Brief Articles
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 8177
tion and partial purification of the rat and human enzyme systems
active in the reduction of N-hydroxymelagatran and benzamidoxime.
Drug Metab. Dispos. 2005, 33, 570–578.
cytochrome b5 reductase, 200 pmol cytochrome b5, 0.5 mM of the
N-hydroxylated substrate, and 1.0 mM NADH in a total volume of
150 µL of 100 mM potassium phosphate buffer pH 6.0. After
preincubation for 3 min at 37°C, the reaction was started by NADH
and terminated after 15 or 20 min by adding aliquots of methanol.
The precipitated proteins were sedimented by centrifugation and
the supernatant was analyzed by HPLC.
(17) Kurian, J. R.; Bajad, S. U.; Miller, J. L.; Chin, N. A.; Trepanier, L. A.
NADH cytochrome b5 reductase and cytochrome b5 catalyze the
microsomal reduction of xenobiotic hydroxylamines and amidoximes
in humans. J. Pharmacol. Exp. Ther. 2004, 311, 1171–1178.
(18) Kurian, J. R.; Chin, N. A.; Longlais, B. J.; Hayes, K. L.; Trepanier,
L. A. Reductive detoxification of arylhydroxylamine carcinogens by
human NADH cytochrome b5 reductase and cytochrome b5. Chem.
Res. Toxicol. 2006, 19, 1366–1373.
(19) Saulter, J. Y.; Kurian, J. R.; Trepanier, L. A.; Tidwell, R. R.; Bridges,
A. S.; Boykin, D. W.; Stephens, C. E.; Anbazhagan, M.; Hall, J. E.
Unusual dehydroxylation of antimicrobial amidoxime prodrugs by
cytochrome b5 and NADH cytochrome b5 reductase. Drug Metab.
Dispos. 2005, 33, 1886–1893.
(20) Clement, B.; Behrens, D.; Amschler, J.; Matschke, K.; Wolf, S.;
Havemeyer, A. Reduction of sulfamethoxazole and dapsone hydroxy-
lamines by a microsomal enzyme system purified from pig liver and
pig and human liver microsomes. Life Sci. 2005, 77, 205–219.
(21) Clement, B.; Mau, S.; Deters, S.; Havemeyer, A. Hepatic, extrahepatic,
microsomal, and mitochondrial activation of the N-hydroxylated
prodrugs benzamidoxime, guanoxabenz, and Ro 48-3656 ([[1-[(2s)-
2-[[4-[(hydroxyamino)iminomethyl]benzoyl]amino]-1-oxopropyl]-4-
piperid inyl]oxy]-acetic acid). Drug Metab. Dispos. 2005, 33, 1740–
1747.
(22) Torres, R. A.; Korzekwa, K. R.; McMasters, D. R.; Fandozzi, C. M.;
Jones, J. P. Use of density functional calculations to predict the
regioselectivity of drugs and molecules metabolized by aldehyde
oxidase. J. Med. Chem. 2007, 50, 4642–4647.
Acknowledgment. We thank Petra Ko¨ster for technical
assistance.
Supporting Information Available: Purification of cytochrome
b5 and NADH cytochrome b5 reductase, enzyme assays, instru-
mentation (HPLC data for all compounds), and references. This
material is available free of charge via the Internet at http://
pubs.acs.org.
References
(1) Clement, B. Reduction of N-hydroxylated compounds: amidoximes
(N-hydroxyamidines) as pro-drugs of amidines. Drug Metab. ReV.
2002, 34, 565–579.
(2) Gustafsson, D.; Nystrom, J.; Carlsson, S.; Bredberg, U.; Eriksson, U.;
Gyzander, E.; Elg, M.; Antonsson, T.; Hoffmann, K.; Ungell, A.;
Sorensen, H.; Nagard, S.; Abrahamsson, A.; Bylund, R. The direct
thrombin inhibitor melagatran and its oral prodrug H 376/95: intestinal
absorption properties, biochemical and pharmacodynamic effects.
Thromb. Res. 2001, 101, 171–181.
(3) Clement, B.; Lopian, K. Characterization of in vitro biotransformation
of new, orally active, direct thrombin inhibitor ximelagatran, an
amidoxime and ester prodrug. Drug Metab. Dispos. 2003, 31, 645–
651.
(23) Sergeev, G. V.; Gilep, A. A.; Estabrook, R. W.; Usanov, S. A.
Expression of outer mitochondrial membrane cytochrome b5 in
Escherichia coli purification of the recombinant protein and studies
of its interaction with electron-transfer partners. Biochemistry (Mos-
cow) 2006, 71, 790–799.
(24) Shirabe, K.; Landi, M. T.; Takeshita, M.; Uziel, G.; Fedrizzi, E.;
Borgese, N. A novel point mutation in a 3′ splice site of the NADH-
cytochrome b5 reductase gene results in immunologically undetectable
enzyme and impaired NADH-dependent ascorbate regeneration in
cultured fibroblasts of a patient with type II hereditary methemoglo-
binemia. Am. J. Hum. Genet. 1995, 57, 302–310.
(25) Wittke, B.; Mackie, I. J.; Machin, S. J.; Timm, U.; Zell, M.; Goggin,
T. Pharmacokinetics and pharmacodynamics of Ro 44-3888 after single
ascending oral doses of sibrafiban, an oral platelet aggregation
inhibitor, in healthy male volunteers. Br. J. Clin. Pharmacol. 1999,
47, 521–530.
