Derivatives of colchicine and the bicyclic colchicine analog Z-methoxy-S-(2',3',4' trimethoxyphenyl)tropone were tested for inhibition of tubulin polymerization. The nature of the tropone substituent had little effect on the efficacy of the colchicine series, with some exceptions. In contrast, the potency of the bicyclic analogs varied greatly with the tropone substituent.Tubulin is the target protein for a variety of antimitotic drugs, which possess a wide range of therapeutic utilities. A number of these agents (including colchicine, steganacin, podophyllotoxin, and combretastatin) exert their biological effects by binding to a single site on the tubulin heterodimer (M, = 100 kdal) which has come to he known as the colchicine binding site. t While the colchicine binding site appears to accommodate a diversity of structures, it is also remarkably sensitive to changes in the tropone C ring of colchicine. Colchicine itself binds to tubulin with moderately high affinity (K, = lo6 - 10' M-t at 37' C)', while isocolchicine, a colchicine analog in which the tropone methoxy and carbonyl substituents are interchanged, binds with 1000 fold less affinity than colchicine. 3 When the C ring of colchicine is a tropolone as in colchiceine, the molecule's affinity for the colchicine site on tubulin is also greatly decreased, and the molecule binds to tubulii at sites other than the colchicine binding site.'Colchicine is believed to interact with tubulin through up to three binding site subdomains: the A ring subdomain, which recognizes the trimethoxyphenyl portion of the molecule, the C ring subdomain, which accommodates the tropone portion of the molecule, and possibly a site related to the substitution pattern at the chiral C-7 on the B ring. 's6 The B ring and substituents are known to affect the kinetic' and spectroscopic features" of colchicinoids binding to tubulin, but in colchicine analogs investigated to date the effect of the B ring on the ligand's binding affinity appears to be minor (l-2 kcal/mol).7-9 In this work we have examined the role of the tropone substituent in colchicine and bicyclic colchicine analogs binding to tubulin. Our results show that the colchicine binding site can accommodate relatively diverse tropone substituents in the colchicine series, but only a more limited range of structures in the absence of the B ring. These results support the hypothesis that the B ring and/or C-7 substituent contribute positively to the affinity of colchicinoids for tubulin, and define the characteristics of the optimal tropone substituent for high affinity biding to tub&.