NorA Efflux Pump Inhibitors: Expanding SAR Knowledge of Pyrazolo[4,3-c][1,2]benzothiazine 5,5-Dioxide Derivatives

Antimicrobial resistance (AMR) represents a significant global concern, driven by the overuse of antibiotics. One of the principal mechanisms contributing to AMR is the activity of microbial efflux pumps (EPs), which expel diverse antibiotics out of bacterial cells, thereby rendering them ineffective. Our research aimed to expand the range of molecular classes that inhibit the Staphylococcus aureus EP NorA. In this study, starting from the hit compound pyrazolo[4,3-c][1,2]benzothiazine 5,5-dioxide 1, previously reported as a NorA efflux pump inhibitor (EPI), we undertook medicinal chemistry efforts, which involved the iterative combination of the design and synthesis of new analogues with data obtained through ethidium bromide efflux inhibition assays. Subsequent synergistic assays with ciprofloxacin (CPX) against the resistant strain SA-1199B led to the identification of three potent compounds (3, 10, and 19). The evaluation of these compounds in combination with CPX against NorA-overexpressing and NorA-knockout strains (SA-K2378 and SA-K1902, respectively) confirmed that the observed synergy with CPX is dependent on the presence of NorA. Additionally, the combination of NorA EPIs with CPX reduced biofilm production in NorA-overexpressing strains. These findings enhance our understanding of the structure-activity relationship of pyrazolobenzothiazine derivatives and support the use of EtBr efflux assays for rapid NorA inhibitors' identification.