Novel N-4-(5-Amino-7-substituted-triazolotriazinepiperazin-1-yl) Norfloxacin Analogues Exhibit Potent and Selective Anticancer Activity via Topoisomerase Inhibition, Cell-Cycle Arrest, and Apoptosis in A 431 Skin Carcinoma Cells

Background: Skin carcinoma is among the most common cancers globally, necessitating the urgent development of innovative chemotherapeutic drugs that exhibit great selectivity and diminished toxicity towards normal cells. This study assessed a series of recently synthesized compounds (4–15) for screening their anticancer efficacy against A 431 human skin carcinoma cells to find effective and selective treatment candidates. Methods: Compounds were synthesized via a one-pot, three-component reaction. Cytotoxicity was evaluated using the MTT assay, with 5-fluorouracil (5-FU) as the reference standard, which exhibited potent activity against A 431 skin cancer cells. The activity of these drugs against normal BJ cells was evaluated, with mechanistic investigations encompassing topoisomerase I/II enzyme inhibition, cell-cycle analysis, and Annexin V–FITC/PI apoptosis assays. Results: Most compounds exhibited dose-dependent cytotoxicity, with compound 14 demonstrating the highest potency (IC50 = 76.7 µg/mL), exceeding that of 5-FU (IC50 = 83.7 µg/mL) while preserving selectivity for BJ cells. Compound 14 exhibited moderate inhibition of topoisomerases I and II (IC50 values of 17.5 and 17.3 µM), as confirmed by docking studies. Flow cytometry indicated G0/G1 phase arrest (64.09% vs. 58.18% in control), while apoptosis assays confirmed induction of both early and late apoptosis, accompanied by significant necrosis. Conclusions: Compound 14 is the most efficacious and selective drug in this series, functioning through topoisomerase inhibition, G0/G1 cell cycle arrest, and apoptosis, thereby representing a strong candidate for subsequent preclinical research.