Design,synthesis And Anti-angiogenesis Study Of A New Type Of VEGFR-2 Inhibitor Containing 1,2,3-triazole Structure

Objective:As an important receptor of VEGF,VEGFR-2 can initiate downstream signal transduction,regulate endothelial cell proliferation,migration and angiogenesis in vivo after being activated by VEGF.Studies have found that VEGFR-2 is highly expressed in a variety of malignant tumors,and the inhibition of VEGFR-2 will result in the inhibition of angiogenesis in tumor tissues,which will lead to tumor cell death.Therefore,VEGFR-2 has always been an ideal target for tumor drug development,with a novel development structure.VEGFR-2 inhibitors with high selectivity and low toxic and side effects are of great significance in cancer treatment.Methods:In this paper,a series of novel VEGFR-2 inhibitors,namely（Z）-3-（4-（1-phenyl-1H-1,2,3-triazol-4-yl）benzylidene）indole-2-one compounds,were designed and synthesized by using indole-2-one as the parent nucleus of the lead compound and based on the analysis and summary of its mechanism of action.In this paper,a synthetic route with easy availability of low cost and high yield was designed.Firstly,different substituted anilines were used as starting materials to obtain substituted indole-2,3-dione（D9-D10）by the synthesis of Isatin by Sandmeyer,and then the substituted indole-2-one（D11-D12）was obtained by reduction.Then,different substituted anilines were diazotized and reacted with sodium azide to obtain various substituted azide benzenes.Then,the key intermediate 4-（1-phenyl-1H-1,2,3-triazol-4-yl）benzaldehyde（D4a-D4n）was obtained by click reaction with 4-acetylbenzaldehyde.Finally,intermediate（D4a-D4n）and intermediate（D11-D12）undergo nucleophilic substitution reaction to obtain the target compound（D13a-D14n）.The structures of the target compounds were characterized by ¹H NMR,¹³C NMR,and HRMS.With sunitinib as a positive control drug,through VEGFR-2 kinase experiment in vitro,and three cell lines,HT-29,MKN-45,and HUVECs,were selected as test cell lines,and CCK-8 method was used to evaluate the antiproliferative activity of the synthesized target compound.The inhibitory effect of compound D13d on HUVECs migration was investigated by Transwell assay.The effect of compound D13d on tubule formation length of HUVECs was verified by tubule formation experiment.The effect of compound D13d on the phosphorylation of VEGFR-2 on HUVECs membrane was analyzed by Western blotting.The inhibitory effect of compound D13d on angiogenesis in vivo was verified by transgenic zebrafish.Results and Conclusion:（1）Twenty-eight indole-2-ones with 1,2,3-triazole structure were designed and synthesized,namely（Z）-3-（4-（1-phenyl-1H-1,2,3-triazole-4-yl）benzylidene）indole-2-ones,all of which have not been reported in the literature.（2）Most of the synthesized target compounds showed good inhibitory activity in VEGFR-2 kinase detection,among which compound D13d had an excellent inhibitory effect on HT-29 and MKN-45 cells and low toxicity to human umbilical vein endothelial cells（HUVECs）.（3）Transwell results showed that compound D13d could effectively inhibit the migration of HUVECs in vitro.In the tubule formation length experiment,the tubule formation length of HUVECs decreased with the increase of compound D13d concentration.Western blotting results showed that the phosphorylation level of VEGFR-2 in HUVECSs decreased with the increase of compound D13d concentration.（4）In vivo angiogenesis experiment,zebrafish model labeled with VEGFR-2confirmed that compound D13d has higher anti-angiogenesis ability than sunitinib.