Diethylcarbamoylating/nitroxylating agents as dual action inhibitors of aldehyde dehydrogenase: A disulfiram-cyanamide merger

Article abstract of DOI:10.1021/jm990235p

Benzenesulfohydroxamic acid (Piloty's acid) was functionalized on the hydroxyl group with the N,N-diethylcarbamoyl group, and the hydroxylamine nitrogen was substituted with acetyl (1a), pivaloyl (1b), benzoyl (1c), and ethoxycarbonyl (1d) groups. Only co

Full text of DOI:10.1021/jm990235p

4016
J . Med. Chem. 1999, 42, 4016-4020
Expedited Articles
Dieth ylca r ba m oyla tin g/Nitr oxyla tin g Agen ts a s Du a l Action In h ibitor s of
Ald eh yd e Deh yd r ogen a se: A Disu lfir a m -Cya n a m id e Mer ger
Terry T. Conway,† Eugene G. DeMaster,† David J . W. Goon,†,§ Frances N. Shirota,† and Herbert T. Nagasawa*,†,‡
Medical Research Laboratories, VA Medical Center, One Veterans’ Drive, Minneapolis, Minnesota 55417, and Department of
Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, Minnesota 55417
Received May 12, 1999
Benzenesulfohydroxamic acid (Piloty’s acid) was functionalized on the hydroxyl group with
the N,N-diethylcarbamoyl group, and the hydroxylamine nitrogen was substituted with acetyl
(1a ), pivaloyl (1b), benzoyl (1c), and ethoxycarbonyl (1d ) groups. Only compound 1d inhibited
yeast aldehyde dehydrogenase (AlDH) in vitro (IC50 169 µM). When administered to rats, 1d
significantly raised blood acetaldehyde levels following ethanol challenge, thus serving as a
diethylcarbamoylating/nitroxylating, dual action inhibitor of AlDH in vivo. A more potent dual
action agent was N-(N,N-diethylcarbamoyl)-O-methylbenzenesulfohydroxamic acid (5c), which
was postulated to release diethylcarbamoylnitroxyl (9), a highly potent diethylcarbamoylating/
nitroxylating agent, following metabolic O-demethylation in vivo. The dual action inhibition
of AlDH exhibited by 1d , and especially 9, constitutes a merger of the mechanism of action of
the alcohol deterrent agents, disulfiram and cyanamide.
Ch a r t 1
In tr od u ction
Disulfiram (DSSD; tetraethylthiuram disulfide; Ant-
abuse) has been used as an alcohol deterrent agent for
nearly 50 years.1 Its mode of action is known to be
mediated by the inhibition of hepatic aldehyde dehy-
drogenase (AlDH; EC 1.2.1.3), the enzyme that converts
ethanol-derived acetaldehyde (AcH) to acetate, resulting
in the elevation of blood AcH levels on consumption of
ethanol. This evokes a disulfiram-ethanol reaction
(DER), a syndrome characterized by facial flushing,
tachycardia, and a general feeling of malaise, ostensibly
leading to alcohol avoidance.2 A similar carbimide-
ethanol reaction (CER) is elicited by cyanamide vis-a`-
vis its calcium salt, calcium carbimide,3,4 an alcohol
deterrent agent prescribed in Europe, Canada, and
J apan. Both DSSD and cyanamide require metabolic
bioactivation in vivo before AlDH inhibition is manifest.
The active metabolite of DSSD has been isolated and
shown to be methyl N,N-diethylthiolcarbamate S-oxide
(DETC-MeSO; for structure, see Chart 2),5 while the
active inhibitor from cyanamide metabolism has been
identified as nitroxyl (HNdO).6
Ch a r t 2
Biliary excretion of S-(N,N-diethylcarbamoyl)glu-
tathione after administration of DSSD to rats7 suggests
that DETC-MeSO can carbamoylate sulfhydryl groups,
in particular, the SH group of the active site Cys-302
residue of AlDH, thus irreversibly inhibiting this en-
zyme. We have previously shown that nitroxyl can react
with sulfhydryl reagents such as glutathione8 and
N-acetyl-L-cysteine (NAC)9 to give the corresponding
sulfinamides. Based on these studies, we postulated that
the inhibition of AlDH by nitroxyl was a consequence
of the nitroxylation of the active-site Cys-302 residue
of the enzyme to an initial enzyme N-hydroxysulfena-
mide intermediate, followed by molecular rearrange-
ment to an enzyme sulfinamide, resulting in irreversible
inhibition.10
† VA Medical Center.
