Multidrug-resistant bacterial infections continue to be a rising global health concern. Herein, we describe the development of a novel class of 3-substituted benzoazepinedione derivatives with promising antibacterial activity. The pivotal compound, benzoazepinedione carboxylate 9, represents a highly electrophilic Michael acceptor, enabling divergent access to a wide range of thia-, aza-, oxa-, and phospha-Michael adducts. Notably, most prepared compounds exhibited potent antibacterial activity against both drug-susceptible and drug-resistant strains of Staphylococcus aureus (MIC90 of up to 2 μg mL-1). The cytotoxicity assessment in the VERO6 cell line revealed that thia-adduct 10d (IC50 of 36.5 μg mL-1) exhibits lower toxicity compared to its parent electrophile 9 (IC50 of 14.3 μg mL-1), which is in agreement with the hypothesis of covalently modified prodrugs. Additionally, stability studies of the prepared compounds in CD3OD and a DMSO-PBS mixture confirmed that thia-Michael adducts 10 are stable under neutral conditions while dynamic under mildly basic conditions. Moreover, 3D reconstructed tissue models (human lung epithelial EpiAirway™ and a human small intestine model) did not exhibit a viability decrease below 80% of the untreated control at all concentrations tested, indicating tolerance to higher concentrations of potential drugs and prodrugs.

Central Laboratories Slovak University of Technology Radlinského 9 812 37 Bratislava Slovakia

Department of Chemistry Faculty of Education Trnava University Priemyselná 4 918 43 Trnava Slovakia

Department of Inorganic Chemistry Faculty of Science Charles University Hlavova 2030 128 40 Prague Czech Republic

Institute of Biochemistry and Microbiology Faculty of Chemical and Food Technology Slovak University of Technology Bratislava Slovakia

Institute of Experimental Pharmacology and Toxicology Centre of Experimental Medicine Slovak Academy of Sciences Bratislava Slovakia

Institute of Organic Chemistry Catalysis and Petrochemistry Slovak University of Technology Radlinského 9 812 37 Bratislava Slovakia

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