Design,Synthesis And Activity Study Of Indole Flavonoid Derivatives Modified By Salicylic Acid

Objective:To design a class of drugs that can simultaneously inhibit tumor tubulin polymerization and reduce the level of anaerobic glycolysis of tumor cells through the principle of molecular hybridization and pharmacophore assembly.Method:Design and synthesis of the target compound:Using3,4,5-trimethoxyphenol as the synthetic raw material,the structure 2chloroacetophenone was obtained by Friedel-Crafts acylation reaction,and indole-3-carbaldehyde/indole-5-carbaldehyde Cyclization to obtain intermediates 3 and 4（indole flavonoids）;salicylic acid and its derivatives undergo substitution reaction with thionyl chloride after methylation treatment to obtain intermediates 7a-7h;under the catalysis of Na H,Compounds 3 and 4 are respectively connected with intermediates 7a-7h through amide bonds to finally generate target compounds 8a-8h and 9a-9h.The structures of 18 compounds have been characterized and confirmed by¹H NMR,¹³C NMR and high resolution mass spectrometry.In vitro anti-tumor activity experiment:1.Select gastric cancer cells（HGC-27）,colon cancer cells（HCT-116）,breast cancer cells（MDA-MB-231）,liver cancer cells（HEPG-2,SMMC-7721）and normal Liver cells（L-02）were used as the research object,and CA4,sodium citrate dihydrate,5-fluorouracil（5-FU）were used as positive control drugs,and 18 new types of salicylic acid were synthesized by MTT colorimetric method.The anti-proliferative activity of the indole flavonoid derivatives of serotonin was screened for the preliminary screening of tumor cells;2.The representative compound 8f at different concentrations（0,1.5μM,3μM,6μM）was detected against SMMC-7721 of liver cancer cells by observing the formation of cancer cell colonies.anti-proliferative activity;3.Use flow cytometry to analyze the effects of various concentrations of 8f on the cell cycle and apoptosis of SMMC-7721;4.Observe the effects of different concentrations of compound 8f on intracellular microtubules by immunofluorescence The influence of protein and cell nucleus;5.Exploring the position of compound 8f in tubulin and its binding to tubulin through computer molecular simulation docking technology;6.Analyzing the effect of compound 8f on tumor cell anaerobic glycolysis through the determination of lactic acid content The impact of solutions.Results:Designed and synthesized 18 new indole flavonoid derivatives modified by salicylic acid.The compound 8f was preliminarily screened out by MTT colorimetry as the compound with the most anti-hepatocarcinoma cell proliferation activity;subsequent colony formation experiments were further verified The excellent anti-proliferative activity of compound 8f;flow cytometry confirmed that compound 8f can induce SMMC-7721 cell apoptosis in a concentration-dependent manner;immunofluorescence results show that compound 8f can destroy the morphology of microtubules and inhibit the polymerization of tubulin,Prevent the construction of a complete microtubule network;through computer molecular docking simulation experiments,it is observed that compound 8f is embedded in the binding pocket of tubulin,and compared with colchicine,8f can form more hydrogen bonds with surrounding amino acid residues,Seems to bind more tightly with tubulin.The determination of lactic acid content confirmed that compound 8f can down-regulate the lactic acid content in liver cancer cells,indicating that compound 8f can effectively reduce the level of anaerobic glycolysis in liver cancer cells.Conclusion:Compound 8f can not only inhibit the polymerization of tubulin in liver cancer cells SMMC-7721,but also down-regulate the level of anaerobic glycolysis in liver cancer cells.It may be a dual anti-tumor drug with great potential.