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

Abstract

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.

Fig. 1.

Crystal structure of S. aureus DHFR (cyan) in complex with compound 11 (green). Compound 11 makes conserved hydrogen bonds with Leu 5 and Asp 27 through the diaminopyrimidine ring and hydrophobic residues as well as significant van der Waals contacts with Leu 20, Ser 49, Ile 50, and Phe 92. Protein Data Bank ID: 6ND2.

Fig. 2.

The proposed metabolic scheme for compound 12 following incubation with mouse liver microsomes.

Fig. 3.

The UV trace at 284 nM for compound 17 metabolite identification in mouse and human hepatocytes. (A) Incubation with human hepatocytes indicates there are 10 distinct metabolites: four m/z 438 (double hydroxylation products); three m/z with MS2 data, indicating a hydroxylation product; two m/z 422 with MS-2 data indicating N-oxide products; one m/z 436 with MS-2 data indicative of methyl conversion to a carboxylic acid. MS-2 spectra are provided in the Supplemental Materials. (B) Similarly, mouse hepatocyte incubation indicates there are eight distinct metabolites: three m/z 438 (double hydroxylation products); two m/z 422 with MS-2 data indicating a hydroxylation product; two m/z 422 with MS-2 data indicating N-oxide products; one m/z 436 with MS-2 data indicative of methyl conversion to a carboxylic acid.

Fig. 4.

The proposed metabolic pathway for compound 17.

Fig. 5.

Time-kill curve determination of S. aureus American Type Culture Collection 43300 exposed to compound 19 at 1×, 5×, 10×, and 100× the MIC. Data suggest that compound 19 demonstrates concentration-dependent bactericidal activity at concentrations >10× the MIC. CFU, colony-forming unit.