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.