Diazen-1-ium-1,2-diolated nitric oxide donor ester prodrugs of 5-(4-carboxymethylphenyl)-1-(4-methanesulfonylphenyl)-3-trifluoromethyl-1H-pyrazole and its aminosulfonyl analog: Synthesis, biological evaluation and nitric oxide release studies

Article abstract of DOI:10.1016/j.bmc.2009.05.046

A new class of hybrid nitric oxide-releasing anti-inflammatory (AI) ester prodrugs (NONO-coxibs) wherein an O²-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate (13a-b), or O²-acetoxymethyl-1-(2-methylpyrrolidin-1-yl)di

Full text of DOI:10.1016/j.bmc.2009.05.046

Bioorganic & Medicinal Chemistry 17 (2009) 5182–5188
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
5-(4-carboxymethylphenyl)-1-(4-methanesulfonylphenyl)-3-trifluoromethyl-
1H-pyrazole and its aminosulfonyl analog: Synthesis, biological evaluation
and nitric oxide release studies
Khaled R. A. Abdellatif, Morshed Alam Chowdhury, Ying Dong, Dipankar Das, Gang Yu, Carlos Velázquez,
*
Mavanur R. Suresh, Edward E. Knaus
Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2N8
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of hybrid nitric oxide-releasing anti-inflammatory (AI) ester prodrugs (NONO-coxibs)
wherein an O2-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-diolate (13a–b), or O2-
acetoxymethyl-1-(2-methylpyrrolidin-1-yl)diazen-1-ium-1,2-diolate (16a–b), NO-donor moiety was
covalently coupled to the COOH group of 5-(4-carboxymethylphenyl)-1-(4-methane(amino) sulfonylphe-
nyl)-3-trifluoromethyl-1H-pyrazole (11a–b) was synthesized. The percentage of NO released from these
diazen-1-ium-1,2-diolates was significantly higher (59.6–74.6% of the theoretical maximal release of 2
molecules of NO/molecule of the parent hybrid ester prodrug) upon incubation in the presence of rat
serum, relative to incubation with phosphate buffer (PBS) at pH 7.4 (5.0–7.2% range). These incubation
studies suggest that both NO and the AI compound would be released from the parent NONO-coxib upon
in vivo cleavage by non-specific serum esterases. All compounds were weak inhibitors of the COX-1 iso-
Received 7 April 2009
Revised 15 May 2009
Accepted 18 May 2009
Available online 27 May 2009
Keywords:
NONO-coxib ester prodrugs
Diazen-1-ium-1,2-diolate nitric oxide donor
Cyclooxygenase inhibition
Anti-inflammatory activity
zyme (IC50 = 8.1–65.2 lM range) and modest inhibitors of the COX-2 isozyme (IC50 = 0.9–4.6 lM range).
The most potent parent aminosulfonyl compound 11b exhibited AI activity that was about sixfold greater
than that for aspirin and threefold greater than that for ibuprofen. The ester prodrugs 13b, 16b exhibited
similar AI activity to that exhibited by the more potent parent acid 11b when the same oral lmol/kg dose
was administered. These studies indicate hybrid ester AI/NO donor prodrugs of this type (NONO-coxibs)
constitute a plausible drug design concept targeted toward the development of selective COX-2 inhibi-
tory AI drugs that are devoid of adverse cardiovascular effects.
