N1-Benzofused Modification of Fluoroquinolones Reduces Activity Against Gram-Negative Bacteria

[Image: see text] The fluoroquinolone class of antibiotics has a well-established structure–activity relationship (SAR) and a long history in the clinic, but the effect of electron-rich benzofused substituents at the N1 position remains poorly explored. Because groups at this position are part of the topoisomerase–DNA binding complex and form a hydrophobic interaction with the major groove of DNA, it was hypothesized that an electron-rich benzofused N1 substituent could enhance this interaction. Molecular modeling techniques were employed to evaluate the binding of certain N1-modified fluoroquinolones to DNA gyrase targets from both Staphylococcus aureus and Klebsiella pneumoniae species compared with ciprofloxacin and norfloxacin. Seven N1-modified fluoroquinolones were subsequently synthesized and tested against a panel of Gram-negative pathogens to determine minimum inhibitory concentration (MIC) values. Gram-negative outer membrane penetration was investigated using the membrane permeabilizer polymyxin B nonapeptide and compound efflux via resistance–nodulation–division-family efflux transporters was evaluated using the known efflux pump inhibitor phenylalanine–arginine β-naphthylamide. Additionally, the target inhibitory activity of representative compound 6e was determined in a cell-free environment. A correlation between N1 substituent hydrophobicity and activity was observed across the MIC panel, with compound activity decreasing with increased hydrophobicity. Those compounds with highest hydrophobicity were inactive because of poor solubility profiles whereas compounds with intermediate hydrophobicity were inactive because of impaired outer membrane penetration, and reduced inhibition of topoisomerase targets, the latter in contrast to modeling predictions. This study adds new information to the fluoroquinolone SAR and suggests limited utility of large hydrophobic substituents at the N1 position of fluoroquinolones.