Diazen-1-ium-1,2-diolated and nitrooxyethyl nitric oxide donor ester prodrugs of anti-inflammatory (E)-2-(aryl)-3-(4-methanesulfonylphenyl)acrylic acids: Synthesis, cyclooxygenase inhibition, and nitric oxide release studies

Article abstract of DOI:10.1016/j.bmc.2007.12.006

A new group of hybrid nitric oxide-releasing anti-inflammatory drugs wherein an O²-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate (11a-d), or 2-nitrooxyethyl (12a-d), ^{{radical dot}}NO-donor moiety is attached directly

Full text of DOI:10.1016/j.bmc.2007.12.006

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3302–3308
Diazen-1-ium-1,2-diolated and nitrooxyethyl nitric oxide donor
ester prodrugs of anti-inflammatory (E)-2-(aryl)-3-
(4-methanesulfonylphenyl)acrylic acids: Synthesis,
cyclooxygenase inhibition, and nitric oxide release studies
Khaled R. A. Abdellatif, Morshed Alam Chowdhury, Ying Dong,
Qiao-Hong Chen and Edward E. Knaus*
Dentistry Pharmacy Centre, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta,
3-118 Edmonton, Alta., Canada T6G 2N8
Received 13 November 2007; accepted 4 December 2007
Available online 8 December 2007
Abstract—A new group of hybrid nitric oxide-releasing anti-inflammatory drugs wherein an O2-acetoxymethyl-1-(N-ethyl-N-meth-
ylamino)diazen-1-ium-1,2-diolate (11a–d), or 2-nitrooxyethyl (12a–d), NO-donor moiety is attached directly to the carboxylic acid
ꢀ
group of (E)-3-(4-methanesulfonylphenyl)-2-(phenyl)acrylic acids were synthesized. The 2-nitrooxyethyl ester prodrugs (12a–d) all
exhibited in vitro inhibitory activity against the cyclooxygenase-2 (COX-2) isozyme (IC50 = 0.07–2.8 lM range). All compounds
ꢀ
releasedꢀa low amount of NO upon incubation with phosphate buffer (PBS) at pH 7.4 (1.0–4.8% range). In comparison, the per-
centage NO released was significantly higher (76.2–83.0% range) when the diazen-1-ium-1,2-diolate ester prodrugs were incubated
in the presence of rat serum, or moderately higher (7.6–10.1% range) when the nitrooxyethyl ester prodrugs were incubated in the
presence of L-cysteine. These incubation studies suggest that both ꢀNO and the parent anti-inflammatory (E)-3-(4-methanesulfonyl-
phenyl)-2-(phenyl)acrylic acid would be released upon in vivo cleavage by non-specific serum esterases in the case of the
diazen-1-ium-1,2-diolate esters (11a–d), or interaction with systemic thiols in the case of the nitrate esters (12a–d). O2-Acetoxy-
methyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate (E)-3-(4-methanesulfonylphenyl)-2-phenylacrylate (11a) released 83%
ꢀ
of the theoretical maximal release of 2 molecules of NO/molecule of the parent hybrid ester prodrug upon incubation with rat
serum. Hybrid ester anti-inflammatory/ꢀNO donor prodrugs offer a potential drug design concept targeted toward the development
of anti-inflammatory drugs that are devoid of adverse ulcerogenic and/or cardiovascular effects.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
gastric injury in experimental models.3,4 A biologically
relevant class of O2-unsubstituted N-diazen-1-ium-1,2-
diolates (NONOates) have the potential to release NO
ꢀ
Inhibition of cyclooxygenase-derived prostaglandin syn-
thesis, the major mechanism by which non-steroidal
anti-inflammatory drugs (NSAIDs) exert their anti-
inflammatory effect, is also responsible for adverse gas-
troduodenal erosions and ulcerations with an incidence
that is near 15% in chronic NSAID users.1 Nitric oxide
(ꢀNO) is also a beneficial mediator of gastrointestinal
mucosal protection that induces many actions similar
to prostaglandins in the gastrointestinal tract.2 In this
(first-order kinetics) without metabolic activation, pos-
sess structural diversity that allows controllable rates
of ꢀNO-release, and are amenable to facile derivatization
chemistry that facilitates targeting of ꢀNO to specific or-
gan and/or tissue sites.5 The concept6 that linking an
ꢀNO-releasing moiety to a NSAID may reduce the gas-
trointestinal toxicity of the latter has been validated
by in vivo ulcerogenicity studies. Accordingly, we
showed that a novel group of hybrid NO-releasing
NONO-NSAID ester prodrugs possessing a 1-(pyrroli-
din-1-yl)diazen-1-ium-1,2-diolate or 1-(N,N-dimethyl-
amino)-diazen-1-ium-1,2-diolate moiety attached via a
one-carbon spacer, or an O2-acetoxymethyl-1-(N-ethyl-
N-methylamino)diazen-1-ium-1,2-diolate moiety at-
tached directly to the carboxylic acid group of aspirin
ꢀ
respect, NO has been shown to reduce the severity of
Keywords: Anti-inflammatory acrylic ester prodrugs; Cyclooxygenase-
1 and -2 inhibition; Diazen-1-ium-1,2-diolate and nitrooxyethyl nitric
oxide donors.
