Diazen-1-ium-1,2-diolated nitric oxide donor ester prodrugs of 1-(4-methanesulfonylphenyl)-5-aryl-1H-pyrazol-3-carboxylic acids: Synthesis, nitric oxide release studies and anti-inflammatory activities

Article abstract of DOI:10.1016/j.bmc.2008.05.028

A new group of hybrid nitric oxide-releasing anti-inflammatory drugs (NONO-coxibs) wherein an O²-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate (11a-c) NO-donor moiety is attached directly to the carboxylic acid group of 1-(4-methanesulfonylphenyl)-5-aryl-1H-pyrazol-3-carboxylic acids were synthesized. The diazen-1-ium-1,2-diolate compounds 11a-c all released a low amount of NO upon incubation with phosphate buffer (PBS) at pH 7.4 (7.7-9.3% range). In comparison, the percentage of NO released was significantly higher (67.5-73.6% of the theoretical maximal release of two molecules of NO/molecule of the parent hybrid ester prodrug) when the diazen-1-ium-1,2-diolate ester prodrugs were incubated in the presence of rat serum. These incubation studies suggest that both NO and the anti-inflammatory 1-(4-methanesulfonylphenyl)-5-(4-H, 4-F or 4-Me-phenyl)-1H-pyrazol-3-carboxylic acid (9a-c) would be released from the parent NONO-coxib upon in vivo cleavage by non-specific serum esterases. The 1-(4-methanesulfonylphenyl)-5-(4-H, 4-F or 4-Me-phenyl)-1H-pyrazol-3-carboxylic acids (9a-c) exhibited AI activities (ID₅₀ = 85.2-104.4 mg/kg po range) between that exhibited by the reference drugs aspirin (ID₅₀ = 128.7 mg/kg po) and celecoxib (ID₅₀ = 10.8 mg/kg po). Hybrid ester anti-inflammatory/NO-donor prodrugs (NONO-coxibs) offers a potential drug design concept targeted toward the development of anti-inflammatory drugs that are devoid of adverse ulcerogenic and/or cardiovascular effects.

Full text of DOI:10.1016/j.bmc.2008.05.028

Bioorganic & Medicinal Chemistry 16 (2008) 6528–6534
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Diazen-1-ium-1,2-diolated nitric oxide donor ester prodrugs
of 1-(4-methanesulfonylphenyl)-5-aryl-1H-pyrazol-3-carboxylic acids:
Synthesis, nitric oxide release studies and anti-inflammatory activities
*
Khaled R. A. Abdellatif, Morshed Alam Chowdhury, Ying Dong, Edward E. Knaus
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alta., Canada T6G 2N8
a r t i c l e i n f o
a b s t r a c t
A new group of hybrid nitric oxide-releasing anti-inflammatory drugs (NONO-coxibs) wherein an O2-
acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate (11a–c) NO-donor moiety is
attached directly to the carboxylic acid group of 1-(4-methanesulfonylphenyl)-5-aryl-1H-pyrazol-3-car-
boxylic acids were synthesized. The diazen-1-ium-1,2-diolate compounds 11a–c all released a low
amount of NO upon incubation with phosphate buffer (PBS) at pH 7.4 (7.7–9.3% range). In comparison,
the percentage of NO released was significantly higher (67.5–73.6% of the theoretical maximal release
of two molecules of NO/molecule of the parent hybrid ester prodrug) when the diazen-1-ium-1,2-diolate
ester prodrugs were incubated in the presence of rat serum. These incubation studies suggest that both
NO and the anti-inflammatory 1-(4-methanesulfonylphenyl)-5-(4-H, 4-F or 4-Me-phenyl)-1H-pyrazol-
3-carboxylic acid (9a–c) would be released from the parent NONO-coxib upon in vivo cleavage by
non-specific serum esterases. The 1-(4-methanesulfonylphenyl)-5-(4-H, 4-F or 4-Me-phenyl)-1H-pyra-
zol-3-carboxylic acids (9a–c) exhibited AI activities (ID50 = 85.2–104.4 mg/kg po range) between that
exhibited by the reference drugs aspirin (ID50 = 128.7 mg/kg po) and celecoxib (ID50 = 10.8 mg/kg po).
Hybrid ester anti-inflammatory/NO-donor prodrugs (NONO-coxibs) offers a potential drug design con-
cept targeted toward the development of anti-inflammatory drugs that are devoid of adverse ulcerogenic
and/or cardiovascular effects.
Article history:
Received 1 April 2008
Revised 9 May 2008
Accepted 13 May 2008
Available online 17 May 2008
Keywords:
1,5-Diarylpyrazoles
NONO-coxib ester prodrugs
Diazen-1-ium-1,2-diolate nitric oxide donor
Anti-inflammatory activity
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
oxib were voluntarily terminated due to adverse cardiovascular
effects associated with their use.11
The development of celecoxib,1 rofecoxib,2 valdecoxib,3 and
etoricoxib4,5 validated the original concept that selective cyclooxy-
genase-2 (COX-2) inhibitors would be effective anti-inflammatory
agents with a diminished gastrointestinal (GI) and renal toxicity.6–9
These drugs preferentially inhibit the inducible COX-2 isozyme
that causes inflammation relative to the constitutive COX-1 iso-
zyme that provides gastroprotection and maintains vascular
homeostasis. This initial apparently safe pharmacological profile
of selective COX-2 inhibitors was relatively short-lived. It was
not long until evidence surfaced suggesting that highly selective
COX-2 inhibitors alter the balance in the COX pathway resulting
in a decrease in the level of the desirable vasodilatory and anti-
aggregatory prostacyclin (PGI2) in conjunction with an increase
in the level of the undesirable prothrombotic thromboxane A2
(TxA2). This alteration resulted in increased incidences of an
adverse cardiovascular thrombotic event such as myocardial
infarction.10 Accordingly, the clinical use of rofecoxib and valdec-
Nitric oxide (NO), like PGI2, plays an important cytoprotective
role in GI homeostasis by helping to maintain mucosal blood flow,
by optimizing mucus secretion, and by inhibiting platelet and
inflammatory-cell activation.12–15 These actions of NO could en-
hance the gastro-sparing features of selective COX-2 inhibitors
and potentially induce peripheral vasodilation to circumvent the
elevation in blood pressure exhibited by selective COX-2 inhibitors
that decrease the physiological level of PGI2. In this regard, hybrid
selective COX-2 inhibitors possessing a NO-donor moiety (NO-cox-
ibs) have been investigated as a method to increase the clinical
safety of COX-2 inhibitors. Examples of NO-coxibs (see Fig. 1) hav-
ing a nitrate ester NO-donor moiety include the oxazole (1) which
exhibits anti-inflammatory activity similar to that of valdecoxib
with anti-thrombotic action at higher doses,16 the pyrazoles which
are selective COX-2 inhibitors (2a–b) that exhibit anti-inflamma-
tory activity and low gastric toxicity (2b),17 and the imidazoles
(3a–b) that exhibit a NO-dependent vasodilator activity.18 In ear-
lier studies, we described novel acrylate ester prodrugs such as
4,19 and related analogs,20 having a NO-donor diazen-1-ium-1,2-
diolate moiety that are effectively cleaved by esterases to release
the parent COX-2 inhibitory acrylic acid and NO.
