CN115991705A - 3- (1H pyrrolo [2,3-b ] pyridin-5-yl) benzoyl derivatives, preparation and application thereof - Google Patents
3- (1H pyrrolo [2,3-b ] pyridin-5-yl) benzoyl derivatives, preparation and application thereof Download PDFInfo
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Abstract
The invention discloses a 3- (1H pyrrolo [2,3-b ] pyridine-5-yl) benzoyl derivative, and preparation and application thereof, and relates to the technical field of pharmaceutical chemistry; the preparation method provided by the invention is simple and easy to implement, has good repeatability, and can obtain the 3- (1H pyrrolo [2,3-b ] pyridine-5-yl) benzoyl derivative with high purity for further research.
Description
Technical field:
the invention relates to the technical field of pharmaceutical chemistry, in particular to a 3- (1H pyrrolo [2,3-b ] pyridine-5-yl) benzoyl derivative, and preparation and application thereof.
The background technology is as follows:
current treatments for cancer are mainly chemotherapy, CAR-T cell immunotherapy and the use of targeted inhibitors. Chemotherapy causes the patient to have reduced immunity, is easy to infect and has great side effect. CAR-T treatment has many adverse effects such as cytokine release syndrome CRS, off-target effects, neurotoxicity, anaphylaxis, graft versus host disease, oncolytic syndrome, etc. Among these, CRS is the most serious, which is a fatal uncontrolled systemic inflammatory response. The increased risk of TRM often makes these patients unacceptable for optimal chemotherapy or stem cell transplantation. Thus, new targeted therapies offer the hope of potent anti-tumor activity, reducing the toxicity of off-target effects.
The cyclin-dependent protease 8 (cdk 8) was originally referred to as protein K35 and was found to be a putative kinase partner for cyclin C. The CDK8 gene is located on human chromosome 13q12.13, transcribed into a 53kDa protein containing 464 amino acids, whose kinase activity is regulated by association with Cyc-C, and the 13q12.13 chromosomal region is amplified in most colon cancers. Among the many cellular functions of CDK8, the most notable is the involvement in transcription. CDK8 and MED12, MED13, cys-C constitute an intermediate complex, a large multi-subunit protein complex that is central in regulating transcription in eukaryotes.
Hahn and his co-workers in the institute of dana-farbo cancer in 2008 proposed CDK8 for the first time as an oncogene for colorectal cancer by regulating β -catenin, and later studies showed that CDK8 was overexpressed in melanoma, breast cancer, acute myeloid leukemia, pancreatic cancer, prostate cancer, and other cancers. Studies have shown that CDK8 kinase activity weakens natural killer cell defenses against malignant cells and inhibits tumor surveillance of pre-cells. CDK8 was validated by gene knockout as having an important role in these cancer survival. These evidence suggests that CDK8 carcinogenesis in these cancers and inhibition of CDK8 protein activity can inhibit tumorigenesis. Thus, potent and selective small molecule CDK8 inhibitors were found to be useful in the treatment of cancer as a new strategy for the treatment of cancer.
The invention comprises the following steps:
the technical problem to be solved by the invention is to design and synthesize a structural framework taking 3- (1H pyrrolo [2,3-b ] pyridin-5-yl) benzoyl as a core by means of a computer-aided drug design technology based on pharmaceutical chemical structure modification to develop a CDK8 inhibitor, and to find a CDK8 inhibitor with high activity through pharmacological evaluation, so as to enrich a small molecule library of targeted CDK 8.
The first object of the 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:
wherein R is 1 Any one of substituents selected from hydrogen, amino, alkylamino, alkyl, alkoxy and the like;
R 2 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.