‡ University of Minnesota.
§ Present address: LecTec Corp., Minnetonka, MN 55343.
Earlier, we showed that chemical carbethoxylating
10.1021/jm990235p This article not subject to U.S. Copyright. Published 1999 by the American Chemical Society
Published on Web 09/15/1999

Dual Action Inhibitors of AlDH
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4017
Sch em e 1
Sch em e 2a
a
(a) 3.3 H2NNH2, then H2O2; (b) Et3N + Ac2O (1a ), t-BuCOCl
(1b), C6H5COCl (1c), EtOCOCl (1d ).
Resu lts
Compounds 2, 3, and 4 were prepared by heating
1-hydroxybenzotriazole, N-hydroxysuccinimide, and N-
hydroxyphthalimide, respectively, with excess diethyl-
carbamoyl chloride and triethylamine in THF under
reflux for 4 h, while compound 5a was readily prepared
by carbethoxylating the sodium salt of O-methylbenze-
nesulfohydroxamic acid (O-methyl Piloty’s acid) with
ethyl chloroformate at room temperature.
agents such as the O-ethoxycarbonylated derivatives of
1-hydroxybenzotriazole, N-hydroxyphthalimide, N-hy-
droxysuccinimide, and N-hydroxysaccharin were rea-
sonably good inhibitors of yeast AlDH,11 a readily
available enzyme with a conserved Cys-302 and a
sequence homology similar to the mammalian liver
mitochondrial enzyme at the C-terminal active-site
region.12 We also demonstrated that prodrugs of nitroxyl
that can also carbethoxylate the enzyme were powerful
dual action inhibitors of yeast AlDH in vitro yet were
totally inactive when tested in rats.11,13 It was postu-
lated that premature hydrolysis of the carbethoxy group,
perhaps in plasma,14 might be responsible for this lack
of in vivo activity.
The syntheses of compounds 1a -d followed Scheme
2. The successful preparation of 6 required 3.3 equiv of
hydrazine which necessitated destroying the excess with
H2O2, while for the preparation of synthon 7, a NaHCO3
suspension was used as the acid scavenger, since use
of triethylamine as base in the acylation reaction
resulted in the formation of the disulfonylated product
(structure not shown). However, all attempts to prepare
N,O-bis(diethylcarbamoyl)benzenesulfohydroxamic acid
(1e) from 7 under the usual conditions with diethylcar-
bamoyl chloride or with the reagent 4-nitrophenyl N,N-
diethylcarbamate (8)15 failed, even with heating under
a variety of conditions, due to the extreme unreactivity
of these carbamoylating reagents and the thermal
instability of 7 (and 6). Diethylcarbamoylation of 6 using
2 as well as the above reagents was also unsuccessful.
