Design,Synthesis And Antitumor Activity Of Cabozantinib Derivatives Containing Naphthyridine Structure

The binding of hepatocyte growth factor(HGF)to c-Met induces a series of effects,including cell proliferation,migration and angiogenesis,which are essential in normal physiology.However,abnormally highly expressed c-Met signaling pathway is related to a variety of human cancers,such as breast,liver,gastric cancer and so on.Therefore,c-Met has attracted great attention for use as an anticancer drug target,triggering a large number of researches and development of inhibitors pertaining to c-Met kinase.In this paper,we have designed three novel series of Cabozantinib derivatives containing naphthyridine fragment(95 compounds)and studied their antitumor activities in vitro,in view of the summary of the essential skeleton of TypeⅡ c-Met inhibitors.Based on the previous research of our group and reports of related literatures,small-molecular c-Met kinase inhibitors can be divided into either Type Ⅰ,Type Ⅱ or Type Ⅲ according to the molecular binding mode with c-Met kinase and whether they are ATP competitive inhibitors.Among them,c-Met inhibitors of Type Ⅱ are multi-target tyrosine kinase inhibitors with the characteristics of low drug resistance,low toxicity and stronger efficacy.In this paper,Cabozantinib was used as the control compound to analyze the basic active skeleton of c-Met inhibitors of Type Ⅱ.Combined with the above analyses,we designed and modified the compounds according to the principles of mosaic,bioisosterism and skeleton transition.First,the moiety A was introduced the active groups pyrrole[2,3-d]pyrimidine,pyrrole[2,3-d]pyridine and pyridinamide structures replaced by different flexible chains.Subsequently,the cyclopropane amide structure of Cabozantinib was replaced by the active fragment of naphthyridine onto the moiety C in order to preserve the intramolecular hydrogen bonding properties of Cabozantinib.In addition,the central and terminal benzene rings with different substituents were introduced into moiety B and moiety D.Finally,three series of Cabozantinib derivatives containing naphthyridine structure were designed and synthesized.Cabozantinib derivatives containing naphthyridine structure from ZJQ-1 to ZJQ-95 were evaluated for cytotoxicity using MTT method against three cancer cell lines.Among them,A549(lung cancer cells)and Hela(cervical cancer cells)cell lines with high and moderate expression of c-Met and MCF-7(breast cancer cells)with low expression of c-Met were selected to evaluate the in vitro cytotoxic activities test of those compounds.Cabozantinib was used as a positive control.Some compounds also were screened for toxicity test on L-O2(human normal hepatocytes cells).Based on the above activity data results,the further research on the promising compounds ZJQ-32,ZJQ-35 and ZJQ-95 mainly included the activity and selectivity of c-Met kinase,concentration dependence,acridine orange(AO)staining,annexin V-FITC/PI flow cytometry,fluorescence quantitative PCR analysis,molecular docking and molecular dynamics simulation.The results showed that compounds ZJQ-14,ZJQ-32,ZJQ-35,ZJQ-37～ZJQ-41 had better inhibitory activity than Cabozantinib against one or more cancer cell lines.Among them,12 compounds such as ZJQ-4,ZJQ-6 and ZJQ-14 and so on,showed low toxicity against L-O2 cell lines.The preferred compounds ZJQ-32 and ZJQ-35 were tested for c-Met kinase activity,the results of which showed that ZJQ-32 and ZJQ-35 had certain selectivity for c-Met kinase over related kinases(PDGFR-β,c-Kit,ALK and so on).The results of acridine orange(AO)staining experiments showed that compounds ZJQ-32,ZJQ-35 and ZJQ-95 could induce apoptosis with obvious effect and ZJQ-35 induced apoptosis in a concentration dependent manner.The annexin V-FITC/PI flow cytometry experiments showed that ZJQ-32 induced early cell apoptosis,while ZJQ-35 and ZJQ-95 induced late cell apoptosis in a concentration dependent manner and arrested cells in G0/G1 and G2/M phase.Through molecular docking and molecular dynamics simulation analysis,the results showed that ZJQ-35 and ZJQ-95 could form four key hydrogen bonds with c-Met kinase.And key amino acids Met1160,Phe1134 and Phe1223 played a major role in the binding free energy.In addition,the Electrostatic energy(EE energy)and van der Waals energy(VDW energy)significantly affected the binding free energy,the Non-polar solvation energy had a weak positive effect,while the Polar solvation energy had a negative effect.According to the summary of antitumor activity data,the structure-activity relationships of Cabozantinib derivatives were initial analysed.We found that the‘5-atom linker’ of compounds played a significant role in the biological activity,and this part had high affinity for the key amino acids of c-Met kinase.The naphthyridine having the possibility to form intramolecular hydrogen was introduced into the ‘5-atom linker’,the overall biological activity of compounds was more potent than that of Cabozantinib.Among them,the antitumor activity of compounds with pyridine was superior to other compounds with pyrrole[2,3-d]pyridine and pyrrole[2,3-d]pyrimidine as the parent core.In addition,the different substituents of terminal benzene ring had a strong influence on the cytotoxic activity,with potent antitumor activity when it was the electron withdrawing substituents.In conclusion,three series of 95 novel Cabozantinib derivatives containing naphthyridine fragment were obtained by structural optimization of Cabozantinib as a positive control.The preliminary analysis and investigation of antitumor activity,structure-activity relationships and mechanism of action in vitro of these compounds were provided an idea of modification for the study of c-Met inhibitors,and established a foundation for further research in the future.