The 3- (1H pyrrolo [2,3-b ] pyridin-5-yl) benzoyl derivative comprises compounds 1-12 with the following structural formula:
a second object of the present invention is to provide a process for preparing the 3- (1H pyrrolo [2,3-b ] pyridin-5-yl) benzoyl derivative, comprising the steps of:
(1)
with 5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -1H-pyrrolo [2,3-b]Pyridine undergoes Suzuki-Miyaura reaction to obtain compounds 1-3;
(2) The compound 1-3 and N-chlorosuccinimide undergo chlorination reaction to obtain a compound 4;
the reaction equation is as follows:
(3) The 5-bromo-7-azaindole undergoes an amino-group protection reaction to obtain an intermediate M1;
(4) Intermediate M1 and
generating Suzuki reaction to obtain an intermediate M2;
(5) The intermediate M2 and N-iodinated succinimide undergo iodination reaction to obtain an intermediate M3;
(6) Intermediates M3 and R 2 B(OH) 2 Generating a Suzuki reaction to obtain an intermediate M4;
(7) The intermediate M4 undergoes amino deprotection reaction to obtain compounds 5-12.
The reaction equation is as follows:
a third object of the present invention is to provide a pharmaceutical composition comprising said 3- (1H pyrrolo [2,3-b ] pyridin-5-yl) benzoyl derivative or a pharmaceutically acceptable salt thereof.
A fourth object of the present invention is to provide a pharmaceutical preparation comprising an active ingredient containing the 3- (1H pyrrolo [2,3-b ] pyridin-5-yl) benzoyl derivative or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients and/or carriers.
A fifth object of the present invention is to provide the use of said 3- (1H pyrrolo [2,3-b ] pyridin-5-yl) benzoyl derivative or a pharmaceutically acceptable salt thereof in the preparation of a CDK8 inhibitor.
The sixth object of the invention is to provide the application of the 3- (1H pyrrolo [2,3-b ] pyridin-5-yl) benzoyl derivative or the pharmaceutically acceptable salt thereof in preparing antitumor drugs.
The tumor is acute myelogenous leukemia, gastric cancer, breast cancer, malignant melanoma, non-small cell lung cancer, colorectal cancer, etc.
The beneficial effects of the invention are as follows: the 3- (1H pyrrolo [2,3-b ] pyridine-5-yl) benzoyl derivative with a novel structure is designed and synthesized, and is subjected to in vitro CDK8 kinase activity and in vitro cell activity screening, and partial compounds are found to have stronger inhibition activity on CDK8 kinase and tumor cells, and have lower toxicity, so that the 3- (1H pyrrolo [2,3-b ] pyridine-5-yl) benzoyl derivative can be applied to research and development of CDK8 inhibitors and antitumor drugs; the preparation method provided by the invention is simple and easy to implement, has good repeatability, and can obtain the 3- (1H pyrrolo [2,3-b ] pyridine-5-yl) benzoyl derivative with high purity for further research.
Description of the drawings:
FIG. 1 is an in vitro mechanism of action study of Compound 2 of the present invention;
FIG. 2 is an acute myeloid leukemia cell apoptosis assay of Compound 2 of the present invention;
FIG. 3 is an acute toxicity test of Compound 2 of the present invention.
The specific embodiment is as follows:
the invention is further described below with reference to specific embodiments and illustrations in order to make the technical means, the creation features, the achievement of the purpose and the effect of the implementation of the invention easy to understand.
Example 1
Synthesis of 3- (1H-pyrrolo [2,3-b ] pyridin-5-yl) benzaldehyde (Compound 1):
to the reaction flask was added 3-bromo-benzaldehyde (0.74 g,4 mmol), 5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -1H-pyrrolo [2,3-b ] pyridine (1.2 g,5 mmol), potassium carbonate (2.2 g,16 mmol), dichloro [1,1' -bis (diphenylphosphine) ferrocene ] palladium (0.15 g,0.02 mmol), 1, 4-dioxane/water (30 mL/5 mL), nitrogen displacement, and the reaction was continued while maintaining the temperature at 80℃for 12 hours. After the reaction is finished, adding water and ethyl acetate, stirring, filtering with diatomite, and washing a filter cake with ethyl acetate; standing the filtrate, separating, extracting the water phase with ethyl acetate, mixing the organic phases, washing with saturated saline solution, drying with anhydrous sodium sulfate, filtering, and spin-drying to obtain black oily substance; column chromatography gave a yellow solid in 75.6% yield and 97.60% purity.
