Design,Synthesis And Antitumor Activity Of PROTAC Based On Multi-target Drug

Cancer has emerged as one of the most serious diseases endangering human health.At present,targeted anti-tumor drugs are currently a hot spot in the research and development of novel cancer treatments.However,targeted drugs focusing on optimize drug binding affinity,which is called"place-driven",could lead to less druggable targets,short half-life,drug resistance,and off-target effects.Proteolysis Targeting Chimeras（PROTACs）are a class of bifunctional molecules composed of target protein ligand,linker and E3 ubiquitinase ligand.They induce the formation of a"protein of interest-PROTACs-E3 ubiquitinase"ternary complex.The protein of interest is then labeled with ubiquitination and is allowed to be recognized and degraded by the ubiquitin-proteasome system（UPS）.Unlike traditional small molecule drugs with"space-driven"acts,PROTACs play the anti-tumor role through"event-driven"methods.Therefore,PROTACs exhibited high catalytic activity,lower drug resistance,lower target affinity requirements,and can target undruggable targets.Based on the research background above,the main work of this paper is as follows:（1）Taking multi-target tyrosine kinase inhibitors including Sunitinib and imatinib and their derivatives as the target protein-ligand,CRBN ligand as E3 ubiquitinase ligand,28novel PROTACs with different hindrance,polar groups positions and sites connecting CRBN ligands of linker were synthesized.The ¹H NMR,¹³C NMR,and HRMS data of key intermediates and target compounds were obtained for structural characterization.（2）The anticancer activity of 28 novel synthesized PROTACs was studied in vitro by the CCK8 method.The experimental results showed that PROTAC 1（based on imatinib derivative and CRBN ligand）and PROTAC 27（based on sunitinib derivative and CRBN ligand）showed significant anti-proliferative activity in tumor cell lines such as K562,A498,and HL60,with IC₅₀ values of 0.1～5μM.No obvious cytotoxicity was observed in NCM460 cells.（3）By analyzing the targets of the target protein ligands and the results of network pharmacology experiments,it is speculated that BCR-ABL may be the target proteins of the new PROTACs.The results of Western blot showed that PROTAC 1 degrades BCR-ABL via the UPS pathway and reduces the expression of c-ABL in K562 cells.PROTAC27 degraded GSPT1 via the UPS pathway in K562 cells and reduced the expression of c-KIT,FLT3,and SRC in A498 cells.In this article,28 novel PROTACs with multi-target drugs as the target protein ligands were synthesized.The anti-tumor activity and mechanism of action were studied by CCK8and Western blot methods.Two PROTACs candidate compounds with anti-tumor potential were found.The effect of linker in PROTACs based on multi-target drug design was studied.This work provides a reference for the development of PROTACs based on multi-target drug design,proves the importance of the selection of linker in the design of PROTACs,and lays the foundation for the application of PROTAC technology in overcoming targeted drug resistance.