Identification of a Hydroxylamine Glucuronide Metabolite of an Oral Hypoglycemic Agent

Abstract

This article is available online at http://dmd.aspetjournals.org ABSTRACT:Glucuronides of piperazine hydroxylamines are rarely reported in the literature, and even more rarely are their structures unambiguously identified. One major metabolite was detected by liquid chromatography/mass spectrometry-radioactivity in urine from monkeys treated with the aryl piperazine oral hypoglycemic agent 9-[(1S,2R)-2-fluoro-1-methylpropyl]-2-methoxy-6-(1-piperazinyl) purine hydrochloride (1). The mass spectrum of this metabolite indicated that it was both monooxygenated and glucuronidated on the piperazine ring. Possible structures included the N-or O-glucuronic acid conjugates of a carbinolamine, hydroxylamine, or N-oxide. Treatment with ␤-glucuronidase gave a monooxygenated derivative of the parent compound. H NMR analysis of either the glucuronic acid conjugateor the monooxygenated product provided insufficient evidence to unambiguously determine their structures. Incubation of 1 with pig liver microsomes resulted in formation of the same monooxygenated derivative derived from ␤-glucuronidase treatment of the glucuronide metabolite. This in vitro system was used to generate sufficient material for analysis by 13 C NMR, and the metabolite was identified as a hydroxylamine derivative 2. Incubation of the hydroxylamine with monkey liver microsomes and uridine diphospho-5-glucuronic acid gave the same glucuronic acid conjugate as that observed in monkey urine. 13

Biosynthesis and Identification of an N-Oxide/N-Glucuronide Metabolite and First Synthesis of an N-O-Glucuronide Metabolite of Lu AA21004

“…The ions at m/z 315, 299, 298, and 240 for 4 and 299, 298, 256, and 240 for 3 correspond to the loss of a glucuronic acid moiety (m/z 315), loss of a glucuronic acid moiety and oxygen (m/z 299), and loss of an O-glucuronic acid moiety and fragmentation of the piperazine ring (m/z 240 and 256). This pattern is consistent with the fragmentation of glucuronic acid conjugate metabolites of a monooxygenated piperazine derivative (Delbressine et al, 1992;Schaber et al, 2001;Miller et al, 2004). Analysis of the (bio)synthesized standards containing 3 and 4 using MicrOTOF MS showed the compounds to have exact mass values of 491.18608 and 491.18325, respectively.…”

“…Organic synthesis of 4 was unsuccessful, but in vitro biosynthesis using human liver microsomes, UDPGA, alamethicin (antibiotic, precautionary measure) and saccharic acid lactone (␤-glucuronidase inhibitor, precautionary measure) (Miller et al, 2004;Picard et al, 2005;Bowalgaha et al, 2007;Wen et al, 2007a,b;Hintikka et al, 2008) and subsequent semipreparative HPLC-MS resulted in the production of approximately 10 mg of the metabolite 4. During evaporation of the fractions collected during semipreparative HPLC-MS, 4 started precipitating and was collected in sufficiently pure form for one-and two-dimensional NMR experiments.…”

“…at ASPET Journals on May 12, 2018 dmd.aspetjournals.org possibility of an N-oxide/N-glucuronide metabolite, but none have described such a metabolite (Straub et al, 1988;Miller et al, 2004).…”

“…Drugs that are piperazine derivatives typically generate piperazine ring carbon alcohols or metabolize at the piperazine nitrogen to generate hydroxylamines/N-oxides as phase I metabolites and Nglucuronides or N-O-glucuronides as phase II metabolites (Delbressine et al, 1992;Kassahun et al, 1996;Schaber et al, 2001;Miller et al, 2004). N-Glucuronides are common metabolic products of drugs containing amino groups or aromatic nitrogen heterocycles and in some cases tertiary, secondary, and primary amines form N-glucuronides from a drug and its phase I metabolites (Dalgaard and Larsen, 1999).…”

Species Differences in the Biotransformation of an α4β2 Nicotinic Acetylcholine Receptor Partial Agonist: The Effects of Distinct Glucuronide Metabolites on Overall Compound Disposition

“…However, because of the significance of M5 in monkeys reported herein, LC/MS/NMR analyses of nonradiolabeled M5 purified from the urine of a monkey administered 1 unequivocally identified M5 as an N-O-glucuronide of 1, consistent with the mechanistic structural rationalization of fragment ions within its product ion spectrum (Fig. 3) and previously characterized alicyclic N-O-glucuronides (Straub et al, 1988;Delbressine et al, 1992;Edlund and Baranczewski, 2004;Miller et al, 2004). In particular, the 1 H spectrum of M5 clearly showed the anomeric proton signal (4.37 ppm) of the glucuronide moiety, whereas the correlation spectroscopy spectrum indicated that this was the most downfield resonance in a five-proton spin system that terminated with a doublet at 3.59 ppm.…”