Multi-Target Anticancer Activity of Structurally Diverse Schiff Bases: Insights into Cell-Cycle Arrest, DNA Damage, Metabolic Signaling, and Biomolecular Binding

Schiff bases are widely studied for their biological activities, yet structure–activity relationships governing their anticancer potential remain insufficiently understood. In this work, eight structurally diverse imine derivatives (A–H) were evaluated for their cytotoxic, biochemical, and biomolecular interactions in human cancer cells. Their antiproliferative effects were assessed in HeLa, A549, and LS174T cell lines, with MRC-5 fibroblasts used as a non-malignant control. Cytotoxicity screening identified three compounds (A, B, and F) with the highest potency, prompting further mechanistic investigation. Cell cycle analysis revealed G1 arrest and accumulation of sub-G1 populations for all three derivatives, with compound B additionally increasing S-phase content and compound F inducing G2/M arrest. All compounds reduced intracellular ROS levels and caused significant DNA damage at subtoxic concentrations. Western blot analysis demonstrated downregulation of HIF-1α and PDK3, suggesting disruption of hypoxia-associated metabolic signaling. Fluorescence quenching experiments showed strong binding of the active compounds to bovine serum albumin (Ka ≈ 106 M−1), and molecular docking supported stable interactions near tryptophan-adjacent binding regions. Collectively, these findings indicate that selected Schiff bases exert multi-target anticancer activity by modulating oxidative stress, DNA integrity, cell-cycle progression, and metabolic adaptation pathways, warranting further investigation of their therapeutic potential.