Design, Synthesis, <i>In vitro</i> , Molecular Docking and Simulation Assessment of Benzimidazole Derivatives Against Liver Cancer by Targeting STAT3, Bcl2 and MDM2

Hepatocellular carcinoma (HCC) is the most common primary liver cancer type and continues to be a significant global health problem because of late diagnosis and inadequate treatment response. In this study, a novel benzimidazole-based chalcone series was synthesized and tested for its anticancer activity on the liver HepG-2 cancer cell line. Compounds were prepared in a DMSO solution and screened for cytotoxicity using the MTT assay. Among them, 4e exhibited the strongest cytotoxicity (IC50 [Formula: see text] 6.68 ± 0.08 [Formula: see text]M), markedly inhibiting cell proliferation and migration, while showing no toxicity against human kidney cells HEK293. A wound healing (scratch) assay showed that cell migration was reduced upon treatment, indicating anti-metastatic activity. RT-qPCR analysis revealed significant downregulation of the key oncogenes Bcl2, STAT3 and MDM2, showing pro-apoptotic and anti-proliferative effects. Additionally, PASS predictions showed similar anticancer probabilities (Pa [Formula: see text] 0.5). Molecular docking studies demonstrated that cancer-related proteins had strong binding affinities to the compounds, forming stable ligand–protein complexes as confirmed by 100 ns molecular dynamics simulations using Desmond. Under physiological conditions, the ligand–protein complexes remained intact throughout the entire simulation, confirming the stability of the interactions. This integrative approach, which combines synthetic chemistry, in vitro testing, gene expression analysis and computational modeling, emphasizes the therapeutic potential of 4e as a promising benzimidazole derivative for future preclinical development against HCC.