* Corresponding author. Tel.: +1 780 492 5993; fax: +1 780 492 1217.
E-mail address: eknaus@pharmacy.ualberta.ca (E.E. Knaus).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.05.028

K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 16 (2008) 6528–6534
6529
N
N
O
N
R
ONO2
O
O
S
S
O
O
H2N
Me
NMI-1093 (1)
2a, R = CH2ONO2
2b, R = C(=NOH)(CH2)3ONO2
N
O
Me
Cl
S
NHCOMe
N
F
O
+
O
N
N
OR
S
H
CO2
O
N
O
Me
O
3a, R = CH2CH2ONO2
3b, R = CH2CH(ONH2)CH2ONO2
4
Figure 1. Some selective cyclooxygenase-2 (COX-2) inhibitors that possess a nitric oxide donor nitrate (1–3), or diazen-1-ium-1,2-diolate (4), moiety.
NO-coxibs that release NO from a nitrooxy group (nitrate ester)
are disadvantaged by the fact that the production of NO requires a
demanding three-electron reduction. The efficacy of this metabolic
activation process can decrease on prolonged use of a nitrate ester
NO-donor drug culminating in nitrate tolerance.21–23 Our group
has introduced and developed the concept of ‘NONO-coxibs’, which
is based on the linkage of a N-diazen-1-ium-1,2-diolate moiety to
the structure of a selective COX-2 inhibitor such as the acrylate
yield (81–91%). It is well documented that this latter reaction oc-
curs in a regiospecific manner to yield the 1,5-diarylpyrazole
product when
a phenylhydrazine hydrochloride is employed
and the reaction is carried out in ethanol at reflux temperature.1
Alkaline hydrolysis of the esters (8a–c) using LiOH gave the
respective acid (9a, 9b or 9c) in good yield (63–91%). Nucleo-
philic displacement of the mesyloxy group present in the mesy-
late 10 upon reaction with the respective sodium carboxylate of
9a, 9b or 9c in hexamethylphosphoramide (HMPA) afforded the
4
19 shown inFigure 1, and other NO-donor analogs thereof.20 These
studies were initiated to address safety and efficacy issues related
to classical selective COX-2 inhibitors, and organic nitrate-based
NO-coxibs. Diazen-1-ium-1,2-diolate ions, after their cleavage
from the parent hybrid NONO-coxib, can release up to two equiv-
alents of NO without further metabolic activation, they are struc-
turally diverse, and they possess a rich derivatization chemistry
that facilitates delivery of NO to specific organ and/or tissue sites.24
These features distinguish the NONO-coxib 4 (Fig. 1) from nitrate-
based NO-coxibs (1–3) which require redox activation before NO is
released. Our ongoing research program is targeted toward the de-
sign of improved anti-inflammatory agents devoid of adverse GI
and cardiovascular effects. We now report the synthesis and nitric
oxide release data for a group of hybrid diazen-1-ium-1,2-diolated
nitric oxide donor ester prodrugs of 1-(4-methanesulfonylphenyl)-
5-aryl-1H-pyrazol-3-carboxylic acids (11a–c), and the anti-inflam-
matory activities for the parent 1-(4-methanesulfonylphenyl)-
5-aryl-1H-pyrazol-3-carboxylic acids (9a–c).
target
O2-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-
ium-1,2-diolate 1-(4-methanesulfonylphenyl)-5-aryl-1H-pyrazol-
3-carboxylates (11a–c).
3. Results and discussion
Three plausible positions on a generic 1,5-diaryl-3-substituted-
pyrazole structure were considered for attachment of an O2-acet-
oxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate
NO-donor moiety via an ester moiety (see Fig. 2). The R1 position
on the N1-phenyl ring requires a COX-2 pharmacophore such as a
MeSO2 or H2NSO2 substituent for potent and selective COX-2
inhibitory activity.1 Ahlstrom et al., in an elegant study that inves-
tigated CYP2C9 structure—metabolism relationships to optimize
the metabolic stability of COX-2 inhibitors, showed that the pyra-
zole ring C-3 position (R2 substituent) has very few steric restric-
tions with respect to COX-2 suggesting that COX-2 inhibition
properties should be retained.25 It was also suggested that a com-
pound containing a R2 carboxyl substituent at the C-3 position may
undergo an electrostatic interaction with Arg120 in the binding
pocket of the COX-2 enzyme. We envisaged that a R3 carboxyl sub-
stituent on the pyrazole C-5 phenyl ring was not tolerable since the
R3 methyl substituent (benzylic carbon) in celecoxib (see structure
in Fig. 2) undergoes sequential metabolic biotransformation
(Me ? CH2OH ? CO2 H ? CO2-glucuronide conjugate) to inactive
metabolites.26 Accordingly, it was decided based on this structural
information to couple the O2-acetoxymethyl-1-(N-ethyl-N-methyl-
amino)diazen-1-ium-1,2-diolate NO-donor moiety to a pyrazole
ring C-3 CO2H group via an ester moiety to prepare the target
NONO-coxib hybrid ester prodrugs (11a–c).
