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 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 , 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 , , , , and , 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 , which exhibited high cytotoxic activity in vitro, a longer half-life than the parental compound in liver microsomes (IC = 0.2 nM, = >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, , , and , confirmed their direct binding to the colchicine site of tubulin and revealed their detailed molecular interactions. Further evaluation of 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 as a potential candidate for development.