Synthesis and biological evaluation of polymethoxy homoisoflavanones as tubulin polymerization inhibitors

Microtubules have long been considered an ideal target for anticancer drugs because of the essential role they play in mitosis, forming the dynamic spindle apparatus. In this work, we reported the discovery of a novel series of homoisoflavonoids analogues bearing 3-benzylidene-6,7,8-trimethoxy-chroman-4-one moiety as tubulin polymerization inhibitors. The cytotoxic activity of the synthesized compounds was evaluated against two human cancer cell lines including A-375 and HL-60. Among them, compound B6 was identified as a potent tubulin polymerization inhibitor with effective anticancer potency, which demonstrated significant cytotoxic activity in vitro with IC₅₀ values below 200 nM. Notably, it displayed remarkable inhibitory potency on A375 (IC₅₀ = 90 nM) melanoma cancer cells, which surpassed those of CA-4 inhibitor. Mechanism explorations demonstrated that compound B6 inhibited tubulin polymerization by targeting the colchicine binding site, thereby disrupting the microtubule network in A375 cancer cells. Molecular docking studies of B6 into the colchicine-binding site of tubulin displayed possible mode of interaction between this compound and tubulin. Most anti-microtubule drugs inhibit cancer cell growth and proliferation by inducing cell cycle arrest during interphase, particularly at the G2/M phase. Additionally, the flow cytometry analysis of A375 and SK-MEL-28 cell lines treated with compound B6 showed that B6 effectively suppress colony formation, induce G2/M phase arrest, and promote apoptosis in A375 cancer cells, as well as regulate the expression levels of cell cycle and apoptosis-related proteins. Finally, the results of in vivo experiments indicated that the treatment with B6 could significantly inhibit tumor growth in a dose-dependent manner. Notably, the tumor weight growth inhibition (TGI) of B6 at 10 mg/kg was higher than that of CA-4 (15 mg/kg) and no significant body weight loss was observed for any of the treatments. All these results demonstrated that B6 showed in vivo anticancer activity and might be a promising hit compound.