The goal of this project is the development of new antineoplastic agents directed against tubulin, a protein critical for cell division and for the maintenance of cellular morphology. Detailed mechanistic and structure-- activity studies were performed with seven classes of compounds. Combretastatins A-2 and A-4 were found to bind to tubulin rapidly and reversibly, but with high affinity, at the colchicine site, and studies to determine the optimal bridge length between phenyl rings were initiated. The interaction with tubulin of the pentapeptide dolastatin 10, a noncompetitive inhibitor of vinca alkaloid and nucleotide binding, was defined with great precision based on properties of stereoisomers and segments of the active molecule. Mechanistic studies were initiated with dolastatins 11, 14, and 15, which do not appear to interact with tubulin in vitro but which disrupt the function of cellular microtubules. Structure-activity and mechanistic studies were continued or initiated with analogs of colchicine, 2-methoxy-5-(2',3',4'trimethoxyphenyl)tropone, derivatives of 2-strylquinazolin-4-one (SQZ), and derivatives of 5,6-diphenylpyridazin-3-one (DPP). The SQZ derivatives were defined as compounds binding rapidly and reversibly, but with low affinity, at the colchicine site. The DPP derivatives appear to bind at a unique binding site on tubulin. The evaluation of the mechanism of action of the antimitotic agent 2,4-dichlorobenzyl thiocyanate was completed with the demonstration that it formed a mixed sulfhydryl with 8-tubulin by alkylating cysteine residue 239.