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). 13 CNMR(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] + calcd for C 14 H 10 N 2 O:239.223.0866；found:223.0866.
Example 2
Synthesis of 3- (1H-pyrrolo [2,3-b ] pyridin-5-yl) benzamide (Compound 2):
the synthesis procedure is as in example 1, except that 3-bromobenzamide is used in place of 3-bromobenzaldehyde.
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] + calcd for C 14 H 11 N 3 O:238.0975；found:238.0975.
Example 3
Synthesis of N-methyl-3- (1H-pyrrolo [2,3-b ] pyridin-5-yl) benzamide (Compound 3):
the procedure is as in example 1, except that 3-bromo-N-methylbenzamide is used in place of 3-bromobenzaldehyde.
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)δ167.05,148.64,142.03,139.55,135.64,129.82,129.53,128.03,127.59,126.75,126.20,125.76,120.19,100.70,26.76.HRMS(ESI):m/z[M+H] + calcd for C 15 H 13 N 3 O:252.1131；found:252.1130.
Example 4
Synthesis of 3- (3-chloro-1H-pyrrolo [2,3-b ] pyridin-5-yl) benzamide (Compound 4):
to the reaction flask were added compound 2 (0.24 g,1 mmol), N-chlorosuccinimide (0.27 g,2 mmol) and N, N-dimethylformamide (10 mL), and the mixture was heated to 50℃to react for 4 hours. After the reaction is finished, adding sodium thiosulfate aqueous solution for quenching; stirring ethyl acetate, filtering with diatomite, and washing filter cake with ethyl acetate; standing for separating, extracting the water phase with ethyl acetate, mixing the organic phases, washing with saturated saline solution, drying with anhydrous sodium sulfate, filtering, and spin-drying to obtain black oily substance; column chromatography gave a yellow solid in 23.5% yield and 97.21% purity.
1 H 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)δ169.37,168.04,153.38,149.72,136.46,135.62,132.02,131.90,129.74,129.65,127.80,125.89,124.15,74.50.HRMS(ESI):m/z[M+H] + calcd for C 14 H 10 N 3 OCl:272.0585；found:272.0580.
Example 5
5.1 Synthesis of 5-bromo-1-tolyl-1H-pyrrolo [2,3-b ] pyridine (intermediate M1):
to the reaction flask were added 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), and the mixture was reacted at 40℃for 6 hours. After the reaction, concentrating, adding ethyl acetate and water, stirring, standing for separating, extracting the water phase with ethyl acetate, mixing the organic phases, washing with saturated common salt water, drying with anhydrous sodium sulfate, and performing column chromatography to obtain white solid with a yield of 85.2%.
5.2 Synthesis of 3- (1-tolyl-1H-pyrrolo [2,3-b ] pyridin-5-yl) benzamide (intermediate M2):
the procedure is as in example 1, except that 3-bromo-benzaldehyde and 5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -1H-pyrrolo [2,3-b ] pyridine are replaced with intermediates M1 and 3-carboxamide phenylboronic acid.
5.3 Synthesis of 3- (3-iodo-1-tolyl-1H-pyrrolo [2,3-b ] pyridin-5-yl) benzamide (intermediate M3):
the procedure is as in example 4, except that compound 3 and N-chlorosuccinimide are replaced with intermediates M2 and N-iodosuccinimide.
5.4 Synthesis of 3- (3-phenyl-1-tolyl-1H-pyrrolo [2,3-b ] pyridin-5-yl) benzamide (intermediate M4):
the procedure is as in example 1, except that intermediate M3 and phenylboronic acid are used in place of 3-bromo-benzaldehyde and 5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -1H-pyrrolo [2,3-b ] pyridine.
5.5 Synthesis of 3- (3-phenyl-1H-pyrrolo [2,3-b ] pyridin-5-yl) benzamide (Compound 5):
to the reaction flask were added intermediate M4 (0.46 g,1 mmol), sodium hydroxide (0.08 g,2 mmol) and ethanol// water (8 mL/1 mL), and the mixture was allowed to react at 80℃for 1h. After the reaction, column chromatography gave a white solid in a yield of 64.5% and a purity of 99.01%.