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
that include rofecoxib (1) and valdecoxib (2) alter the natural bal-
ance in the COX pathway (see structures in Fig. 1). In this regard,
Non-steroidal anti-inflammatory drugs (NSAIDs) represent one
of the most common classes of medications used worldwide, with
an estimated usage of >30 million individuals per day.1 The bene-
ficial anti-inflammatory (AI) and analgesic effects of these drugs
have been linked to inhibition of the inducible cyclooxygenase-2
(COX-2) isozyme, whereas adverse gastrointestinal irritation and
ulcerative effects are believed to be mainly due to inhibition of
the constitutive cyclooxygenase isoform (COX-1). Therefore, COX-
2 selective inhibitors (coxibs) such as celecoxib (CelebrexÒ, 1),
rofecoxib (VioxxÒ, 2), valdecoxib (BextraÒ, 3), etoricoxib (ArcoxiaÒ,
4) and lumiracoxib (PrexigeÒ, 5) have been developed for the long
term treatment of patients suffering from chronic pain and inflam-
mation.2 Unfortunately, some selective COX-2 inhibitory drugs
the amount of the desirable vasodilatory and anti-aggregatory
prostacyclin (PGI2) produced is decreased together with a simulta-
neous increase in the level of the undesirable vasoconstrictory and
prothrombotic thromboxane A2 (TxA2).3–5 These two adverse bio-
chemical changes in the COX pathway are believed to be responsi-
ble for the increased incidences of high blood pressure and
myocardial infarction that ultimately prompted the withdrawal
of rofecoxib (VioxxÒ) and valdecoxib (BextraÒ).6,7 Nitric oxide
(NO) is an effective vasodilation agent that also inhibits platelet
aggregation and adhesion.8 Therefore, NO release provides an
attractive method to suppress vascular side effects associated with
NSAID use.9 In this regard, we described novel hybrid ester pro-
drugs having 2-nitrooxyethyl (6), diazen-1-ium-1,2-diolate ( 7,
8)10,11 and other NO-donor analogs12 that are effectively cleaved
by esterases to release the parent COX-2 inhibitory compound
and NO. NO-coxibs that release NO from a nitrooxy group (nitrate
* 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 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.046

K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 17 (2009) 5182–5188
5183
Me
O
O
NH2
O
Me
S
S
NH2
S
O
O
N
O
O
N
O
O
Me
Valdecoxib (3)
N
F3C
Celecoxib (1)
Rofecoxib (2)
O
O
SO2Me
Me
S
R
COOH
Me
Cl
NH
O
Cl
Cl
H
C
O
ONO2
N
N
Me
R = H, F, Br, OMe
6
Lumiracoxib (5)
Etoricoxib (4)
O
N
O
N
+
O
O
N
CH3
O
O
S
R
Me
O
O
O
O
N
O
H
C
N
N
O
O
Me
N
SO2R
O
Me
N
F3C
R = H, F, Br, OMe
7
R = NH2, Me
8
Figure 1. Chemical structures of the selective cyclooxygenase-2 (COX-2) inhibitors celecoxib (1), rofecoxib (2), valdecoxib (3), etoricoxib (4), lumiracoxib (5), nitrooxyethyl
ester prodrugs (6) and diazen-1-ium-1,2-diolated ester prodrugs (7, 8).
ester) are disadvantaged by the fact that the production of NO re-
quires a demanding three-electron reduction. The efficacy of this
metabolic activation process can decrease on prolonged use of a ni-
trate ester NO-donor drug culminating in nitrate tolerance.13–15
We now report a group of hybrid ester prodrugs in which an (i)
O2-acetoxymethyl-1-(N-ethyl-N-methylamino)diazen-1-ium-1,2-
diolate (13a–b), or (ii) O2-acetoxymethyl-1-[2-methylpyrrolidin-1-
yl]diazen-1-ium-1,2-diolate (16a–b), NO-donor moiety is attached
directly to the carboxylic acid group of 5-(4-carboxymethylphe-
nyl)-1-(4-methanesulfonylphenyl)-3-trifluoromethyl-1H-pyrazole
(11a), and its aminosulfonyl analog (11b). It is expected that these
hybrid NO-donor prodrugs will be devoid of adverse ulcerogenic
and cardiovascular effects based on their ability to release cytopro-
tective and vasodilatory NO.
ylphosphoramide (HMPA) afforded the target products 13a (84%
yield) and 13b (32% yield). Elaboration of the carboxylic acids
11a–b using oxalyl chloride gave the more reactive acid chlorides
(14a–b) which upon subsequent reaction with a pure enantiomer
of O2-acetoxymethyl-1-(2-hydroxymethylpyrrolidin-1-yl)diazen-
1-ium-1,2-diolate (15) derived from L
-prolinol24 in dry THF, in
the presence of the non-nucleophilic base triethylamine, furnished
the two target ester prodrugs 16a–b in good yield (51–69%).
3. Results and discussion
Three positions on the structure of celecoxib (1) were consid-
ered for attachment of an O2-acetoxymethyl-1-(N-ethyl-N-methyl-
amino)diazen-1-ium-1,2-diolate,
or
O2-acetoxymethyl-1-(2-
methylpyrrolidin-1-yl)diazen-1-ium-1,2-diolate, NO donor moiety
via an ester linkage. The para-position on the N1-phenyl ring was
not selected since a COX-2 pharmacophore such as a MeSO2 or
H2NSO2 substituent is required at this location for potent and
selective COX-2 inhibitory activity.16 Although the pyrazole ring
C-3 position has very few steric restrictions with respect to COX-
2 inhibition properties, the electronegative CF3 substituent was re-
tained since it generally provides optimal COX-2 potency.17 The C-
5 para-methylphenyl substituent (benzylic carbon) in celecoxib
undergoes sequential metabolic biotransformation (Me ?
