Conformationally constrained 6‑aryl‑1‑(3,4,5‑trimethoxyphenyl)-1H-pyrazolo[3,4‑b]pyrazines as novel microtubule destabilizers: design, synthesis, and antitumor evaluation.

A novel class of 6-aryl-1-(3,4,5-trimethoxyphenyl)-1H-pyrazolo[3,4-b]pyrazine derivatives was designed and synthesized as tubulin polymerization disruptors. Among them, compound 10t emerged as the most potent agent, exhibiting IC₅。 values of 0.13-0.18 µM against HeLa, SGC‑7901, and MCF‑7 cancer cell lines. Functional studies revealed that 10t effectively inhibits tubulin assembly in vitro, destabilizes cellular microtubule networks, induces G₂/M cell‑cycle arrest, and triggers apoptosis. Molecular docking indicated that 10t binds to the colchicine site on tubulin, forming a unique hydrogen bond with β‑Asn349 that is not observed with the reference compound CA‑4. Importantly, 10t showed significantly lower cytotoxicity toward normal HUVECs than CA‑4, suggesting an improved safety profile. Collectively, these findings establish the pyrazolo[3,4‑b]pyrazine scaffold as a promising platform for microtubule‑targeting anticancer agents and identify 10t as a compelling lead for further development.