Ligand-based design and synthesis of novel 3-spiro-2-oxindoline derivatives as potent VEGFR-2 inhibitors with antiproliferative activity and cell cycle arrest.

Rational design, synthesis, and biological evaluation of a novel series of 3-spiro-2-oxindoline derivatives as potential VEGFR-2 inhibitors were performed. Guided by the pharmacophoric requirements of type II VEGFR-2 inhibitors, the compounds were designed to incorporate key structural features required for VEGFR-2 recognition, with particular emphasis on aromatic linker integration to optimize binding interactions within the kinase active site. The synthesized derivatives were fully characterized and evaluated for their antiproliferative activity against HepG2 and HCT-116 cancer cell lines. Several compounds exhibited potent cytotoxic activity and favorable selectivity toward cancer cells compared with normal cells. Among them, compounds 4, 5, and 6g emerged as the most active derivatives and demonstrated VEGFR-2 inhibitory activity comparable to that of sorafenib. Mechanistic studies revealed that these compounds induced G0/G1 cell cycle arrest and apoptosis, as confirmed by flow cytometric cell cycle and Annexin V-FITC/PI analyses. Western blot analysis further demonstrated upregulation of p21 and hypophosphorylated retinoblastoma protein (Rb), accompanied by downregulation of Cyclin D1 and p-FAK, indicating disruption of signaling pathways involved in cell proliferation and survival. Molecular docking and triplicate molecular dynamics simulations provided a structural rationale for the biochemical VEGFR-2 inhibitory activity of compounds 4, 5, and 6g by identifying binding modes compatible with stable occupancy of the isolated kinase domain. These computational findings were interpreted as supportive of a VEGFR-2 binding hypothesis rather than as direct evidence of intracellular target engagement or predictors of antiproliferative potency. In addition, in silico ADMET profiling predicted favorable pharmacokinetic and safety properties. Collectively, these results validate the design strategy and identify compound 6g as a promising lead within this spirooxindoline series, while providing mechanistic insights into the structural features associated with VEGFR-2-targeted anticancer activity.