2908 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Conway et al.
by the dropwise addition of a solution of K2CO3 (3.45 g, 25
mmol) in 4 mL of water. After 15 min the ice-bath was
removed, and the reaction mixture was stirred for another 20
min. The precipitate was removed by filtration and washed
with methanol (2 × 1 mL). The combined filtrates were cooled
(5 °C) for 15 min and refiltered. Concentration of the solvent
in vacuo (<30 °C) to approximately 2 mL, and cooling the
residue gave crude 8 as white crystals. Recrystallization from
a small volume of methanol gave 0.80 g (26% yield) of 8, mp
94.5-98 °C; Rf 0.32 (EtOAc-hexane 1:2). NMR (CDCl3):3.73
(s, 3H, OCH3), 2.97 (s, 3H, O2SCH3), 3.56 (s, 1H, NH). Anal.
(C2H7NO3S) C, H, N, S.
Su p p or tin g In for m a tion Ava ila ble: Table 2, Blood AcH
and Ethanol Levels in the Rat after Pretreatment with
Prodrugs of NB and Nitroxyl, and Table 3, Inhibition of
Glyceraldehyde-3-phosphate Dehydrogenase (GADPH) by NB,
Cpd 1a , Piloty’s Acid and Angeli’s Salt (2 pages). Ordering
information is given on any current masthead page.
Refer en ces
(1) (a) Shirota, F. N.; DeMaster, E. G.; Nagasawa, H. T. Cyanide is
a product of the catalase mediated oxidation of the alcohol
deterrent agent, cyanamide. Toxicol. Lett. 1987, 37, 7-12. (b)
Shirota, F. N.; DeMaster, E. G.; Kwon, C.-H.; Nagasawa, H. T.
Metabolism of cyanamide to cyanide and an inhibitor of aldehyde
dehydrogenase (AlDH) by rat liver microsomes. Alcohol Alcohol-
ism 1987, Suppl. 1, 219-223. (c) Nagasawa, H. T.; DeMaster,
E. G.; Redfern, B.; Shirota, F. N.; Goon, D. J . W. Evidence for
nitroxyl in the catalase-mediated bioactivation of the alcohol
deterrent agent, cyanamide. J . Med. Chem. 1990, 33, 3120-
3122.
In h ibition of Yea st AlDH. The test compounds were
evaluated for inhibitory activity against yeast AlDH as previ-
ously described.24
In h ibition of Glycer a ld eh yd e-3-p h osp h a te Deh yd r o-
gen a se (GAP DH). The primary reaction mix containing 20
mM sodium arsenate (pH 7.4), 1.0 mM EDTA, and 0.5 mM
NAD+ was preincubated for 5 min at 37 °C followed by the
addition of GAPDH (10 µL) and inhibitor in 5 µL of DMSO for
a total of 0.1 mL. In the case of Angeli’s salt, 2 µL of 5 mM
solution (in 20 mM KOH) was added where indicated. After
10 min, a 20 µL aliquot of the primary mix was added to a
cuvette containing 0.5 mM NAD+, 1.0 mM EDTA, 20 mM
sodium arsenate, 50 mM Tris-Cl (pH 8.6) and 1.0 mM
glyceraldehyde-3-phosphate. This secondary reaction was
initiated by the addition of 20 µL (giving a final volume of 1.0
mL) of the primary reaction mix containing GAPDH. Enzyme
activity was determined spectrophotometrically by following
the increase in absorbance at 340 nm with time. The data
presented (Table 3, Supporting Information) represent tripli-
cate incubations.
An a lysis of N2O follow in g Action of P or cin e Liver
Ester a se on 7a , 7b, a n d 7c. Reaction vessels (25 mL)
containing 1.25 mL of 80 mM potassium phosphate buffer (pH
8.0) and 0.525 mL of H2O were prepared in triplicate for each
sample. The compounds to be tested were dissolved in THF
at a concentration of 100 mM, and an aliquot (200 µL) was
added, followed by the addition of 25 µL of porcine liver
esterase (19 mg protein/mL; 335 units/mg protein) to initiate
the reaction. The vessels were immediately stoppered and
placed in a 37 °C shaking water bath for 10 min before
sampling for N2O determination by headspace gas chroma-
tography.1c Controls contained 25 µL of H2O in place of porcine
liver esterase.
P h a r m a cologica l Eva lu a tion s in Vivo. These studies
were performed in adherance with guidelines established in
the Guide for the Care and Use of Laboratory Animals
published by the U.S. Department of Health and Human
Resources (NIH Publication 85-23, revised 1985). Animals
were housed in facilities accredited by the American Associa-
tion for the Accreditation of Laboratory Animal Care (AAA-
LAC), and the research protocol was approved by the subcom-
mittee on Animal Studies of the Minneapolis VA Medical
Center. This committee is vigorous in enforcing its charge of
minimizing the use of animals in research.
Dr u g Ad m in istr a tion P r otocol. Sprague-Dawley male
rats (Biolab Corp., St. Paul, MN) weighing 170-220 g were
fasted ∼24 h prior to the time of sacrifice. All drugs were
dissolved or suspended in 2% carboxymethylcellulose (CMC).
