Over the years, bacterial pathogens that cause common or severe infections have rapidly developed antimicrobial resistance (AMR) to newly marketed antibiotics. Specifically, ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter sp.) pathogens exhibit strong AMR to various antibiotics, with multiple mechanisms in nosocomial infections [[1], [2], [3]]. Gram-positive bacterial pathogen S. aureus is often observed in hospital-acquired and community-associated infections and is a major cause of severe pulmonary and systemic infections. S. aureus is an opportunistic pathogen causing various human conditions, including skin and soft tissue infections, boils, sinusitis, pneumonia, meningitis, osteomyelitis, endocarditis, and sepsis [[3], [4], [5], [6]].

β-lactam antibiotics, such as penicillin and methicillin, are generally used to treat S. aureus infections. However, AMR against β-lactams has become increasingly prevalent in clinical S. aureus isolates, as exemplified by the emergence of methicillin-resistant S. aureus (MRSA) strains [7,8]. As MRSA strains consistently induce multidrug resistance against multiple classes of widely used antibiotics, including macrolides, quinolones, aminoglycosides, tetracyclines, and vancomycin, their emergence and rapid spread pose serious concerns regarding increased morbidity and mortality worldwide [5,6,9]. Therefore, development of new antibiotics with novel antibacterial mechanisms and potent efficacy is necessary to effectively target MRSA strains and prevent their transmission.

To identify novel antibacterial agents to mitigate the emergence and spread of AMR strains, we focused on new antibacterial mechanisms using key pharmacophores to increase the generation of reactive oxygen species (ROS). ROS-mediated bacterial lethality is a promising strategy to combat bacterial infections as ROS affect multiple pathways, making it difficult for bacteria to develop targeted resistance mechanisms against ROS-induced damage [10,11]. Specifically, 1,4-naphthoquinones (NQs) are attractive scaffolds with ROS-generating properties, promising biological activities, and therapeutic applications in various human diseases. Quinones are converted into semi-quinones and further reduced to dihydroquinones via NAD(P)H:quinone oxidoreductase or cytochrome P450 and its reductase-mediated enzymatic reduction. Under normoxic conditions, semi-quinones and dihydroquinones spontaneously transform into their corresponding quinones, in which oxygen is reduced to generate superoxide radical anions, leading to ROS production [[12], [13], [14]].

Recent advances in ROS and relevant medicinal chemistry have increased the interest to rationally harnessing NQ pharmacophores for drug discovery and development of novel antibiotics. Recently, we identified a new antibacterial agent, 1-(2-methoxyethyl)-2-methyl-3-(hexyl)-4,9-dioxo-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide (c5), harboring a NQ moiety fused with an imidazolium ring, as the key pharmacophore for ROS generation during aerobic metabolism. c5 was developed via a drug-redirecting strategy from its congeneric anti-tumor compound, YM155, that exhibited selective antibacterial activities against gram-positive bacteria, especially MRSA with a moderate minimum inhibitory concentration (MIC) of 3.13–6.25 μg/mL [15]. This suggests the potential of new c5-related chemical entities as novel antibiotics. Therefore, development of new NQ-based antibacterial agents is important to overcome the limitations of currently available therapeutics against pathogenic gram-positive bacteria.

In this study, we aimed to develop novel NQ-based chemical entities and optimize c5 to improve its pharmaceutical properties as a potent antibacterial candidate against gram-positive bacteria, particularly MRSA. Additionally, we explored the antibacterial mechanism of the newly developed compound, conducted in vivo toxicological and efficacy studies in a Drosophila animal model, and investigated the emergence of bacterial resistance.