6‑Chloropyridinyl Benzoate Fragments as Selective Anti-Melanoma Scaffolds Triggering ROS-Mediated Mitochondrial Apoptosis

Fragment-like molecules represent valuable starting points in anticancer drug discovery due to their structural simplicity and favorable physicochemical properties. In this context, the combination of phenotypic drug discovery (PDD) with fragment-based drug discovery (FBDD) offers an effective strategy for the rapid identification of bioactive scaffolds. Herein, a small in-house library of 6-chloropyridinyl benzoate fragments was designed to explore the antiproliferative potential of this underexplored scaffold. The compounds were first evaluated in silico, confirming favorable physicochemical and ADME profiles consistent with fragment-like chemical space, and were subsequently synthesized through simple one-pot procedures. Phenotypic screening against a panel of human cancer cell lines (A-375, HCT-116, and MCF-7) revealed a selective antiproliferative effect toward melanoma cells. Two derivatives, 7d and 7k, emerged as the most active compounds, displaying low micromolar activity against A-375 cells (IC₅₀ = 1.59 ± 0.07 μM and 1.08 ± 0.03 μM, respectively) and high selectivity (SI > 60) compared to noncancerous 16-HBE cells. Mechanistic investigations demonstrated that these fragments induce apoptosis through an ROS-dependent mitochondrial pathway, associated with mitochondrial dysfunction and cell cycle arrest at the G1-S transition. Density functional theory (DFT) analysis further suggested that the enhanced electrophilic character and electron-accepting ability of the most active derivatives may contribute to their biological activity. Overall, this study identifies chloropyridinyl benzoate fragments as a previously unexplored scaffold with selective antimelanoma activity and highlights their potential as promising starting points for fragment-to-lead optimization.