*
Corresponding author. Tel.: +1 780 492 5993; fax: +1 780 492
1217; e-mail: eknaus@pharmacy.ualberta.ca
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.006

K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 16 (2008) 3302–3308
3303
(1a–c), ibuprofen (2a–c) and indomethacin (3a–c)
2. Chemistry
showed approximately equipotent anti-inflammatory
activity to the parent NSAID, extensive in vitro ester
cleavage by nonspecific serum esterase, and no (aspirin
and ibuprofen), or minimal (indomethacin), in vivo ulc-
erogenicity7,8 (see structures in Fig. 1). Animal studies
carried out by others have shown that nitrate-based
NO-NSAIDs such as NO-naproxen (4),9 NO-flurbipro-
fen (5),10,11 NO-diclofenac (6),12 and NO-aspirin (7)13
are gastrointestinal sparing while simultaneously sup-
pressing prostaglandin synthesis similar to the parent
drugs.14–16
A group of (E)-2-(phenyl)-3-(4-methanesulfonylphenyl)
acrylate esters possessing an O2-acetoxymethyl-1-(N-
ethyl-N-methylamino)diazen-1-ium-1,2-diolate
ester
moiety (11a–d) were synthesized using the reaction se-
quence illustrated in Scheme 1. Accordingly, nucleo-
philic displacement of the mesyloxy group present in
the mesylate 9 by the respective sodium salt of the ac-
rylic acid (8a–d) in hexamethylphosphoramide (HMPA)
afforded the target product (11a–d) in moderate yield
(32–48%). The corresponding 2-nitrooxyethyl acrylate
esters 12a–d were prepared in higher yields (76–94%)
by the K2CO3 mediated coupling of the acrylic acids
(8a–d) with 2-nitrooxyethyl bromide (10) in DMF.
A novel class of acyclic (E)-2-(phenyl)-3-(4-metha-
nesulfonylphenyl)acrylic acids (8) that exhibit COX
isozyme and/or 5-lipoxygenase (5-LOX) inhibitory
activities were reported recently.17 We now present
a group of hybrid ester prodrugs in which (i) an
O2-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-
1-ium-1,2-diolate (11a–d), or (ii) 2-nitrooxyethyl (12a–
d), ꢀNO-donor moiety is attached directly to the
carboxylic acid group of a select group of (E)-3-(4-
methanesulfonylphenyl)-2-(phenyl)acrylic acids (8,
R = 4-H, 4-OMe, 4-F, 4-Br). It is expected that these
3. Results and discussion
A group of acyclic (E)-3-(4-methanesulfonylphenyl)-
2-(phenyl)acrylic acids that exhibited interesting
combinations of COX-1/COX-2 and/or 5-LOX/15-
LOX isozyme inhibitory activities were recently
reported.17 Four compounds from within this group,
having a 4-H, 4-OMe, 4-F or 4-Br substituent on the
C-2 phenyl ring, were selected for elaboration to hybrid
ester prodrugs wherein an O2-acetoxymethyl-1-(N-ethyl-
N-methylamino)diazen-1-ium-1,2-diolate (11a–d), or
ꢀ
hybrid NO-donor prodrugs will be devoid of adverse
ulcerogenic and cardiovascular effects based on their
ability to release cytoprotective and vasodilatory
ꢀNO.