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] + calcd for C 20 H 15 N 3 O:314.1288；found:314.1286.
Example 6
Synthesis of 3- (3- (1-methyl-1H-pyrazol-4-yl) -1H-pyrrolo [2,3-b ] pyridin-5-yl) benzamide (Compound 6):
the procedure is as in example 5, except that 1-methyl-1H-pyrazole-4-boronic acid is used in place of phenylboronic acid.
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)δ168.35,148.81,142.40,139.52,136.65,135.41,130.41,129.41,128.29,127.56,126.63,126.40,125.99,123.26,117.85,115.62,107.36,60.23.HRMS(ESI):m/z[M+H] + calcd for C 18 H 15 N 5 O:318.1349；found:318.1346.
Example 7
Synthesis of 3- (3- (4-fluorophenyl) -1H-pyrrolo [2,3-b ] pyridin-5-yl) benzamide (Compound 7):
the synthesis procedure is as in example 5, except that 4-fluorobenzeneboronic acid is used instead of phenylboronic acid.
1 H 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＝24.2Hz,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] + calcd for C 20 H 14 N 3 OF:332.1194；found:332.1198.
Example 8
Synthesis of 3- (3- (4-chlorophenyl) -1H-pyrrolo [2,3-b ] pyridin-5-yl) benzamide (Compound 8):
the procedure is as in example 5, except that phenylboronic acid is replaced with 4-chlorophenylboronic acid.
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). 13 C 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] + calcd for C 20 H 14 N 3 OCl:348.0898；found:348.0893.
Example 9
Synthesis of 3- (3- (furan-3-yl) -1H-pyrrolo [2,3-b ] pyridin-5-yl) benzamide (Compound 9):
the synthesis procedure is as in example 5, except that phenylboronic acid is replaced with 3-furanboronic acid.
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 C NMR(101MHz,DMSO-d6-d6)δ168.35,148.97,143.80,142.53,139.33,138.25,135.42,130.44,129.42,128.45,126.69,126.37(2C),124.80,119.74,117.62,109.81,107.05.HRMS(ESI):m/z[M+H] + calcd for C 18 H 13 N 3 O 2 :239.0927；found:304.1084.
Example 10
Synthesis of 3- (3- (thiophen-3-yl) -1H-pyrrolo [2,3-b ] pyridin-5-yl) benzamide (Compound 10):
the synthesis procedure is as in example 5, except that 3-thiopheneboronic acid is used instead of phenylboronic acid.
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] + calcd for C 18 H 13 N 3 OS : 320.0852；found:320.0853.
Example 11
Synthesis of 3- (3- (pyridin-4-yl) -1H-pyrrolo [2,3-b ] pyridin-5-yl) benzaldehyde (Compound 11):
the procedure is as in example 5, except that 4-pyridineboronic acid and 3-formylphenylboronic acid are used in place of phenylboronic acid and 3-carbamoylphenylboronic acid.
1 H 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] + calcd for C 19 H 13 N 3 O : 300.1131；found:300.1128.
Example 12
Synthesis of 3- (3- (furan-3-yl) -1H-pyrrolo [2,3-b ] pyridin-5-yl) benzaldehyde (Compound 12):
the procedure is as in example 5, except that 3-furanboronic acid and 3-formylphenylboronic acid are used in place of phenylboronic acid and 3-carbamoylphenylboronic acid.
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,1H),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). 13 CNMR(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] + calcd for C 18 H 12 N 2 O 2: 289.0972；found:289.0971.
