Design,Synthesis And Biological Evaluation Of Small-molecular PROTAC Targeting BTK In B Cell Lymphoma

B cell lymphoma is a non-Hodgkin’s lymphoma(NHL)with poor clinical curability and prognosis,which accounts for about 85%-90%of NHL.The B cell receptor(BCR)signaling pathway is a key pathway for survival,proliferation and apoptosis of B cell,Bruton’s tyrosine kinase(BTK)exerts biological functions through BCR pathway and triggers multiple downstream signaling pathways.Ibrutinib(IBN)is the first generation irreversible and covalent inhibitor,approved to treat relapsed/refractory MCL.Although IBN has acquried many clinical achievements,some patients show resistance to IBN,while even have serious side effects such as bleeding and thrombocytopenia.Proteolysis Targeting Chimeras(PROTAC)technology is an emerging technology developed rapidly in recent years,which works through the ubiquitin-proteasome system.Compared with traditional small molecule inhibitors,PROTAC molecules can overcome drug resistance caused by mutation or overexpression of the target protein,improve selectivity for specific target protein and catalyze degradation of the target protein.PROTAC molecules for BTK can selectively degrade BTK and significantly degrade expression level of BTKC481S mutant in cells,which is significant for B cell lymphoma.The design of target compounds was conducted based on literature research and results of our group on BTK inhibitors in paper.Ibrutinib was selected as the ligand for binding with target protein,and pomalidomide was used as the molecular ligand for CRBN.In order to explore influence of difference of Linker on the biological activity,ligands at both ends are connected with flexible alkyl chains and piperazine is located in the middle to alleviate steric clashes.Which leads to efficient PROTAC molecules.Subsequently,16 compounds in PA series were designed and synthesized.Furthermore,piperidine and pyrrole were introduced into ibrutinib to explore effect of PROTAC spatial configuration,and 8 compounds of PC series were designed and synthesized.The anti-proliferation experiment showed that compounds have significantly inhibition effect on Jeko-1 cells than K562 and HEL,and most of them have good antiproliferative activity(75%-85%)in vitro on Jeko-1.Specially,compound PC7(IC50=3.59 ±0.13 μM)was better than IBN(IC50=4.70 ± 0.12μM).Western Blot revealed that some compounds significantly degrade BTK at different concentrations(0.5,5,and 15 μM)while IBN and Poma have no degradation,which proves that PROTAC molecules can efficiently degrade BTK protein in cells.In addition,compounds of PC series with piperidine and pyrrole have more significant degradation than PA series when the length of linker is same,which is more conducive to form stable ternary complexes.The compound PA7,PC5,and PC7 can degrade BTK in concentrationdependent and time-dependent.Moreover,DC50 of PA7 is 0.45 μM,Dmax is 72.84%after 24 h(0.5 μM),DC50 of PC5 is 0.25 μM(Dmax=91.86%),and DC50 of optimal compound PC7 is 0.10 μM(Dmax=94.44%).At the same time,compound PC7 can significantly degrade BTK protein in Z138 cells with DC50 of 0.02 μM,Mino cells with DC50 of 0.09 μM.The preferred compounds have strong inhibition on BTK(≥80%),and changes in the length of linkers have little effect on BTK.Otherwise,compounds were selected to execute affinity experiment with BTK and molecular docking was used to further explore optimal linker length of BTK protein and CRBN protein in space.We have designed and synthesized 24 PROTAC molecules targeting BTK.The activity results show that compound PC7 is an effective BTK degrading agent,which provides reference for structural optimization and modification of PROTACs latter.