(26) Lanteri, C. A.; Trumpower, B. L.; Tidwell, R. R.; Meshnick, S. R.
DB75, a novel trypanocidal agent, disrupts mitochondrial function in
Saccharomyces cereVisiae. Antimicrob. Agents Chemother. 2004, 48,
3968–3974.
(27) Stuehr, D. J.; Kwon, N. S.; Nathan, C. F.; Griffith, O. W.; Feldman,
P. L.; Wiseman, J. N-omega-Hydroxy-L-arginine is an intermediate
in the biosynthesis of nitric oxide from L-arginine. J. Biol. Chem. 1991,
266, 6259–6263.
(28) Kru¨ger, P. Ueber Abko¨mmlinge des Benzenylamidoxims. Ber. Dtsch.
Chem. Ges. 1885, 18, 1055–1060.
(29) Clement, B.; Bu¨renheide, A.; Rieckert, W.; Schwarz, J. Diacetyldia-
midoximeester of pentamidine, a prodrug for treatment of protozoal
diseases: synthesis, in vitro and in vivo biotransformation. ChemMed-
Chem 2006, 1, 1260–1267.
(30) Clement, B.; Immel, M.; Raether, W. Metabolic N-hydroxylation of
diminazene in vitro. Arzneimittelforschung 1992, 42, 1497–1504.
(31) Bailey, D. M.; DeGrazia, C. G. Hydroxyguanidines. A new class of
antihypertensive agents. J. Med. Chem. 1973, 16, 151–156.
(32) Palmer, T.; Santini, C. L.; IobbiNivol, C.; Eaves, D. J.; Boxer, D. H.;
Giordano, G. Involvement of the narJ and mob gene products in distinct
steps in the biosynthesis of the molybdoenzyme nitrate reductase in
Escherichia coli. Mol. Microbiol. 1996, 20, 875–884.
(4) Peterlin-Masic, L.; Cesar, J.; Zega, A. Metabolism-directed optimi-
sation of antithrombotics: the prodrug principle. Curr. Pharm. Des.
2006, 12, 73–91.
(5) Clement, B.; Raether, W. Amidoximes of pentamidine: synthesis,
trypanocidal and leishmanicidal activity. Arzneimittelforschung 1985,
35, 1009–1014.
(6) Bray, P. G.; Barrett, M. P.; Ward, S. A.; de Koning, H. P. Pentamidine
uptake and resistance in pathogenic protozoa: past, present and future.
Trends Parasitol. 2003, 19, 232–239.
(7) Clement, B.; Demesmaeker, M.; Linne, S. Microsomal catalyzed
N-hydroxylation of guanabenz and reduction of the N-hydroxylated
metabolite: characterization of the two reactions and genotoxic
potential of guanoxabenz. Chem. Res. Toxicol. 1996, 9, 682–688.
(8) Kadlubar, F. F.; McKee, E. M.; Ziegler, D. M. Reduced pyridine
nucleotide-dependent N-hydroxy amine oxidase and reductase activities
of hepatic microsomes. Arch. Biochem. Biophys. 1973, 156, 46–57.
(9) Kadlubar, F. F.; Ziegler, D. M. Properties of a NADH-dependent
N-hydroxy amine reductase isolated from pig liver microsomes. Arch.
Biochem. Biophys. 1974, 162, 83–92.
(10) Bernheim, M. L.; Hochstein, P. Reduction of hydroxylamine by rat
liver mitochondria. Arch. Biochem. Biophys. 1968, 124, 436–442.
(11) Bernheim, M. L. The hydroxylamine reductase of mitochondria. Arch.
Biochem. Biophys. 1969, 134, 408–413.
(12) Bernheim, M. L. The reduction of hydroxylamine and some aryl
hydroxamates by liver mitochondria from mammals and birds.
Enzymologia 1972, 43, 167–176.
(13) Clement, B.; Lomb, R.; Mo¨ller, W. Isolation and characterization of
the protein components of the liver microsomal O2-insensitive NADH-
benzamidoxime reductase. J. Biol. Chem. 1997, 272, 19615–19620.
(14) Havemeyer, A.; Bittner, F.; Wollers, S.; Mendel, R.; Kunze, T.;
Clement, B. Identification of the missing component in the mitochon-
drial benzamidoxime prodrug-converting system as a novel molyb-
denum enzyme. J. Biol. Chem. 2006, 281, 34796–34802.
(15) Nason, A.; Lee, K. Y.; Pan, S. S.; Ketchum, P. A.; Lamberti, A.;
DeVries, J. In vitro formation of assimilatory reduced nicotinamide
adenine dinucleotide phosphate: nitrate reductase from a Neurospora
mutant and a component of molybdenum enzymes. Proc. Natl. Acad.
Sci. U.S.A 1971, 68, 3242–3246.
(33) Johnson, J. L.; Rajagopalan, K. V. Structural and metabolic relationship
between the molybdenum cofactor and urothione. Proc. Natl. Acad.
Sci. U.S.A 1982, 79, 6856–6860.
(34) Schwarz, G.; Boxer, D. H.; Mendel, R. R. Molybdenum cofactor
biosynthesis. The plant protein Cnx1 binds molybdopterin with high
affinity. J. Biol. Chem. 1997, 272, 26811–26814.
(16) Andersson, S.; Hofmann, Y.; Nordling, A.; Li, X. Q.; Nivelius, S.;
Andersson, T. B.; Ingelman-Sundberg, M.; Johansson, I. Characteriza-
JM8010417