Novel 4,6-pyrimidine analogues were designed and synthesized as colchicine binding site inhibitors (CBSIs) with potent antiproliferative activities. Among them, compound 17j has the most potent activities against 6 human cancer cell lines with IC50 values from 1.1 nM to 4.4 nM, which was 76 times higher than the lead compound 3 in A549 cells. The co-crystal structure of 17j in complex with tubulin confirms the key binding mode at the colchicine binding site. Moreover, 17j inhibited the tubulin polymerization in biochemical assays, depolymerized cellular microtubules, induced the G2/M arrest, inhibited the cell migration, and promoted the initiation of apoptosis. In vivo, 17j effectively inhibits primary tumor growth with tumor growth inhibition rates of 42.51% (5 mg/kg) and 65.42% (10 mg/kg) in A549 xenograft model. Taken together, 17j represents a promising new generation of CBSIs.Copyright © 2023 Elsevier Masson SAS. All rights reserved.