Antimicrobial Activity, Preliminary Safety, and Pharmacokinetics Assessment of 2–3: A Pleuromutilin-Derived Compound

<i>Staphylococcus aureus</i> poses a global threat to livestock health and public health security, necessitating a novel antibiotic. Pleuromutilin, a natural antibiotic, has served as a promising foundation for developing new antibacterial agents through structural modification. This study aims to evaluate the antibacterial potential, safety, and pharmacokinetic profile of a novel pleuromutilin derivative 2-3 (22-[2-(L-prolylamino)phenylsulfanyl]-22-deoxypleuromutilin). The compound 2-3 exhibited potent antibacterial activity (MIC = 0.25 μg/mL), concentration-dependent bactericidal effects, and prolonged post-antibiotic effects (PAEs). Safety assessments revealed low cytotoxicity (CC₅₀ = 62.63 μg/mL) and no observable hemolytic activity. In vitro metabolic studies indicated species-dependent clearance, primarily mediated by CYP3A4. Pharmacokinetic in rats showed rapid absorption and elimination, with oral and intramuscular bioavailability of 16.03% ± 8.82% and 53.36% ± 12.27%, respectively. Notably, 2-3 demonstrated superior efficacy over tiamulin in a neutropenic murine thigh infection model. Molecular docking revealed a stronger binding free energy between 2-3 and the 50S ribosomal subunit compared to tiamulin. Collectively, these results highlight 2-3 as a promising clinical candidate against <i>S. aureus</i> infections, characterized by enhanced efficacy and a favorable safety profile.