OAc
OR
O
O
Cl
O
-CH2O
N
+
;
a, R =
c, R =
-CH2O
N
+
Me
b, R =
N
N
N
N
O
1 (NONO-Aspirin)
N
M
e
CH3
CH3
OR
O
OR
MeO
N
O
O
Me
+
N
N
O
i-Bu
O
Me
O
3 (NONO-Indomethacin)
2 (NONO-Ibuprofen)
O
O
Me
Me
OR
OR
Cl
H
N
OR
ONO2
R =
O
MeO
F
Cl
6 (NO-Diclofenac)
4 (NO-Naproxen)
5 (NO-Flurbiprofen)
O
O
O
O
O
3
R
S
ONO2
4
Me
H
CO2H
O
CH3
7 (NO-Aspirin)
8, R = 4-H, 4-F, 3-Br, 4-Br, 4-OH,
4-OMe, 4-OAc, 4-NHCOMe
Figure 1. Chemical structures of some diazen-1-ium-1,2-diolate ester prodrugs of aspirin (1a–c), ibuprofen (2a–c) and indomethacin (3a–c), some
nitrooxybutyl ester prodrugs of naproxen (4), flurbiprofen (5) and diclofenac (6), the 3-(nitrooxymethylphenyl) ester of aspirin (7), and (E)-3-(4-
methanesulfonylphenyl)-2-(phenyl)acrylic acids (8).

3304
K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 16 (2008) 3302–3308
O
slow ꢀNO release.20 In contrast, the effect of non-specific
O
esterases present in rat serum on the ꢀNO release proper-
ties of compounds 11a–d was substantially higher (76.2–
83.0% range). These data indicate the non-specific serum
esterases present in rat serum cleave these hybrid pro-
drug esters more effectively than PBS at pH 7.4. From
a mechanistic perspective, the hybrid ester prodrugs
11a–d cannot release ꢀNO prior to cleavage of the termi-
nal O2-acetoxymethyl ester group. This requirement is
consistent with the observation that O2-sodium 1-[N-(2-
hydroxyethyl)-N-methylamino]diazen-1-ium-1,2-diolate
(13), which does not possess an ester group that requires
prior ester cleavage, releases 84.9% and 85% of the the-
S
R
Me
O
N
O
O
O
Me
H
C
O
N
N
Me
O
11a, R = H
11b, R = OMe
11c, R = F
Me
O
11d, R = Br
(a), 9
O
O
O
O
S
R
N
N
Me
+
ꢀ
O
N
oretical maximal release of two molecules of NO/mole-
Me
OH
cule of the parent NO donor in PBS and serum,
respectively. A plausible mechanism for the ester
hydrolysis of hybrid ester prodrugs containing an O2-
acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-
1,2-diolate ester moiety, and the subsequent release of
H
C
O
8a, R = H
8b, R = OMe
8c, R = F
O
9
8d, R = Br
(b), 10
O
S
Me
ꢀ
two molecules of NO, was described in an earlier
O
ꢀ
study.8 The hybrid ester NO-donor prodrugs 11a–d
O
O
S
R
were designed with a one-carbon methylene spacer be-
tween the terminal acetoxy group and the diazen-1-
ium-1,2-diolate O2-atom, such that the O2-(hydroxy-
methyl)diazen-1-ium-1,2-diolate compound formed
after terminal acetoxy cleavage would spontaneously
eliminate formaldehyde to produce the free diazen-1-
ium-1,2-diolate compound (NONOate) that can subse-
quently fragment to release two molecules of ꢀNO
(NONO-donor). Comparative studies showed that the
Me
Br
O
H
C
ONO2
O2NO
12a, R = H
12b, R = OMe
12c, R = F
O
10
12d, R = Br
Scheme 1. Reagents and conditions: (a) Na2CO3, hexamethylphos-
phoramide (HMPA), 25 ꢁC, 72 h; (b) K2CO3, dimethylformamide
(DMF), 25 ꢁC, 36 h.
ꢀ
extent of NO release upon incubation of the hybrid
nitrooxyethyl ester prodrugs (12a–d) in PBS at a pH
of 7 was lower (1.0–3.3% range) than the correspond-
ing O2-acetoxymethyl-1-(N-ethyl-N-methylamino)dia-
zen-1-ium-1,2-diolate ester prodrugs (11a–d, 3.5–4.8%
ꢀ
2-nitrooxyethyl (12a–d), NO-donor moiety is attached
directly to the carboxylic acid group of the anti-inflam-
matory compound (8a–d). In vitro structure-activity
relationships acquired for the hybrid nitrooxy ester pro-
drugs (12a–d) showed that they exhibited medium-to-
high COX-1 (IC50 = 0.37–6.2 lM range), and COX-2
(IC50 = 0.07–2.8 lM range), inhibitory activities (see
data in Table 1). The OMe (12b) and F (12c) analogs
were non-selective COX-2 inhibitors, whereas the H
(12a) and Br (12d) analogs showed COX-2 selectivity in-
dexes of 34.4 and 5.3, respectively. Esterase cleavage of
the acrylic acid ester moiety in the ester prodrugs (11a–
d) would liberate the parent acrylic acids (8a–d) for
which the COX-1 and COX-2 inhibitory data previously
acquired17 are listed in Table 1.
