Integrated design, synthesis, biological evaluation, and computational mechanistic insights of novel benzanilide derivatives as VEGFR-2 targeted anticancer agents

Abstract

VEGFR-2 is a key regulator of tumor angiogenesis and a validated target in anticancer drug discovery. In this study, a series of novel benzanilide derivatives (7a–7f and 9a–9b) were designed, synthesized, and evaluated as potential VEGFR-2 inhibitors. The design strategy was guided by key pharmacophoric requirements for VEGFR-2 inhibition. All compounds were synthesized successfully and characterized, then screened for in vitro cytotoxic activity against MCF-7, MDA-MB-231, HePG-2, and HCT-116 cancer cell lines, with WI-38 and WISH normal cells used for selectivity assessment. Among the tested compounds, 7e emerged as the most potent derivative, exhibiting IC50 values ranging from 8.49 to 11.21 µM across cancer cell lines, comparable to sorafenib. Importantly, 7e also demonstrated favorable selectivity toward cancer cells with a promising selectivity index profile. Mechanistic studies revealed that compound 7e significantly inhibited VEGFR-2 kinase activity (IC50 = 1.79 ± 0.05 µM), supporting its targeted anti-angiogenic mechanism. Flow cytometry analysis showed that 7e induced G0/G1 cell cycle arrest and markedly increased apoptotic cell populations. This was further confirmed by upregulation of Bax, Caspase-3, and Caspase-8, alongside downregulation of Bcl-2, indicating activation of both intrinsic and extrinsic apoptotic pathways. Interestingly, 7e inhibited cancer cell migration, suggesting a potential anti-angiogenetic effect. Density functional theory (DFT) calculations revealed that 7e possesses a conjugated electronic system with a suitable frontier orbital distribution, moderate HOMO–LUMO gap, and a favorable electrostatic potential for receptor interaction. Molecular docking and dynamics simulations demonstrated stable binding of 7e within the VEGFR-2 active site, supported by consistent RMSD, hydrogen bonding, and compact structural behavior. MM-GBSA calculations confirmed favorable binding free energy, while per-residue decomposition and protein–ligand interaction fingerprint analysis identified key stabilizing interactions. Essential dynamics and free energy landscape mapping further confirmed a stable and energetically favorable conformational state. ADMET and toxicity predictions indicated acceptable pharmacokinetic properties, low mutagenic risk, and an overall favorable safety profile. Collectively, these findings identify compound 7e as a promising VEGFR-2-targeted anticancer lead with strong enzymatic inhibition, potent cytotoxicity, and a well-supported mechanistic profile integrating experimental and computational evidence.