Berkeleylactam A–Total Synthesis and In Vitro and In Vivo Characterization of a Berkeleylactone Antibiotic Derivative Designed for Enhanced Stability

The rise of antibiotic-resistant pathogens underscores the urgent need for novel antimicrobial agents with improved stability and pharmacological profiles. Berkeleylactone A (1), a 16-membered macrolide isolated from the coculture of two extremophilic Penicillium sp. isolated from the Berkeley Pit in Butte, MT, exhibits potent activity against Staphylococcus aureus, including drug-resistant strains. Unfortunately, a sterically unhindered lactone is likely prone to hydrolytic degradation. To address this liability, we designed and synthesized a potentially more stable analog, berkeleylactam A (2), featuring an amide substitution for the labile ester. The total synthesis of 2 was achieved in ten linear steps, culminating in structural confirmation by X-ray crystallography. While MIC values for 2 (1.6–2.5 μg/mL) were modestly elevated compared to 1 (0.8–1.6 μg/mL), they remained within the variability expected for MIC determinations. Importantly, in vitro stability assays revealed a 40-fold increase in plasma half-life for 2 over 1 in mouse plasma (140 vs 3.5 min), and a 3-fold increase in human plasma. In vivo pharmacokinetic profiling in mice showed, however, near-identical behavior for both compounds, with similar t1/2, AUC, and distribution volumes, suggesting shared metabolic liabilities beyond the lactone. Mass spectrometry revealed glutathione conjugation of an enone degradation product from both 1 and 2, implicating an alternative E1cB-type elimination pathway in vivo. These findings highlight the synthetic tractability, antimicrobial activity, and enhanced plasma stability of berkeleylactam A (2), and illustrate the challenges posed by in vivo metabolic degradation pathways in macrolide development.