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

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. ... mehrFlow 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