Benzodioxin-Annulated Naphthalimides as Potent DNA Replication Stress Inducers with Dual p53-Dependent and Independent Antitumor Activity

<b>Background/Objectives:</b> The development of small-molecule agents that selectively target DNA replication remains a central strategy in anticancer drug discovery. In this study, we report the biological characterization of a novel 6-nitro-benzodioxin-naphthalimide (NI) derivative (compound <b>5a</b>), evaluated as a potential DNA-targeted anticancer lead. <b>Methods/Results:</b> The antiproliferative activity of <b>5a</b> was assessed in a small panel of human lung carcinoma cell models (A549, H1299) and a non-malignant control (MRC-5), revealing pronounced cytotoxic effects in tumor cells, accompanied by favorable selectivity indices. Mechanistic investigations demonstrated that treatment with <b>5a</b> results in strong inhibition of DNA synthesis, as evidenced by a marked reduction in EdU incorporation and a robust induction of the DNA damage marker γH2AX. These effects were associated with cell-cycle perturbations characterized by accumulation in G₁ and G₂/M phases, followed by activation of apoptotic pathways. Importantly, clonogenic survival assays confirmed that even transient exposure to <b>5a</b> leads to a sustained loss of proliferative capacity, indicating irreversible long-term cellular damage. These results support a replication stress-driven mechanism of action for compound <b>5a</b>, consistent with interference in DNA-associated processes during S phase. <b>Conclusions:</b> While the precise molecular initiating event remains to be elucidated, the observed biological profile positions <b>5a</b> as a promising DNA-targeted lead structure with potential for further pharmaceutical optimization. These findings provide a solid foundation for the continued development of naphthalimide-based compounds as anticancer agents within a pharmaceutically relevant framework.