Heterocyclic-Fused Pyrimidines as Novel Tubulin Polymerization Inhibitors Targeting the Colchicine Binding Site: Structural Basis and Antitumor Efficacy

We report the design, synthesis, and biological evaluation of heterocyclic-fused pyrimidines as tubulin polymerization inhibitors targeting the colchicine binding site with significantly improved therapeutic index. Additionally, for the first time, we report high-resolution X-ray crystal structures for the best compounds in this scaffold, <b>4a</b>, <b>4b</b>, <b>6a</b>, and <b>8b</b>. These structures not only confirm their direct binding to the colchicine site in tubulin and reveal their detailed molecular interactions but also contrast the previously published proposed binding mode. Compounds <b>4a</b> and <b>6a</b> significantly inhibited tumor growth in an A375 melanoma xenograft model and were accompanied by elevated levels of apoptosis and disruption of tumor vasculature. Finally, we demonstrated that compound <b>4a</b> significantly overcame clinically relevant multidrug resistance in a paclitaxel resistant PC-3/TxR prostate cancer xenograft model. Collectively, these studies provide preclinical and structural proof of concept to support the continued development of this scaffold as a new generation of tubulin inhibitors