ꢀ
range). The percentage of NO released from the nitro-
oxyethyl compounds 12a–d was greater in the presence
of 5 mM L-cysteine (7.6–10.1% range) than in the ab-
sence of L-cysteineꢀ. This latter observation is consistent
with reports that NO release from organic nitrates is
facilitated by thiols.21,22
ꢀ
Two disadvantages of NO-releasing NSAIDs having a
nitrooxyalkyl group are (i) that production of NO
ꢀ
from organic nitrate esters requires a three-electron
reduction, and (ii) this metabolic activation decreases
in efficiency on continued use of the drugs contribut-
ing to ‘nitrate tolerance’.23 In contrast, O2-unsubstit-
ued N-diazen-1-ium-1,2-diolates have the potential to
ꢀ
ꢀ
The rate of NO release from diazen-1-ium-1,2-diolates
release up to 2 equivalents of NO with half-lives that
can be decreased by chemical modification involving
the attachment of alkyl substituents to the O2-posi-
tion.18 Thus, O2-substituted-diazen-1-ium-1,2-diolates
are stable compounds that hydrolyze slowly even in
acidic solution.19 The percent ꢀNO released from the hy-
brid O2-acetoxymethyl-1-(N-ethyl-N-methylamino)dia-
zen-1-ium-1,2-diolate ester prodrugs (11a–d) upon
incubation in phosphate-buffered-saline (PBS at pH
7.4), and in the presence of rat serum, was determined
by quantitation of nitrite using the Griess reaction ꢀ(see
data in Table 1). In this regard, the percentage of NO
released upon incubation of 11a–d in PBS at pH 7.4 var-
ied over a narrow 3.5–4.8% range which is indicative of
correlate well with their pharmacological durations of
action. In earlier studies, we validated the drug design
concept that hybrid NSAID ester prodrugs (NSAID-
NONOates) possessing
a
diazen-1-ium-1,2-diolate
ꢀNO donor moiety constitute a highly effective method
to abolish NSAID induced gastrointestinal toxicity
(ulcerogencity).7,8 It is equally probable that incorpo-
ration of a vasodilator diazen-1-ium-1,2-diolate NO
donor moiety into selective COX-2 inhibitors such as
rofecoxib and/or valdecoxib may provide a therapeutic
approach to counteract the adverse cardiovascular ef-
fects that led to their withdrawal from the clinical
market.24,25

K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 16 (2008) 3302–3308
3305
Table 1. In vitro COX-1 and COX-2 inhibition, and percent (%) nitric oxide release, data for acrylic acids (8a–d), diazeniumdiolate acrylate esters
(11a–d), nitrooxyethyl acrylate esters (12a–d), O2-sodium 1-[N-(2-hydroxyethyl)-N-methylamino]diazen-1-ium-1,2-diolate (13), and glycerol trinitrate
(14)
O
O
O
N
O
O
O
S
R
O
S
R
HO
ONa
S
R
Me
Me
N
N
Me
O
N
13
Me
O
O
O
Me
O
CH ONO
CHONO
CH ONO
H
C
O
N
N
H
C
O
ONO
2
2
2
OH
H
C
O
Me
O
2
8
11
12
14, GTN)
2
2
ꢀ
Compound
R
IC50 (lM)a
COX-2 S.I.b
% NO releasedc
COX-1
COX-2
PBSd
Serume
L-Cysteinef
8a
8b
H
1.5g
31.6g
0.4g
0.88g
—
3.0g
1.9g
36.0g
3.6g
—
0.50g
16.6g
—
—
—
—
—
—
—
—
OMe
F
8c
8d
—
—
—
—
Br
H
0.24g
—
11a
11b
4.8
3.9
3.6
3.5
3.0
1.4
3.3
1.0
84.9
2.8
—
83.0
77.6
82.1
76.2
—
—
—
OMe
F
—
—
—
—
—
—
11c
11d
—
—
Br
H
—
6.2
—
—
12a
12b
0.18
1.4
34.4
0.78
0.57
5.3
8.5
9.2
7.6
10.1
—
OMe
F
1.1
1.6
—
—
12c
12d
2.8
Br
—
0.37
—
0.07
—
—
13
—
—
85.0
—
14 (GTN)
Aspirin
Celecoxib
Rofecoxib
—
—
—
—
2.4
10.1
—
0.35
33.1
>100
0.15
473
>200
—
—
—
—
0.07
0.50
—
—
—
—
—
a Values are means of two determinations acquired using an ovine COX-1/COX-2 assay kit (Catalog No. 560101, Cayman Chemicals Inc., Ann
Arbor, MI, USA) and the deviation from the mean is <10% of the mean value.