The inhibition of yeast AlDH by these diethylcarbam-
oyl compounds was examined in vitro by procedures
described previously.16 Except for 1d (IC50 169 µM), the
remaining compounds of this series (Chart 1, except 6),
including compound 7, did not inhibit this enzyme at a
concentration of 0.5 mM (or 0.25 mM for 1c due to
aqueous insolubility). The inactivity of compounds 5a ,b,
the negative controls for compound 1d in this system,
supports our conclusion that the inhibition of the
enzyme by 1d must be due to diethylcarbamoylation
rather than to carbethoxylation (ethoxycarbonylation)
of the active-site residue. It appears that the N-
carbethoxy group in a mixed sulfonic/carboxylic imide
structure as in 1d and 5a ,b may not be as reactive as
we had anticipated. The 4-chloro analogue 5b was
shown previously to inhibit rat liver mitochondrial
AlDH in vivo following O-demethylation and the release
of nitroxyl, since blood AcH levels of rats pretreated with
5b were increased significantly over controls when
subsequently challenged with ethanol.13
We now describe a series of compounds designed not
only to diethylcarbamoylate the active-site Cys-302 of
AlDH, thus mimicking the mechanism of action of
DETC-MeSO, the active metabolite of DSSD,5 but also
to release nitroxyl, the putative inhibitor of AlDH
produced by the catalase-mediated oxidation of the
alcohol deterrent agent, cyanamide.6
Compounds 1a -e (Chart 1) were designed to be such
dual action inhibitors of AlDH, since, theoretically, they
can diethylcarbamoylate the enzyme by initial esterase
action intrinsic to this enzyme, followed by release of
an equivalent of nitroxyl to nitroxylate any uncarbam-
oylated enzyme thiol at the active site (Scheme 1). The
diethylcarbamoyl group being much less reactive than
the carbethoxy group, extrahepatic reactions would be
expected to be minimal. Compounds 2-4 are the O-
diethylcarbamoyl analogues of the corresponding O-
ethoxycarbonyl derivatives of 1-hydroxybenzotriazole,
N-hydroxysuccinimide, and N-hydroxyphthalimide, re-
spectively. The latter ethoxycarbonyl compounds were
shown previously to inhibit yeast AlDH in vitro by
carbethoxylating the enzyme.11 Compound 5a was
intended to be the negative control for compound 1d in
vitro, since 5a cannot nitroxylate in the absence of an
O-demethylase (not found in AlDH) but can still carb-
ethoxylate the enzyme based on the intrinsic reactivity
of imides in general. Compound 5b, which liberates
nitroxyl following O-demethylation,13 served as a posi-
tive control for the in vivo studies.
Compound 1d was administered to rats as a suspen-
sion in 2% carboxymethylcellulose (CMC), and blood
AcH levels were measured after ethanol challenge and

4018 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
Conway et al.
Sch em e 3
had shown earlier that such a liver-specific group indeed
existed in the form of the O-methyl ether of sulfo-
hydroxamic acid derivatives,13,22 as exemplified here by
compound 5b. Accordingly, the O-methylated, N-dieth-
ylcarbamoylated derivative of Piloty’s acid (compound
5c) was prepared and tested in rats for its in vivo effect
on AlDH.
F igu r e 1. Blood AcH levels 1 h after ethanol administration
in rats pretreated with compounds 1d and 5c. Compound 5b,
the positive control, is a known in vivo inhibitor of AlDH in
rats. N ) 3, except for 5b where N ) 4. There is no statistical
difference between the AcH levels for 1d and 5b.
As shown in Figure 1, compound 5c was highly
effective in inhibiting the liver mitochondrial AlDH of
rats, as reflected by the pronounced elevation in blood
AcH levels following ethanol challenge. We envision the
biochemical mechanism leading to this pharmacological
end result to proceed as in Scheme 3. Specifically, the
diethylcarbamoylnitroxyl intermediate 9, produced fol-
lowing the hepatic cytochrome P-450-catalyzed O-de-
methylation of 5c and subsequent elimination of ben-
zenesulfinic acid, is now sufficiently active to diethyl-
carbamoylate the active-site enzyme thiol, thereby
generating an equivalent quantity of nitroxyl to react
further with any uninhibited enzyme. Although estima-
tion of the relative degrees of carbamoylation versus
nitroxylation of the enzyme by 5c would be speculative,
dual action is clearly indicated by the nearly 6-fold
increase in activity compared to 5b , a latent nitrox-
ylating agent13 (Figure 1).
