Combretastatin A4-aminophosphate Hybrids:Design,Synthesis And Evaluation Of The Anti-HCC Activity In Vitro

Background:Microtubules are the basic skeleton of cells,which can control cell morphology,cell movement,mitosis,transport of vesicles and localization of membranous organelles through dynamic changes.As a tubulin inhibitor,CA4 binds to colchicine binding sites,has a very powerful anti-tumor effect,and can also destroy tumor blood vessels,which is a promising anti-tumor drug.But,it has problems such as insufficient activity in the body and easy to form tumor remnant circles.Studies have found that matrix metalloproteinases can affect the migration capacity of tumor cells,so the development of dual-target anti-tumor compounds that act on tubulin and matrix metalloproteinases may be effective in overcoming the shortcomings of CA4 itself.Objective: Combretastatin A4-aminophosphate hybrids was designed and synthesised.It was evaluated for biological activity and its structure-activity relationship was studied.Screening compounds with high activity and effective inhibition of tumor metastasis.Method: Different substituted aromatic aldehydes were reacted with aminophenylacetic acid,then treated with diethyl subphosphate,and a series of CA4 derivatives were synthesized by reaction with CA4 through esterification.Cell activity screening by MMT method selected high activity and selective compounds for subsequent experiments.The strength of the inhibition of tubulin polymerization was observed by tubulin polymerization and fluorescence experiments.The effects of the compounds on the cell cycle,cell apoptosis,reactive oxygen species,and mitochondrial membrane potential were analyzed using flow cytometry.The effect of the compounds on the expression levels of MMP2 and MMP9 protein in the cells was evaluated by Western blot.Cell scratch assay and infection assay to analyze the effects of candidate compounds on cell migration and invasion ability.Result: A novel series of Combretastatin A4-aminophosphate hybrids was designed and synthesized,in cytotoxic activity screening experiments,compound 15 g exhibited the strongest anti-proliferative activity against Hep G2 cells and a higher selectivity for LO2 in normal cells.Mechanism studies indicated that 15 g could inhibit tubulin polymerization,cause G2/M phase arrest,concurrent induce apoptosis via the mitochondrial dependent apoptosis pathway,and cause reactive oxidative stress generation in Hep G2 cells.Furthermore,15 g also exerted strong anti-metastasis activities by simultaneously reducing the protein level of MMP2 and MMP9 in Hep G2 cells.Conclusion: A series of novel conjugates comprising tubulin and MMPs inhibitors were designed,synthesized and evaluated for their antitumor activity.It shows that this coupling method can effectively and safely improve the ability of CA4 against HCC metastasis.