Structure-based Design,synthesis And Biological Evaluation Of Novel XPO1 Inhibitor And Proteolytic Targeting Chimera

Multiple myeloma（MM）is an incurable hematological malignancy.The current treatment regimens for MM are prone to relapse and short duration of remission,therefore,it urgently demands new therapeutic strategies for patients who are not sensitive to the current regimens.The inhibition of exportin 1（XPO1）has emerged as a novel and effective strategy for the treatment of MM.XPO1 mediates the nuclear export of a variety of proteins and RNAs,including tumor suppressor protein（TSP）.TSP must be located in the nucleus to suppress tumorigenesis.However,XPO1 is frequently overexpressed in malignant tumors,resulting in excessive nuclear export of TSP and thus unable to exert tumor suppression.Therefore,inhibition of XPO1 or direct degradation of XPO1 is a way to keep TSP in the nucleus and play the tumor suppressive role.Selinexor（KPT-330）,a selective XPO1 inhibitor,has been approved by FDA for the treatment of multiple myeloma and diffuse Large B-cell lymphoma.Based on the perspectives of protein inhibition and degradation,the design,synthesis and antitumor activity evaluation of novel XPO1 inhibitors and proteolytic targeted chimeras（PROTACs）were performed in this thesis.The details are as follows:Design,synthesis and bioactivity studies of novel XPO1 inhibitors:As a second-generation XPO1 inhibitor,KPT-8602 has a larger therapeutic window and a weaker blood-brain barrier penetration.Currently,a number of phase II clinical trials are being conducted with KPT-8602.As to the molecular mechanism,KPT-8602 can form covalent bond with Cys539 residue of XPO1 and bind reversibly to the protein.In order to enhance the interaction between target compounds and XPO1,we designed and synthesized four series and 33 target compounds by structural modification of KPT-8602.Among them,compound D4 showed significant anti-proliferative activity(IC₅₀=24 nM)against MM.1S cells,and also displayed good inhibitory effect on eight cell lines of four different types of lymphoma.The results of immunofluorescence assay and fluorescence probe competitive binding assay showed that D4 could effectively inhibit the nuclear export function of XPO1,and its inhibitory activity was attributed to the occupation of D4 in the NES-binding region of XPO1 protein.In addition,compound D4 showed good metabolic stability in rat plasma and liver microsomes,and pharmacokinetic data showed that D4had a high drug exposure and good bioavailability in SD rats（F=34.6%）.Overall,as a highly active oral drug,D4 has great potential for development in the treatment of multiple myeloma and lymphoma.Design,synthesis and bioactivity of novel proteolytic targeting chimeras for XPO1:PROTAC has shown great potential in the field of drug development in recent years,and PROTAC molecule has many advantages compared with small molecule drugs.Studies have demonstrated that XPO1 protein can be degraded through the ubiquitin-proteasome pathway,so it is feasible to develop XPO1 degraders based on PROTAC technology.As an efficient inhibitor of XPO1,KPT-276 has a smaller relative molecular weight than D4.Therefore,KPT-276 was used as the XPO1 ligand in this thesis,flexible hydrophobic carbon chain and rigid chain containing piperidine were used as the linker,and different ligands of E3 ligases（CRBN and VHL）were employed,to designed and synthesized ten XPO1-PROTAC molecules.The target compounds with flexible hydrophobic carbon chains（E1-E4,E8,E9）displayed moderate inhibitory activity inMM.1S cells,but no degradation of XPO1 protein was observed.We assumed that the anti-proliferation activity was attributed to the inhibition effect of PROTAC molecules over XPO1 rather than the degradation effect.The study in this thesis provides a reference for systematically exploring the structure-activity relationship over linkers and E3 ligands of XPO1-PROTAC.