Synthesis And Antitumor Effect Of Combretastatin A-4 Analogues Of Benzo[b]Furans

Objective: By analyzing the structure activity relationship of compound CA-4,synthesis of benzo[b]furan derivatives of CA-4 by chemical modification of its structure.The in vitro antitumor activity of these compounds was studied and molecular docking with tubulin(PDB: 1SA0)for analyzing binding manner with the colchicine binding site of tubulin.Methods:(1)The synthesis strategy was based on a alpha bromo reaction,iodine substitution reaction,classical Rap-stereomer reaction Sonogashira and Heck reaction,the formation of the target compound as a starting compound from 2-hydroxy-4-methoxybenzaldehyde and 2-bromo-1-(3,4,5-trimethox-yphenyl)ethanone.Synthesis of a series of analogues of CA-4,these are the ring B of CA-4 was replaced by benzo[b]furans and the double bond was replaced by carbonyl group,A ring was not replaced.(2)In vitro growth inhibitory of synthetic compounds on human lung cancer cell line A549,human Liver cancer cell line Bel-7404,human colon cancer cell line SW620 and human cervical cancer cell line HeLa by conventional MTT.(3)To characterize the mode of cell death induced by compound 12,we performed an Annexin V-APC/7-AAD Apoptosis Detection Kit.(4)Molecular docking towards the best active compounds 5 and 12 with tubulin by the software of autodock vina.Verify the activity of the compounds and analysis binding mode of compounds with the colchicine binding site of tubulin.Results:(1)In this study,ten benzo[b]furan derivatives of CA-4 were prepared,compound 5-13 are new compounds,yield was between 24.7%-83.6%.Their structure were confirmed by 1H NMR and 13 C NMR spectrosco,and new compounds also confirmed by HRM.(2)The compounds all have a certain inhibitory effect on four kinds of tumor cell,all of compounds showed higher activity for A549 and HeLa cells.The compounds 5-10 were aromatic alkyne derivative.Among them,5,6 and 10 showed higher activity than 7-11 and compound 5 had the best activity.Compound 11-13 had a good antitumor activity.Compound 12 showed higher activity than 11 and 13,it was as potent as CA-4 against these four tumor cell lines.IC50 were 0.14 and 0.12?M.Cell-cycle perturbations of HeLa cells that the percentage of cells(cell accumulation)at the G2/M phase increased from 11.5 to 79.65 % if the concentration of compound 12 was increased from 0.01 to 10?M,and showed dose dependency.To characterize the mode of HeLa cells death induced by compound 12,and the results indicated that the compound 12 significantly induce apoptosis in HeLa cells.When the compound 12 was administered 0.1?M,the results showed that the proportion of cells in the early and late stage of apoptosis was about 27.4%.When it was 1?M,the apoptosis was about 66.4%,and this accumulation was concentration dependent.(3)Molecular docking studies shows that the 3,4,5-trimethoxyphenyl group of 12 was competed with colchicine for combine with the A site of tubulin protein and displayed hydrogen-bonding interactions with two amino acid residues Gln-11 and Tyr-224 on the A chain of the tubulin.Compound 6 was not competed with colchicine for combine with the A site of tubulin protein and displayed hydrogen-bonding interactions with two amino acid residues GLN-11 and TYR-224 on the A chain and one amino acid residues GYS-254 on the B chain.Conclusion: The ten benzo[b]furans derivatives of CA-4 were synthesized successfully.Their structure were confirmed by 1H NMR and 13 C NMR spectrosco,and new compounds also confirmed by HRM.The ten derivatives were subjected to in vitro growth inhibitory assay against four tumor cells by conventional MTT method.Compound 12 showed highest activity and it was as potent as CA-4 against these four tumor cell lines.Further cycle-cell studies show that compound 12 can block He La cells in phase G2/M;The compound 12 significantly induce apoptosis in HeLa cells.The results of molecular docking showed that compound 12 combine with tubulin were more stable.