In vitro activity: D-64131, an aroylindole analog, is a novel, oral and potent inhibitor of tubulin polymerization. As a mitotic inhibitor, D-64131 competitively binds to the colchicine binding site of αβ-Tubulin. It displayed high cytotoxicity of IC(50) = 20 to 75 nM against the human HeLa/KB cervical, SK-OV-3 ovarian, and U373 astrocytoma carcinoma cell lines. The inhibition of proliferation correlated with the arrest in the G2/M phase of the cell cycle. D-64131 prevents growth of tumor models in mice following oral administration in vivo. D-64131 has significant potential to be used for cancer treatment.

Tubulin Binding Assay: The tubulin binding assay was performed according to Tahit et al. using biotin-labeled tubulin, streptavidin-coated yttrium SPA beads, and [3H]colchicine (1 mCi/ml; specific activity, 76.5 Ci/mmol). Briefly, the binding mixture includes 0.08 μm [3H]colchicine, 1 mm GTP, and 0.5 μg of biotin-tubulin in G-PEM buffer, pH 6.9 (80 mm PIPES, 1 mm MgCl2, 1 mm EGTA, and 5% glycerol) in 100-μl final volume. The test compound and [3H]colchicine were added before tubulin. After incubation at 37°C for 2 h, 20 μl of SPA beads (80 μg in P-GEM buffer) were added. After further incubation for 30 min under agitation at room temperature, the SPA beads were allowed to settle down for 45 min, and scintillation counting was done on a MicroBeta Trilux counting device.

Cell Assay: D-64131 prevents growth of tumor models in mice following oral administration in vivo. D-64131 was freshly dissolved in DMSO and subsequently diluted with PBS containing 0.05% v/v Tween 80 to obtain a final DMSO concentration of 10%. Outbred nude mice, 6–8 weeks of age, of NMRI genetic background were used for all experiments. For the experiments, the human amelanoic melanoma MEXF 989 tumor xenograft model was chosen based on in vitroselectivity of D-64131 toward melanoma (data not shown) and engrafted from tumors in serial passage growing s.c. in nude mice. Fragments of ~25 mg were implanted s.c. in both flanks of the animals. Details of the animal experiments and data analysis were described by Mahboobi et al. The MEXF 989 tumor-bearing nude mice were treated p.o. with doses of 200 and 400 mg/kg/day on days 1–5, 8–9, and 15–18. In two independent experiments, the drug doses and treatment schedule used were determined as being well tolerated in non-tumor bearing nude mice before initiation of tumor experiments.