A Pyrimidine-Based Tubulin Inhibitor Shows Potent Anti-Glioblastoma Activity in vitro and in vivo

Background: Glioblastoma (GBM) presents as an aggressive and treatment-resistant brain tumor with few effective therapies. We targeted the colchicine-binding site on tubulin—a validated but underutilized site—to develop novel small-molecule inhibitors as alternatives to temozolomide (TMZ), the current standard treatment. Methods: We synthesized a focused library of pyrimidine- and dihydroquinoxalinone-based analogs and tested nine compounds for cytotoxicity in GBM cell lines using the Sulforhodamine B (SRB) assay. We identified compound 4 as the most promising compound and evaluated its effects on cell growth using live-cell imaging, assessed apoptosis via cell death ELISA, and tested its anticancer activity in GBM xenografts in vivo. Results: Several compounds demonstrated nanomolar IC₅₀ values and outperformed TMZ. We identified compound 4—a pyrimidine analog with a secondary amine—as the lead candidate. Compound 4 reduced cell viability in a dose-dependent manner and induced complete growth arrest within 48 hours at concentrations as low as 3–10 nM in LN229 and MT330 GBM cell lines. ELISA confirmed that compound 4 triggered dose-dependent apoptosis, whereas TMZ failed to induce apoptosis even at micromolar concentrations. In vivo, compound 4 significantly inhibited GBM xenograft growth in immunocompromised mice by 66%. Conclusions: Compound 4 acts as a potent tubulin inhibitor with strong anti-GBM activity and merits further preclinical development.