b In vitro COX-2 selectivity index (COX-1 IC50/COX-2 IC50).
c Percent of nitric oxide released based on a theoretical maximum release of (i) 2 mol of NO/mol of the diazen-1-ium-1,2-diolate test compounds
(11a–d, 13), (ii) 1 mol of NO/mol of the nitrooxyethyl test compounds (12a–d), and (iii) 3 mol of NO/mol of glycerol trinitrate (14). The result is the
mean value of 3 measurements (n = 3) where variation from the mean % value was 60.2%.
d A solution of the test compound (2.4 mL of a 1.0 · 10À2 mM solution in phosphate buffer at pH 7.4) was incubated at 37 ꢁC for 1.5 h.
e A solution of the test compound (2.4 mL of a 1.0 · 10À2 mM solution in phosphate buffer at pH 7.4 to which 90 lL rat serum had been added) was
incubated at 37 ꢁC for 1.5 h.
f A solution of the test compound (2.4 mL of a 1.0 · 10À2 mM solution in phosphate buffer at pH 7.4 which contained 5.0 mM L-cysteine) was
incubated at 37 ꢁC for 1.5 h.
g Data taken from the literature.17
ꢀ
4. Conclusions
of NO in the 76.2–83.0 % range, (iv) L-cysteine moder-
ately enhances the release of NO (7.6–10.1% range) from
the nitrooxyethyl esters (12a–d) that requires a metabol-
ically demanding three-electron reduction for the release
A group of hybrid ester prodrugs in which an O2-acet-
oxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,
2-diolate (11a–d), or 2-nitrooxyethyl (12a–d), NO-donor
moiety is attached directly to the carboxylic acid group
of (E)-3-(4-methanesulfonylphenyl)-2-(4-H, 4-OMe, 4-F
or 4-Br substituted-phenyl)acrylic acids were synthe-
sized for comparative biological evaluation. Structure-
activity and biological stability studies showed that (i)
the nitrooxyethyl ester prodrugs (12a–d) retain in vitro
COX-1 and COX-2 inhibitory activity, (ii) both classes
of prodrugs (11a–d, 12a–d) are relatively stable in phos-
phate-buffered saline at pH 7 where ꢀNO release is in the
1.0–4.8 % range, and specifically (iii) the O2-acetoxy-
methyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-
diolates (11a–d) undergo extensive ester cleavage by rat
serum esterase(s) that is followed by a significant release
ꢀ
of NO, and (v) hybrid ester NO donor prodrugs offer a
potential drug design concept for the development of
NSAIDs that are devoid of adverse ulcerogenic and/or
cardiovascular side effects.
5. Experimental
Melting points were determined on a Thomas–Hoover
capillary apparatus and are uncorrected. Infrared (IR)
spectra were recorded as films on NaCl plates using a
1
Nicolet 550 Series II Magna FT-IR spectrometer. H
NMR spectra were measured on a Bruker AM-300 spec-
trometer in CDCl3 with TMS as the internal standard,

3306
K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 16 (2008) 3302–3308
where J (coupling constant) values are estimated in
Hertz (Hz). Mass spectra (MS) were recorded on a
Water’s Micromass ZQ 4000 mass spectrometer using
the ESI ionization mode. Microanalyses were performed
for C, H, N by the Microanalytical Service Laboratory,
Department of Chemistry, University of Alberta. Silica
gel column chromatography was performed using
Merck silica gel 60 ASTM (70–230 mesh). All other re-
agents, purchased from the Aldrich Chemical Company
(Milwaukee, WI), were used without further purification.
