Structure-Guided In Vitro to In Vivo Pharmacokinetic Optimization of Propargyl-Linked Antifolates

• Published in: Drug metabolism and disposition Vol. 47; no. 9; pp. 995 - 1003
• Main Authors: Lombardo, M.N., G-Dayanandan, N., Keshipeddy, S., Zhou, W., Si, D., Reeve, S.M., Alverson, J., Barney, P., Walker, L., Hoody, J., Priestley, N.D., Obach, R.S., Wright, D.L.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01.09.2019; American Society for Pharmacology and Experimental Therapeutics, Inc; American Society for Pharmacology and Experimental Therapeutics (ASPET); The American Society for Pharmacology and Experimental Therapeutics
• Subjects: Animals; Anti-Bacterial Agents - chemistry; Anti-Bacterial Agents - pharmacokinetics; Antibacterial activity; Antibiotics; Bacterial Proteins - antagonists & inhibitors; Bacterial Proteins - chemistry; Bacterial Proteins - metabolism; Bactericidal activity; BASIC BIOLOGICAL SCIENCES; Cell Line; Crystal structure; Crystallography, X-Ray; Cytochrome P-450 CYP3A - chemistry; Cytochrome P-450 CYP3A - metabolism; Cytochrome P450; Cytochromes P450; Demethylation; Design; Deuterium; Drug Design; Drug development; Drug Interactions; Drug Resistance, Bacterial; Enzyme Assays; Female; Fluorine; Folic Acid Antagonists - chemistry; Folic Acid Antagonists - pharmacokinetics; Hepatocytes; Humans; Inhibitory Concentration 50; Lead compounds; Male; Metabolic Clearance Rate; Methicillin-Resistant Staphylococcus aureus - drug effects; Mice; Microbial Sensitivity Tests; Microsomes; Microsomes, Liver - metabolism; Models, Biological; Multidrug resistance; Nitrogen; Optimization; Organic chemistry; Oxidation; Pharmacodynamics; Pharmacokinetics; Pharmacology; Recombinant Proteins - metabolism; Structure-Activity Relationship; Tetrahydrofolate Dehydrogenase - chemistry; Tetrahydrofolate Dehydrogenase - metabolism; PLA; MIC; MS; CL; IS; ACN; AUC; DHFR; LC-MS/MS; CLint; P450; HFBA; HPLC; HPMC
• ISSN: 0090-9556; 1521-009X; 1521-009X
• DOI: 10.1124/dmd.119.086504

Pharmacokinetic/pharmacodynamic properties are strongly correlated with the in vivo efficacy of antibiotics. Propargyl-linked antifolates, a novel class of antibiotics, demonstrate potent antibacterial activity against both Gram-positive and Gram-negative pathogenic bacteria, including multidrug-resistant Staphylococcus aureus. Here, we report our efforts to optimize the pharmacokinetic profile of this class to best match the established pharmacodynamic properties. High-resolution crystal structures were used in combination with in vitro pharmacokinetic models to design compounds that not only are metabolically stable in vivo but also retain potent antibacterial activity. The initial lead compound was prone to both N-oxidation and demethylation, which resulted in an abbreviated in vivo half-life (∼20 minutes) in mice. Stability of leads toward mouse liver microsomes was primarily used to guide medicinal chemistry efforts so robust efficacy could be demonstrated in a mouse disease model. Structure-based drug design guided mitigation of N-oxide formation through substitutions of sterically demanding groups adjacent to the pyridyl nitrogen. Additionally, deuterium and fluorine substitutions were evaluated for their effect on the rate of oxidative demethylation. The resulting compound was characterized and demonstrated to have a low projected clearance in humans with limited potential for drug-drug interactions as predicted by cytochrome P450 inhibition as well as an in vivo exposure profile that optimizes the potential for bactericidal activity, highlighting how structural data, merged with substitutions to introduce metabolic stability, are a powerful approach to drug design.