Pro-Apoptotic and Autophagy-Inducing Activity of Novel 1,4-Naphthoquinone Derivatives against Oral Squamous Cell Carcinoma without ROS Generation

Abstract Oral squamous cell carcinoma (OSCC) remains a major global health burden, and current platinum-based chemotherapeutics are constrained by systemic toxicity and acquired resistance, motivating the search for novel antitumor agents. Naphthoquinones are a privileged scaffold in anticancer drug discovery. Here, we report the synthesis of 18 1,4-naphthoquinone esters (1–18), 14 of which are novel, and their first evaluation as anticancer agents against three OSCC cell lines (SCC-4, SCC-9, and SCC-25) and primary human gingival fibroblasts. Derivative 14 emerged as the lead compound, displaying a geometric mean IC50 of 5.0 μM and a geometric mean selectivity index of 9.15, outperforming, in terms of selectivity, the chemotherapeutic standards carboplatin, lapachol, and shikonin. Mechanistically, 14 induced cell death through a combined apoptotic–autophagic pathway, evidenced by membrane blebbing, G2/M arrest, sub-G1 DNA fragmentation, caspase 3/7 activation, and LC3-puncta formation, with wortmannin-mediated rescue indicating that autophagy contributes to cell death, and without detectable induction of reactive oxygen species. This phenotype was accompanied by pronounced disorganization of the actin and tubulin cytoskeletal networks, evidenced by confocal fluorescence microscopy and reduced anisotropy of both networks. Derivative 14 induced less than 5% hemolysis at concentrations up to 1000 μM and was well tolerated in C57BL/6 mice at intraperitoneal doses up to 600 mg/kg, with no mortality or morphological alterations. In silico analyses showed no violations of Lipinski’s rule of five and a topological polar surface area within the range associated with good membrane permeability, while reverse molecular docking pointed to the DNA-binding domains of topoisomerase IIα and IIβ as plausible molecular targets, with binding modes and energies comparable to doxorubicin. Together, these findings establish 14 as a selective and well-tolerated 1,4-naphthoquinone derivative, with a proposed mechanism of action, warranting further preclinical development against OSCC.