Structure–Function Insights into Quinuclidine-3-One BisQACs: Synthesis, Modulation of Bacterial Resistance, Structure–Activity Relationship, and Biological Profiling

<b>Background</b>: The increasing prevalence of antibiotic-resistant bacterial strains highlights the urgent need for new membrane-targeting antimicrobial agents. Bisquaternary ammonium compounds (bisQACs) have attracted attention for their ability to disrupt bacterial membranes more effectively than monoquaternary analogs. Quinuclidine, known for its health-beneficial properties, has previously been explored for monoQAC derivatization, but studies using natural scaffolds to generate bisQACs remain limited. <b>Methods</b>: Here, we synthesized twelve novel quinuclidine-based bisQACs, systematically varying alkyl chain and linker lengths to investigate structure-activity relationships. <b>Results:</b> Several compounds, including 2(QC₁₆)₃, 2(QC₁₆)₄, 2(QC₁₄)₆, and 2(QC₁₆)₆, exhibited strong activity against <i>Staphylococcus aureus</i> (including MRSA), <i>Listeria monocytogenes</i>, and <i>Escherichia coli</i>, with 2(QC₁₆)₆ being the most potent (MICs 5-38 µM). While cytotoxicity was observed on human RPE1 and HEK293 cells, selectivity indices indicated a favorable therapeutic window relative to reference QACs. <b>Conclusions:</b> These compounds also inhibited biofilm formation and induced rapid bacterial killing through a membrane-disruptive mode of action. Molecular docking showed that alkyl chain and linker variations modulate binding to the QacR efflux regulator, revealing a lower potential for efflux-mediated resistance. Overall, quinuclidine-based bisQACs represent promising leads for potent, selectively active next-generation antimicrobials with a reduced likelihood of resistance development.