Based on our prior antitumor hits, 32 novel N-alkyl-N-substituted phenylpyridin-2-amine derivatives were designed, synthesized and evaluated for cytotoxic activity against A549, KB, KB(VIN), and DU145 human tumor cell lines (HTCL). Subsequently, three new leads (6a, 7g, and 8c) with submicromolar GI(50) values of 0.19-0.41 μM in the cellular assays were discovered, and these compounds also significantly inhibited tubulin assembly (IC(50) 1.4-1.7 μM) and competitively inhibited colchicine binding to tubulin with effects similar to those of the clinical candidate CA-4 in the same assays. These promising results indicate that these tertiary diarylamine derivatives represent a novel class of tubulin polymerization inhibitors targeting the colchicine binding site and showing significant anti-proliferative activity.