2. Chemistry
A group of 1-(4-methanesulfonylphenyl)-5-aryl-1H-pyrazol-3-
carboxylate esters possessing an O2-acetoxymethyl-1-(N-ethyl-N-
methylamino)diazen-1-ium-1,2-diolate ester moiety (11a–c)
were synthesized using the reaction sequence illustrated in
Scheme 1. Accordingly, Claisen condensation of acetophenone
(5a), 4-fluoroacetophenone (5b), or 4-methylacetophenone (5c)
with diethyl oxalate gave the respective 2,4-diketo ester (6a–
c). Subsequent reaction of the ester 6a, 6b, or 6c with (4-meth-
ylsulfonylphenyl)hydrazine hydrochloride (7) in ethanol at reflux
furnished the corresponding pyrazole ester (8a, 8b or 8c) in high

6530
K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 16 (2008) 6528–6534
R
Me
R
R
O
S
O
O
S
Me
a
b
+
O
N
OH
N
OMe
NH
O
O
O
Me
NH2
7
HCl
O
OMe
6a, R = H
8a, R = H
8b, R = F
8c, R = Me
5a, R = H
5b, R = F
5c, R = Me
6b, R = F
6c, R = Me
c
Me
O
O
S
R
R
O
O
S
O
+
Me
Me
N
+
N
Me
S
N
O
O
N
d
O
N
N
N
O
O
O
O
OH
O
N
O
O
Me
O
9a, R = H
9b, R = F
9c, R = Me
+
N
N
O
Me
O
Me
11a, R = H
11b, R = F
11c, R = Me
10
Scheme 1. Reagents and conditions: (a) diethyl oxalate, NaOMe, MeOH, reflux, 2 h; (b) EtOH, reflux, 3 h; (c) THF/MeOH, LiOH (2 M), 25 °C, 15 h; (d) Na2CO3, hexamethyl-
phosphoramide (HMPA), 25 °C, 96 h.
R3
higher (67.5–73.6% range). These data indicate that the non-spe-
cific serum esterases present in rat serum cleave these hybrid
prodrug esters more effectively than PBS at pH 7.4. The hybrid
ester prodrugs 11a–c cannot release NO prior to cleavage of
the acetoxy moiety present in the terminal O2-acetoxymethyl-
R1
1-(N-methylamino)diazen-1-ium-1,2-diolate NO-donor moiety.
This requirement is consistent with the observation that the
N
O2-sodium diazen-1-ium-1,2-diolate 12, which does not possess
an ester group that requires prior ester cleavage, releases
84.5% and 85% of the theoretical maximal release of two mole-
cules of NO/molecule of the parent NO donor. Two plausible
pathways for the ester hydrolysis of hybrid ester prodrugs con-
N
R2
Figure 2. Generic 1,5-diaryl-3-substituted-pyrazole selective COX-2 structure ba-
sed on the structure of celecoxib (R1 = SO2NH2, R2 = CF3, R3 = Me).
taining
an
O2-acetoxymethyl-1-[N-(2-ethoxy)-N-methyl-
amino]diazen-1-ium-1,2-diolate moiety, and the subsequent
release of acetic acid, formaldehyde, two molecules of NO, and
N-methylethanolamine was described in an earlier study.29 The
hybrid ester NO-donor prodrugs 11a–c were designed with a
one-carbon methylene spacer between the terminal acetoxy
group and the diazen-1-ium-1,2-diolate O2-atom, such that the
O2-(hydroxymethyl)diazen-1-ium-1,2-diolate compound formed
after cleavage of the acetoxy group would spontaneously elimi-
nate formaldehyde to produce the free diazen-1-ium-1,2-diolate
compound that can subsequently fragment to release two mole-
cules of NO. In contrast, cleavage of the second ester group at-
tached directly to the C-3 position of the pyrazole ring, that
releases the parent coxib 9a–c, can occur either prior to, or after,
NO release has occured.
The percent of NO released from the hybrid ester prodrugs
(11a–c) upon incubation in phosphate-buffered saline (PBS at
pH 7.4), and in the presence of rat serum, was determined
(see data in Table 1). One type of chemical modification used
to control the rate of NO release from diazen-1-ium-1,2-diolates
is the attachment of alkyl substituents to the O2-position.27 O2-
substituted-diazen-1-ium-1,2-diolates are stable compounds that
hydrolyze slowly even in acidic solution.28 Consistent with these
observations, when compounds 11a–c were incubated for 1.5 h
in PBS at pH 7.4, the percentage of NO released varied from
7.7% to 9.3% which is indicative of slow NO release. On the other
hand, the effect of non-specific esterases present in rat serum on
the NO release properties of compounds 11a–c was substantially

K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 16 (2008) 6528–6534
6531
Table 1
Percentage (%) of nitric oxide release data for the diazeniumdiolate pyrazole esters (11a–c) and O2-sodium 1-[N-(2-hydroxyethyl)-N-methylamino]diazen-1-ium-1,2-diolate (12),
and in vivo anti-inflammatory activities for the 1-(4-methanesulfonylphenyl)-5-(4-substituted-phenyl)-1H-pyrazol-3-carboxylic acids (9a–c)
R
R
O
Me
S
O
O
N
Me
S
N
O
O
N
N
O
O
HO
ONa
N
O
N
O
O
Me
N
N
+
N
N
O
Me
OH
Me
O
9a-c
12
11a-c
Compound
R
% NO releaseda
AI activityd: ID50 (mg/kg)
PBSb
Serumc
9a
9b
9c
11a
11b
11c
12
Celecoxib
Aspirin
H
F
Me
H
F
Me
—
—
—
—
—
—
7.7
9.3
9.2
84.5
—
—
—
—
67.5
73.6
70.9
85.0
—
104.4
104.1
85.2
—
—
—
—
10.8
128.7
—
a
Percent of nitric oxide released based on a theoretical maximum release of 2 mol of NO/mol of the diazen-1-ium-1,2-diolate test compounds (11a–c, 12). The result is the
mean value of three measurements (n = 3) where variation from the mean % value was 60.5%.