It is noted that intermediate 9 bears a formal struc-
tural resemblance to DETC-MeSO (Chart 2) and also
to nitrosyl cyanide (OdN-CN) produced in the oxida-
tion of cyanamide by catalase/H2O2;23 indeed, 9 closely
resembles carbamoylnitroxyl (10; Chart 2) the partial
hydrolysis product of nitrosyl cyanide.23 Thus, the
mechanism of inhibition of AlDH by disulfiram and the
mode of action of cyanamide appears now to have
merged in this postulated mechanism of inhibition of
AlDH by diethylcarbamoylnitroxyl (9) generated from
its prodrug 5c.
compared to that of 5b, the positive control (Figure 1).
It can be seen that 1d was comparable to 5b in raising
ethanol-derived blood AcH in rats, a measure of the
inhibition (>70%) of hepatic mitochondrial AlDH.17
Discu ssion
The lack of inhibitory activity of compounds 2-4
against yeast AlDH confirmed our premise that the
diethylcarbamoyl group is much less electrophilic than
the ethoxycarbonyl group,18 since the corresponding
ethoxycarbonyl analogues of 2-4 were found to inhibit
this enzyme, albeit with IC50’s in the millimolar range.11
The similar lack of activity of compounds 1a -c against
yeast AlDH was, however, surprising in view of the
activity of prodrug 1d , and we are unable to offer any
explanation for this at this time. We considered the
possibility that 1d might be carbethoxylating rather
than diethylcarbamoylating the enzyme. However, com-
pounds 5a as well as 5b were inactive in this system,
and we were unable to prepare the N,O-bis(diethylcar-
bamoyl) compound (1e) which might have resolved this
question.19 Nevertheless, the AlDH inhibitory activity
of 1d in rats fulfilled our goal of sufficiently attenuating
the initial bioactivation step (Scheme 1) for the prodrug
to be acted on by the liver enzyme and providingsfor
the first timesa diethylcarbamoylating/nitroxylating,
dual action agent that inhibits AlDH in vivo.
DETC-MeSO has an activated diethylcarbamoyl group
that readily reacts with glutathione and the active-site
thiol of AlDH. The electrophilicity of this group in-
creases significantly when the sulfoxide moiety is
oxidized further to the sulfone, i.e., to DETC-MeSO2.20,21
However, it is clear from the present results that the
diethylcarbamoyl group attached to the hydroxyl group
of a sulfohydroxamic acid (compounds 1a -c) or an
N-hydroxyimide (compounds 3 and 4) as carbamate
esters, is not sufficiently electrophilic to react with the
enzyme thiol. Thus, while 1d did exhibit the desired
properties that we had been seeking, a reevaluation of
our structural design parameters and the hypothesis on
which they were based appeared to be in order.
Exp er im en ta l Section
Ch em istr y. Melting points were taken on a Fisher-J ohns
hot-stage melting point apparatus and are uncorrected. IR
spectra were recorded on a Nicollet FT-IR spectrophotometer.
1H NMR spectra were recorded on a Varian Gemini 300-MHz
NMR spectrometer. Chemical shifts (δ) are in parts per million
(ppm) relative to Si(CH3)4, and coupling constants (J ) are in
hertz. Microanalyses were performed by M-H-W Laboratories,
Phoenix, AZ. Thin-layer chromatography (TLC) was performed
using Analtech silica gel GF Uniplates, and the products were
visualized either by fluorescence quenching observed under
UV light or by exposure to iodine vapor in an iodine chamber.
Thick layer chromatography was performed using Analtech
1000-µm preadsorbent silica gel GF Uniplates. Organic sol-
vents extracts were dried over Na2SO4 for 15-20 min before
filtration.
At this juncture, we sought a much more liver-specific
functional group for the initial bioactivation step. We

Dual Action Inhibitors of AlDH
J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20 4019
Rea gen ts a n d Ch em ica ls. N-Hydroxyphthalimide, 1-hy-
droxysuccinimide, 1-hydroxybenzotriazole, benzenesulfonyl
chloride, ethyl chloroformate, pivaloyl chloride, benzoyl chlo-
ride, p-(diethylamino)benzoic acid, thionyl chloride, diethyl-
carbamoyl chloride, and sodium hydride were purchased from
Aldrich Chemical Co. (Milwaukee, WI). N-Carbethoxyl-O-
methyl-4-chlorobenzenesulfohydroxamic acid (5b) was pre-
pared as described.14 Yeast AlDH, NAD+, and glucose-6-
phosphate dehydrogenase were purchased from Sigma Chemical
Co. (St. Louis, MO).
