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A new class of antimitotic agents, derivatives of 2-styrylquinazolin-4(3H)-one (SQZ), was recently described [J. Med. Chem. 33:1721-1728 (1990)]. Because they appeared to interact at a new ligand binding site on tubulin, we attempted to determine their mechanism of action as inhibitors of tubulin polymerization. Although in initial studies inhibition of colchicine binding was negligible, substantial and competitive inhibition of this reaction could be demonstrated with very short incubation times (less than 5 min), provided that a relatively low colchicine to tubulin ratio was used. The initial apparent failure to inhibit colchicine binding resulted from extremely rapid binding to tubulin and dissociation from tubulin by the SQZ derivatives, in comparison with the slow, temperature-dependent, poorly reversible binding of colchicine. The most inhibitory of the SQZ derivatives in the colchicine binding assay was 6-methyl-2-styrylquinazolin-4(3H)-one (NSC 379310), and its interaction with tubulin, particularly as an inhibitor of colchicine binding, was compared with that of 2-methoxy-5-(2',3',4'-trimethoxyphenyl)tropone (MTPT), because the binding parameters of MTPT with tubulin have been well described. The data indicate that NSC 379310 binds to tubulin and dissociates from the protein about 3 times as rapidly as MTPT. The other SQZ derivatives with equal or greater potency as inhibitors of tubulin polymerization but apparently less potency as inhibitors of colchicine binding presumably bind to and/or dissociate from tubulin even more rapidly than does NSC 379310.

A novel series of 2-styrylquinazolin-4(3H-ones which inhibited tubulin polymerization and the growth of L1210 murine leukemia cells was discovered. Extensive structure-activity relationship studies suggest that the entire quinazolinone structure was required, but activity was further enhanced by halide or small hydrophobic substituents at position 6. These analogues did not substantially interfere with the binding of radiolabeled colchicine, vinblastine, or GTP to tubulin and weakly stimulated GTP hydrolysis uncoupled from polymerization. Several analogues have shown in vivo tumor growth inhibitory activity in the L1210 leukemia model, with the lead compound 5o exhibiting good antitumor activity against murine solid tumors as well as human tumor xenografts.