| Background and objectivesChemotherapy has always played a very important role in the treatment of tumors.However,due to the low selectivity of most chemotherapeutic drugs,it is easy to produce drug resistance and many unbearable side effects,resulting in the little effect of tumor treatment.Therefore,it is of great significance to develop drugs that are safe,effective,and highly selective for tumor cells.The development of novel tubulin polymerization inhibitors provides basis and lead compounds for the development of chemotherapeutic drugs.Methods1.Synthesis of compound 413: According to the principle of splicing,compound413 was obtained by chemical synthesis method.2.Evaluation of anti-tumor activity: The MTT assay was used to screen the antitumor activity of various human tumor cells to evaluate the anti-tumor activity of compound 413 in vitro and determine the in vitro anti-tumor model.3.Target verification: The tubulin polymerization experiment,immunofluorescence experiment,molecular docking experiment,and iodoacetamide experiment were conducted to verify the tubulin inhibitory activity of compound 413 and explore the binding sites on tubulin of compound 413.4.Research on the anti-tumor mechanism in vitro: The kinase spectrum selectivity evaluation experiment was used to evaluate the kinase selectivity of compound 413,and discuss the potential mechanism of action of compound 413.The western Blot assay was used to verify the inhibitory effect of compound 413 on the cell signaling pathway.The flow cytometry and Western Blot assay were used to investigate the apoptotic effect of compound 413 on tumor cells.The growth curve,clone formation assay,and flow cytometry were conducted to detect the antiproliferative effect of compound 413 on tumor cells.The scratch assay,Transwell assay,and Western Blot assay were carried out to investigate the effects of compound 413 on the migration and invasion of tumor cells.5.Research on the anti-tumor mechanism in vivo: By establishing an anti-tumor model in nude mice,investigating the anti-tumor activity of compound 413 in vivo,and studying the anti-tumor mechanism in vivo.Results1.The 1-methyl-1H-indole group is connected to the coumarin-2-one group through the N atom,and compound 413 is obtained by separation and purification.The structure is determined by mass spectrometry(MS),nuclear magnetic resonance(NMR),and infrared spectroscopy(IR).2.It was found that compound 413 generally has a strong inhibitory effect on tumor cells,and has the most significant effect on gastric cancer cells through the screening of tumor cell activity in vitro.What’s more,there was little toxicity on human gastric epithelial cells.3.Through the verification of the polymerization activity of compound 413,it was found that compound 413 has a strong inhibitory effect on the polymerization of microtubules and acted on the colchicine binding site of tubulin.4.In the study of anti-tumor activity in vitro,it was found that compound 413 can regulate the expression of gastric cancer cell MAPK signaling pathway-related proteins,induce gastric cancer cell apoptosis,inhibit gastric cancer cell proliferation,block gastric cancer cell cycle,and inhibit gastric cancer cell migration and invasion.5.The results of anti-tumor experiments in vivo showed that compound 413 could significantly inhibit tumor growth without obvious toxic effects,and the anti-tumor mechanism in vivo was consistent with that in vitro.Conclusions1.Compound 413 was obtained by chemical synthesis.2.Compound 413 generally has good anti-tumor activity against tumor cells in vitro,and has selective inhibitory properties against gastric cancer cells.3.Compound 413 exerts an anti-tumor effect by inhibiting the polymerization of tubulin and inhibiting the MAPK signaling pathway. |
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