Gen er a l P r oced u r e for th e P r ep a r a tion of 2-4. O-
(Diet h ylca r ba m oyl)-N-h yd r oxyp h t h a lim id e (4). N-Hy-
droxyphthalimide (48.9 g; 0.3 mol) was dissolved in 900 mL
of THF, and the solution was cooled in an ice bath under an
atmosphere of N2. Et3N (43.2 mL; 0.31 mol) was added over
ca. 10 min (red color). Diethylcarbamoyl chloride (38.0 mL;
0.30 mol) was added dropwise over 30 min. The ice bath was
then removed, and the reaction mixture was heated under
reflux for 4 h (red color faded to yellow). The reaction mixture
was allowed to cool to room temperature, EtOAc (500 mL) was
added, and the mixture was washed twice with 1 N HCl (100
mL), twice with 1 N NaHCO3 (100 mL, red color), and once
with water (100 mL). After drying, the solvents were removed
on a rotary evaporator in vacuo at 40 °C to give a colorless
solid (60.9 g; 77.4% yield; mp 121.5-122 °C) which was used
directly in the next reaction. An analytical sample, mp 126-
127 °C, was obtained by recrystallization from EtOAc; Rf )
0.95 (EtOAc:hexane, 2/1). 1H NMR: 7.74-7.88 (m, 4H, aryl-
H), 3.48 (q, 2H, J ) 6.9, CH2N), 3.37 (q, 2H, J ) 7.05, CH2N),
1.331 (t, 3H, J ) 6.81, CH3), 1.198 (t, 3H, J ) 6.96, CH3). 13C
NMR: 150.823 (CdO carbamate), 161.615 (CdO imide).
FTIR: (KBr) νCdO 1762; 1746 cm-1. Anal. (C13H14N2O4) C, H,
N.
colorless crystals; mp 71.5-72 °C; Rf ) 0.52 (toluene/EtOAc,
50:10). 1H NMR: 7.52-7.96 (m, 5H, aryl H), 9.00 (s, 1H, NH),
3.22 (q, 2H, CH2N, J ) 7.14), 3.11 (q, 2H, CH2N, J ) 7.08),
1.11 (t, 3H, CH3, J ) 7.14), 0.88 (t, 3H, CH3, J ) 7.08). FTIR:
νCdO 1727 cm-1. Anal. (C11H16N2O4S) C,H,N,S.
Gen er a l P r oced u r e for th e P r ep a r a tion of Com p ou n d s
1a -d . N-Ben zoyl-O-(d iet h ylca r b a m oyl)b en zen esu lfo-
h yd r oxa m ic Acid (1c). Et3N (408 µL; 2.93 mmol) was added
to a solution of 7 (723 mg; 2.66 mmol) and benzoyl chloride
(340 µL; 2.93 mmol) dissolved in 27 mL of CH2Cl2. The reaction
mixture was stirred at room temperature for 24 h at which
time TLC (toluene/EtOAc, 50:10) showed complete reaction of
7 (Rf ) 0.52). The reaction mixture was evaporated to dryness
and the residue was flash chromatographed using toluene/
EtOAc (50:10) as eluant to give 700 mg (70% yield) of a
colorless oil which crystallized on standing at 0 °C. Recrys-
tallization from toluene-hexane gave 1c as colorless crystals;
mp 71-72 °C; Rf ) 0.70 (toluene/EtOAc, 50:10). 1H NMR:
7.35-8.11 (m, 10H, aryl H), 3.23 (brd q, 4H, J ) 6.93), 1.04
(brd t, 6H, J ) 6.93). FTIR: (KBr) νCdO 1763, 1697 cm-1. Anal.
