Design, Synthesis And Structure-activity Relationship Study Of Tetrapeptide Ethylene Oxide Proteasome Inhibitors

The ubiquitin-proteasome pathway(UPP)is an important pathway of protein metabolism in human body,which is responsible for the degradation of more than 80%abnormal intracellular proteins.Its target proteins mainly include tumor suppressor,activating transcription factor,cell cycle regulators and so on.Overdegradation of p21,p53,κB and other suppressive factors by the proteasome will lead to a variety of tumors.Therefore,by inhibiting the proteasome activity to affect tumor cells has become an important method to treat cancers.Carfilzomib was approved by FDA in 2012 as the second generation of proteasome inhibitor for the treatment of multiple myeloma.Compared with other proteasome inhibitors,the peptidyl epoxyketone compound carfilzomib has a higher selectivity and specificity.But it still caused some side effects,including peripheral neurotoxicity,neutrophil,thrombocytopenia and so on.So we selected carfilzomib as lead compound to design epoxyketone derivatives with novel structure,higher activity,less toxicity.Three series of novel compounds were designed according to molecular docking results of carfilzomib with 20S proteasome and the experience of our group in the study of proteasome inhibitors.Twenty novel target compounds were synthesized by nucleophilic substitution,addition,selective reduction,Sharpless asymmetric epoxidation,hydrolysis,amide,etc.The structures of the compounds were confirmed by HRMS,1H-NMR and 13C-NMR.In addition,the activities of twenty compounds were investigated in inhibiting proteasome.The structure-activity relationship(SAR)were discussed and the biological results showed that the activities of 5 compounds were similar to carfilzomib,D187(IC50=17.21 nM),D213(15.07 nM),D214(17.89 nM),D225(23.60 nM)and D227(11.64 nM),carfilzomib(11.42 nM).These five active compounds were further used to evaluate the cytotoxicities of human multiple myeloma cell lines U266,RPMI 8226 and ARH-77.Pharmacological data indicated that compounds D187(RPMI 8226:25.52 nM;ARH-77:73.60 nM;U266:36.94 nM)and D213(RPMI 8226:14.69 nM;ARH-77:41.75 nM;U266:23.18 nM)were as active as positive drug carfilzomib(RPMI 8226:39.09 nM;ARH-77:89.92 nM;U266:35.72 nM).Compounds D187 and D213 were selected to carry out molecular dynamics studies.The differences of the activity of small molecules were predicted from five different aspects including the changes of overall structure,secondary structure and the comparison of gyration radius,the changes of solvent accessible surface area of complex,active site,which would provide some theoretical guidance for the further structural optimization.