Herein, we report the hit-to-lead identification of a drug-like pleuromutilin conjugate 16, based on a triaromatic hit reported in 2020. The lead arose as the clear candidate from a hit-optimization campaign in which Gram-positive antibacterial activity, solubility, and P-gp affinity were optimized. Conjugate 16 was extensively evaluated for its in vitro ADMET performance which, apart from solubility, was overall on par with lefamulin. This evaluation included Caco-2 cell permeability, plasma protein binding, hERG inhibition, cytotoxicity, metabolism in microsomes and CYP3A4, resistance induction, and time-kill kinetics. Intravenous pharmacokinetics of 16 proved satisfactory in both mice and pigs; however, oral bioavailability was limited likely due to insufficient solubility. The in vivo efficacy was evaluated in mice, systemically infected with Staphylococcus aureus, where 16 showed rapid reduction in blood bacteriaemia. Through our comprehensive studies, lead 16 has emerged as a highly promising and safe antibiotic candidate for the treatment of Gram-positive bacterial infections.

Original languageEnglish
JournalJournal of Medicinal Chemistry
Issue number5
Pages (from-to)3692-3710
Publication statusPublished - 14. Mar 2024


  • Animals
  • Anti-Bacterial Agents/therapeutic use
  • Biological Availability
  • Caco-2 Cells
  • Diterpenes/pharmacology
  • Humans
  • Mice
  • Microbial Sensitivity Tests
  • Pleuromutilins
  • Polycyclic Compounds/pharmacology
  • Staphylococcal Infections/drug therapy
  • Swine


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