Investigation of ROS-mediated oxidative stress and membrane disruption caused by Cu(II)-isatin semicarbazone against S. aureus and A. calcoaceticus.

Five isatin-based semicarbazones (H1L-H5L) and their Cu(II) complexes (1-5) were synthesized and characterized by elemental analysis, NMR, ESR, mass spectrometry, and single-crystal X-ray diffraction. ESR data (g∥ = 2.32-2.48; g⊥ = 2.11-2.14) confirmed axial geometry for the complexes. Antitubercular screening identified compound 1 as highly potent (MIC = 1.6 μg/mL), significantly outperforming its free ligand (MIC = 50 μg/mL), consistent with strong HSA binding (Kb = 4.88 × 104 M-1). Among the antibacterial evaluations, compound 5 displayed broad-spectrum activity, surpassing standard drugs, particularly against S. aureus and A. calcoaceticus. A strong HSA binding affinity (Kb = 1.77 × 104 M-1) in compound 5 was observed. It inhibited biofilm formation, showed rapid bactericidal action within 3 h, and delayed resistance development for up to 30 passages. Mechanistic studies revealed membrane disruption, cytoplasmic leakage, metabolic impairment, and ROS-mediated oxidative stress, including GSH oxidation to GSSG. These results identify compound 5 as a promising antibacterial scaffold with potent bactericidal and anti-biofilm properties suitable for further in vivo investigation.