Bacterial infection poses a serious threat to human life due to its rapidly growing resistance to antibacterial drugs, which is a significant public health issue. Annually, over 700,000 people die of untreatable infections in the world [1], [2]. In 2017, the World Health Organization (WHO) announced some worrisome drug-resistant bacterial strains encompassing Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species [3], [4]. Among these, methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-intermediate-resistant Staphylococcus aureus (VISA) have been categorized into the high-priority species [5]. In addition, the increasing prevalence of vancomycin-resistant Enterococci (VRE) strains and the rise of resistance to current therapeutic antibacterial agents are also of particular concern [6]. Consequently, there is a clear and continuous need for the discovery and development of novel antibacterials [7], [8].

DNA gyrase is essential in all bacteria except in humans and this makes it an attractive and viable therapeutic target for novel antibacterials discovery [9]. It is a heterotetrameric protein composed of two GyrA subunits and two GyrB subunits. GyrA is involved primarily in breaking and reuniting double-stranded DNA, GyrB is responsible for providing energy for the catalytic function of the enzyme through ATP hydrolysis [10], [11]. Because of its great therapeutic potential, GyrB inhibitors have long been actively studied in the field of antibacterial drugs [12]. In recent years, despite the wide array of new structurally diverse GyrB inhibitors such as ethyl ureas [13], pyrazolopyridones [14], pyrrolamides [15], quercetin diacylglycosides [16] have been developed by industry and academia, none of them have been approved for clinical use except novobiocin (cf. Fig. 1), which was withdrawn in 2011 due to safety and efficacy. Fobrepodacin (SPR720) [17] and DS-2969b [18] (cf. Fig. 1) are the only two compounds currently in phase I clinical trials. Therefore, the discovery and exploitation of novel chemotypes of GyrB inhibitors with stronger antibacterial activities, less toxicity, and good pharmacokinetic profiles is still needed.

It is noteworthy that the quinolone-based GyrB inhibitors have been attracting great attention over the recent years [19], [20]. Shiroya et al, 2013 synthesized a set of 2-quinolone derivatives and among them, compound 1 (cf. Fig. 1) was found to be the most active against S. aureus and E. coli with inhibition zone (DIZ) values of 13.5 mm and 12.8 mm, respectively. Further, the computational study revealed that compound 1 showed good interactions within the active site of S. aureus GyrB [21]. Ushiyama et al, 2020 discovered a collection of (methylamino)-2-oxo-1,2-dihydroquinoline-type antibacterial agents. In particular, TP0480066 (cf. Fig. 1) was the most potent compound achieving MIC90 values of 0.25 and 0.015 μg/mL against MRSA and VRE, respectively. Mechanistically, the compound TP0480066 could exert the antibacterial activity via inhibition of E. coli topoisomerase IV and DNA gyrase [22]. Similarly, Saleh et al, 2021 synthesized a series of 6-hydroxyquinolinone analogs and evaluated their antibacterial activities against a range of bacterial strains. Interestingly, the intermediate 2 (cf. Fig. 1) showed the most potency against MRSA, having inhibition zone of 19 mm. Molecular docking study indicated that intermediate 2 could bind to key amino acid residues of S. aureus GyrB [23].

In our previous study, a series of N-quinazolinone-4-hydroxy-2quinolone-3-carboxamides as novel DNA gyrase B inhibitors were discovered. Of note, the compound f1 (cf. Fig. 1) with modest potency, good in vitro metabolic stability and low cytotoxicity has been identified as a promising antibacterial agent worth further exploring. As part of our ongoing work on the development of GyrB inhibitors, we focused our attention on investigating the effects of bioisosteric replacements on biological activities and properties. In this study, we present the synthesis of a new series of 4-hydroxy-2-quinolone-3-carboxamide-based inhibitors, for which we evaluated their inhibitory effects against GyrB. In addition, the inhibitors that possessed good potencies against GyrB were employed for in vitro antibacterial evaluation against the selected Gram-positive bacteria strains. Furthermore, for the most promising antibacterial agent, we performed in-depth studies for cytotoxicity, metabolic stability, in vivo pharmacokinetic profiles, and molecular modeling.