(C18H20N2O7S) C,H,N,S.
N -Ac e t y l -O -( d i e t h y l c a r b a m o y l ) b e n z e n e s u l f o -
h yd r oxa m ic Acid (1a ). This compound was prepared using
acetic anhydride as described above for 1c. Colorless solid; mp
93-94 °C (benzene-hexane); 58.4% yield; Rf ) 0.57 (hexanes-
EtOAc, 2:1). 1H NMR: 7.52-8.05 (m, aryl-H, 5H), 3.36 (q, 4H,
J ) 7.11, (CH2)2), 2.12 (s, 3H, CH3CO), 1.234 (t, 3H, J ) 7.11,
CH3), 1.164 (t, 3H, J ) 7.11, CH3). FTIR: (KBr) νCdO 1708,
1768 cm-1. Anal. (C13H18N2O5S) C,H,N,S.
N -P i v a lo y l-O -(d i e t h y lc a r b a m o y l)b e n z e n e s u lfo -
h yd r oxa m ic Acid (1b). Prepared as described above for 1c
except that pivaloyl chloride was used. Colorless solid; mp
110-111 °C (toluene-hexane); 60.1% yield; Rf ) 0.56 (toluene-
EtOAc, 50:5). 1H NMR: 7.51-8.16 (m, 5H, aryl-H), 3.46 (dq,
4H, J ) 7.20, 2(CH2N)), 1.33 (t, 3H, J ) 7.20, CH3 carbamate),
1.23 (J ) 7.31, t, 3H, CH3 carbamate), 1.18 (s, 9H, (CH3)3.
FTIR: (KBr) νCdO 1754, 1694 cm-1. Anal. (C14H20N2O7S)
C,H,N,S.
O-(Dieth ylca r ba m oyl)-1-h yd r oxysu ccin im id e (3). Pre-
pared from 1-hydroxysuccinimide as described above for 4.
Colorless solid (57% yield); mp 109-111 °C (recrystallized from
1
EtOAc-hexane); Rf ) 0.27 (hexane:THF, 3:1). H NMR: 3.40
(q, J ) 8 Hz, 4H), 2.80 (s, 4H), 1.23 (t, J ) 8 Hz, 6H). FTIR:
(KBr) νCdO 1756, 1745 cm-1. Anal. (C9H14N2O4) C, H, N.
N-(E t h oxyca r b on yl)-O-(d iet h ylca r b a m oyl)b en zen e-
su lfoh yd r oxa m ic Acid (1d ). Prepared as described above for
1c except that ethyl chloroformate was used. Colorless oil
obtained by flash chromatography using toluene/EtOAc (50:
10) as eluant; 83.2% yield; Rf ) 0.68 (toluene/ETOAc, 50:10),
Rf ) 0.71 (toluene/EtOAc/Et3N, 50:10:1), Rf ) 0.55 (toluene/
O-(Dieth ylca r ba m oyl)-1-h yd r oxyben zotr ia zole (2). Pre-
pared from 1-hydroxybenzotriazole as described above for 4.
Colorless solid (81.6% yield); mp 71-72 °C (recrystallized from
EtOAc-hexane); Rf ) 0.48 (hexane:THF, 3/1). 1H NMR: 8.17-
7.83 and 7.57-7.13 (2m, 4H), 3.52 (vbrm, 4H), 1.32 (vbrm, 6H).
FTIR: (KBr) νCdO 1769 cm-1. Anal. (C11H14N2O4) C,H,N.
1
EtOAc/AcOH, 50:10:2). H NMR: 7.52-8.10 (m, 5H, aryl H),
4.17 (q, 2H, J ) 7.00, CH2O), 3.37 (q, 4H, J ) 6.95, CH2N),
1.19 (t, 3H, J ) 7.01, CH3), 1.25 (brd t, 6H, 2CH3). 13C NMR:
151.73 (CdO, carbamate), 149.513 (CdO, carbethoxy). FTIR:
(KBr) νCdO 1759, 1759 cm-1. λmax (EtOH): 224.5 nm (ꢀ 12 200).
