Polymerization of
tubulin dimers to form microtubules is one of the key events in cell proliferation. The inhibition of this event has long been recognized as a potential treatment option for various types of
cancer. Compound 1e was previously developed by our team as a potent inhibitor of
tubulin polymerization that binds to the
colchicine site. To further improve the potency and therapeutic properties of compound 1e, we hypothesized based on the X-ray crystal structure that modification of the
pyrimidine dihydroquinoxalinone scaffold with additional hetero-atom (N, O, and S) substituents could allow the resulting new compounds to bind more tightly to the
colchicine site and display greater efficacy in
cancer therapy. We therefore synthesized a series of new
pyrimidine dihydroquinoxalinone derivatives, compounds 10, 12b-c, 12e, 12h, and 12j-l, and evaluated their cytotoxicity and relative ability to inhibit proliferation, resulting in the discovery of new
tubulin-polymerization inhibitors. Among these, the most potent new inhibitor was compound 12k, which exhibited high cytotoxic activity in vitro, a longer half-life than the parental compound in liver microsomes (IC50 = 0.2 nM, t 1/2 = >300 min), and significant potency against a wide range of
cancer cell lines including those from
melanoma and breast, pancreatic, and
prostate cancers. High-resolution X-ray crystal structures of the best compounds in this scaffold series, 12e, 12j, and 12k, confirmed their direct binding to the
colchicine site of
tubulin and revealed their detailed molecular interactions. Further evaluation of 12k in vivo using a highly
taxane-resistant
prostate cancer xenograft model, PC-3/TxR, demonstrated the strong
tumor growth inhibition at the low dose of 2.5 mg/kg (i.v., twice per week). Collectively, these results strongly support further preclinical evaluations of 12k as a potential candidate for development.