Design,Synthesis And Biological Evaluation Of Small Molecule Degradation Agent Targeting Bcr-Abl^T315I Based On PROTAC Principle

Chronic myeloid leukemia?CML?is a clonal myeloproliferative neoplasm that originates from hematopoietic stem cells?HSCs?,with an annual incidence of about 1.6cases per 100,000 adults.Abnormal Bcr-Abl fusion protein is the main molecular mechanism that causes the pathogenesis of CML.The first-generation Abl kinase inhibitor imatinib significantly improved the survival time of CML patients,but the rapid emergence of target protein mutation resistance limits its clinical efficacy.Although the second-generation Abl inhibitors such as nilotinib and dasatinib,etc.can overcome most Abl mutation resistance,they are ineffective against“gatekeeper”T315I mutations and have potential cardiotoxicity.The third-generation Abl inhibitor ponatinib is effective against T315I mutation resistance,but the side effects such as“fatal thrombosis and severe vascular stenosis disease”caused by the drug have been recorded as black box warnings in Europe and the United States,which limits its clinical application.In addition,ponatinib has poor effect on P-Loop mutations such as Bcr-Abl^E255K/V.At the same time,with the clinical use of ponatinib,some CML-BP and Ph+ALL patients are prone to produce T315I-inclusive compound mutations?E255K/T315I,etc.?,which are resistant to all Abl inhibitors including ponatinib.Therefore,CML clinical drug resistance is still a medical problem that has not been completely solved,and there is an urgent need for new drugs that are safe,efficient and overcome drug resistance.Proteolytic target chimeras?PROTACs?is a new technology developed in recent years that uses bifunctional small molecules to achieve selective degradation of target proteins.Currently,molecules based on this technology that target AR and ER have entered clinical research.At low drug doses,PROATC binds to the target protein of transiently and induces its degradation,so it is less susceptible to increased expression level of target protein and resistance mutations occurring in target protein and have the advantage of overcoming the potential resistance of small molecule inhibitors.Several Abl PROTAC degraders have been reported,while no Bcr-Abl^T315I mutant PROTACs.Therefore,we designed and synthesized a series of PROTAC degradants,based on PROTAC technology with the ABl ^T315I inhibitor GZD824,and studied their degradation activity in vitro and anti-tumor activity in vitro and in vivo.We first synthesized a series of PROTACs 7a-7f containing different E3 ligase ligands,and studied the anti-proliferative activity against different CML cell lines and Bcr-Abl^T315Idegradation activity.The results showed that only compound 7a linked to pomalidomide?a CRBN E3 ligand?had moderate degradation activity(DR_{300n M}=39.01%)and comparatively strong antiproliferative activity(IC₅₀=83.2 n M).After fixing the ligand end of E3 ligase to pomalidomide,the structure-activity relationships of two types of linkers?PEG and linear alkyl chain?was discussed by changing the linker of 7a,two types of PROTACs molecules 7k-7g,7l-7s were designed and synthesized.Representative compound 7o?linker is 6 carbon atoms?showed the strongest degradation effect on Bcr-Abl^T315I in Ba F3 cells(D_{100n M}=69.89%,D_{300n M}=94.23%)and better antiproliferative activity against Ba F3 cells containing Bcr-Abl^T315I(IC₅₀=26.8 n M).The mechanism of action study showed that compound 7o induced the degradation of Bcr-Abl^T315I through the E3 ubiquitin proteasome pathway in a time-dependent manner.Furthermore,the degradation time of compound 7o on Bcr-Abl^T315I-bearing Ba/F3 cells and Bcr-Abl^WT-bearing K562 cells was different.The maximum degradation of Bcr-Abl^T315I was observed at 24h,while the significant degradation of Bcr-Abl^WT was only observed at 36h.Pharmacokinetic results of intraperitoneal?IP?administration of 7o proved that its blood concentration was much higher than that of Bcr-Abl^T315I in vivo concentration.In vivo activity showed that compound 7o significantly inhibited the growth of Ba/F3-Bcr-Abl-T315I xenograft tumor?TGI=55.6%?,and significantly prolonged the median survival time of mice.The research of this subject provides a certain research basis for overcoming the clinical resistance of CML.