Design, synthesis, and integrated biological and molecular modeling studies of ciprofloxacin–amino acid derivatives as multi-target antimicrobials

Fluoroquinolones are broad-spectrum antibacterials that exert their effects by inhibiting DNA gyrase and topoisomerase IV, enzymes crucial for bacterial DNA replication. Here, we report the synthesis of 60 novel ciprofloxacin-amino acid derivatives, designed to inhibit multiple bacterial targets by the so-called polypharmacology approach. Some of the synthesized compounds retained or improved the antibacterial potency against Gram-positive, Gram-negative, and mycobacterial species. Notably, some derivatives demonstrated promising activity (MIC <10 μM) against multidrug-resistant strains. The most active derivatives were selected for in silico, in vitro, and in vivo mechanistic studies. Molecular modeling, enzyme assay, and phenotypic analysis supported the polypharmacological mode of action. These findings indicate inhibition of DNA gyrase, topoisomerase IV and reduced off-target potential on human topoisomerase II, along with engagement of LpxC and urease as supportive targets. Interference with bacterial cell division and transient membrane disruption in Gram-positive bacteria and aberrant penicillin binding protein activity and possible effects on the outer membrane in Gram-negative bacteria may additionally contribute to the observed antibacterial activity. Compounds 10b and 12b showed the most potent antibacterial activity, with MIC values ranging from 0.2 to 5.47 μM against the evaluated strains. Cytotoxicity studies further revealed favorable selectivity, with SI values of 10-25. Overall, these findings underscore new ciprofloxacin derivatives as promising multi-target antimicrobial agents with potential to combat multidrug-resistant pathogens.