Discovery and biological evaluation of nitrofuranyl–pyrazolopyrimidine hybrid conjugates as potent antimicrobial agents targeting <i>Staphylococcus aureus</i> and methicillin-resistant <i>S. aureus</i>

Nitrofuran and pyrazolopyrimidine-based compounds possess a broad antimicrobial spectrum including Gram-positive and Gram-negative bacteria. In the present work, a series of conjugates of these scaffolds was synthesized and evaluated for antimicrobial activity against <i>Staphylococcus aureus</i> and methicillin-resistant <i>S. aureus</i> (MRSA). Many compounds showed MIC values of ≤2 μg ml^-1, with compound 35 demonstrating excellent activity (MICs: 0.7 and 0.15 μg ml^-1 against <i>S. aureus</i> and MRSA, respectively) and safety up to 50 μg ml^-1 in HepG2 cells. Compound 35 also exhibited no hemolytic activity, biofilm eradication, and effectiveness against efflux-pump-overexpressing strains (NorA, TetK, MsrA) without resistance development. It showed synergistic effects with vancomycin (<i>S. aureus</i>) and rifampicin (MRSA). Mechanistic studies revealed that compound 35 exhibits good membrane-targeting abilities, as evidenced by DAPI/PI staining and scanning electron microscopy (SEM). In an intracellular model, it reduced bacterial load efficiently in both <i>S. aureus</i> and MRSA strains. With a strong <i>in vitro</i> profile, compound 35 demonstrated favorable oral pharmacokinetics at 30 mg kg^-1 and potent <i>in vivo</i> anti-MRSA activity, highlighting its potential against antibiotic-resistant infections.