CH2OH ? CO2H ? CO2–glucuronide conjugate).18 Accordingly, we
decided based on these structural information to couple the NO do-
nor moiety to a pyrazole ring C-5 para-C6H4–CH2COOH group via
an ester moiety to prepare the target NONO-coxib hybrid ester pro-
drugs (13a–b, 16a–b).
2. Chemistry
The two 5-(4-carboxymethylphenyl)-1-[4-methane(amino)sul-
fonylphenyl]-3-trifluoromethyl-1H-pyrazoles (11a–b), and the es-
ter prodrugs 13a–b, 16a–b, were synthesized using the reaction
sequence illustrated in Scheme 1. Accordingly, the bromomethyl
compounds 9a–b were converted to the respective cyanomethyl
analogs 10a–b in high yield (72–91%) upon heating under reflux
with NaCN in ethanol for 2 h. Alkaline hydrolysis of the nitrile
10b bearing a SO2NH2 group using aqueous NaOH afforded the cor-
responding acid 11b in 72% yield. In contrast, a similar hydrolysis
of 10a having a SO2Me substituent did not furnish the acid 11a.
The failure of this latter reaction is attributed to the low solubility
of the SO2Me compound 10a relative to the more soluble SO2NH2
compound 10b. On the other hand, hydrolysis of 10a using 6 N
HCl in dioxane yielded the acid 11a in 27% yield. Nucleophilic dis-
placement of the mesyloxy group present in the mesylate 12 by the
respective sodium salt of the carboxylic acids 11a–b in hexameth-
In vitro COX-1 enzyme inhibition studies (Table 1) showed that
the parent acetic acid (CH2CO2H) compounds 11a–b, and the two
types of diazen-1-ium-1,2-diolate derivatives 13a–b and 16a–b,
like the reference drug celecoxib (COX-1 IC50 = 7.7 lM), were weak

5184
K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 17 (2009) 5182–5188
CH2Br
CH2CN
CH2COOH
a
b
N
N
SO2R
N
N
SO2R
N
N
SO2R
F3C
F3C
F3C
9a-b
10a-b
11a-b
a, R = Me, b, R = NH2
c, 12
O
d
N
N
O
N
O
CH2COCl
O
O
O
N
N
O
O
O
Me
O
N
Me
O
O
e, 15
Me
N
N
SO2R
N
SO2R
N
N
SO2R
F3C
N
F3C
F3C
16a-b
14a-b
13a-b
O
N
O
O
O
Me
N
O
O
+
N
N
N
N
S
O
O
Me
Me
O
O
O
Me
HOH2C
12
15
Scheme 1. Reagents and conditions: (a) NaCN, EtOH, reflux, 2 h; (b) HCl, dioxane, reflux, 16 h for 10a, NaOH, H2O/THF, reflux, 16 h for 10b; (c) Na2CO3,
hexamethylphosphoramide (HMPA), 25 °C, 96 h; (d) (COCl)2, CH2Cl2, 25 °C, 12 h; (e) Et3N, THF, 25 °C, 48 h.