Doses of 0.5 mmol/kg were given ip as 1.0 mL/100 g body
weight except where noted. Ethanol was administered 2 h
later and was given as a 20% (w/v) solution, 1.0 mL/100 g of
body weight. The animals were sacrificed 1 h following ethanol
administration, and blood was collected for AcH and ethanol
measurements.
(2) (a) DeMaster, E. G.; Shirota, F. N.; Nagasawa, H. T. The
metabolic activation of cyanamide by liver mitochondria:
a
requirement for inhibition of aldehyde dehydrogenase enzymes.
Biochem. Biophys. Res. Commun. 1982, 107, 1333-1339. (b)
DeMaster, E. G.; Shirota, F. N.; Nagasawa, H. T. The metabolic
activation of cyanamide to an inhibitor of aldehyde dehydroge-
nase is catalyzed by catalase. Biochem. Biophys. Res. Commun.
1984, 122, 358-365. (c) Svanas, G. W.; Weiner, H. Enzymatic
requirement for cyanamide inactivation of rat liver aldehyde
dehydrogenase. Biochem. Pharmacol. 1985, 34, 1197-1204.
(3) (a) DeMaster, E. G.; Redfern, Nagasawa, H. T. Mechanisms of
inhibition of aldehyde dehydrogenase by nitroxyl, the active
metabolite of the alcohol deterrent agent cyanamide. Biochem.
Pharmacol. (in press). (b) Wong, P. S.-Y.; Hyun, J .; Fukuto, J .
M.; Shirota, F. N.; DeMaster, E. G.; Shoeman, D. W.; Nagasawa,
H. T. The reaction between S-nitrosothiols and thiols: Genera-
tion of nitroxyl (HNO) and subsequent chemistry. Biochemistry
1998 (in press).
(4) Smith, P. A. S.; Hein, G. E. The alleged role of nitroxyl in certain
reactions of aldehydes and alkyl halides. J . Am. Chem. Soc.
1960, 82, 5731-5740.
(5) Nagasawa, H. T.; Yost, Y.; Elberling, J . A.; Shirota, F. N.;
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dehydrogenase. Biochem. Pharmacol. 1993, 45, 2129-2134.
(6) NA readily dimerizes in protic solvents to an insoluble dimer
which is inactive.
(7) (a) Lee, M. J . C.; Elberling, J . A.; Nagasawa, H. T. N1-
Hydroxylated derivatives of chlorpropamide and its analogues
as inhibitors of aldehyde dehydrogenase in vivo. J . Med. Chem.
1992, 35, 3641-3647. (b) Lee, M. J . C.; Nagasawa, H. T.;
Elberling, J . A.; DeMaster, E. G. Prodrugs of nitroxyl as
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3648-3652.
(8) Shirota, F. N.; DeMaster, E. G.; Elberling, J . A.; Nagasawa, H.
T. Metabolic depropargylation and its relationship to aldehyde
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673.
(9) Vogel, E. Practical Organic Chemistry, 3rd ed.; J ohn Wiley and
Sons: New York, 1956; pp 629-630.
(10) Piloty, O. An account for oxidation of hydroxylamines with
Benzenesulfonyl chloride (in German). Chem. Ber. 1896, 29,
1559-1567.
(11) (a) Wajer, T. A. J . W.; Gelak, H. W.; Engberts, J . B. F. N.;
DeBoer, T. J . C-Nitroso compounds. Part XVI. The reaction of
C-nitroso compounds with benzenesulfinic acid. An ESR study
of alkyl and aryl benzenesulfonyl nitroxides. Rec. Trav. Chim.
Pays Bas 1970, 89, 696-704. (b) Darchen, A.; Monet, C.
Arenesulfinic acids. Nitroso protecting reagents applicable to
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(12) Shchukin, G. I.; Volodarskii, L. B. Reaction of amides of
derivatives of 3-imidazoline and 3-imidazoline 3-oxide with
sodium hypobromite. Izy. Akad. Nauk. SSSR, Ser. Khim. 1979,
1, 228-31.
(13) Chavis, C.; Dumont, F.; Wightman, R. H.; Ziegler, J . C.; Imbach,
J . L. Synthesis of 2′,3′-differentiated ribonucleosides via glyco-
sylation reactions with 2-O-Me or 2-O-TBDMS ribofuranose
derivatives. 1. Pyrimidine series. J . Org. Chem. 1982, 47, 202.-
206.
Mea su r em en t of Blood AcH a n d Eth a n ol Levels. Blood
AcH and ethanol levels were measured as previously de-
scribed.25
(14) Ohno, K.; Nishiyama, H.; Nagase, H. A mild methylation of
alcohols with diazomethane catalyzed by silica gel. Tetrahedron
Lett. 1979, 45, 4405-4406.
(15) (a) Santos, P. F.; Lobo, A. M.; Prabhahar, S. A convenient method
for the preparation of N-aryl and N-alkyl-O-methyl hydroxyl-
amines. Synth. Commun. 1995, 25, 3509-3518. (b) Branco, P.
Ack n ow led gm en t. This work was supported by the
Department of Veterans Affairs and, in part (M.J .C.L.),
by Grant RO1AA07317, National Institutes of Health,
USPHS.