Phenylalanine- and tryptophan-based hydrazone hybrids targeting the VEGF axis and caspase-mediated apoptosis: Design, synthesis, and biological evaluation

Lung Cancer remains a leading cause of cancer-related mortality worldwide, highlighting the urgent need for effective and selective therapeutic agents. In this study, novel phenylalanine and tryptophan-derived hydrazone hybrids (9a-h and 10a-h) against Lung Cancer were designed, synthesized and characterized via analytic and spectral methods (FT-IR, ¹H NMR, ¹³C NMR APT, and LC-MS/HR-MS). Cytotoxic activities of synthesized compounds were evaluated against human Non-Small Cell Lung Cancer (NSCLC) (A549 and PC9) cells and human healthy lung epithelial BEAS-2B cells. Among the hydrazone hybrids, phenylalanine derivative compound 9d exhibited the most potent and selective cytotoxic effect against NSCLC cells, with IC₅₀ values of 15.60 μM for A549 and 22.49 μM for PC9, and high selectivity indices (SI = 76.92 and 53.36, respectively). Mechanistic studies revealed that these compounds induce Apoptosis in A549 cells, with a concomitant decrease in VEGF-A levels and downregulation of VEGFR-1 and 2 expression; this indicates a direct modulation of the tumor-autonomous VEGF signaling axis. Additionally, the compounds significantly activated Caspase-3/7, 8, and 9 Enzymes, confirming the triggering of both intrinsic and extrinsic apoptotic pathways. Furthermore, intracellular reactive oxygen species (ROS) accumulation and mitochondrial membrane potential (MMP) depolarization were distinctly induced by compounds 10d and 10g, suggesting an additional mitochondrial stress-mediated mechanism of action within this series. Molecular docking simulations were performed to evaluate binding affinity within the VEGFR-2 active site. In vitro and in silico results identified compound 9d as a promising lead structure for further optimization of selective Lung Cancer therapeutics. Future studies were targeted at further optimizing drug-like properties and exploring downstream signaling kinetics.