Hz,1H),8.52(d,J=1.8Hz,1H),8.42(s,1 H),8.37(s,1H),8.19(d,J=7.8Hz,1H),7.90(t,J=4.6Hz,2H),7.73(dd,J=13.4,5.7Hz,2H),7.03(s,1H). 13CNMR(101MHz,DMSO)δ193.82,149.15,143.79,142.44,140.30,138.36,137.34,133.66,130.24, 129.54,127.76,127.50,126.41,124.90,119.67,117.61,109.80,107.14.HRMS(ESI):m / z[M+H] + Calculate for C 18 H 12 N2O 2: 289.0972; found:289.0971.

[0102] Example 13

[0103] Evaluation of in vitro CDK8 kinase inhibitory activity and in vitro anti-tumor activity:

[0104] The inhibitory activity of the above compounds 1-12 on CDK8 kinase was tested by ADP-Glo ​​Kinase Assay (Promega) using a 384-well plate. The active CDK8 kinase was diluted in a mixture (5ng CDK8 kinase, 0.5μg substrate, 50μM DTT, 1μL buffer, dd H2O was added to each well to 3μL), and then 1uL of a 1uM compound solution was added to each well (the final concentration of the compound was 200nM), followed by 1uL of ATP (adenosine triphosphate) to a final concentration of 50μM. After incubation at room temperature for 1h, ADP-Glo ​​solution and kinase detection reagent were added, and the data were collected by an ELISA instrument. SEL-120 34A was selected as the positive control, and the results are shown in Table 1.

[0105] Table 1 CDK8 kinase inhibitory activity of compounds 1-12 at a concentration of 200 nM

[0106]

[0107]

[0108] As can be seen from Table 1, compounds 1-12 of the present invention all showed certain CDK8 kinase inhibitory activity at a concentration of 200 nM.

[0109] The compounds with higher inhibition rates were preferably subjected to MTT test to test the inhibitory activity of these compounds on human acute myeloid leukemia cells, human gastric cancer cells, human breast cancer cells, human malignant melanoma cells, human non-small cell lung cancer cells, human colorectal cancer cells, and human gastric mucosal cells, and sorafenib was selected as the positive control.

[0110] MTT test: MOLM-13, MV4-11, MGC-803, MDA-MB-231, A375, A549, HCT-116, SW-480, HT-29, GES-1 cells were seeded at 6000 cells / well in a 96-well plate and cultured in an incubator at 37°C and 5% CO2 for 24 h; the culture medium was discarded, and then 100 μL of compound solution of each concentration (concentrations were 100, 20, 4, 0.8, and 0.016 μM) was added and cultured for 48 h; MTT (5 mg / mL, 20 μL) was added and incubated at 37°C for 4 h; the culture medium was removed, and 150 μL DMSO was added to dissolve; the absorbance at 492 nm was measured by a microplate reader (PerkinElmer Envision), and the GI was calculated. 50 The results are shown in Table 2.

[0111] Table 2 Inhibitory activity of compounds on CDK8 kinase and tumor cells

[0112]

[0113]

[0114] As can be seen from Table 2, compounds 2, 9 and 10 have good inhibitory activity against CDK8 kinase and tumor cells.

[0115] Embodiment 14

[0116] PK assay of compounds 2 and 9:

[0117] The pharmacokinetic properties of compounds 2 and 9 in SD rats were evaluated by intravenous (2 mg / kg) and oral (10 mg / kg) routes. Plasma samples were collected at 3 min, 5 min, 10 min, 15 min, 30 min, 45 min, 1 h, 90 min, 2 h, 4 h, 6 h, 8 h and 24 h after administration. Samples were analyzed using HPLC, and time and plasma concentration analysis was performed using DAS2.0.

[0118] The results are shown in Tables 3 and 4.

[0119] Table 3 PK test results of compound 2

[0120]

[0121] Table 4 PK test results of compound 9

[0122]

[0123] It can be seen from Table 3 and Table 4 that compounds 2 and 9 have good bioavailability.

