BACKGROUND Combretastatin A-4 (CA-4), which is an excellent antineoplastic agent, was isolated from Combretum caffrum. To date, structural modification studies of CA-4 have focused predominantly on the construction of new therapeutic agents for drug discovery. As a part of our ongoing work towards the modification of natural products, we have focused on the 3'-O-substituent groups in the B-ring of CA-4 under the hypothesis that these novel derivatives will possess good bioactivities and behave as effective antiproliferative pro-drugs. RESULTS A series of novel CA-4 derivatives, which contained a 3'-O-substituted carbonic ether moiety, were synthesized and evaluated for their antitumor activities against four tumor cell lines, including MDA-MB-231, MCF-7, K562 and A549 cells. These derivatives exhibited clear antitumor activities, and CA-4E, in particular, showed the highest bioactivity of all of the derivatives tested against all four tumor cell lines, with IC50 values in the range of 1 to 180 nM. Based on its high bioactivity, CA-4E was subsequently selected to investigate the antitumor mechanism of these synthetic compounds. The cell cycle results demonstrated that CA-4E induced time- and dose-dependent G2/M arrest in a similar manner to CA-4, although its effect was more powerful than that of CA-4, and the apoptosis data showed that CA-4E induced cellular apoptosis in a dose-dependent manner. CONCLUSIONS The newly synthesized CA-4 derivatives exhibited good antitumor activities in vitro, with CA-4E, in particular, showing the highest bioactivity of all of the compounds tested. Furthermore, CA-4E induced time- and dose-dependent G2/M arrest and cellular apoptosis in a dose-dependent manner. Taken together, these results suggest that CA-4E should be subjected to further investigation as a potential anticancer drug candidate.