Structure-guided design of potent tetrazolo[1,5-a]pyrimidine-based tubulin inhibitors with in vivo antitumor activity.

Inhibitors targeting the colchicine-binding site of tubulin (CBSIs) represent a promising class of antitumor agents, offering the potential to overcome multidrug resistance. Through high-throughput virtual screening (HTVS) via molecular docking (Glide) followed by structural optimization, a novel hit compound Q1 (tetrazolo[1,5-a]pyrimidine scaffold; SKOV3 IC50 = 2.20 ± 0.05 μM) was identified from the ChemDiv library. Molecular dynamics (MD) simulations revealed that the cyclohexane moiety of Q1 exhibited high flexibility, which was identified as a key factor limiting its potency. Three stages of structural modification (Parts A-C) yielded 66 derivatives. The racemic lead compound rac-Q31 (hereinafter referred to as Q31 for brevity), which bears an optimized 3,5-dimethylphenyl group, exhibited potent antitumor activity with an IC50 of 24.6 ± 1.4 nM against SKOV3 cells. A co-crystal structure (PDB: 9LSE) confirmed that Q31 binds stably within the colchicine site, forming a hydrogen bond with α-Thr179 and key hydrophobic interactions, including those with α-Leu240 and β-Ile316. (S)-Q31 showed enhanced activity (SKOV3 IC50 = 17.2 ± 1.8 nM). In an SKOV3 xenograft model, (S)-Q31 exhibited significant antitumor efficacy (tumor growth inhibition, TGI = 74.12 % at 4 mg/kg, i.v.) with good tolerability. Mechanistically, (S)-Q31 inhibited microtubule polymerization, arrested the cell cycle at the G2/M phase, and induced apoptosis. This study establishes (S)-Q31 as a promising CBSI lead compound and provides structural insights for novel microtubule inhibitor development.