inhibitors of COX-1 (IC50 = 8.1–65.2
compounds 11a–b, 13a–b and 16a–b were more potent inhibitors
of COX-2 (IC50 = 0.9–4.6 M range) than COX-1 that resulted in a
higher selectivity for the COX-2 isozyme (COX-2 selectivity indexes
in the 1.8–28.3 range). The differences in COX-2 inhibitory poten-
cies between compounds having SO2Me and SO2NH2 pharmaco-
phores was small. Among the group of compounds 11a–b, 13a–b
and 16a–b, the proline diazen-1-ium-1,2-diolate derivatives 16a–
l
M range). In comparison,
[N-(2-hydroxyethyl)-N-methylamino]diazen-1-ium-1,2-diolate (17)
and O2-sodium 1-[2-(hydroxymethyl)pyrrolidin-1-yl]diazen-1-ium-
1,2-diolate (18), which do not possess an ester group that requires
prior ester cleavage, released 84.5–85% of the theoretical maximal
release of two molecules of NO/molecule of the parent NO-donor
compound in both PBS and serum. Two plausible pathways for
the ester hydrolysis of hybrid O2-acetoxymethyl-1-(N-ethyl-N-
methylamino) diazen-1-ium-1,2-diolate ester prodrugs and the
subsequent release of acetic acid, formaldehyde, two molecules
of NO, and 2-(N-methylamino)ethanol were described in an earlier
publication.22 Similar esterase cleavage and NO release pathways,
that are applicable to the O2-acetoxymethyl-1-[2-(methyl)pyrroli-
din-1-yl)]diazen-1-ium-1,2-diolate esters (16a–b), are illustrated
in Figure 2. Prodrugs 16a–b were designed (i) such that the car-
boxyl group of the parent compounds 11a–b is covalently attached
directly to the alcohol substituent of the diazenium-1,2-diolate
(15), and (ii) subsequent cleavage of the ester groups and release
of NO would furnish the parent COX-2 inhibitor (11a–b) and the
l
b were the most potent inhibitors of COX-2 (IC50 = 0.9
lM) relative
to the reference drug celecoxib (COX-2 IC50 = 0.12 M).
l
The percent NO released from the hybrid ester prodrugs 13a–b,
16a–b 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). The rate of NO release from diazen-1-ium-1,2-diolates
can be controlled by chemical modification such as attachment of
an alkyl substituent to the O2-position.19 O2-substituted-diazen-1-
ium-1,2-diolates are stable compounds that hydrolyze slowly even
in acidic solution.20 Consistent with these observations, when
compounds 13a–b, 16a–b were incubated for 1.5 h in PBS at pH
7.4, the percentage of NO released varied from 5.0% to 7.2% which
is indicative of slow NO release.21 In contrast, the effect of non-spe-
cific esterases present in rat serum on the NO release properties of
compounds 13a–b, 16a–b was substantially higher (59.6–74.6%
range). These data indicate the non-specific serum esterases pres-
ent in rat serum cleave these hybrid prodrug esters more effec-
tively than PBS at pH 7.4. From a mechanistic perspective, the
hybrid ester prodrugs 13a–b, 16a–b can not release NO prior to
cleavage of the terminal O2-acetoxymethyl ester group. This
requirement is consistent with the observation that O2-sodium 1-
natural amino alcohol L-prolinol.
The anti-inflammatory (AI) activities exhibited by the 5-(4-
carboxymethylphenyl)-1-[4-methane(amino)sulfonylphenyl]-3-tri-
fluoromethyl-1H-pyrazoles (11a–b) were determined using
a
carrageenan-induced rat foot paw edema model (see data in Table
1). The carboxymethyl compound 11a exhibited moderate AI activ-
ity (ED50 = 215.8
by the reference drugs aspirin (ED50 = 714
fen (ED50 = 327 mol/kg po), but less than that of celecoxib
(ED50 = 30.9 mol/kg po). The more potent aminosulfonyl analog
11b exhibited higher AI activity (ED50 = 113.8 mol/kg po) that
lmol/kg po) that was greater than that exhibited
lmol/kg po) and ibupro-
l
l
l

K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 17 (2009) 5182–5188
5185
Table 1
In vitro COX-1 and COX-2 inhibition, percent (%) nitric oxide release and anti-inflammatory (AI) data for 5-(4-carboxymethylphenyl)-1-(4-methane(amino)sulfonylphenyl)-3-
trifluoromethyl-1H-pyrazoles (11a–b), diazeniumdiolate esters (13a–b, 16a–b), O2-sodium 1-[N-(hydroxyethyl)-N-methylamino]diazen-1-ium-1,2-diolate (17), O2-sodium 1-[2-
(hydroxymethyl)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate (18) and the reference drugs, celecoxib, aspirin and ibuprofen
N
O
O
N
HO
O
N
N
N
O
O
O
O
O
O
O
Me
ONa
N
N
Me
CH2COOH
O
N
N
O
Me
Me
O
17
N
N
N
N
SO2R
N
N
SO2R
N
O
N
N
SO2R
ONa
F3C
F3C
HOH2C
F3C
13a; R = CH3, 13b; R = NH2
18
11a; R = CH3,11b; R = NH2
16a; R = CH3,16b; R = NH2
COX-2 S.I.b
% NO releasedc
PBSd Serume
AI activityf
a
Compound
IC50
(l
M)
COX-1
COX-2
ED50
(l
mol/kg)
% Inhibition (113.8 lmol/kg)
11a
11b
13a
13b
16a
16b
17
18
65.2
8.1
8.2
35.4
11.8
13.4
—
2.3
1.8
4.6
3.5
0.9
0.9
—
28.3
4.5
1.8
10.1
13.1
14.4
—
—
—
—
—
215.8
113.8
—
—
—
—
—
—
—
50.0
—
55.8
—
46.5
—
—
—
—
—
6.9
7.2
5.0
5.4
84.5
84.5
—
72.2
74.6
62.9
59.6
84.8
85.0
—
—
—
—
Celecoxib
Aspirin
Ibuprofen
7.7
0.3
2.9
0.12
2.4g
1.1g
110
0.13
2.64
30.9
714
327
—
—
—
—
a
The in vitro test compound concentration required to produce 50% inhibition of ovine COX-1 or human recombinant COX-2. The result (IC50, lM) is the mean of two
determinations acquired using the enzyme immuno assay kit (Catalog No. 560131, 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).
