Design,Synthesis And Biological Activity Evaluation Of CDK9 Inhibitor And Degradation Agent

Cyclin-Dependent Kinases(CDK),combined with the regulatory subunit family of cyclins,form a functional heterodimeric complex that regulates the life functions of several cells,including proliferation,differentiation,DNA repair,and apoptosis.As a member of the transcriptional CDK subfamily,CDK9 is responsible for the transcriptional function of cells.Therefore,targeting CDK9 can effectively inhibit the proliferation of tumor cells in vivo and induce apoptosis of tumor cells in vivo.So far,more and more CDK9 inhibitors have shown good antitumor activity in vitro,including pyrazoles,pyrimidines and flavonoids.In this paper,2-phenyl-N-(5-cyclopropyl-1H-pyrazol-3-yl)acetamide was selected as a lead compound,and CDK9 inhibitors were designed and synthesized based on its structure-activity relationship.After screening for CDK9 inhibitory activity in vitro,the compound with better activity were combined with the PROTAC technology to synthesize CDK9 degradation agent.The specific research contents and results are as follows:In this paper,a total of 57 CDK9 inhibitors were designed based on structure of lead compound.By changing the position of the double bond of the pyrazole parent ring,five class I compounds were obtained.The position of the amide bond at the C3 position was changed to obtain three class I compounds.The C5 position of the pyrazole parent ring was replaced by a methyl group,and the benzene ring structure was substituted to obtain 11 class I compounds;the C5 position was replaced by a t-butyl group,and the benzene ring structure was substituted to obtain 13 class I compounds;the C5-position cyclopropyl group was retained to further verify the activity of different substituents of the benzene ring structure,and 7 class I compounds were obtained.Secondly,the pyrazole parent ring was replaced with a structurally similar isoxazole,and 18 class II compounds were obtained.34 compounds were selected for CDK9 inhibitory activity testing,and a total of 8 compounds with inhibition rate greater than 40%.By comparison,it was found that the activity of class I compounds was significantly higher than that of class II compounds,indicating that pyrazole ring is more suitable for the development of CDK9 inhibitors than isoxazole;the activity of compounds obtained by substitution of methyl group at C5 position is generally lower than that of tert-butyl group.The compound obtained by substituting with a cyclopropyl group may be because the cyclopropyl group and the tert-butyl group were better bonded to the hydrophobic pocket;when the structural modification of the benzene ring at the C3 position was carried out,it was found that the substitution at the meta position of the benzene ring is obtained.The activity of the compound is significantly lower than that of the compound obtained by the para-substitution,and the strong electron-donating group in the para-position has better activity than the strong electronwithdrawing group;the ligand of the E3 ligase and CDK9 inhibitor were connected by a suitable Linker to obtain class P compounds,8 compounds were selected for Western blotting experiments.According to the experimental results,the selection of Linker will affect the degradation of PROTAC to some extent.the tert-butyl group and cyclopropyl group at the C5 position of the pyrazole parent ring can produce good degradation activity,but methyl group and ethyl group show little degradation activity,further verifying that the tert-butyl group and the cyclopropyl group can bind better to the hydrophobic pocket;the C3 position was substituted with an amino group,and the synthesized PROTAC has a certain degradation effect at 50 ?M.The subsequent optimization based on this structure has certain feasibility and provides a certain theoretical basis for the development of related CDK9 degradation agents in the future.