b
A solution of the test compound (2.4 mL of a 1.0 Â 10À2 mM solution in phosphate buffer at pH 7.4) was incubated at 37 °C for 1.5 h.
c
A solution of the test compound (2.4 mL of a 1.0 Â 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.
d
Inhibitory activity in a carrageenan-induced rat paw edema assay. The results are expressed as the ID50 value (mg/kg) at 3 h after oral administration of the test
compound.
The anti-inflammatory (AI) activities exhibited by the parent 1-
(4-methanesulfonylphenyl)-5-aryl-1H-pyrazol-3-carboxylic acids
(9a, R = H; 9b, R = F; 9c, R = Me), that would be released upon
cleavage of the ester group attached directly to the C-3 position
of the pyrazole ring, were determined using a carrageenan-induced
rat foot paw edema model (see data in Table 1). The relative po-
tency profile with respect to the R-substituent was Me > F ꢀ H.
The AI activities exhibited by the hybrid ester prodrugs 11a–c were
not determined in this study since it was previously reported that
the same hybrid ester prodrug analogs of aspirin, ibuprofen and
indomethacin exhibited similar AI activities to aspirin, ibuprofen
and indomethacin for comparable ID50 lmol/kg oral dosage regi-
mens.29 Compounds 9a–c exhibited AI activities (ID50 = 85.2–
104.4 mg/kg po range) between that exhibited by the reference
stability, NO release, and AI studies showed that (i) the NONO-cox-
ib prodrugs (11a–c) are relatively stable in phosphate-buffered sal-
ine at pH 7 where NO release is in the 7.7–9.3% range, (ii) the O2-
acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-dio-
lates (11a–c) undergo extensive cleavage of the terminal acetoxy
group by rat serum esterase(s) that is followed by a significant re-
lease of NO in the 67.5–73.6% range, and (iii) the moderate AI activ-
ity exhibited by the parent compounds 9a–c (ID50 = 85.2–
104.4 mg/kg po range) suggest that an alternative linker group to
the ester moiety attached directly to the C-3 position of the pyra-
zole ring is required to provide more potent AI activity.
5. Experimental
drugs aspirin (ID50 = 128.7 mg/kg po) and celecoxib (ID50
=
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 Nicolet 550 Series II Magna FT-IR
spectrometer. 1H NMR spectra were measured on a Bruker AM-
300 spectrometer in D2O, CDCl3 or DMSO-d6 with TMS as internal
standard, where J (coupling constant) values are estimated in Hertz
(Hz). Microanalyses were performed for C, H, N (Micro Analytical
Service Laboratory, Department of Chemistry, University of Alber-
ta) and were within 0.4% of theoretical values. Silica gel column
chromatography was performed using Merck silica gel 60 ASTM
(70–230 mesh). All other reagents, purchased from the Aldrich
Chemical Company (Milwaukee, WI), were used without further
purification. (4-Methylsulfonylphenyl)hydrazine hydrochloride
(7),30 and O2-acetoxymethyl-1-[N-(2-methylsulfonyloxyethyl)-N-
methylamino]diazen-1-ium-1,2-diolate (10)31 were prepared
according to literature procedures.
10.8 mg/kg po). These AI data are consistent with a qualitative
study which showed that ethyl 5-(4-methylphenyl)-1-(4-sulfa-
moylphenyl)-1H-pyrazole-3-carboxylate (66% and 43% inhibition
of COX-1 and COX-2) is a less potent inhibitor of the COX isozymes
than celecoxib (96% inhibition of both COX-1 and COX-2) at a
100 lM compound concentration.25
4. Conclusions
A group of hybrid ester prodrugs (NONO-coxibs) in which an
O2-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-
diolate (11a–c) NO-donor moiety is attached directly to the
carboxylic acid group of 1-(4-methanesulfonylphenyl)-5-(4-H,
4-F or 4-Me-phenyl)-1H-pyrazol-3-carboxylic acids (9a–c) were
synthesized for comparative biological evaluation. Biological

6532
K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 16 (2008) 6528–6534
5.1. General method for preparation of methyl 2-hydroxy-4-
oxo-4-aryl-2-butenoates (6a–c)
(dd, J = 8.5, 3.7 Hz, 2H, fluorophenyl H-2, H-6), 7.56 (d, J = 8.5 Hz,
2H, methanesulfonylphenyl H-2, H-6), 7.96 (d, J = 8.9 Hz, 2H, meth-
anesulfonylphenyl H-3, H-5).
A solution of diethyl oxalate (1.63 mL, 12.0 mmol) and either
acetophenone 5a, 4-fluoroacetophenone 5b, or 4-methylacetophe-
none 5c (6.0 mmol) in methanol (10 mL) was added dropwise to a
solution of NaOMe in MeOH (2.6 mL of 25% w/v, 12.0 mmol), and
the reaction was allowed to proceed at reflux for 2 h. After cooling
to 25 °C, the reaction mixture was poured into water (40 mL), acid-
ified with HCl (1 mL of 37% w/v), and extracted with diethyl ether
(3Â 100 mL). The combined organic extracts were washed with
brine (30 mL), dried over MgSO4, filtered, and the solvent was re-
moved in vacuo to afford the respective product 6a–c. Physical
and spectral data for 6a–c are listed below.
