Design And Synthesis Of A 2-Amino-pyridine Derivative As A Potent CDK8 Inhibitor For Anti-tumor Therapy

Cyclin-dependent kinases（CDKs）belong to the serine/threonine protein kinase family.It was initially found that CDKs could bind to specific cyclins and regulate the cell cycle.Later,it was found that other CDKs related to other physiological processes,such as participation in transcriptional regulation and DNA damage repair.CDK8 is one of CDKs that participates in transcriptional regulation via forming a kinase module of mediator complex with C（cyclin C,CNCC）,Med12 and Med13.Conformational changes of mediator complex induced by interacting with kinase module destroy the binding of the mediator complex to RNA Pol II and block subsequent transcription.CDK8 has attracted much attention due to its role in transcription and its close correlation with tumorigenesis.At present,only three CDK8 inhibitors,BCD-115,SEL120-34A and TSN084,have entered the clinical trial phase.Therefore,it is necessary to develop novel and selective,safe and effective CDK8 inhibitors.In this study,a novel CDK8 inhibitor was designed and synthesized based on the analysis of the sorafenib/CDK8 co-crystal complex.The urea group forms hydrogen bond interaction with Glu66 and Asp173,the benzene ring formsπ-πinteraction with Phe97,the N-methylpyridinium amide in the hinge region forms hydrogen bond interaction with Ala100,and the 4-chloro-3-（trifluoromethyl）benzene is located in the hydrophobic cavity.Therefore,we retained the diaryl urea moiety of sorafenib,explored the fragments that combined with the hinge region and occupied the hydrophobic pocket,designed and synthesized a series of 2-aminopyridine derivatives,and screened out HIT compounds by evaluating their inhibition on CDK8 kinase.Compound 29 showed strong inhibition of CDK8 with an IC₅₀ value of 46 n M and showed good kinase selectivity.Cellular thermal shift assay showed that the temperature of CDK8 protein degradation rose from 55℃to 61℃by treated with compound 29.In addition,compound 29 inhibited the phosphorylation of STAT1 SER727 which was the biomarker of CDK8.It was proved that compound 29 could bind to endogenous and exogenous CDK8 protein,indicated that compound 29 could bind to CDK8.WNT pathway is important for the occurrence and development of colorectal cancer.Compound 29 inhibited the activation of the classical WNT pathway and inhibitedβ-catenin mediated transcription.In addition,compound 29induced G1/S phase arrest in HCT-116 cells and inhibited the expression of CDK6 and p-Rb S780 that regulate the transition of G1/S phase.After the evaluation of compound 29 in vitro,we examined the toxicity,pharmacokinetics,and anti-tumor activity of compound 29 in vivo.The acute toxicity assay showed that compound 29 did not cause obvious tissue damage.In vivo pharmacokinetic studies have demonstrated an oral bioavailability of approximately 31.7%for compound 29.Finally,the antitumor activity of compound 29 was evaluated by using a mouse subcutaneous xenografts model.The results showed that the compound significantly inhibited the growth of tumors.In addition,the expression levels ofβ-catenin,E2F1 and c-Myc in tumor tissues were significantly reduced,indicating that compound 29 exerted antitumor activity through the WNT/β-catenin signaling pathway related to CDK8 in vivo.In summary,a series of new CDK8 inhibitors were designed and synthesized based on sorafenib.Compound 29 was screened out on the basis of reasonable drug design and structure-activity relationship analysis,and showed good activity in vivo and in vitro.We hope that the discovery of compound 29 could enrich the understanding of the function of CDK8 and help develop effective drug of colorectal cancer.