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 (13a–b, 16a–b, 17 and 18).
c
The result is the mean value of 3 measurements (n = 3) where variation from the mean% value was 6 0.2%.
d
A solution of the test compound (2.4 mL of a 1.0 ꢀ 10ꢁ2 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 ꢀ 10ꢁ2 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
Inhibitory activity in a carrageenan-induced rat paw edema assay. The results are expressed as the% inhibition of inflammation at 3 h after oral administration of the test
compound at the specified dose ( mol/kg).
l
g
Data acquired using ovine COX-2 (Catalog No. 56101, Cayman Chemical Inc.).
O
esterase
N
N
+
O
N
+
N
R
O
N
R
O
O
H
CH3CO2H
+
N
CH3
O
O
O
O
O
O
16a-b
CH2O
O
R
C
OH
2 NO
Parent
acid (11a-b)
+
N
+
esterase
N
O
N
O
R
R
N
O
H
O
O
O
N
HO
H
L-Prolinol
Figure 2. Theoretical metabolic activation (esterase hydrolysis) and nitric oxide release from the O2-acetoxymethyl-1-[2-methylpyrrolidin-1-yl]diazen-1-ium-1,2-diolate
esters (16a–b). The sequence in which ester cleavage and nitric oxide release may also occur in a reverse order.
was about sixfold greater than that exhibited by aspirin and three-
fold greater than that exhibited by the ibuprofen. The ester pro-
drugs 13b, 16b exhibited similar in vivo anti-inflammatory
activity (55.8%, 46.5% inhibition, respectively) to that exhibited
by the more potent parent acid 11b (50.0% inhibition) when the
ties in oral AI activity between the parent acid 11b and the corre-
sponding NONO-coxib esters 13b and 16b suggest that 13a–b and
16a–b (i) act as classical prodrugs that require metabolic activation
by esterase-mediated hydrolysis, and (ii) the AI data provides cre-
dence for the drug design concept that covalent attachment of a
NO donor moiety directly to a suitably positioned para-C6H4–
same oral dose (113.8 lmol/kg) was administered. These similari-

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K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 17 (2009) 5182–5188
CH2COOH group present in a selective COX-2 inhibitor offers a po-
tential method to circumvent adverse cardiovascular effects.
2H, cyanomethylphenyl H-3, H-5), 7.39 (dd, J = 6.1, 1.8 Hz, 2H,
cyanomethylphenyl H-2, H-6), 7.53 (dd, J = 6.7, 1.7 Hz, 2H, metha-
nesulfonylphenyl H-2, H-6), 7.97 (dd, J = 6.7, 1.7 Hz, 2H, metha-
nesulfonylphenyl H-3, H-5); MS 406.0 (M+1).