5.2.3. Methyl 1-(4-methanesulfonylphenyl)-5-(4-methylphen-
yl)-1H-pyrazole-3-carboxylate (8c)
Yield, 91%; white powder; IR (film) 2952 (C–H aromatic), 2922
(C–H aliphatic), 1732 (CO2), 1317, 1153 (SO2) cmÀ1 1H NMR
;
(CDCl3) d 2.39 (s, 3H, phenyl CH3), 3.07 (s, 3H, SO2CH3), 3.99 (s,
3H, OCH3), 7.04 (s, 1H, pyrazole H-4), 7.11 (d, J = 8.1 Hz, 2H, meth-
ylphenyl H-3, H-5), 7.18 (d, J = 8.1 Hz, 2H, methylphenyl H-2, H-6),
7.57 (d, J = 8.7 Hz, 2H, methanesulfonylphenyl H-2, H-6), 7.93 (d,
J = 8.7 Hz, 2H, methanesulfonylphenyl H-3, H-5).
5.3. General method for preparation of 1-(4-
methanesulfonylphenyl)-5-aryl-1H-pyrazol-3-carboxylic acids
(9a–c)
5.1.1. Methyl 2-hydroxy-4-oxo-4-phenyl-2-butenoate (6a)
Yield, 98%; brown solid; IR (film) 3001 (C–H aromatic), 2954 (C–
H aliphatic), 1735 (CO2), 1685 (CO) cmÀ1; 1H NMR (CDCl3) d 3.98 (s,
3H, OCH3), 7.10 (s, 1H, butenoate H-3), 7.52 (dd, J = 8.0, 8.0 Hz, 2H,
phenyl H-3, H-5), 7.63 (dd, J = 8.0, 8.0 Hz, 1H, phenyl H-4), 8.02 (d,
J = 8.0 Hz, 2H, phenyl H-2, H-6), 15.3 (br s, 1H, OH, D2O
exchangeable).
The ester 8a, 8b, or 8c (1.40 mmol) was added to a stirred solu-
tion comprising THF (50 mL), MeOH (50 mL), and LiOH (50 mL of
2 N). This mixture was stirred for 15 h at 25 °C, NaOH (200 mL of
1N) was added, and the mixture was extracted with EtOAc
(200 mL). The aqueous phase was acidified with concentrated
HCl (38 mL) to pH 1.0 prior to extraction with EtOAc (300 mL),
the organic fraction was dried (MgSO4), filtered, and the solvent
was removed in vacuo to furnish the respective acid (9a, 9b, or
9c) for which the physical and spectral data are listed below.
5.1.2. Methyl 4-(4-fluorophenyl)-2-hydroxy-4-oxo-2-butenoate
(6b)
Yield, 89%; brown solid; IR (film) 3060 (C–H aromatic), 2959 (C–H
aliphatic), 1733 (CO2), 1685 (CO) cmÀ1 1H NMR (CDCl3) d 4.13 (s,
;
3H, OCH3), 7.47 (dd, J = 7.9, 7.9 Hz, 2H, fluorophenyl H-3, H-5), 7.51
(s, 1H, butenoate H-3), 8.10 (dd, J = 7.9, 3.7 Hz, fluorophenyl H-2, H-6).
5.3.1. 1-(4-Methanesulfonylphenyl)-5-phenyl-1H-pyrazol-3-
carboxylic acid (9a)
5.1.3. Methyl 2-hydroxy-4-(4-methylphenyl)-4-oxo-2-
butenoate (6c)
Yield, 91%; pale brown powder; mp 181–182 °C; IR (film) 3525–
3140 (OH), 2965 (C–H aromatic), 2927 (C–H aliphatic), 1717 (CO2),
Yield, 97%; white solid; IR (film) 3007 (C–H aromatic), 2952 (C–
H aliphatic), 1731 (CO2), 1684 (CO) cmÀ1; 1H NMR (CDCl3) d 2.63 (s,
3H, phenyl CH3), 4.13 (s, 3H, OCH3), 7.45 (s, 1H, butenoate H-3),
7.50 (d, J = 8.5 Hz, 2H, methylphenyl H-3, H-5), 8.10 (d, J = 8.5 Hz,
2H, methylphenyl H-2, H-6).
1317, 1154 (SO2) cmÀ1 1H NMR (CDCl3 + DMSO-d6) d 3.09 (s, 3H,
;
SO2CH3), 7.14 (s, 1H, pyrazole H-4), 7.26 (m, 3H, phenyl H-3, H-4,
H-5), 7.42 (m, 2H, phenyl H-2, H-6), 7.76 (d, J = 8.5 Hz, 2H, metha-
nesulfonylphenyl H-2, H-6), 7.96 (d, J = 8.5 Hz, 2H, methanesulfo-
nylphenyl H-3, H-5); MS 365.04 (M+Na).
5.2. General method for preparation of methyl 1-(4-methane-
sulfonylphenyl)-5-aryl-1H-pyrazole-3-carboxylates (8a–c)
5.3.2. 5-(4-Fluorophenyl)-1-(4-methanesulfonylphenyl)-1H-
pyrazol-3-carboxylic acid (9b)
Yield, 63%; pale brown powder; mp 213–215 °C; IR (film) 3583–
3280 (OH), 2970 (C–H aromatic), 2930 (C–H aliphatic), 1704 (CO2),
(4-Methylsulfonylphenyl)hydrazine hydrochloride (7, 0.979 g,
4.4 mmol) was added to a stirred solution of the dione 6a, 6b, or
6c (4.0 mmol) in EtOH (50 mL), and the reaction mixture was
heated at reflux with stirring for 3 h. After cooling to 25 °C, the sol-
vent was removed in vacuo. The residue was dissolved in EtOAc
(50 mL), washed with water and brine, the organic fraction was
dried (MgSO4), filtered, and solvent was removed in vacuo to afford
8a–c for which the physical and spectral data are listed below.
