In vitro activity: Combretastatin A4 inhibits the growth of MDA-MB-231, A549, Hela, HL-60, SF295, HCT-8, MDA-MB435, PC3M, OVCAR-8, NCI-H358M, and lymphocyte cells with IC50 of 2.8, 3.8, 0.9, 2.1, 6.2, 5.3, 7.9, 4.7, 0.37, 8, and 3.2 nM, respectively. 1 μM Combretastatin A4 inhibits tubulin polymerization by 35%, and 10 μM nearly completely blocks tubulin polymerization. Combretastatin A4 demonstrates great relative binding capacity, reaching 78% of colchicine binding

Kinase Assay: Colchicine (1.2 μ M) is incubated with tubulin (1.3 mg/mL) in the incubation buffer (80 mM PIPES, 2.0 mM MgCl2, 0.5 mM EGTA, pH 6.9) at 37℃ for 1 h. Varying concentrations (0.1 – 125 μ M) of Combretastatin A4 are used to compete with colchicine originally bound to tubulin. After incubation, the filtrate is obtained. The ability of the analogue to inhibit the binding of colchicine is expressed as a percentage of control binding in the absence of any competitor.

Cell Assay: MDA-MB-231, A549, and HeLa cells are grown in DMEM medium (115 units/mL of penicillin G, 115 μg/mL of streptomycin, and 10% fetal bovine serum). Cells are seeded in 96-well plates (5000 cells/well) containing 50 μL of growth medium for 24 h. After medium removal, 100 μL of fresh medium containing individual analogue compounds at different concentrations is added to each well and incubated at 37 ℃ for 72 h. After 24 h of culture, the cells are supplemented with 50 μL of analogue compounds dissolved in DMSO (less than 0.25% in each preparation). After 72 h of incubation, 20 μL of resazurin is added for 2 h before recording fluorescence at 560 nm (excitation) and 590 nm (emission) using a Victor microtiter plate fluorimeter. The IC50 is defined as the compound concentration required to inhibit cell proliferation by 50% in comparison with cells treated with the maximum amount of DMSO (0.25%) and considered as 100% viability.