4. Conclusions
5.2. 4-[5-(4-Cyanomethylphenyl)-3-trifluoromethyl-1H-
pyrazol-1-yl]benzenesulfonamide (10b)
A novel type of hybrid ester prodrugs (NONO-coxibs) (13a–b,
16a–b) in which an O2-acetoxymethyl 1-(N-ethyl-N-methyl-
amino)diazen-1-ium-1,2-diolate or O2-acetoxymethyl-1-(2-meth-
ylpyrrolidin-1-yl)diazen-1-ium-1,2-diolate, NO-donor moiety was
covalently coupled to the COOH group of 5-(4-carboxymethylphe-
nyl)-1-(4-methane(amino)sulfonylphenyl)-3-trifluoromethyl-1H-
pyrazoles (11a–b) were synthesized for evaluation as COX-1/
COX-2 isozyme inhibitors, NO donors, and as AI agents. Struc-
ture–activity and biological stability studies showed that these
ester prodrugs (i) retain COX-1 and COX-2 inhibitory activity with
moderate selectivity for the COX-2 isozyme, (ii) are relatively sta-
ble in phosphate-buffered saline at pH 7 where NO release is in the
5.0–7.2% range, (iii) undergo extensive cleavage of the terminal
acetoxy group by rat serum esterase(s) that is followed by a signif-
icant release of NO in the 59.6–74.6% range, and (iv) the relatively
potent AI activity exhibited by the carboxymethyl compound 11b
The title compound 10b was synthesized, using a similar proce-
dure to that described for the preparation of 10a, in 72% yield as a
white powder; mp 82–84 °C; IR (film) 3348, 3264 (NH2), 3025
(CN), 3027 (C–H aromatic), 2965 (C–H aliphatic), 1339, 1163
(SO2) cmꢁ1 1H NMR (CDCl3) d 3.81 (s, 2H, CH2CN, 4.91 (br s, 2H,
;
NH2, exchanges with D2O), 6.80 (s, 1H, pyrazole H-4), 7.27 (d,
J = 8.6 Hz, 2H, cyanomethylphenyl H-3, H-5), 7.38 (d, J = 8.6, 2H,
cyanomethylphenyl H-2, H-6), 7.48 (dd, J = 6.7, 1.9 Hz, 2H, amin-
osulfonylphenyl H-2, H-6), 7.94 (dd, J = 6.7, 1.9 Hz, 2H, aminosulfo-
nylphenyl H-3, H-5); MS 407.02 (M+1), 428.97 (M+Na).
5.3. 5-(4-Carboxymethylphenyl)-1-(4-methanesulfonylphenyl)-
3-trifluoromethyl-1H-pyraz-ole (11a)
(ED50 = 113.8 lmol/kg po range) and the approximate equipotency
A solution of 6 N HCl (35 mL) was added to a solution of 5-(4-
cyanomethylphenyl)-1-(4-methanesulfonylphenyl)-3-trifluoro-
methyl-1H-pyrazole (10a, 1.22 g, 3 mmol) in dioxane (15 mL) and
the reaction was allowed to proceed at reflux for 16 h. After cooling
to 25 °C, the solvent was removed in vacuo, the residue was mixed
with cold water (10 mL), and this mixture was extracted with
EtOAc (2 ꢀ 25 mL). The organic layer was extracted with 1 N NaOH
(2 ꢀ 15 mL), the aqueous NaOH layer was acidified with 1 N HCl to
pH 1, and the acidic mixture was extracted with EtOAc
(2 ꢀ 25 mL). The combined organic extracts were dried (Na2SO4)
and the solvent was removed in vacuo. The residue was purified
by silica gel column chromatography using EtOAc/hexane (2:1, v/
v) as eluent to furnish the product that was recrystallized from
EtOAc/hexane to give 11a as a white powder (0.34 g, 27%): mp
168–170 °C; IR (film) 3350 (broad OH), 3027 (C–H aromatic),
of its hybrid ester prodrugs 13b, 16b support the drug design con-
cept that covalent attachment of the NO donor moiety directly to a
suitably positioned para-C6H4–CH2COOH group present in a selec-
tive COX-2 inhibitor offers a rational approach to circumvent ad-
verse cardiovascular 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 Nicolet 550 Series II Magna FT-IR
spectrometer. 1H NMR and 13C NMR spectra were measured on a
Bruker AM-300 spectrometer in CDCl3, or CDCl3 + DMSO-d6, with
TMS as the internal standard. Nominal mass, positive polarity, elec-
trospray spectra were acquired using a Waters Micromass ZQ mass
spectrometer. Microanalyses were performed for C, H, N (Micro
Analytical 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 reagents,
purchased from the Aldrich Chemical Company (Milwaukee, WI),
were used without further purification. 5-(4-Bromomethylphe-
nyl)-1-(4-methanesulfonylphenyl)-3-trifluoromethyl-1H-pyrazole
(9a),11 4-[5-(4-bromomethylphenyl)-3-trifluoromethyl-1H-pyra-
zol-1-yl]benzenesulfonamide (9b)11 O2-acetoxymethyl-1-[N-(2-
methylsulfonyloxyethyl)-N-methylamino]diazen-1-ium-1,2-
diolate (12)23, and O2-acetoxymethyl-1-(2-hydroxymethylpyrroli-
din-1-yl)diazen-1-ium-1,2-diolate (15)24 were prepared according
to literature procedures.