Anal. (C14H20N2O6S) C,H,N,S.
O-(Dieth ylca r ba m oyl)h yd r oxyla m in e (6). O-(Dieth yl-
ca r ba m oyl)ben zen esu lfoh yd r oxa m ic Acid (7). Compound
4 (2.62 g; 10 mmol) was dissolved in 100 mL of acetonitrile.
The reaction vessel was flushed with N2 for 5 min, and
anhydrous N2H2 (3.3 equiv; 1.06 mL; 33 mmol) was added.
Caution: Anhydrous N2H2 should be stored and handled under
N2. A white solid formed in a pale-yellow liquid suspension.
The mixture was stirred at room temperature for 30 min and
then allowed to stand in an ice bath for 3 h. TLC (toluene/
EtOAc, 50:10) showed that the reaction was complete. The
precipitate was collected and washed twice with 20 mL of ice-
cold CH3CN. [This precipitate, the hydrazine salt of phthal-
hydrazide, weighed 1.92 g (99% yield) after drying in vacuo.]
The CH3CN filtrate was cooled in an ice bath, and 30% w/w
H2O2 (2.66 mL, 10.3 mmol) was added in four portions over 5
min when gas evolution ensued. After 1 h of stirring, at which
point gas evolution had stopped and tests for H2O2 (iodide-
starch paper) and N2H2 (salicylaldehyde/yellow fluorescence
under long-wave UV light) were negative, NaHCO3 (840 mg,
10 mmol) was added, followed by benzenesulfonyl chloride
(1.28 mL, 10 mmol), and the reaction mixture was allowed to
proceed at room temperature overnight. TLC at this time
showed almost complete loss of 6 (Rf ) 0.30; EtOAc/hexane,
1:2) and formation of 7 (Rf ) 0.56; EtOAc/hexane, 1:2). The
mixture was diluted with 5 volumes of saturated NaCl and
extracted three times with ether, and the combined ether
extracts were washed once with saturated NaCl, dried, and
evaporated to dryness to give 7 (2.68 g, 98.5% yield) as a
slightly yellow oil. Crystallization from toluene-hexane gave
N-(E t h oxyca r b on yl)-O-m e t h ylb e n ze n e su lfoh yd r ox-
a m ic Acid (5a ). Prepared from O-methylbenzenesulfohydrox-
amic acid24 using the method described for 5b.13 This com-
pound was purified by flash chromatography (silica gel 60)
using toluene/EtOAc (50:10) as eluant (18.3% yield, colorless
1
oil); Rf ) 0.65 (toluene/EtOAc, 50:10). H NMR: 7.506-8.011
(m, 5H, aryl H), 4.21 (q, J ) 7.11, 2H, CH2), 4.02 (s, 3H, OCH3),
1.25 (t, J ) 7.14, 3H, CH3). FTIR: (KBr) νCdO 1751.6 cm-1
Anal. (C10H13NO3S) C,H,N,S.
.
N-(N,N-Dieth ylca r ba m oyl)-O-m eth ylben zen esu lfoh y-
d r oxa m ic Acid (5c). O-Methylbenzenesulfohydroxamic acid
(1.87 g; 10 mmol) was dissolved in 10 mL of dry dimethylac-
etamide in an oven-dried three-necked 25-mL flask, equipped
with a magnetic stirrer and a bubbler-guarded water-cooled
condenser. A N2 atmosphere was established in the flask.
