Design,Synthesis And Antitumor Activity Of 4-phenoxypyridine/pyrimidine C-Met Inhibitors

HGF/c-Met signaling pathway plays an important role in cell movement,growth,metastasis,invasion,wound healing,tissue regeneration,angiogenesis and other biological changes,but its overexpression often leads to the occurrence,development,invasion and metastasis of tumors.Therefore,c-Met/HGF signaling pathway is an effective target for cancer treatment.Class II c-Met inhibitors have the characteristics of multi-targets,anti-tolerance and potent efficacy,and gradually become the research hotspot of c-Met inhibitors.Based on the previous research of our research group and the structural characteristics of Class II c-Met inhibitors,the active skeleton of Class II c-Met inhibitors is concluded to moieties A,B,C and D.Moieties A,B,C and D are composed of pyridine/pyrimidine（heteroaromatic ring）structures,benzene ring structure with single/unsubstituted group,active fragment with 1～2 hydrogen-bond donor/acceptor protons and meets the"five atom rule",benzene ring structure without/multi substitution,respectively.Combined with the above active skeleton,Golvatinib was modified as the lead compound.Firstly,the pyridine structure of moiety A was retained and introduced the amide flexible chain to obtain the fragments of pyridine amides,and then obtained the fragments of pyrrole pyrimidines through the skeleton transition principle.Secondly,the active segments bearing pyridazinone,triazole and naridinone structure were introduced to moiety C based on the basic modifications rules such as pharmacophore heterozygosity,bioelectronic emission rule and molecular docking simulation.Thirdly,different substituents were introduced to moieties B and D.Three series of 684-phenoxypyridine（pyrimidine）c-Met inhibitors were designed.Based on the method of reverse synthesis analysis and literature review,the synthesis routes of key intermediates,side chains and target compounds were finally synthesized.The key intermediates of 7a～7d synthesized from pyridinic acid by multi-step reaction and the side chain of 12a～12f prepared from aniline analogues by multi-step reaction were connected to give the target compounds XHH-1～XHH-14.The key intermediates of 7a～7e and 8a～8e the side chain of 16a～a6i prepared from phenylboric acid analogues by multi-step reaction were connected to give the target compounds XHH-15～XHH-50.The key intermediates of 19a～19c synthesized from4-chloro-7-H-pyrrole[2,3-d]pyrimidine by multi-step reaction and the side chain of26a～26h prepared from aniline analogues by multi-step reaction were connected to give the target compounds XHH-51～XHH-68.The structures of all the target compounds were confirmed by ¹H NMR,and some of them were confirmed by ¹³C NMR and TOF Ms.The cytotoxic activities of 68 target compounds against Hela（cervical cancer cell）,A549（human lung cancer cell）and MCF-7（breast cancer cell）cells were tested in vitro using the MTT method.Most compounds with excellent cytotoxic activity were selected to evaluate the kinase activity against c-Met.Some of them with outstanding inhibitory activity have been further studied,including concentration dependence test,AO staining test,cell flow cycle test and molecular docking simulation.Most of the target compounds showed moderate to potent inhibitory activity against cancer cell lines with overexpression of c-Met,some of them showed the equal or stronger inhibitory activity than the control Golvatinib,and 11 target compounds showed more potent inhibitory activity against 1～3 cell lines than Golvatinib.Among of them,XHH-9 exhibited 2.60～6.95-folds stronger inhibitory activity against A549,He La and MCF-7 cell lines than Golvatinib,and its inhibitory activities against the three cell lines were 1.03,1.15 and 2.59μM respectively.Some compounds,such as XHH-7～XHH-10,XHH-48～XHH-50,XHH-58～XHH-59 and XHH-64～XHH-65(IC₅₀ values of 1.03～13.60μM),possessed more efficiently inhibitory activity than Golvatinib were selected the c-Met kinase activity.And those compounds showed moderate to strong c-Met kinase inhibitory activity.XHH-9 and XHH-64 showed prominent c-Met kinase activity with IC₅₀ values of 0.807 and 0.612μM,respectively.In addition,XHH-9and XHH-49 could inhibit cell lines in a concentration-dependent manner in the concentration-dependent test,and induce the apoptosis of cells in a dose-dependent manner in the AO staining and flow cycle experiments.We also performed the molecular docking of XHH-9,XHH-49 and XHH-64 with c-Met（PDB code:3LQ8）.The results showed that all of them had strong interaction with c-met kinase with an extended conformation.Based on the in vitro antitumor activity and molecular docking of target compounds,the structure-activity relationships（SAR）of the target compound is summarized as follows:the pyridylamide and pyrrolidine structures of key structure can form a double tooth or two hydrogen bonds with Met1160,which is one of the key segments to maintain the biological activity of the compounds,and theπ-πconjugation between the middle benzene ring and Phe1223 is formed,and the introduction of F atom is conducive to the enhancement of the biological activity of the compounds,and the 5-atom linkers usually form two hydrogen bonds with Asp1222 and Lys1110.In this part,triazole,pyridazinone and naridinone structures with potent biological activity and hydrogen bond receptor/donor atom were introduced to improve the biological activity of the compounds,and the terminal-benzene ring extends into the hydrophobic back pocket,and the introduction of the end benzene ring as a single electron withdrawing substituent is obviously beneficial to the biologist activity,however,the introduction of double substituents（electron acceptor/donor）or large structural segments significantly weakened the biological activity of the compounds.In conclusion,three series of 68 novel 4-phenoxypyridine（pyrimidine）c-Met inhibitors have been obtained by the modification of Golvatinib as the lead compound,and their structure-activity relationship and mechanism of action have been preliminarily analyzed and discussed,which lays a foundation for further study of these inhibitors.