Design,Synthesis And Anti-tumor Activities Of Novel 4-phenoxy Quinolines As C-met Kinase Inhibitiors

The HGF/c-Met signal pathway,which exists widely in various kinds of cells,plays a crucial role in the growth of tissues and organs.However,the overexpression of HGF/c-Met usually leads to the invasion and metastasis of tumor cells.Therefore,developing inhibitors of c-Met have emerged as a new approach for the treatment of cancer.In this paper,the c-Met kinase and the HGF/c-Met signal pathway was introduced,and the recent development of small-molecule c-Met kinase inhibitors(TKIs)was summarized.According to the structure-activity relationships(SARs)of small-molecule Type Ⅱc-Met inhibitors bearing 4-phenoxyquinolines reported in literatures,taking foretinib as the leading compound,we introduced 1,2,4-triazolone,quinoxalinone and imidazole moieties into 4-phenoxy-6,7-disubstituted quinolines on the basis of combination principles and local modification.Thus,three series of quinoline derivatives,86 compounds,were designed and synthesized,in which the 4-phenoxy-6,7-disubstituted quinoline framework was preserved,and substituted 1,2,4-triazolone,quinoxalinone and imidazole groups were introduced to the C-4 position,respectively.There were three series of compounds:compounds containing 1,2,4-triazolone moiety(L-1～L-41),compounds possessing quinoxalinone moiety(J-1～J-28),and compounds bearing imidazole moiety(Y-1～Y-17).All the target compounds have not been reported by other groups and their chemical structures were confirmed by MS,1H NMR.Some of them were further identified by 13C NMR and IR.All the target compounds were evaluated for their antiproliferative activities against HT-29(human colon cancer),H460(human lung cancer),A549(human lung adenocarcinoma),MKN-45(human gastric cancer),and U87MG(human glioblastoma)using the standard MTT assay in vitro,with foretinib as the positive control.Most compounds showed moderate to excellent cytotoxic activity against the different cancer cells with potencies in the single-digit μM range,and 55 of them were more potent than foretinib against one or more cell lines.Among them,the most promising compounds L-22,L-28,J-7,J-23 and J-26 exhibited more potent antitumour activity against all tested cell lines than the positive control.In addition,25 of them were further screened for their activity against c-Met kinase by the Homogeneous Time Resolved Fluorescence(HTRF)assay,using foretinib as the positive control.The results showed that some compounds displayed preferable c-Met kinase inhibition,with IC50 values ranging from 0.90～42.40 nM.Compounds J-7 and J-26 displayed 1.57 and 1.15 fold increase against c-Met kinase,respectively,compared with that of foretinib.To examine the selectivity of compounds L-22 and J-23 on c-Met over other kinases,they were screened against c-Kit、Flt-3、PDGFRα、VEGFR-2、Ron and EGFR tyrosine kinases.Compound L-22 also exhibited high inhibitory effects against c-Kit and Flt-3 with IC50 values less than 10 nM,and demonstrated moderate selectivity against PDGFRα,Ron and VEGFR-2 kinase,and this compound showed barely inhibited kinase activity against epidermal growth factor receptor(EGFR)kinase.Compound J-23 also exhibited high inhibitory effects against c-Kit and PDGFRα,and showed moderate-to-excellent selectivity against Ron,VEGFR-2,Flt-3,EGFR and ALK kinase.These data indicated that compounds L-22 and J-23 may be promising multitarget inhibitors of tyrosine kinases.As migration is an important characteristic for metastatic cancers,the effect of compounds L-22 and J-23 on A549 cancer cells was investigated by wound healing assay,and the results showed that compounds L-22 and J-23 could significantly inhibit cellular migration.The fluorescence microscopy after double staining with acridine orange/ethidium bromide(AO/EB)demonstrated that compounds L-22 and J-23 induced dose-dependent apoptosis of H460 cells.To elucidate whether the cytotoxicity induced by the derivatives was due to cell cycle arrest,flow cytometry was used to evaluate the cell cycle distribution in H460 cells.The results indicated that treatment of H460 cells with compound J-23 resulted in cell cycle arrest in G2/M phase in a dose-dependent manner.To further elucidate the binding mode of compounds L-22 and J-23,a detail docking analysis was performed.The co-crystal structure of foretinib(GSK1363089)with c-Met was selected as the docking model(PDB ID code:3LQ8).The N atom of quinoline,the 2-N atom of triazole ring and NH of amide of L-22 formed three hydrogen-bonding interactions with MET1160 and ASP1222.Besides,the π-π hydrophobic interaction is also formed between the 2-fluorophenyl ring and Phe1134.Moreover,the π-sigma interaction was formed between the quinoline in L-22 with ILE1084.The N atom of the quinoline,NH and O atom of amide of J-23 formed three hydrogen-bonding interactions with MET 1160,ASP1222 and LYS1110,respectively,and three π-sigma interaction between quinoline with ILE1084 and MET1211 had beenformed.Docking model showed that the conformation of J-23 is well consistent with foretinib’s binding conformation.According to the result of anti-tumor activity of the target compounds,the preliminary structure-activity relationships were summarized,which would provide useful information for discovery of 4-phenoxyquinolines as the small-molecule Type Ⅱ c-Met inhibitors.