Example 13
In vitro CDK8 kinase inhibitory activity and in vitro antitumor activity evaluation:
the inhibitory activity of the above compounds 1-12 on CDK8 Kinase was tested by ADP-Glo Kinase Assay (Promega) using 384 well plates. Active CDK8 kinase was diluted in the mixture (5 ng CDK8 kinase, 0.5. Mu.g substrate, 50. Mu.M DTT, 1. Mu.L buffer, dd H was added to each well) 2 O to 3. Mu.L) and then 1. Mu.L of a 1. Mu.M compound solution (200 nM final compound concentration) was added to each well, followed by 1. Mu.L of ATP (adenosine triphosphate) to give a final concentration of 50. Mu.M. After incubation for 1h at room temperature, ADP-Glo solution and kinase detection reagent were added and the data collected by a microplate reader. SEL-120 34a was selected as a positive control and the results are shown in table 1.
CDK8 kinase inhibitory Activity of Compounds 1-12 at 200nM concentration
As can be seen from Table 1, compounds 1-12 of the present invention all showed a certain CDK8 kinase inhibitory activity at a concentration of 200 nM.
Preferably, the compounds with higher inhibition rate are subjected to MTT test, and the inhibition activity of the 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 mucosa cells is tested, and sorafenib is selected as a positive control.
MTT test: MOLM-13, MV4-11, MGC-803, MDA-MB-231, A375, A549, HCT-116, SW-480, HT-29, GES-1 cells were seeded in 96-well plates at 6000 cells/well, and at 37deg.C, 5% CO 2 Culturing for 24 hours in an incubator; the medium is discarded and thenAdding 100. Mu.L of compound solution (with the concentration of 100, 20, 4, 0.8 and 0.016. Mu.M) at each concentration, and culturing for 48h; MTT (5 mg/mL, 20. Mu.L) was added and incubated at 37℃for 4h; removing the culture medium, and adding 150 mu L of DMSO for dissolution; measuring absorbance at 492nm by a microplate reader (PerkinElmer Envision), and calculating GI 50 Values, results are shown in Table 2.
Inhibitory Activity of Compounds of Table 2 on CDK8 kinase and tumor cells
As can be seen from Table 2, compounds 2, 9 and 10 all have better inhibitory activity against CDK8 kinase and tumor cells.
Example 14
PK assay for compounds 2 and 9:
the pharmacokinetic profile of compounds 2 and 9 in SD rats was assessed by intravenous (2 mg/kg) and oral (10 mg/kg) routes. Plasma samples were collected 3min,5min,10min,15min,30min,45min,1h,90min,2h,4h,6h,8h and 24h post-dose. Samples were analyzed using HPLC and time and plasma concentration analysis was performed using DAS 2.0.
The results are shown in tables 3 and 4.
TABLE 3 PK assay results for Compound 2
TABLE 4 PK assay results for Compound 9
As can be seen from tables 3 and 4, compounds 2 and 9 have better bioavailability.
Example 15
In vitro mechanism of action study of compound 2:
HCT-116 cells were plated at 1.5X10 cells per dish 6 The density of individual cells was inoculated into 60mm dishes, after 12h of incubation, the cells were treated with DMSO or 10ng/mL IFN-gamma, after 1h, the cells were treated with different concentrations of Compound 2 for 12h; cells were lysed using RIPA containing PMSF and phosphatase inhibitors, and then cell samples were treated by western blotting to analyze the levels of total STAT1, phosphorylated STAT 1S 727, Y701 and GAPDH.
HL-60 cells were plated at 2×10 cells per dish 6 The density of individual cells was inoculated into a 60mm dish, and after 12h incubation, the cells were treated with different concentrations of compound 2 for 12h; cells were lysed using RIPA containing PMSF and a phosphatase inhibitor, and then cell samples were treated by western blotting to analyze the levels of total STAT5, phosphorylated STAT 5S 726, and GAPDH.
The results are shown in FIG. 1.
As can be seen from fig. 1, compound 2 was able to inhibit STAT 1S 727 and STAT 5S 726 phosphorylation.
