Design, Synthesis, Activity Evaluation And Structure-activity Relationship Of CDK8 Inhibitors

Cyclin-dependent kinases（CDKs）are a family of serine/threonine protein kinases,which are very important factors in regulating cell cycle.Some CDK family members are involved in transcriptional regulation,and a few members are involved in other functions.Almost all CDKs need to form complexes with corresponding cyclins to exert physiological functions.The primary way CDK8 regulates transcription is to form a kinase module with CCNC,Med12 and Med13 as part of a mediator complex.CDK8is overexpressed in more than 60%of colorectal cancers,and it mediates abnormal activation of the Wnt/β-catenin signaling pathway,which is manifested by excessive activation and increased levels ofβ-catenin,so CDK8 has become an effective therapeutic target for colorectal cancer.The reported CDK8 inhibitors show excellent anti-proliferative and enzyme inhibitory effects,and it is of great significance to target CDK8 to develop therapeutic drugs.There are only two CDK8/19 inhibitors in the world that have entered the clinical trial stage.Although the development of CDK8inhibitors can be traced in terms of enzyme inhibitory efficacy,anti-proliferative activity and toxicity,due to the complexity of CDK8/CNCC structural biology,its mechanism of action as a tumor drug therapy target is also complex and diverse.And the existing CDK8 inhibitors’structure are single,so the development of new structures and new skeletons of CDK8 inhibitors is still necessary.In this study,we designed and synthesized novel CDK8 inhibitors with the help of the latest research results on the structural biology of CDK8.First,we constructed pharmacophore based on ligand-receptor interaction,and then screened commercial compound libraries.The Hit compound 8018-3781 was obtained through further docking screening and interaction analysis,which has strong interactions with CDK8key amino acid residues such as Lys52,Phe97,Arg356 and Tyr32,especially Arg356which is critical for CDK8 selectivity.Therefore,in this study,8018-3781 was used as a Hit compound.By replacing,substituting,and extending the benzene ring connected by the sulfonamide linker,and then adding different alkyl groups to the N atom of benzimidazolone,Forty nine novel 1,3,6-trimethyl-benzimidazolone-5-sulfonamide derivatives were designed and synthesized,which were characterized by ~1H-NMR,¹³C-NMR and ESI-MS.The inhibition rate of CDK8 kinase was used as the criterion to evaluate the activity of the compounds.Among them,compound 51b with the pyrrole structure has the best enzyme inhibitory activity(IC₅₀=72.7 n M).The docking analysis results show that,in addition to the interaction with the above CDK8 typical amino acid residues,the-NH of the amide forms a hydrogen-bonding interaction with Val27,and the-NH of the pyrrole ring forms a hydrogen-bonding interaction with Arg29.51b inhibits CDK8-mediated phosphorylation of STAT1 at Ser727 in HCT-116,suggesting that it can affect the biological function of CDK8 in tumor cells.51b regulates the Wnt/β-catenin signaling pathway by inhibiting the expression of Wnt protein and inducing the degradation ofβ-catenin protein.At the same time,51b can also play an anti-tumor cell proliferation effect by arresting the cell cycle and increasing the level of reactive oxygen species in cells.The phosphorylation levels of cell cycle-related proteins Rb and CTD were down-regulated by 51b.Further research found that 51b could significantly reduce the transcriptional activity of the TCF family and have an impact on the expression of downstream target genes.In conclusion,compound 51b is a novel and potent CDK8 lead compound that can be used to further develop a highly potent and selective CDK8 inhibitor for the treatment of colorectal cancer.