[0124] Embodiment 15

[0125] In vitro study of the mechanism of action of compound 2:

[0126] HCT-116 cells were cultured at 1.5 × 10 6 The cells were seeded at a density of 10 cells / mL into a 60 mm culture dish and treated with DMSO or 10 ng / mL IFN-γ after 12 h of culture. One hour later, the cells were treated with different concentrations of compound 2 for 12 h. The cells were lysed using RIPA containing PMSF and phosphatase inhibitors, and the cell samples were processed by western blotting to analyze the levels of total STAT1, phosphorylated STAT1 S727, Y701 and GAPDH.

[0127] HL-60 cells were cultured at 2 × 10 6 The cells were seeded at a density of 10 cells / mL into a 60 mm culture dish and treated with different concentrations of compound 2 for 12 h after incubation for 12 h. The cells were lysed using RIPA containing PMSF and phosphatase inhibitors, and the cell samples were processed by western blotting to analyze the levels of total STAT5, phosphorylated STAT5 S726, and GAPDH.

[0128] Results Figure 1 .

[0129] from Figure 1 It can be seen that compound 2 can inhibit the phosphorylation of STAT1 S727 and STAT5 S726.

[0130] Example 16

[0131] Acute myeloid leukemia cell apoptosis assay of compound 2:

[0132] HL-60 cells were cultured at 2 × 10 6 The cells were inoculated into a 60 mm culture dish at a density of 10 cells / mL. After incubation for 24 h, the cells were treated with different concentrations of compound 2 for 48 h. The cells were collected and washed three times with PBS, and then resuspended with 400 μL 1× Annexin V binding solution. Subsequently, 5 μL Annexin V-FITC solution was added to the sample, and the sample was incubated in a dark area at 2 to 8 ° C for 15 min. Then 10 μL PI was added, and the sample was analyzed by flow cytometry after incubation at 2 to 8 ° C for 5 min. The results are shown in Figure 2 .

[0133] from Figure 2 It can be seen that compound 2 can induce apoptosis of acute myeloid leukemia cells.

[0134] Embodiment 17

[0135] Acute toxicity test of compound 2:

[0136] The mice were randomly divided into a normal group, a 1000mg / kg administration group, and a 2000mg / kg administration group. The mice in the experimental group were gavaged once and observed for 14 consecutive days. The normal group was given an equal amount of saline. Throughout the 14-day process, changes in the weight of the mice were recorded, and physiological phenomena such as loose hair, lethargy, hyperactivity, anorexia, and convulsions were observed. After the experiment, the mice were anesthetized and killed, and pathological sections of the main organs were examined. The histopathological sections also showed that the 2000mg / kg administration group did not show any toxic results within 14 days. Figure 3 .

[0137] from Figure 3 It can be seen that compound 2 has lower in vivo toxicity.

[0138] In summary, compounds 2 and 9 of the present invention exhibited good CDK8 kinase inhibitory activity and anti-acute myeloid leukemia tumor cell proliferation activity, good bioavailability, and had potential drug development value.

[0139] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, which fall within the scope of the present invention to be protected. The scope of protection of the present invention is defined by the attached claims and their equivalents.

Claims

1. Use of a 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative or a pharmaceutically acceptable salt thereof in the preparation of a CDK8 inhibitor, wherein the structural formula of the 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative is as follows:

2. A pharmaceutical composition comprising a 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative or a pharmaceutically acceptable salt thereof, wherein the structural formula of the 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative is as follows:

3. A pharmaceutical preparation comprising an active ingredient and a pharmaceutically acceptable excipient and / or carrier, wherein the active ingredient contains a 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative or a pharmaceutically acceptable salt thereof, and the structural formula of the 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative is as follows:

4. Use of a 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative or a pharmaceutically acceptable salt thereof in the preparation of an anti-tumor drug, wherein the structural formula of the 3-(1H pyrrolo[2,3-b]pyridin-5-yl)benzoyl derivative is as follows: The tumor is acute myeloid leukemia.

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