Novel Antibacterial 4-Piperazinylquinoline Hybrid Derivatives Against Staphylococcus aureus: Design, Synthesis, and In Vitro and In Silico Insights

Molecular hybridization, which consists of the combination of two or more pharmacophores into a single molecule, is an innovative approach in drug design to afford new chemical entities with enhanced biological activity. In the present study, this strategy was pursued to develop a new series of 6,7-dimethoxy-4-piperazinylquinoline-3-carbonitrile derivatives (<b>5a</b>-<b>k</b>) with potential antibiotic activity by combining the quinoline, the piperazinyl, and the benzoylamino moieties, three recurrent frameworks in antimicrobial research. Initial in silico evaluations were conducted on the designed compounds, highlighting favorable ADMET and drug-likeness properties, which were synthesized through a multistep strategy, isolated, and fully characterized. The whole set was tested in vitro against <i>Staphylococcus aureus</i> ATCC 25923 and <i>Pseudomonas aeruginosa</i> ATCC 10145 representative Gram-positive and Gram-negative strains, respectively. Notably, <b>5k</b> exhibited potent and selective activity against <i>S. aureus</i> (MIC 10 μM), with a dose- and time-dependent response and capability to affect cell membrane integrity. On the other hand, no significant activity was observed against <i>P. aeruginosa</i>. Further in silico docking and molecular dynamics studies highlighted strong interactions of <b>5k</b> with bacterial enzymes, such as tyrosyl-tRNA synthetase, pyruvate kinase, and DNA gyrase B, suggesting potential modes of action. These findings underscore the value of the hybridization approach in producing new antimicrobial agents, guiding future optimization for broader-spectrum activity.