Synthesis And Evaluation of Piperic Acid and 4-Ethylpiperic Acid Amide Derivatives as Nora Efflux Pump Inhibitors Against Multidrug-Resistant Staphylococcus Aureus

Abstract

The rising prevalence of multidrug-resistant (MDR) bacterial infections, particularly caused by Staphylococcus aureus, underscores the urgent need for novel therapeutic strategies. Among various resistance mechanisms, the NorA efflux pump plays a pivotal role in reducing intracellular concentrations of fluoroquinolones, such as ciprofloxacin, thereby limiting their antibacterial efficacy. Efflux pump inhibitors (EPIs) offer a promising approach to combat such resistance by restoring antibiotic sensitivity. In this study, a series of twenty novel amide derivatives of piperic acid (PA) and 4-ethylpiperic acid (EPA) were synthesized via EDCI-mediated coupling with structurally diverse α-, β-, and γ-amino acids. The synthesized compounds were structurally characterized using ^1H and ^13C NMR spectroscopy and high-resolution mass spectrometry (HRMS), confirming their purity and structural integrity. Biological evaluation was conducted using the NorA-overexpressing S. aureus SA-1199B strain. MIC determination by checkerboard assay revealed significant potentiation of ciprofloxacin activity in the presence of selected compounds, with compounds 19 and 20 exhibiting strong synergy (FICI = 0.1875), reducing ciprofloxacin MIC from 8 µg/mL to 0.5 µg/mL. Ethidium bromide (EtBr) accumulation and efflux assays demonstrated that these compounds effectively inhibited NorA-mediated efflux, showing comparable or superior activity to natural piperine and standard EPI reserpine. EPA-based derivatives consistently showed enhanced EPI activity compared to their PA counterparts, suggesting that subtle structural modifications in the parent scaffold significantly influence potency. The findings highlight the potential of EPA-amide derivatives, particularly those bearing hydrophobic or aromatic amino acid residues, as promising NorA-targeted EPIs. These scaffolds provide a foundation for further structural optimization and development as adjuvants in antimicrobial therapy against resistant bacterial pathogens.