The (E)-3-(4-methanesulfonylphenyl)-2-(phenyl)acrylic
acids (8a–d, R = 4-H, 4-OMe, 4-F, 4-Br),12 O2-acetoxy-
methyl-1-[N-(2-methylsulfonyloxyethyl)-N-methylamino]-
diazen-1-ium-1,2-diolate (9),26 2-bromoethylnitrate (10)22,
O2-sodium 1-[N-(2-hydroxyethyl)-N-methylamino]diazen-
1-ium-1,2-diolate (13),26 and glycerol trinitrate (14)27 were
prepared according to literature procedures. Nitric oxide
gas was purchased from BOC Scientific (Burlington,
Ontario).
3H, COCH3), 3.01 (s, 3H, NCH3), 3.03 (s, 3H,
CH3SO2), 3.74 (t, J = 5.2 Hz, 2H, CH2N), 3.85 (s, 3H,
OCH3), 4.42 (t, J = 5.2 Hz, 2H, CO2CH2), 5.76 (s, 2H,
OCH2O), 6.91 (dd, J = 7.1, 2.0 Hz, 2H, 4-methoxyphenyl
H-3, H-5), 7.11 (dd, J = 7.1, 2.0 Hz, 2H, 4-methoxy-
phenyl H-2, H-6), 7.27 (d, J = 8.2 Hz, 2H, 4-metha-
nesulfonylphenyl H-2, H-6), 7.75 (d, J = 8.2 Hz, 2H, 4-
methanesulfonylphenyl H-3, H-5), 7.80 (s, 1H, H-3);
MS 543.93 (M+Na). Anal. Calcd for C23H27N3O9S: C,
51.58; H, 5.38; N, 7.85. Found: C, 51.84; H, 5.82; N,
7.84.
5.1.3. O2-Acetoxymethyl-1-(N-ethyl-N- methylamino)dia-
zen-1-ium-1,2-diolate (E)-2-(4-fluorophenyl)-3-(4-metha-
nesulfonylphenyl)acrylate (11c). Yield, 36%; yellow
gum; IR (film) 2965 (C–H aromatic), 2927 (C–H ali-
phatic), 1735, 1717 (CO2), 1307, 1148 (SO2), 1220,
1025 (N@N–O) cmÀ1 1H NMR (CDCl3) d 2.10 (s,
;
3H, COCH3), 3.02 (s, 3H, NCH3), 3.08 (s, 3H,
CH3SO2), 3.72 (t, J = 5.2 Hz, 2H, CH2N), 4.45 (t,
J = 5.2 Hz, 2H. CO2CH2), 5.78 (s, 2H, OCH2O), 7.01–
7.17 (m, 4H, fluorophenyl hydrogens), 7.22 (d,
J = 8.2 Hz, 2H, 4-methanesulfonylphenyl H-2, H-6),
7.76 (d, J = 8.2 Hz, 2H, 4-methanesulfonylphenyl H-3,
H-5), 7.91 (s, 1H, H-3); MS 531.90 (M+Na). Anal.
Calcd for C22H24FN3O8S: C, 51.86; H, 4.75; N, 8.25.
Found: C, 51.96; H, 5.01; N, 7.95.
5.1. General method for preparation of the O2-acetoxy-
methyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-dio-
late acrylate esters (11a–d)
Sodium carboxylates of the respective acrylic acids 8a–d
(R = H, OMe, F, Br) were prepared in situ by stirring
each acid (2.5 mmol) in a suspension of sodium carbon-
ate (0.27 g, 2.5 mmol) and HMPA (3.5 mL) for 19 h at
25 ꢁC. A solution of O2-acetoxymethyl-1-[N-(2-meth-
ylsulfonyloxyethyl)-N-methylamino]diazen-1-ium-1,2-
diolate (9, 2.5 mmol) in HMPA (1.5 mL) was then
added, and the reaction was allowed to proceed for
72 h at 25 ꢁC. EtOAc (30 mL) was added, the mixture
was washed with water (5 · 15 mL), the organic phase
was dried (Na2SO4), and the solvent was removed in va-
cuo. The residue obtained was purified by silica gel col-
umn chromatography using EtOAc/hexane (2:1, v/v) as
the eluent. Physical and spectral data for 11a–d are listed
below.