2963 (C–H aliphatic), 1733 (CO2), 1320, 1156 (SO2) cmꢁ1 1H
;
NMR (CDCl3) d 3.08 (s, 3H, SO2CH3), 3.72 (s, 2H, CH2CO2H, 6.79
(s, 1H, pyrazole H-4), 7.22 (d, J = 8.2 Hz, 2H, benzyl H-3, H-5),
7.34 (d, J = 8.2 Hz, 2H, benzyl H-2, H-6), 7.55 (d, J = 8.8 Hz, 2H,
methanesulfonylphenyl H-2, H-6), 7.96 (d, J = 8.8 Hz, 2H, metha-
nesulfonylphenyl H-3, H-5); 13C NMR (CDCl3) d 40.5, 44.5, 104.1,
120.9, 125.7, 127.6, 128.6, 129.0, 130.2, 134.9, 139.9, 143.3,
144.1, 144.6, 176.2; MS 425.08 (M+1).
5.4. 5-(4-Carboxymethylphenyl)-1-(4-aminosulfonylphenyl)-3-
trifluoromethyl-1H-pyrazole (11b)
A solution of 1 N NaOH (18 mL, 18 mmol) was added to a solu-
tion of 4-[5-(4-cyanomethylphenyl)-3-trifluoromethyl-1H-pyra-
zol-1-yl]benzenesulfonamide (10b, 1.22 g, 3 mmol) in THF
(5 mL), and the resulting mixture was heated under reflux for
16 h. After cooling to 25 °C, the solvent was removed in vacuo,
the residue was diluted with cold water (10 mL), the aqueous alka-
line layer was washed with ether (10 mL), the aqueous alkaline
layer was acidified with 1 N HCl to pH 1, and the mixture was ex-
tracted with EtOAc (2 ꢀ 25 mL). The combined organic extracts
were dried (Na2SO4) and the solvent was removed in vacuo. The
residue was recrystallized from EtOAc/hexane to give 11b as yel-
low crystals (0.92 g, 72%): mp 145–148 °C; IR (film) 3352, 3254
(NH2), 3030 (C–H aromatic), 2926 (C–H aliphatic), 1717 (CO2),
5.1. 5-(4-Cyanomethylphenyl)-1-(4-methanesulfonylphenyl)-3-
trifluoromethyl-1H-pyrazole (10a)
A solution of the bromomethyl compound 9a (2.3 g, 5 mmol)
and NaCN (0.368 g, 7.5 mmol) in ethanol (50 mL) was heated under
reflux for 2 h. After removal of the solvent in vacuo, the residue
was dissolved in EtOAc (50 mL), this solution was washed with
water, the organic layer was dried (Na2SO4), and the solvent was
evaporated in vacuo. The residue was purified by silica gel column
chromatography using EtOAc/hexane (1:1, v/v) as eluent to give
10a as a yellow powder (1.84 g, 91%): mp 171–174 °C; IR (film)
3024 (CN), 3030 (C–H aromatic), 2928 (C–H aliphatic), 1318,
1339, 1160 (SO2) cmꢁ1 1H NMR (CDCl3) d 3.72 (s, 2H, CH2CO2H),
;
5.18 (br s, 2H, NH2, exchanges with D2O), 6.77 (s, 1H, pyrazole H-
4), 7.20 (d, J = 7.9 Hz, 2H, benzyl H-3, H-5), 7.30 (d, J = 7.9 Hz, 2H,
benzyl H-2, H-6), 7.45 (dd, J = 6.7, 1.9 Hz, 2H, aminosulfonylphenyl
;
1154 (SO2) cmꢁ1 1H NMR (CDCl3) d 3.08 (s, 3H, SO2CH3), 3.81 (s,
2H, CH2CN, 6.81 (s, 1H, pyrazole H-4), 7.27 (dd, J = 6.1, 1.8 Hz,

K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 17 (2009) 5182–5188
5187
H-2, H-6), 7.88 (d, J = 6.7, 1.9 Hz, 2H, aminosulfonylphenyl H-3, H-
5); 13C NMR (CDCl3 + DMSO-d6) d 40.1, 105.4, 120.3, 124.4, 126.2,
126.3, 128.0, 129.2, 135.4, 140.6, 142.3, 142.8, 144.0, 171.9; MS
426.07 (M+1).
