Unveiling Benzoxazole-Substituted Thiazolyl-Pyrazole Derivatives Inducing Apoptosis by Targeting β-Tubulin and Caspase-3

In the present study, the biological activities of novel compounds (BP-1 to BP-6), designed as antitubulin agents, were systematically evaluated, with a particular focus on their effects on the triple-negative breast cancer cell line MDA-MB-231 and non-cancerous cell line MCF-10A. BP-2 and BP-6 demonstrated micromolar-range antiproliferative activity against MDA-MB-231 cancer cells, with IC₅₀ values of 8 and 4 µM, respectively, comparable to nocodazole (3 µM), and showed selective cytotoxicity over normal MCF-10A cells, with selectivity indices of approximately 3.3 and 8. In vitro analyses revealed that BP-2 and more notably BP-6 significantly inhibited cell proliferation in a time- and dose-dependent manner, disrupted microtubule organization through the downregulation of β-tubulin expression, and induced apoptosis, as evidenced by increased levels of Cleaved Caspase-3 and distinct apoptotic morphological changes. Among the tested compounds, BP-6 exhibited the most potent antiproliferative and proapoptotic activity, with an IC₅₀ value close to that of NOC. Molecular docking supported these findings by showing strong binding affinities of BP-6 to both β-tubulin and Caspase-3, indicating a dual-targeted mechanism. Furthermore, molecular dynamics simulations confirmed the stable binding and dynamic integrity of BP-6 within both β-Tubulin and Caspase-3 targets, underscoring its potential as a robust candidate for anticancer drug development.