Design, synthesis, and cytotoxicity evaluation of 1,2,3-triazole-linked β-carboline–benzimidazole hybrids as tubulin polymerization inhibitors

In pursuit of novel chemotherapeutic agents, a new series of 3-((4-((1H-benzo[d]imidazol)methyl)-1H-1,2,3-triazol-1-yl)methyl)-9H-pyrido[3,4-b]indole derivatives 14a–s and 15a–d were rationally designed and synthesized. The design incorporates key pharmacophoric motifs such as β-carboline–benzimidazole to enhance cytotoxic potential. In vitro evaluation revealed that several derivatives exhibited potent cytotoxic effects, with IC₅₀ values below 10 μM against multiple human cancer cell lines. Notably, compounds 14d and 14o demonstrated the highest activity against MCF-7 (breast cancer) cells, with an IC₅₀ of 5.61 ± 1.24 μM and 6.84 ± 1.53 μM, accompanied by high selectivity toward malignant over normal cells. Apoptosis assays confirmed classical hallmarks of programmed cell death, including apoptotic body formation, nuclear condensation and fragmentation, membrane blebbing, and horseshoe-shaped nuclei, alongside elevated intracellular ROS levels. Flow cytometry further established that 14d and 14o induced early apoptosis and triggered significant G2/M phase arrest. Enzyme-based assays revealed that both 14d and 14o inhibited tubulin polymerization with IC₅₀ values of 7.63 μM and 8.77 μM, respectively, implicating disruption of microtubule dynamics as the primary mechanism of action. Molecular docking studies corroborated these findings, showing favorable binding of 14d and 14o within the tubulin active site. Moreover, molecular dynamics simulations (100 ns) confirmed the stability of the 14d–tubulin complex, with RMSD values consistently within the optimal 2–4 Å range, indicating a durable interaction. Collectively, these results highlight compound 14d as a promising lead scaffold for the development of next-generation anticancer therapeutics targeting microtubule function.