1299, 1073 (SO2) cmÀ1 1H NMR (CDCl3 + DMSO-d6) d 3.05 (s, 3H,
;
SO2CH3), 6.92 (s, 1H, pyrazole H-4), 7.03 (dd, J = 8.5, 8.5 Hz, 2H,
fluorophenyl H-3, H-5), 7.19 (dd, J = 8.5, 3.7 Hz, 2H, fluorophenyl
H-3, H-5), 7.49 (d, J = 8.5 Hz, 2H, methanesulfonylphenyl H-2, H-
6), 7.87 (d, J = 8.5 Hz, 2H, methanesulfonylphenyl H-3, H-5),
12.98 (br s, 1H, COOH); MS 383.05 (M+Na).
5.3.3. 1-(4-Methanesulfonylphenyl)-5-(4-methylphenyl)-1H-
pyrazol-3-carboxylic acid (9c)
5.2.1. Methyl 1-(4-methanesulfonylphenyl)-5-phenyl-1H-pyra
zole-3-carboxylate (8a)
Yield, 82%; white powder; mp 242–243 °C; IR (film) 3566–3306
(OH), 2964 (C–H aromatic), 2913 (C–H aliphatic), 1700 (CO2), 1314,
1153 (SO2) cmÀ1; 1H NMR (CDCl3 + DMSO-d6) d 2.30 (s, 3H, phenyl
CH3), 3.04 (s, 3H, SO2CH3), 6.89 (s, 1H, pyrazole H-4), 7.08 (m, 4H,
methylphenyl H-2, H-3, H-5, H-6), 7.49 (d, J = 8.6 Hz, 2H, metha-
nesulfonylphenyl H-2, H-6), 7.84 (d, J = 8.6 Hz, 2H, methanesulfo-
nylphenyl H-3, H-5), 12.57 (br s, 1H, COOH); MS 379.01 (M+Na).
Yield, 81%; pale brown powder; IR (film) 2960 (C–H aromatic),
2925 (C–H aliphatic), 1730 (CO2), 1322, 1155 (SO2) cmÀ1 1H
;
NMR (CDCl3) d 3.09 (s, 3H, SO2CH3), 4.00 (s, 3H, OCH3), 7.09 (s,
1H, pyrazole H-4), 7.23–7.28 (m, 3H, phenyl H-3, H-4, H-5), 7.40
(d, J = 7.3 Hz, 2H, phenyl H-2, H-6), 7.57 (d, J = 8.9 Hz, 2H, metha-
nesulfonylphenyl H-2, H-6), 7.94 (d, J = 8.9 Hz, 2H, methanesulfo-
nylphenyl H-3, H-5).
5.4. General method for preparation of O2-acetoxymethyl-1-(N-
ethyl-N-methylamino)diazen-1-ium-1,2-diolate pyrazole esters
(11a–c)
5.2.2. Methyl 5-(4-fluorophenyl)-1-(4-methanesulfonylphenyl)
-1H-pyrazole-3-carboxylate (8b)
Yield, 65%; pale brown powder; IR (film) 2955 (C–H aromatic),
2926 (C–H aliphatic), 1735 (CO2), 1319, 1154 (SO2) cmÀ1
(CDCl3) d 3.10 (s, 3H, SO2CH3), 4.00 (s, 3H, OCH3), 7.06 (s, 1H, pyr-
azole H-4), 7.11 (dd, J = 8.5, 8.5 Hz, 2H, fluorophenyl H-3, H-5), 7.23
;
1H NMR
The sodium salt of each acid 9a, 9b, or 9c (R = H, F, Me) was pre-
pared in situ by stirring the acid (8a, 8b, or 8c, 2.5 mmol) in a sus-
pension of sodium carbonate (0.27 g, 2.5 mmol) and HMPA

K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 16 (2008) 6528–6534
6533
(3.5 mL) for 24 h at 25 °C. A solution of O2-acetoxymethyl-1-[N-(2-
methylsulfonyloxyethyl)-N-methylamino]diazen-1-ium-1,2-dio-
late (10, 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 (5Â 15 mL), the
organic phase was dried (Na2SO4), and the solvent from the organic
fraction was removed in vacuo. The residue obtained was purified
by silica gel column chromatography using EtOAc:hexane (2:1, v/v)
as eluent to afford the respective product 11a, 11b, or 11c for
which the physical and spectral data are listed below.
O2-sodium 1-[N-(2-hydroxyethyl)-N-methylamino]diazen-1-ium-
1,2-diolate (12) using the reported procedures.32
5.6. In vivo anti-inflammatory assay
The test compounds 9a–c, and the reference drugs celecoxib
and aspirin, were evaluated using the in vivo carrageenan-induced
foot paw edema model reported previously.33
Acknowledgment
5.4.1. O2 -Acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-
ium-1,2-diolate 1-(4-methanesulfonylphenyl)-5-phenyl-1H-
pyrazol-3-carboxylate (11a)
We are grateful to the Canadian Institutes of Health Research
(CIHR) (MOP-14712) for financial support of this research.
Yield, 39%; white powder; mp 95–97 °C; IR (film) 2970 (C–H
aromatic), 2920 (C–H aliphatic), 1744 (CO2), 1322, 1148 (SO2),
1226, 1070 (N@N–O) cmÀ1; 1H NMR (CDCl3) d 2.09 (s, 3H, COCH3),
3.08 (s, 3H, CH3SO2), 3.20 (s, 3H, NCH3), 3.86, (t, J = 5.7 Hz, 2H,
CH2N), 4.60 (t, J = 5.7 Hz, 2H, CO2CH2), 5.77 (s, 2H, OCH2O), 7.05
(s, 1H, pyrazole H-4), 7.25 (m, 3H, phenyl H-3, H-4, H-5), 7.40
(m, 2H, phenyl H-2, H-6), 7.56 (d, J = 9.1 Hz, 2H, methanesulfonyl-
phenyl H-2, H-6), 7.95 (d, J = 9.1 Hz, 2H, methanesulfonylphenyl H-
3, H-5); MS 554.07 (M+Na). Anal. Calcd for C23H25N5O8S: C, 51.97;
H, 4.74; N, 13.18. Found: C, 51.71; H, 4.99; N, 12.94.
