Urinary metabolites of a novel quinoxaline non-nucleoside reverse transcriptase inhibitor in rabbit, mouse and human: identification of fluorine NIH shift metabolites using NMR and tandem MS

Abstract

1. The urinary metabolites of (S)-2-ethyl-7-fluoro-3-oxo-3,4-dihydro-2H-quinoxaline-carboxylic acid isopropylester (GW420867X) have been investigated in samples obtained following oral administration to rabbit, mouse and human. GW420867X underwent extensive biotransformation to form hydroxylated metabolites and glucuronide conjugates on the aromatic ring, and on the ethyl and isopropyl side-chains in all species. In rabbit urine, a minor metabolite was detected and characterized as a cysteine adduct that was not observed in mouse or man. 2. The hydroxylated metabolites and corresponding glucuronide conjugates were isolated by semi-preparative HPLC and characterized using NMR, LC-NMR and LC-MS/MS. The relative proportions of fluorine-containing metabolites were determined in animal species by 19F-NMR signal integration. 3. The fluorine atom of the aromatic ring underwent NIH shift rearrangement in the metabolites isolated and characterized in rabbit, mouse and human urine. 4. The characterization of the NIH shift metabolites in urine enabled the detection and confirmation of the presence of these metabolites in human plasma.

Metabolism, Excretion, and Mass Balance of the HIV-1 Integrase Inhibitor Dolutegravir in Humans

“…However, 19 F chemical shifts vary with concentration, solvent, and endogenous components; therefore, the observed 19 F chemical shift for M4 could not be used to definitively identify a specific regioisomer. In dolutegravir, a large H-F coupling constant was observed between the ortho-fluorine and the benzylic protons.…”

“…We estimate that Ͻ10 g of M4 was isolated for NMR analysis. The 1 H NMR spectrum acquired with 19 F decoupling displayed two singlet aromatic resonances at 6.65 and 7.54 ppm. The absence of observable 1 H-1 H coupling is consistent with these two protons having a 1,4 relationship on the aromatic ring.…”

“…The NMR experiments were conducted on either an Avance II 700-MHz (5-mm TCI cryoprobe) or 600-MHz (5-mm TFI cryoprobe) NMR spectrometer (Bruker, Billerica, MA). The experiments conducted included 1 H with and without 19 F decoupling, 19 F, 2D-TOCSY (2-dimensional total correlation spectroscopy), 2D-ROESY (rotating-frame Overhauser effect spectroscopy), 1 H- 13 C gHSQC (gradient-selected heteronuclear single-quantum coherence), 1 H-13 C gHMBC (gradient-selected heteronuclear multiple-bond coherence), and 1 H-19 F gHSQC as implemented in TopSpin 2.0.4 software (Bruker).…”

The role of fluorine in medicinal chemistry

“…This shift can therefore result in cases where the introduction of fluorine into a compound does not prevent oxidation at that site. Dear et al [26] investigated the urinary metabolites of (S)-2-ethyl-7-fluoro-3-oxo-3,4-dihydro-2H-quinoxaline-carboxylic acid isopropylester (GW420867X), and found that fluorine underwent Figure 4. The use of fluorine substitution to extend the biological half-life, illustrated by (a) the platelet aggregating agent thromboxane A 2 and its 7,7-difluoro derivative, and (b) natural prostacyclin and 10,10-difluoro-13-dehydro-prostacyclin.…”

Characterization by Liquid Chromatography-Nuclear Magnetic Resonance Spectroscopy and Liquid Chromatography-Mass Spectrometry of Two Coupled Oxidative-Conjugative Metabolic Pathways for 7-Ethoxycoumarin in Human Liver Microsomes Treated with Alamethicin

“…However, this methodology has only recently come of age, as articles attest to growing application in the field of drug metabolism (Lindon et al, 1996(Lindon et al, , 1997. Most recently, challenging regiochemistry problems related to acetylenic metabolism (Mutlib et al, 1999(Mutlib et al, , 2000, NIH shift metabolites (Dear et al, 2000), and quinoline oxidation/glucuronidation (Ehlhardt et al, 1998) have been deciphered with LC-NMR. This article reports the application of LC-NMR and LC-MS for the characterization of two sets of coupled phase I and phase II metabolites formed from human liver microsomes perforated with alamethicin and supplemented with the substrate 7-EC and the cofactors NADPH and UDP-glucuronic acid (UDPGA), respectively.…”