A Rationally Engineered Norfloxacin-Phenoxazine Hybrid as a Dual-Function Anticancer Agent: DNA Intercalation Synergizes with Topoisomerase II Inhibition against Murine Breast Tumor.

Breast cancer therapy is compromised by acquired resistance and systemic toxicity, necessitating new therapeutic strategies. Herein, a series of hybrid molecules were synthesized by conjugating nononcogenic FDA-approved norfloxacin with DNA-targeting phenoxazine/phenothiazine through alkyl, amide, and triazole linkers. Photophysical studies demonstrated DNA intercalation, while molecular docking revealed stable interactions with topoisomerase II. The hybrids showed selective cytotoxicity against breast cancer cells, with compound 4 displaying superior potency (IC50 = 3.55 ± 0.28 μM in MCF-7), surpassing the parent drugs and tamoxifen. Cellular studies demonstrated G0/G1 arrest, apoptosis, and DNA damage, as evidenced by γH2AX foci formation. Enzyme-based topoisomerase II inhibition, together with cellular TARDIS assays analysis, indicated stabilization of topo II-DNA cleavage complexes. In vivo, compounds 4 and 10 produced significant tumor regression without detectable hepatic or renal toxicity. Immunofluorescence analysis confirmed elevated cleaved caspase-3, γH2AX, favoring DNA damage-mediated apoptosis. These findings highlight the emergence of norfloxacin-based hybrids as promising anticancer agents.