Design,synthesis And Bioactivity Study Of Novel C-Met Inhibitors Based On Drug Resistance Reversal Strategy

c-Met,also known as hepatocyte growth factor(HGF)receptor,belongs to the family of receptor tyrosine kinases(RTKs).HGF binding with c-Met can regulate a variety of downstream signaling pathways and participate in various physiological processes,including cell proliferation and survival,apoptosis and angiogenesis,etc.The dysregulation of HGF/c-Met signaling will cause cancer.In addition,the overexpression of c-Met is highly associated with poor prognosis and the resistance to EGFR inhibitors.Crizotinib and Cabozintinib are the c-Met small molecule inhibitors approved by FDA for the clinic cancer treatment with robust antitumor efficacy.However,the fast emergence of the acquired drug resistance remarkably compromises the efficacy.Currently,several mechanisms of c-Met drug-resistance have been reported,such as gene amplification and overexpression,mutation of c-Met,and activation of bypass pathway.Therefore,developing new strategies to overcome the drug resistance problem according to the mechanism is an urgent need for the effective application of the molecular targeted tumor therapy.There are two main parts in this thesis.The first part is to design and develop c-Met degraders using PROTAC strategy for the drug resistance caused by gene amplification and overexpression of c-Met and mutations.The second part is aimed to reverse the drug resistance caused by the activation of the c-Met bypass pathway e.g.c-Myc upregulation,and attempt to design c-Met/BRD4 dual inhibitors,in which the BRD4 inhibitors suppress the expression of c-Myc.Proteolysis targeting chimeras(PROTACs)is an emerging technology that can induce the degradation of the targeted protein through the ubiquitin-proteasome system.PROTAC molecule is a bifunctional molecule that binds to the target protein and recruits the E3 ubiquitin ligase to ubiquitinate the target protein and degrades the target protein through the proteasome.Compared with small molecules which can only inhibit one function of the target protein,PROTACs molecules can induce the degradation of the whole proteins which may provide opportunity to overcome the drug resistance problem.Hence,we started the design and SAR study of c-Met targeting PROTACs by using the new structure c-Met small molecule inhibitor LXM-262 which was originally developed by our lab.The systematic optimizing of the linkers(length,polarity,link mode)and the ligand of E3 ubiquitin ligase(CRBN,VHL),a total of 37 c-Met-targeted PROTAC bifunctional molecules were synthesized and evaluated in the enzyme and cell assays.All these compounds showed potent inhibitory activities against c-Met function and the Met-dependent tumor cell growth.Among them,the lead compound 2-28 composed of the ligand of VHL and the polyethylene glycol chain as linker exhibited potent c-Met kinase inhibition activity and anti-proliferative activity(c-Met IC50=129 nM;EBC-1 IC50=0.36μM).Furthermore,2-28 demonstrated distinct c-Met protein degradation activity in cell experiments(DC50=10 nM).Significantly,the c-Met degrader 2-28(EBC-1/D228Y IC50=0.24 μM)displayed a superior therapeutic advantage to the c-Met inhibitor LXM-262(EBC-1/D228Y IC50=0.7 μM)in the c-Met inhibitor acquired drug-resistant tumor cell lines.A series of mechanism verification experiments indicated that compound 2-28 was indeed an effective c-Met degrader,and the degradation of c-Met was achieved through the ubiquitin proteasome pathway.Further pharmacokinetic evaluation showed that compound 2-28 was rapidly metabolized after intravenous administration with a half-life of only 0.1 hours.Due to its large molecular weight,PROTAC molecules usually possesses poor physicochemical and pharmacokinetic properties.Therefore,in order to produce drug-like small molecule c-Met degraders,we applied the "molecular glue" concept to the design of the small molecule PROTACss.Based on the pharmacophore hybridization strategy of c-Met inhibitors and the ligand of CRBN,we designed and synthesized a series of chimeras targeting both c-Met and CRBN.Among them,compound 2-37(c-Met IC50=3 nM,EBC-1 IC50=0.13 μM)displayed improved anti-proliferative activity compared to the inhibitor LXM-262 and the degrader 2-28.Furthermore,the antiproliferative activity of compound 2-37(IC50=167.4 nM)against the drug-resistant tumor cell lines is more potent than LXM-262(IC50=641.1 nM)by nearly 4-fold.However,the Western blotting experiment indicated that compound 2-37 did not directly degrade the c-Met protein.The reason why compound 2-37 had remarkably enhanced cellular activity might be due to the increase of enzyme inhibitory activity by which 2-37 just played the role of potent c-Met inhibitor.Another possibility was that 2-37 still functioned as a degrader but degraded other key proteins rather than c-Met.We are currently using proteomics experiments to study its mechanism of action.The second part of this thesis is focused on the drug resistance caused by the activation of the c-Met bypass pathway,and attempt to design c-Met/BRD4 dual inhibitors.It has been reported that up-regulation of c-Myc is one of the reason of c-Met acquired resistance,and down-regulation of c-Myc can reverse the problem.BRD4 mediates c-Myc transcription,and its inhibitors can effectively down-regulate c-Myc expression.Therefore,the combination of c-Met inhibitors and BRD4 inhibitors offers a certain synergistic effect in overcoming the acquired resistance of c-Met.Considering the potential problems of drug-drug interactions in non-covalent combination therapy,we expect that small molecule c-Met/BRD4 dual inhibitors can solve the problem of acquired resistance of c-Met caused by c-Myc up-regulation.After preliminary exploration,we designed and synthesized 29 compounds by using scaffold hopping and hybridization strategies.Most compounds showed potent BRD4 inhibitory activity,of which compound 3-26(BRD4 IC50=98 nM)is only two times less potent than the positive control JQ1.However,this series showed weak inhibitory activity against c-Met.The subsequent work will be focused on improving the activity of c-Met based on the autodocked binding mode with c-Met.In summary,we aimed to provide new strategies to tackle off the problem of acquired drug resistance of c-Met inhibitors as molecular targeted antitumor therapies.According to two main resistance mechanisms,we designed and developed PROTAC-based c-Met degraders and c-Met/BRD4 dual inhibitors to reverse the c-Met mutation and bypass activation-caused drug-resistance,respectively.Our efforts have generated promising positive results.In particular,by using the PROTAC strategy,we obtain degraders that can effectively degrade wild type and drug-resistant mutated c-Met protein.Our study has provided promising new lead compounds and new mechanism for the design and development of efficacious anti-tumor drugs reversing the drug-resistance.