Design,Synthesis And Anti-tumor Evalution Of 5,6,7-Trimethoxy Flavonoid Methyl Salicylate Derivatives

Objective:Designingandsynthesizingthe trimethoxyphenyl-containing flavonoid methyl salicylate derivatives based on the principle of pharmacophore combination,in order to screening for an excellent anti-tumor lead compounds that can inhibit tumor blood vessels and glycolysis,through the preliminary investigation of its anti-tumor activity and mechanism of actionMethods:The 5,6,7-trimethoxy-4'-hydroxyflavonoid were obtained through total synthesis with 3,4,5-trimethoxyphenol as raw material,and then a series of methyl salicylate derivatives were connected with 1,2-dibromoethane,1,3-dibromopropane,and1,4-dibromobutane,respectively.The obtained product were purified by silica gel column chromatography to obtain 24 novel target compounds.Their structures were characterized by the melting point measurement?MP?,nuclear magnetic resonance hydrogen spectrum?¹H NMR?,nuclear magnetic resonance carbon spectroscopy(¹³C NMR),and mass spectrometry?MS?.The anti-proliferative activity of all target compounds on against human gastric cancer cells?SGC-7901,MGC-803,HGC-27?,human colon cancer cells?HCT-116?,and human liver cancer cells?BEL-7402?and cytotoxicity to normal human gastric mucosal cells?GES-1?were determined by the tetramethyl azozolium blue colorimetric?MTT?method.Based on preliminary MTT assay results,the compound7f was screened for human gastric cancer cells?HGC-27?with the further research on antitumor activity and its mechanism of action.Including the wound healing assay and colony formation assay were used to determine the effect of the compound 7f on the HGC-27 cell colony formation and cell migration.Hoechst 33258 staining was used to qualitatively detect the effect of compound 7f on the apoptosis of HGC-27 cells.Flow cytometry was used to quantitatively determine the effect of compound 7f on the apoptosis and cycle of HGC-27 cells.The effect of compound 7f on the microtubule network morphology of HGC-27 cells was analyzed by immunofluorescence staining and molecular docking simulation experiments were performed to analyze the binding of compound 7f to tubulin.The lactate assay kit was used to determine whether the compound 7f could effectively reduce the lactic acid level of HGC-27cells.Further western blot analysis was used to detect the effects of compound 7f on the expression of glycolysis-related proteins HIF-1?,HK-2,PFK and PK and tumor angiogenesis-related proteins VEGF.Results:Successfully synthesized and identified 24 novel flavonoid methyl salicylate derivatives,among which compound 7f,5-Bromo-2-{3-[4-?5,6,7-trimethoxy-4-oxo-4H-chromen-2-yl?-phenoxy]-p ropoxy}-benzoic acid methyl ester exhibited excellent anti-proliferative activity against HGC-27 cells and MGC-803 cells with IC₅₀ values of10.26±6.94?M and 17.17±3.03?M,and had a better inhibitory effect than the positive control 5-FU(IC₅₀=17.11±2.64 and 45.51±13.31),respectively.The results of colony formation assay and wound healing assay proved that the compound 7f effectively inhibited the proliferation and migration of HGC-27 cells.Apoptosis and cell cycle results showed that compound 7f can inhibited the proliferation of HGC-27 cells by blocking HGC-27 cells at the G2/M phase and inducing apoptosis.The results of immunofluorescence analysis showed that compound 7f caused the microtubules of HGC-27 cells to aggregated or even disappeared,and the formation of cell spindles were reduced.The results of molecular docking simulation analysis showed that the binding site of compound 7f with tubulin was located between?1-tubulin and?1-tubulin subunits,and it formed obvious hydrogen bonding with multiple amino acid residues in the domain,which the way of binding tubulin is similar to colchicine.Lactic acid determination experiment results show that compound 7f effectively reduces the metabolite of anaerobic glycolysis lactic acid in HGC-27 cells.Further western blot analysis confirmed that compound 7f could effectively down-regulated the expression of glycolysis-related proteins HIF-1?,PFK and PK and tumor angiogenesis-related proteins VEGF.Conclusion:First,the concept of pharmacophore combination that simultaneously targets tumor vascular growth and glycolysis levels is correct and effective.Furthermore,compound 7f may be a potential tubulin inhibitor,and it can effectively reduce the glycolysis level of tumor cells by down-regulating the expression of glycolysis-related proteins in tumor cells,thereby achieving significant antitumor activity.