Example 16
Acute myeloid leukemia apoptosis assay of compound 2:
HL-60 cells were plated at 2×10 cells per dish 6 The density of individual cells was inoculated into 60mm dishes and after incubation for 24h, the cells were treated with different concentrations of compound 2 for 48h; cells were collected and washed three times with PBS, then resuspended with 400 μl of 1 x Annexin V binding solution; subsequently, 5. Mu.L of Annexin V-FITC solution was added to the sample, and the sample was incubated in a dark area at 2-8deg.C for 15min; then 10. Mu.L PI was added and the samples were analyzed by flow cytometry after incubation at 2-8℃for 5 min. The results are shown in FIG. 2.
As can be seen from fig. 2, compound 2 was able to induce apoptosis in acute myeloid leukemia cells.
Example 17
Acute toxicity test of compound 2:
mice were randomly divided into normal, 1000mg/kg and 2000mg/kg groups. The mice of the experimental group were gavaged once and observed continuously for 14 days. Normal groups were given equal amounts of physiological saline. Throughout the course of 14 days, changes in the body weight of the mice were recorded, and physiological phenomena such as hair relaxation, somnolence, hyperactivity, anorexia, and tics were observed. After the end of the experiment, the mice were anesthetized and sacrificed and the pathological sections of the major organs were examined. Histopathological sections also showed that the 2000mg/kg dosing group did not show any toxicity results over 14 days as shown in FIG. 3.
As can be seen from fig. 3, compound 2 has lower in vivo toxicity.
In conclusion, the compounds 2 and 9 of the invention have better CDK8 kinase inhibition activity and acute myeloid leukemia tumor cell proliferation resistance activity, good bioavailability and potential drug development value.
The foregoing has shown and described the basic principles and main features of the present invention and the advantages of the present invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (7)
1. A 3- (1H pyrrolo [2,3-b ] pyridin-5-yl) benzoyl derivative, characterized by the structure shown in formula I:
wherein R is 1 Any one group selected from hydrogen, amino, alkylamino, alkyl and alkoxy; r is R 2 Any one of the groups selected from hydrogen, halogen, phenyl, substituted phenyl, pyrazolyl, substituted pyrazolyl, furyl, substituted furyl, thienyl, substituted thienyl, pyridyl and substituted pyridyl.
2. A process for the preparation of 3- (1H pyrrolo [2,3-b ] pyridin-5-yl) benzoyl derivatives according to claim 1, comprising the steps of:
(1)
with 5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) -1H-pyrrolo [2,3-b]Pyridine undergoes Suzuki-Miyaura reaction to obtain compounds 1-3;
(2) The compound 1-3 and N-chlorosuccinimide undergo chlorination reaction to obtain a compound 4;
the reaction equation is as follows:
(3) The 5-bromo-7-azaindole undergoes an amino-group protection reaction to obtain an intermediate M1;
(4) Intermediate M1 and
generating Suzuki reaction to obtain an intermediate M2;
(5) The intermediate M2 and N-iodinated succinimide undergo iodination reaction to obtain an intermediate M3;
(6) Intermediates M3 and R 2 B(OH) 2 Generating a Suzuki reaction to obtain an intermediate M4;
(7) The intermediate M4 undergoes amino deprotection reaction to obtain compounds 5-12;
the reaction equation is as follows:
3. a pharmaceutical composition comprising the 3- (1H pyrrolo [2,3-b ] pyridin-5-yl) benzoyl derivative of claim 1, or a pharmaceutically acceptable salt thereof.
4. A pharmaceutical formulation comprising an active ingredient containing a 3- (1H pyrrolo [2,3-b ] pyridin-5-yl) benzoyl derivative of claim 1, or a pharmaceutically acceptable salt thereof, and pharmaceutically acceptable excipients and/or carriers.
5. Use of a 3- (1H pyrrolo [2,3-b ] pyridin-5-yl) benzoyl derivative or a pharmaceutically acceptable salt thereof according to claim 1, in the preparation of a CDK8 inhibitor.
6. Use of a 3- (1H pyrrolo [2,3-b ] pyridin-5-yl) benzoyl derivative or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an antitumor medicament.
7. The use according to claim 8, characterized in that: the tumor is acute myelogenous leukemia, gastric cancer, breast cancer, malignant melanoma, non-small cell lung cancer and colorectal cancer.
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