5.1.4. O2-Acetoxymethyl-1-(N-ethyl-N-methylamino)dia-
zen-1-ium-1,2-diolate (E)-2-(4-bromophenyl)-3-(4-metha-
nesulfonylphenyl)acrylate (11d). Yield, 39%; yellow gum;
IR (film) 2965 (C–H aromatic), 2927 (C–H aliphatic),
1754, 1715 (CO2), 1304, 1152 (SO2), 1239, 1073
(N@N–O) cmÀ1 1H NMR (CDCl3) d 2.10 (s, 3H,
;
COCH3), 3.01 (s, 3H, NCH3), 3.04 (s, 3H, CH3SO2),
3.73 (t, J = 5.2 Hz, 2H, CH2N), 4.42 (t, J = 5.2 Hz,
2H, CO2CH2), 5.76 (s, 2H, OCH2O), 7.08 (d,
J = 8.2 Hz, 2H, 4-bromophenyl H-3, H-5), 7.26 (d,
J = 8.5 Hz, 2H, 4-methanesulfonylphenyl H-2, H-6),
7.52 (d, J = 8.2 Hz, 2H, 4-bromophenyl H-2, H-6),
7.77 (d, J = 8.5 Hz, 2H, 4-methanesulfonylphenyl H-3,
H-5), 7.80 (s, 1H, H-3); MS 591.83 (M+Na). Anal.
Calcd for C22H24BrN3O8S: C, 46.32; H, 4.24; N, 7.37.
Found: C, 46.79; H, 4.53; N, 7.23.
5.1.1. O2-Acetoxymethyl-1-(N-ethyl-N- methylamino)dia-
zen-1-ium-1,2-diolate (E)-3-(4-methanesulfonylphenyl)-2-
phenylacrylate (11a). Yield, 48%; white powder; mp
94–96 ꢁC; IR (film) 2972 (C–H aromatic), 2930 (C–H
aliphatic), 1753, 1709 (CO2), 1369, 1151 (SO2), 1237,
1065 (N@N–O) cmÀ1
;
1H NMR (CDCl3) d 2.10 (s,
5.2. General method for preparation of the 2-nitrooxy-
ethyl acrylate esters (12a–d)
3H, COCH3), 2.94 (s, 3H, NCH3), 3.01 (s, 3H,
CH3SO2), 3.72 (t, J = 5.2 Hz, 2H, CH2N), 4.40 (t,
J = 5.2 Hz, 2H, CO2CH2), 5.76 (s, 2H, OCH2O), 7.17–
7.27 (m, 3H, phenyl, H-3, H-4, H-5), 7.30 (d,
J = 8.3 Hz, 2H, 4-methanesulfonylphenyl H-2, H-6),
7.39–7.41 (m, 2H, phenyl H-2, H-6), 7.75 (d,
J = 8.3 Hz, 2H, 4-methanesulfonylphenyl H-3, H-5),
7.85 (s, 1H, H-3); MS 513.90 (M+Na). Anal. Calcd for
C22H25N3O8S: C, 53.76; H, 5.13; N, 8.55. Found: C,
53.67; H, 5.23; N, 8.43.
A solution of 2-nitrooxyethyl bromide (10, 0.22 mmol),
the respective acrylic acid (8a–d, 0.2 mmol), and K2CO3
(0.24 mmol) in dry DMF (10 mL) was stirred at 25 ꢁC
for 36 h. Water (40 mL) was added, the mixture was ex-
tracted with EtOAc (3 · 300 mL), the extract was
washed with water (2 · 40 mL) and then brine
(40 mL), the organic phase was dried (Na2SO4), and
the solvent was removed in vacuo. The residue obtained
was purified by silica gel column chromatography using
EtOAc–hexane (1:1, v/v) as eluent. Physical and spectral
data for 12a–d are listed below.
5.1.2. O2-Acetoxymethyl-1-(N-ethyl-N- methylamino)dia-
zen-1-ium-1,2-diolate (E)-3-(4-methanesulfonylphenyl)-2-
(4-methoxyphenyl)acrylate (11b). Yield, 32%; yellow
gum; IR (film) 2970 (C–H aromatic), 2927 (C–H ali-
phatic), 1733, 1712 (CO2), 1372, 1154 (SO2), 1254,
5.2.1. 2-Nitrooxyethyl (E)-3-(4-methanesulfonylphenyl)-
2-phenylacrylate (12a). Yield, 94%; white powder; mp
102–104 ꢁC; IR (film) 2963 (C–H aromatic), 2926 (C–
1069 (N@N–O) cmÀ1 1H NMR (CDCl3) d 2.10 (s,
;

K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 16 (2008) 3302–3308
3307
H aliphatic), 1715 (CO2), 1632, 1284 (ONO2), 1306,
1150 (SO2) cmÀ1 1H NMR (CDCl3) d 3.02 (s, 3H,
5.4. In vitro nitric oxide release assay
;
CH3SO2), 4.48–4.54 (m, 2H, CO2CH2), 4.74 (t, J = 4.5
Hz, 2H, CH2ONO2), 7.15–7.25 (m, 3H, phenyl, H-3,
H-4, H-5), 7.29 (d, J = 8.3 Hz, 2H, methanesulfonylphe-
nyl H-2, H-6), 7.34–7.41 (m, 2H, phenyl H-2, H-6), 7.73
(d, J = 8.2 Hz, 2H, 4-methanesulfonylphenyl H-3, H-5),
7.86 (s, 1H, H-3); MS 413.93 (M+Na). Anal. Calcd for
C18H17NO7S: C, 54.48; H, 5.13; N, 3.55. Found: C,
54.87; H, 5.19; N, 3.17.
