Optimization of 4‑(<i>N</i>‑Cycloamino)phenylquinazolines as a Novel Class of Tubulin-Polymerization Inhibitors Targeting the Colchicine Site

The 6-methoxy-1,2,3,4-tetrahydroquinoline moiety in prior leads 2-chloro- and 2-methyl-4-(6-methoxy-3,4-dihydroquinolin-1­(2<i>H</i>)-yl)­quinazoline (<b>1a</b> and <b>1b</b>) was modified to produce 4-(<i>N-</i>cycloamino)­quinazolines (<b>4a</b>–<b>c</b> and <b>5a</b>–<b>m</b>). The new compounds were evaluated in cytotoxicity and tubulin inhibition assays, resulting in the discovery of new tubulin-polymerization inhibitors. 7-Methoxy-4-(2-methylquinazolin-4-yl)-3,4-dihydroquinoxalin- 2­(1<i>H</i>)-one (<b>5f</b>), the most potent compound, exhibited high in vitro cytotoxic activity (GI₅₀ 1.9–3.2 nM), significant potency against tubulin assembly (IC₅₀ 0.77 μM), and substantial inhibition of colchicine binding (99% at 5 μM). In mechanism studies, <b>5f</b> caused cell arrest in G2/M phase, disrupted microtubule formation, and competed mostly at the colchicine site on tubulin. Compound <b>5f</b> and N-methylated analogue <b>5g</b> were evaluated in nude mouse MCF7 xenograft models to validate their antitumor activity. Compound <b>5g</b> displayed significant in vivo activity (tumor inhibitory rate 51%) at a dose of 4 mg/kg without obvious toxicity, whereas <b>5f</b> unexpectedly resulted in toxicity and death at the same dose