Design, Synthesis and Biological Investigation of 2-Anilino Triazolopyrimidines as Tubulin Polymerization Inhibitors with Anticancer Activities

Abstract

A further investigation aiming to generate new potential antitumor agents led us to synthesize a new series of twenty-two compounds characterized by the presence of the 7-(3′,4′,5′-trimethoxyphenyl)-[1,2,4]triazolo[1,5-a]pyrimidine pharmacophore modified at its 2-position. Among the synthesized compounds, three were significantly more active than the others. These bore the substituents p-toluidino (3d), p-ethylanilino (3h) and 3′,4′-dimethylanilino (3f), and these compounds had IC50 values of 30–43, 160–240 and 67–160 nM, respectively, on HeLa, A549 and HT-29 cancer cells. The p-toluidino derivative 3d was the most potent inhibitor of tubulin polymerization (IC50: 0.45 µM) and strongly inhibited the binding of colchicine to tubulin (72% inhibition), with antiproliferative activity superior to CA-4 against A549 and HeLa cancer cell lines. In vitro investigation showed that compound 3d was able to block treated cells in the G2/M phase of the cell cycle and to induce apoptosis following the intrinsic pathway, further confirmed by mitochondrial depolarization and caspase-9 activation. In vivo experiments conducted on the zebrafish model showed good activity of 3d in reducing the mass of a HeLa cell xenograft. These effects occurred at nontoxic concentrations to the animal, indicating that 3d merits further developmental studies.

Keywords

• colchicine binding site
• antitumor activity
• [1,2,4]triazolo[1,5-<i>a</i>]pyrimidine
• apoptosis
• microtubule-targeting agents