References and notes
1. Penning, T. D.; Tally, J. J.; Bertenshaw, S. R.; Carter, J. S.; Collins, P. W.; Doctor, S.;
Graneto, M. J.; Lee, L. F.; Malecha, J. W.; Miyashiro, J. M.; Rogers, R. S.; Rogier, D.
J.; Yu, S. S.; Anderson, G. D.; Burton, E. G.; Cogburn, J. N.; Gregory, S. A.; Koboldt,
C. M.; Perkins, W. E.; Seibert, K.; Veenhuizen, A. W.; Zhang, Y. Y.; Isakson, P. C.
J. Med. Chem. 1997, 40, 1347.
2. Prasit, P.; Wang, Z.; Brideau, C.; Chan, C. C.; Charleson, S.; Cromlish, W.; Ethier,
D.; Evans, J. F.; Ford-Hutchinson, A. W.; Gauthier, J. Y.; Gordon, R.; Guay, J.;
Gresser, M.; Kargman, S.; Kennedy, B.; Leblanc, Y.; Leger, S.; Mancini, J.; O’Neill,
G. P.; Quellet, M.; Percival, M. D.; Perrier, H.; Riendeau, D.; Rodger, I.; Tagari, P.;
Therien, M.; Vickers, P.; Wong, E.; Xu, L. J.; Young, R. N.; Zamboni, R.; Boyce, S.;
Rupniak, N.; Forrest, M.; Visco, D.; Patrick, D. Bioorg. Med. Chem. Lett. 1999, 9,
1773.
3. Talley, J. A.; Brown, D. L.; Carter, J. S.; Masferrer, M. J.; Perkins, W. E.; Rogers, R.
S.; Shaffer, A. F.; Zhang, Y. Y.; Zweifel, B. S.; Seibert, K. J. Med. Chem. 2000, 43,
775.
4. Riendeau, D.; Percival, M. D.; Brideau, C.; Charleson, S.; Dube, D.; Ethier, D.;
Falgueyret, J.-P.; Friesen, R. W.; Gordon, R.; Greig, G.; Guay, I.; Manacini, J.;
Ouellet, M.; Wong, E.; Xu, L.; Boyce, S.; Visco, D.; Girad, Y.; Prasit, P.; Zamboni,
R.; Rodger, J. W.; Gresser, M.; Ford-Hutchinson, A. W.; Young, R. N.; Chan, C.-C.
J. Pharmacol. Exp. Ther. 2001, 296, 558.
5. Davies, I. W.; Marcoux, J.-F.; Corley, E. G.; Journet, M.; Cai, D.-W.; Palucki, M.;
Wu, J.; Larsen, R. D.; Rossen, K.; Pye, P. J.; Dimichele, L.; Dormer, P.; Reider, P. J.
J. Org. Chem. 2000, 65, 8415.
6. Turini, M. E.; DuBois, R. N. Ann. Rev. Med. 2002, 53, 35.
7. Rodrigues, C. R.; Veloso, M. P.; Verli, H.; Fraga, C. A.; Miranda, A. L.; Barreiro, E. J.
Curr. Med. Chem. 2002, 9, 849.
8. Bombardier, C.; Laine, L.; Reicin, A.; Shapiro, D.; Burgos-Vargas, R.; Davies, B.;
Day, R.; Ferraz, M. B.; Hawkey, C. J.; Hochberg, M. C.; Kvien, T. K.; Schnitzer, T. J.
N. Engl. J. Med. 2000, 343, 1520.
9. Silverstein, F. E.; Faich, G.; Goldstein, J. L.; Simon, L. S.; Pincus, T.; Whelton,
A.; Makuch, R.; Eisen, G.; Agrawa, N. M.; Stenson, F. W.; Burr, A. M.; Zhao,
W. W.; Kent, J. D.; Lefkwith, J. B.; Verburg, K. M.; Geis, G. S. JAMA 2000, 284,
1247.
10. Charlier, C.; Michaux, C. Eur. J. Med. Chem. 2003, 38, 645.
11. Dogné, J.-M.; Supuran, C. T.; Pratico, D. J. Med. Chem. 2005, 48, 2251.
12. Wallace, J. L.; Reuter, B.; Cicala, C.; McKnight, W.; Cirino, G.; Gisham, M. B.
Gastroenterology 1994, 107, 173.
13. Wallace, J. L.; Reuter, B.; Cicala, C.; McKnight, W.; Grisham, M. B.; Cirino, G. Eur.
J. Pharmacol. 1994, 257, 249.
14. Elliott, S. N.; McKnight, W.; Cirino, G.; Wallace, J. L. Gastroenterology 1995, 109,
524–530.
15. Muscara, M. N.; McNight, W.; Del Soldato, P.; Wallace, J. L. Pharmacol. Lett.
1998, 62, 235.
16. Dhawan, V.; Schwalb, D. J.; Shumway, M. J.; Warren, M. C.; Wexler, R. S.;
Zemtseva, I. S.; Zifcak, B. M.; Janero, D. R. Free Radic. Biol. Med. 2005, 39, 1191.
17. Ranatunge, R. R.; Augustyniak, M.; Bandarage, U. K.; Earl, R. A.; Ellis, J. L.;
Garvey, D. S.; Janero, D. R.; Letts, L. G.; Martino, A. M.; Murty, M. G.; Richardson,
S. K.; Schroeder, J. D.; Shumway, M. J.; Tam, S. W.; Trocha, M.; Young, D. V. J.
Med. Chem. 2004, 47, 2180.
18. Chegaev, K.; Lazzarato, L.; Tosco, P.; Cena, C.; Marini, E.; Rolando, B.; Carrupt,
P.-A.; Fruttero, R.; Gasco, A. J. Med. Chem. 2007, 50, 1449.
19. Abdellatif, K. R. A.; Dong, Y.; Chen, Q.-H.; Chowdhury, M. A.; Knaus, E. E. Bioorg.
Med. Chem. 2007, 15, 6796.
