P450-Mediated Metabolism of 1-[3-(Aminomethyl)phenyl]-N-[3-fluoro-2‘-(methylsulfonyl)- [1,1‘-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole- 5-carboxamide (DPC 423) and Its Analogues to Aldoximes. Characterization of Glutathione Conjugates of Postulated Intermediates Derived from Aldoximes

The in vivo and in vitro disposition of DPC 423, a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa, has recently been described. Several metabolites, some of which were considered potentially reactive, were identified in rats. A novel GSH adduct, the struct...

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Bibliographic Details
Published in:Chemical research in toxicology 2002-01, Vol.15 (1), p.63-75
Main Authors: Mutlib, Abdul E, Chen, Shiang-Yuan, Espina, Robert J, Shockcor, John, Prakash, Shimoga R, Gan, Liang-Shang
Format: Article
Language:English
Subjects:
acetaldoxime
Animals
Cytochrome P-450 Enzyme System - metabolism
cytochrome P450
Dose-Response Relationship, Drug
DPC 423
Factor Xa Inhibitors
Fibrinolytic Agents - analysis
Fibrinolytic Agents - metabolism
Glutathione - metabolism
Male
Microsomes, Liver - metabolism
Oximes - metabolism
Pyrazoles - analysis
Pyrazoles - metabolism
Rats
Rats, Sprague-Dawley
Sulfones - analysis
Sulfones - metabolism
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Summary:The in vivo and in vitro disposition of DPC 423, a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa, has recently been described. Several metabolites, some of which were considered potentially reactive, were identified in rats. A novel GSH adduct, the structure of which was not determined conclusively, was isolated from bile of rats dosed with DPC 423. Herein, we describe the complete structural elucidation of this unique GSH conjugate employing LC/MS and high-field NMR. Similar GSH adducts of DPC 602, [13CD2]DPC 602, and SX 737, all structural analogues of DPC 423, were isolated, characterized spectroscopically, and shown to have identical mass fragmentation pathways. The structures of these conjugates were initially suspected to be either an amide with N−S bond or a nitrogen−oxygen juxtaposed amide with a C−S bond. Studies conducted with [13CD2]DPC 602 indicated an aldoxime structure. The concluding evidence came from HMBC NMR spectrum of the conjugate, which showed strong correlation of the cysteine methylene protons with the imino carbon. Further spectroscopic studies with chemically prepared GSH adduct from benzaldehyde oxime confirmed this pattern of correlation. In vivo and in vitro studies with the synthetic oxime intermediate from DPC 423 showed an adduct identical to the one isolated from the bile of rats dosed with DPC 423. This supported the intermediacy of an aldoxime as a precursor to the GSH adducts. It is postulated that the benzylamine moiety of DPC 423 (and its analogues) is oxidized to a hydroxylamine, which is subsequently converted to a nitroso intermediate. Subsequent rearrangement of the nitroso leads to an aldoxime which in turn is metabolized by P450 to a reactive intermediate. The formation of oxime from DPC 423 (and its analogues) was found to be mediated by rat CYP 3A1/2, which were also responsible for converting the oxime to the GSH trappable reactive intermediate. It is postulated that the aldoxime produces a radical or a nitrile oxide intermediate that reacts with GSH and hence produces this unusual GSH adduct. On the basis of synthetic analogy, it is more likely that the nitrile oxide resulting from two-electron oxidation of the aldoxime is the reactive intermediate. Intramolecular kinetic isotope effects were studied with [13CD2]DPC 602 to assess the importance of the metabolic cleavage of the aminomethyl carbon−hydrogen bond in forming this GSH adduct. The lack of isotope effect i
ISSN:0893-228X
1520-5010
DOI:10.1021/tx0101189