Under slow N2 flow, solid NaH (368 mg; 15.3 mmol) was added
all at once. The flask was resealed and stirred at room
temperature for 2 h at which time the flask contained a cream-
colored slurry. Diethylcarbamoyl chloride (1.94 mL; 15.3 mmol)
was added, and the reaction mixture was warmed and stirred
at 40 °C (water bath) for 19 h. The reaction mixture was then
diluted with 100 mL of saturated NaCl and extracted with
ether (4 × 16 mL). The combined ether extracts were washed
with saturated NaCl, and the organic layer was dried and

4020 J ournal of Medicinal Chemistry, 1999, Vol. 42, No. 20
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cloning of the mitochondrial aldehyde dehydrogenase gene of
Saccharomyces cerevisiae by genetic complementation. J . Bac-
teriol. 1991, 173, 3199-3208.
evaporated to dryness in vacuo at room temperature to provide
3.09 g of a thick light yellow oil. This oil was flash chromato-
graphed on silica gel 60 (160 g) using toluene as solvent.
Fractions showing a single spot at Rf ) 0.32 (toluene/EtOAc,
50:10) were combined and evaporated in vacuo at room
temperature to give 1.88 g (66% yield) of 5c as a clear, colorless
oil; Rf ) 0.32 (toluene/EtOAc, 50:10). 1H NMR: 7.50-7.87 (m,
5H, aryl H), 3.30-3.80 (brd, 4H, CH2N), 3.90 (s, 3H, CH3O),
1.22 (t, 3H, CH3, J ) 7.14). FTIR: (KBr) νCdO 1706 cm-1; λmax
(EtOH): 223 nm (ꢀ 15 900). Anal. (C12H18N2O4S) C,H,N,S.
In h ibition of Yea st AlDH. The test compounds were
evaluated for inhibitory activity against yeast AlDH as previ-
ously described.16
P h a r m a cologica l Eva lu a tion s in Vivo. These studies
were performed in adherence 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 (Harlan Sprague-Dawley, Indianapolis, IN) weighing
165-180 g were fasted ∼24 h prior to the time of sacrifice. All
drugs were finely ground and suspended in 2% carboxymeth-
ylcellulose (CMC). Doses of 1.0 mmol/kg were given ip as 1.0
mL/100 g of body weight. Ethanol was administered 1 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. Unlike the prodrugs of nitrosobenzene,20 no
evidence of toxicity was seen with these compounds (1d , 5b,c)
over this short period.
(13) 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.
(14) For example, the N,O-bis(ethoxycarbonyl) derivative of 4-chlo-
robenzenesulfohydroxamic acid is much more readily hydrolyzed
by human plasma than by porcine liver esterase (M. J . C. Lee
and H. T. Nagasawa, unpublished).
(15) Nesynov, R. P.; Pelkis, P. S. Synthesis of p-nitrophenyl esters
of substituted carbamic acids. Obshch. Khim. 1964, 34, 3467-
3469.
(16) DeMaster, E. G.; Shirota, F. N.; Nagasawa, H. T. Catalase
mediated conversion of cyanamide to an inhibitor of aldehyde
dehydrogenase. Alcohol 1985, 2, 117-121.
(17) Shirota, F. N.; DeMaster, E. G.; Elberling, J . A.; Nagasawa, H.
T. Metabolic depropargylation and its relationship to aldehyde
dehydrogenase inhibition in vivo. J . Med. Chem. 1980, 23, 669-
673.
(18) For example, N,N-diethylcarbamoyl chloride is considerably less
reactive than ethyl chloroformate.
(19) We also addressed the possibility that the positions of the
ethoxycarbonyl and diethylcarbamoyl groups might be reversed
as in 1f. This is theoretically possible if 7 underwent an O-to-N
acyl migration during the carbethoxylation step. However, 1d ,
prepared unambiguously by carbethoxylation of 6 followed by
benzenesulfonylation of the resulting product (structure not
shown), had physiochemical properties which were indistin-
guishable from those of 1d prepared via Scheme 2 (see Support-
ing Information). These data also exclude another possible
isomeric structure for 1d , viz., 1g.
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
Ack n ow led gm en t. This work was supported by the
Department of Veterans Affairs. We thank Dr. M. J . C.
Lee for the preparation of O-methyl Piloty’s acid.
Su p p or tin g In for m a tion Ava ila ble: Experimental pro-
cedures for the independent synthesis of 1d from 6. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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