Synthesis, characterization, and biological evaluation of aliphatic-substituted benzimidazole derivatives: induction of apoptosis, cell cycle arrest, and molecular docking in breast cancer cells

Abstract

A new series of aliphatic-substituted benzimidazole derivatives was synthesized and structurally characterized to evaluate their potential anticancer activity. Among the synthesized compounds, compound 4 exhibited the most potent cytotoxic effects against MCF-7 and MDA-MB-231 breast cancer cell lines, with IC₅₀ values comparable to those of cisplatin, while displaying lower toxicity toward normal breast epithelial cells (MCF-10A). Flow cytometric analysis revealed that treatment with compound 4 resulted in significant accumulation of cells in the S phase, indicating inhibition of DNA synthesis and replication. Furthermore, Annexin V/PI double-staining analysis demonstrated a marked increase in both early and late apoptotic cell populations, confirming the activation of apoptotic pathways. Molecular docking studies supported these experimental findings by revealing strong interactions of compound 4 with key regulatory proteins involved in apoptosis and cell cycle progression, including Bcl-2, Bcl-xL, CDK2, and Cyclin E. The compound exhibited the highest binding affinity toward CDK2 (–164.055 kcal/mol), forming hydrogen bonds with critical residues (LEU134, ASP145, GLN131, and LYS33) within the ATP-binding pocket, suggesting potential inhibition of kinase activity. Interactions with Bcl-2 and Bcl-xL occurred within the BH3-binding grooves, which may impair their anti-apoptotic functions and promote mitochondrial-mediated apoptosis. Collectively, the in vitro and in silico results indicate that this newly synthesized benzimidazole derivative exerts its anticancer effects through a dual mechanism involving cell cycle arrest and apoptosis induction. The selective cytotoxicity and multitarget interaction profile of compound 4 highlight its potential as a promising lead compound for the development of novel therapeutic agents against breast cancer.