A novel series of acetyl-β-D-thio-glycoside modified shikonin derivatives were designed and synthesized and investigated for inhibition of cell proliferation against MG63, MCF-7, B16-F10, HepG2, MDA-231, L02, VERO and MCF-10A cell lines. The biological study showed that most single, di- and tri-substituted shikonin derivatives exhibited better anti-proliferative activities against the five cancer cell lines but lower cytotoxic activity against normal cells than shikonin itself. Notably, compared to shikonin, IIb displayed much stronger anti-proliferative effect among them. Furthermore, the inhibition of tubulin polymerization results indicated that IIb showed the most potent anti-tubulin activity (IC50 = 4.67 ± 0.433 μM), which was compared with shikonin (IC50 = 16.8 ± 0.625 μM) and colchicine (IC50 = 3.83 ± 0.424 μM). Docking simulation, confocal microscopy and western bolt results further confirmed that IIb can cause cell arrest in G2/M phase and induce cell apoptosis via binding to the active site of tubulin and inhibiting tubulin polymerization.
