Design,Synthesis And Antitumor Activity Evaluation Of 7-Azaindole Derivatives As Novel CDK8 Inhibitors

Cyclin-dependent kinase 8（CDK8）is a member of the family of cyclin-dependent kinases（CDKs）,which belong to the serine/threonine kinase family.CDK8 has many biological functions,among which the most prominent one is to form mediating complex with CYCC,Med 12 and Med 13 to regulate RNA polymerase II（RNAP II）-mediated transcription.CDK8 is involved in the regulation of many signaling pathways and is closely related to the occurrence of many cancers,such as melanoma,prostate cancer,colorectal cancer,breast cancer,and acute myeloid leukemia.These results indicate that CDK8 is a promising drug target,especially for cancer therapy.Therefore,the development of novel CDK8 inhibitors is of great significance and broad prospects.This study is based on de novo drug design to identify novel and efficient CDK8inhibitors.First,based on the pharmacophore,the highly active fragment H1(5-phenyl-1H-pyrrolo[2,3-b]pyridine,CDK8 IC₅₀=35.2 n M)was found.A novel CDK8 type II inhibitor was discovered by fragment growth method and structure optimization.In the follow-up study,a novel CDK8 typeⅠinhibitor was found based on the structure optimization of compound H1.Part I:Design,synthesis and antitumor activity evaluation of 7-azaindole derivatives as novel CDK8 II inhibitorsIn this study,a total of 91 compounds were designed and synthesized.Compound 22（3-（3-（1H-Pyrrolo[2,3-b]pyridin-5-yl）phenyl）-N-（4-methyl-3-（trifluoromethyl）phenyl）acrylamide）not only showed best inhibitory activity against CDK8(CDK8 IC₅₀=48.6±1.5 n M),but exhibited good selectivity against 63selected kinases,including CDK8 inhibitors off-target kinases GSK and JAK,sorafenib and TSN-084 multi-kinase targets EFGR,FLT-3,TRK,etc.The results of CETSA showed that compound 22 could bind to CDK8 very well.Western blot analysis showed that compound 22 could phosphorylate STAT1^Ser727 with an EC₅₀value of 12.0μM,it is significantly lower than the positive drug CCT251545(EC₅₀=24 n M).These results indicated that compound 22 was a highly active and selective CDK8 II inhibitor.MTT assays showed compound 22 had good antiproliferative activity against human colorectal cancer cell lines HCT-116,HT-29,SW-480 and mouse colon cancer cell line CT-26(GI₅₀<5μM),furthermore,it has low toxicity to normal cells GES1(GI₅₀>60μM)with a>15-fold selectivity,which is noticeably superior to the selectivity of positive drugs sorafenib（SI<5）,CCT251545（SI<10）,and SEL120-34A（SI<5）,indicating compound 22 not only has a larger therapeutic window,but also has lower cytotoxicity than them.The acute toxicity test of compound 22 at a single dose of 1000 mg/kg proved that it had low toxicity in vivo.The luciferase reporter plasmid found that target compounds could targete CDK8 to exert biological activities affectingβ-catenin-mediated TCF/LEF transcriptional activity.We further studied the mechanism of action,and proved that compound 22could inhibit the expression ofβ-catenin and cause the accumulation ofβ-catenin in the cytoplasm instead of transferring to the nucleus.Western blot analysis further demonstrated that compound 22 indirectly inhibited the activity ofβ-catenin by inhibiting the activity of CDK8,resulting in the down-regulation of WNT/β-catenin signaling pathway,such as the expression of c-Myc and Cyclin D1 related to cell cycle.Compound 22 could significantly induce HCT-116 cell cycle arrest in G2/M and S phases,but did not induce apoptosis.We determined the pharmacodynamics of compound 22,which showed that compound 22 had good bioavailability（38.9%）.In a mouse xeno-colorectal cancer tumor model,compound 22 showed a good anti-tumor effect with a dose dependency.Immunohistochemistry showed the expression ofβ-catenin in CT-26 tumor tissue was inhibited in a dose-dependent manner.Western blot analysis also displayed STAT1 SER727 phosphorylation and a decrease in oncogene C-myc expression in tumor tissues were inhibited in a dose-dependent manner.These studies have shown that compound 22 is a CDK8 type II inhibitor with high activity,high selectivity,low toxicity,clear mechanism of action,and good in vivo anti colorectal cancer activity.It is important to guide significance for the design of novel CDK8 type II inhibitors and the development of drugs for the treatment of colorectal cancer.Part II:Design,synthesis and antitumor activity evaluation of 7-azaindole derivatives as novel CDK8 type I inhibitorsIn this study,54 compounds were designed and synthesized,and compound 43（3-（1H-pyrrolo[2,3-b]pyridin-5-yl）benzamide）,showing potent inhibition against CDK8(CDK8 IC₅₀=51.9±1.2 n M),good selectivity over other CDK members,and good anti-AML cell proliferation activity in vitro(molm-13 GC₅₀=1.57±0.59μM).Mechanistic studies showed that compound 43 could target CDK8 and then inhibit phosphorylation of STAT1^S727 and STAT5^S726,leading to the apoptosis of AML cells.Pharmacokinetic evaluation exhibited that compound 43 had relatively good bioavailability（F=28.00%）.the acute toxicity test indicatd that compound 43 had low toxicity in vivo.The growth of AML tumors was inhibited in a dose-dependent manner in the AML-Balb/C mouse xeno-tumor model.Immunohistochemistry showed that the expression of Ki67 in tumor tissues of experimental group decreased in a dose-dependent manner.Western blot results showed that STAT5^S726 and STAT1^S727 in tumor tissue were inhibited in a dose dependent manner.These results suggest that compound 43 is a potent,selective and low toxic CDK8 type I inhibitor with good anti AML activity in vivo,providing guidance and reference for the further development of new CDK8 inhibitors for the treatment of AML.This paper has completed the complete and systematic work of"design,synthesis and activity evaluation of 7-azaindole derivatives as new CDK8 inhibitors",further enriching the structural types of CDK8 inhibitors.These results encourage us to continue to develop more effective CDK8 inhibitors with novel structures and conduct in-depth mechanism research,which has important reference value and guiding significance for the development of CDK8 inhibitors and the development of anti-tumor drugs.