Through the syntheses of its C-1 desvinyl, C-7 methylene, C-7 exocyclic ethylidene, and various C-3 phenylmethyl analogues, the structure−activity relationship of antimitotic ottelione A (4) against tubulin and various cancer cells was established. The results indicated that compound 4 was a colchicine-competitive inhibitor and that the C-1 vinyl group is unnecessary for its potency, whereas the C-7 exocyclic double bond is essential, possibly because of its irreversible interaction with tubulin. Further optimization of the substituents on the phenylmethyl group at the C-3 position generated compound 10g with a 3′-fluoro-4′-methoxyphenylmethyl substituent, which was 6−38-fold more active against MCF-7, NCI-H460, and COLO205 cancer cells relative to 4. Results from in vitro tubulin polymerization assay confirmed the potency of compounds 4, 10g, and 11a. KEYWORDS: ottelione A, antimitotic, microtubule, tubulin, anticancer A fter the success of antimitotic taxanes and vinca alkaloids as anticancer drugs, 1,2 antimitotic agents that target the colchicine-binding site to inhibit tubulin polymerization have attracted much attention. Many structurally diverse compounds binding to the colchicine-binding site have been discovered from natural or synthetic sources. 3,4 Despite no compound of this class having been approved for clinical use, the prototype colchicine (1, Figure 1) has been used to treat gout for many years.5 Combretastatin A-4 (2) 6−9 and ABT-751 (3) 10,11 in Figure 1 are representative examples of colchicine-competitive inhibitors currently in clinical trials to treat various cancers.Ottelione A (4, Figure 1), isolated from the fresh water plant Ottelia alismoides collected in the Nile Delta, 12 is among the most potent natural products that possess in vitro antiproliferative activity. Its IC 50 values lie in the pM−nM range against a panel of 60 human cancer cell lines through acting as a colchicine-competitive inhibitor. 12,13 Because of its promising antitumor activity and intriguing core structure with four stereogenic centers, several synthetic groups have investigated the total synthesis of 4, 14−21 but no analogue has been reported and assayed for anticancer activity, perhaps because of the many steps in its preparation.We already achieved an enantioselective total synthesis of 4 using our α-carbonyl radical cyclization method. 21 The bicyclo[4.3.0]nonane core structure was constructed via radical cyclization. The C-3 3′-hydroxy-4′-methoxyphenylmethyl group, C-1 vinyl group, and C-7 exocyclic double bond were then sequentially introduced. We thus envisaged that our scheme could be adaptable for the preparation of various analogues of 4, which would be useful to establish its structure−activity relationship.As shown in Scheme 1, we first prepared compounds 10a−g and 11a−c according to our scheme for the total synthesis of 4. For economy, these compounds were all synthesized in their racemic forms. Compound 5, prepared via α-carbonyl radical cyclization, 21 served as the starting material. H...