5.4.2. O2-Acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-
ium-1,2-diolate 5-(4-fluorophenyl)-1-(4-methanesulfonylphen-
yl)-1H-pyrazol-3-carboxylate (11b)
Yield, 22%; white powder; mp 120–122 °C; IR (film) 2972 (C–H
aromatic), 2928 (C–H aliphatic), 1730 (CO2), 1317, 1157 (SO2),
1221, 1065 (N@N–O) cmÀ1; 1H NMR (CDCl3) d 2.09 (s, 3H, COCH3),
3.08 (s, 3H, CH3SO2), 3.19 (s, 3H, NCH3), 3.85 (t, J = 5.7 Hz, 2H,
CH2N), 4.60 (t, J = 5.7 Hz, 2H, CO2CH2), 5.77 (s, 2H, OCH2O), 7.03
(s, 1H, pyrazole H-4), 7.09 (dd, J = 8.5, 8.5 Hz, 2H, fluorophenyl H-
3, H-5), 7.24 (dd, J = 8.5, 3.7 Hz, 2H, fluorophenyl H-2, H-6), 7.55
(d, J = 8.5 Hz, 2H, methanesulfonylphenyl H-2, H-6), 7.96 (d,
J = 8.5 Hz, 2H, methanesulfonylphenyl H-3, H-5); MS 572.01
(M+Na). Anal. Calcd for C23H24FN5O8S: C, 50.27; H, 4.40; N, 12.74.
Found: C, 50.28; H, 4.47; N, 12.53.
5.4.3. O2-Acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-
ium-1,2-diolate 1-(4-methanesulfonylphenyl)-5-(4-
methylphenyl)-1H-pyrazol-3-carboxylate (11c)
Yield, 28%; white crystals; mp 75–77 °C; IR (film) 2970 (C–H
aromatic), 2920 (C–H aliphatic), 1738 (CO2), 1318, 1153 (SO2),
1221, 1065 (N@N–O) cmÀ1
; d 2.10 (s, 3H,
1H NMR (CDCl3)
COCH3), 2.39 (s, 3H, phenyl CH3), 3.08 (s, 3H, CH3SO2), 3.20 (s,
3H, NCH3), 3.86, (t, J = 5.5 Hz, 2H, CH2N), 4.59 (t, J = 5.5 Hz, 2H,
CO2CH2), 5.77 (s, 2H, OCH2O), 7.01 (s, 1H, pyrazole H-4), 7.11
(d, J = 8.3 Hz, 2H, methylphenyl H-3, H-5), 7.18 (d, J = 8.3 Hz,
2H, methylphenyl H-2, H-6), 7.56 (d, J = 8.5 Hz, 2H, metha-
nesulfonylphenyl H-2, H-6), 7.94 (d, J = 8.5 Hz, 2H, methanesulfo-
nylphenyl H-3, H-5),; MS 546.07 (M+1). Anal. Calcd for
C24H27N5O8S.1/2H2O: C, 51.98; H, 5.00; N, 12.63. Found: C,
51.85; H, 4.90; N, 12.94.
5.5. Nitric oxide release assays
20. Abdellatif, K. R. A.; Chowdhury, M. A.; Dong, Y.; Chen, Q.-H.; Knaus, E. E. Bioorg.
Med. Chem. 2008, 16, 3302.
In vitro nitric oxide release, upon incubation of the test com-
pound at 37 °C for 1.5 h with either 2.4 mL of a 1.0 Â 10À2 mM
solution in phosphate buffer at pH 7.4, or with 2.4 mL of a
1.0 Â 10À2 mM solution in phosphate buffer at pH 7.4 to which
90 lL rat serum had been added, 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 ac-
quired for test compounds (11a–c), and the reference compound
21. Csont, T.; Ferdinandy, P. Pharmacol. Ther. 2005, 105, 57.
22. Fung, H. L.; Bauer, J. A. Cardiovasc. Drugs Ther. 1994, 8, 489.
23. Hu, R.; Siu, C. W.; Lau, E. O.; Wang, W. Q.; Lau, C.-P.; Tse, H.-F. Int. J. Cardiol.
2007, 120, 351.
24. Keefer, L. K. Annu. Rev. Pharmacol. Toxicol. 2003, 43, 585.
25. Ahlstrm, M. M.; Ridderstrm, M.; Zamora, I.; Luthman, K. J. Med. Chem. 2007, 50,
4444.
26. Borne, R.; Levi, M.; Wilson, N. In Foye’s Principles of Medicinal Chemistry; Lemke,
T. L., Williams, D. A., Roche, V. F., Zito, S. W., Eds., 6th ed.; Lippincott Williams
and Wilkins: Philadelphia, 2008; p 987. Chapter 36.

6534
K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 16 (2008) 6528–6534
27. Saavedra, J. E.; Dunams, T. M.; Flippen-Anderson, J. L.; Keefer, L. K. J. Org. Chem.
1992, 57, 6134.
28. Hrabie, J. A.; Klose, J. R. J. Org. Chem. 1993, 58, 1472.
29. Velázquez, C. A.; Praveen Rao, P. N.; Citro, M. L.; Keefer, L. K.; Knaus, E. E. Bioorg.
Med. Chem. 2007, 15, 4767.
30. Pommery, N.; Taverne, T.; Telliez, A.; Goossens, L.; Charlier, C.; Pommery, J.;
Goossens,J.-F.;Houssin,R.;Durant,F.;Henichart,J.-P.J.Med.Chem.2004,47,6195.
31. Velázquez, C. A.; Knaus, E. E. Bioorg. Med. Chem. 2004, 12, 3831.
32. Velázquez, C.; Vo, D.; Knaus, E. E. Drug Dev. Res. 2003, 60, 204.
33. Winter, C. A.; Risley, E. A.; Nuss, G. W. Proc. Soc. Exp. Biol. Med. 1962, 111, 544.