CB694, a novel antimitotic with antitumor activities

During the course of a mechanism-based screening program aimed at identifying new antimitotic agents, a novel microtubule depolymerizing piperazine derivative, 1-(5-chloro-2-methoxybenzoyl)-4-(3-chlorophenyl) piperazine, was identified. The compound, designated CB694, caused inhibition of proliferation of a wide range of cancer cell lines, with an average IC ₅₀ of 85 nM. A multidrug-resistant cell line was sensitive to inhibition by CB694, suggesting that this compound is a poor substrate for transport by P-glycoprotein. CB694 caused formation of abnormal mitotic structures in HeLa cells. Specifically, CB694 caused a concentration-dependent increase in bipolar spindles with lagging chromosomes and, with slightly higher concentrations, formation of multipolar mitotic spindles. These mitotic abnormalities occurred at concentrations that did not cause significant changes in the appearance or quantity of interphase microtubules. Coincident with the formation of abnormal mitotic spindles, CB694 caused G₂/M arrest. CB694 inhibited the assembly of purified tubulin with an IC₅₀ of 2.3 μM. Colchicine binding was strongly inhibited by CB694, suggesting that it binds to tubulin at the colchicine site. Bcl-2 phosphorylation and activation of ERK and JNK and caspase 3-dependent cleavage of PARP were observed in MDA-MB-435 cells treated with CB694. CB694 caused phosphorylation of Aurora A within 8 hr of treatment, and increases in Aurora A protein levels were coincident with mitotic accumulation. The efficacy of CB694 against a syngeneic murine transplantable solid tumor, Mammary 16/C, was also evaluated. CB694 was well tolerated and showed antitumor activity.