In vitro nitric oxide release, upon incubation of the test
compound at 37 ꢁC for 1.5 h with either 2.4 mL of a
1.0 · 10À2 mM solution in phosphate buffer at pH
7.4, with 2.4 mL of a 1.0 · 10À2 mM solution in phos-
phate buffer at pH 7.4 to which 90 lL rat serum had
been added, or with 2.4 mL of a 1.0 · 10À2 mM solu-
tion in phosphate buffer at pH 7.4 which contained
5.0 mM L-cysteine, was determined by quantification
of nitrite produced by the reaction of nitric oxide with
oxygen and water using the Griess reaction. Nitric
oxide release data were acquired for test compounds
(11a–d; 12a–d), and the reference compounds O2-so-
dium 1-[N-(2-hydroxyethyl)-N-methylamino]diazen-1-
ium-1,2-diolate (13) and glycerol trinitrate (14), using
the reported procedures.29
5.2.2. 2-Nitrooxyethyl (E)-2-(4-methoxyphenyl)-3-(4-
methanesulfonylphenyl)acrylate (12b). Yield, 76%; white
powder; mp 58–60 ꢁC; IR (film) 2962 (C–H aromatic),
2927 (C–H aliphatic), 1719 (CO2), 1633, 1280 (ONO2),
1308, 1149 (SO2) cmÀ1 1H NMR (CDCl3) d 3.03 (s,
;
3H, CH3SO2), 3.85 (s, 3H, OCH3), 4.48–4.54 (m, 2H,
CO2CH2), 4.73–4.76 (m, 2H, CH2ONO2), 6.90 (dd,
J = 7.1, 2.0 Hz, 2H, 4-methoxyphenyl H-3, H-5), 7.10
(dd, J = 7.1, 2.0 Hz, 2H, 4-methoxyphenyl H-2, H-6),
7.27 (d, J = 8.5 Hz, 2H, 4-methanesulfonylphenyl H-2,
H-6), 7.75 (d, J = 8.5 Hz, 2H, 4-methanesulfonylphenyl
H-3, H-5), 7.80 (s, 1H, H-3); MS 443.89 (M+Na). Anal.
Calcd for C19H19NO8S: C, 54.17; H, 4.54; N, 3.32.
Found: C, 54.77; H, 4.31; N, 3.24.
Acknowledgments
We are grateful to the Canadian Institutes of Health Re-
search (CIHR) (MOP-14712) for financial support of
this research. We are also grateful to Benisuef University
for a postdoctoral fellowship award to one of us
(K.A.L.).
5.2.3. 2-Nitrooxyethyl (E)-2-(4-fluorophenyl)-3-(4-metha-
nesulfonylphenyl)acrylate (12c). Yield, 94%; white pow-
der; mp 130–132 ꢁC; IR (film) 2962 (C–H aromatic),
2926 (C–H aliphatic), 1717 (CO2), 1634, 1284 (ONO2),
References and notes
1312, 1151 (SO2) cmÀ1 1H NMR (CDCl3) d 3.03 (s,
;
3H, CH3SO2), 4.50–4.54 (m, 2H, CO2CH2), 4.73–4.76
(m, 2H, CH2ONO2), 7.05–7.19 (m, 4H, fluorophenyl
hydrogens), 7.24 (d, J = 8.2 Hz, 2H, 4-methanesulfonyl-
phenyl H-2, H-6), 7.77 (d, J = 8.2 Hz, 2H, 4-metha-
nesulfonylphenyl H-3, H-5), 7.87 (s, 1H, H-3); MS
431.94 (M+Na). Anal. Calcd for C18H16FNO7S: C,
52.81; H, 3.94; N, 3.42. Found: C, 53.31; H, 4.24; N,
3.27.
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7.54
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