(20 mL) under argon at 25 °C. The reaction mixture was stirred
for 12 h at 25 °C, the solvent was removed in vacuo, the crude
product was washed with hexane (3 ꢀ 25 mL), and dried under
vacuum to give 14a or 14b. Physical and spectral data for 14a–b
are listed below.
5.5. General method for preparation of the O2-acetoxymethyl-
1-(N-ethyl-N-methylamino) diazen-1-ium-1,2-diolate esters
(13a–b)
5.6.1. 5-(4-Chlorocarbonylmethylphenyl)-1-(4-
methanesulfonylphenyl)-3-trifluoromethyl-1H-pyrazole (14a)
Yield, 90%; pale yellow solid; IR (film) 3028 (C–H aromatic),
2964 (C–H aliphatic), 1794 (CO), 1322, 1157 (SO2) cmꢁ1 1H NMR
;
Sodium carboxylates of the respective acids 11a–b were pre-
pared in situ by stirring each acid (2.5 mmol) in a suspension of so-
dium carbonate (0.27 g, 2.5 mmol) and HMPA (3.5 mL) for 24 h at
25 °C. A solution of O2-acetoxymethyl-1-[N-(2-methylsulfonyloxy-
ethyl)-N-methylamino]-diazen-1-ium-1,2-diolate (12, 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 vacuo. The residue ob-
tained was purified by silica gel column chromatography using
EtOAc /hexane (2:1, v/v) as eluent. Physical and spectral data for
13a–b are listed below.
(CDCl3) d 3.08 (s, 3H, SO2CH3), 4.20 (s, 2H, CH2CO), 6.81 (s, 1H, pyr-
azole H-4), 7.26 (d, J = 8.5 Hz, 2H, benzyl H-3, H-5), 7.32 (d,
J = 8.5 Hz, 2H, benzyl H-2, H-6), 7.54 (dd, J = 6.7, 1.8 Hz, 2H, meth-
anesulfonylphenyl H-2, H-6), 7.96 (dd, J = 6.7, 1.8 Hz, 2H, metha-
nesulfonylphenyl H-3, H-5); MS 443.04 (M), 445.04 (M+2).
5.6.2. 5-(4-Chlorocarbonylmethylphenyl)-1-(4-
aminosulfonylphenyl)-3-trifluoromethyl-1H-pyrazole (14b)
Yield, 89%; pale yellow solid; IR (film) 3340, 3270 (NH2), 3064
(C–H aromatic), 2924 (C–H aliphatic), 1792 (CO), 1337, 1162
(SO2) cmꢁ1 1H NMR (CDCl3) d 4.19 (s, 2H, CH2CO), 4.88 (br s, 2H,
;
NH2, exchanges with D2O), 6.80 (s, 1H, pyrazole H-4), 7.25 (d,
J = 8.3 Hz, 2H, benzyl H-3, H-5), 7.31 (d, J = 8.3 Hz, 2H, benzyl H-
2, H-6), 7.48 (d, J = 8.8 Hz, 2H, aminosulfonylphenyl H-2, H-6),
7.91 (d, J = 8.8 Hz, 2H, aminosulfonylphenyl H-3, H-5); MS 444.04
(M), 446.04 (M+2).
5.5.1. O2-Acetoxymethyl-1-{N-[2-(2-(4-(1-(4-
methanesulfonylphenyl)-3-trifluoromethyl-1H-pyrazol-5-
yl)phenyl)acetoxy)ethyl]-N-methylamino}diazen-1-ium-1,2-
diolate (13a)
5.7. General procedure for the synthesis of O2-acetoxymethyl-
1-[2-methylpyrrolidin-1-yl] diazen-1-ium-1,2-diolate esters
(16a–b)
Yield, 84%; pale yellow gum; IR (film) 3026 (C–H aromatic),
2963 (C–H aliphatic), 1734, 1718 (CO2), 1318, 1156 (SO2), 1236,
1099 (N@N–O) cmꢁ1 1H NMR (CDCl3) d 2.11 (s, 3H, COCH3), 3.07
;
(s, 3H, SO2CH3), 3.08 (s, 3H, NCH3), 3.67 (t, J = 5.2 Hz, 2H, CH2N),
3.68 (s, 2H, CH2COO, 4.33 (t, J = 5.2 Hz, 2H, CO2CH2), 5.79 (s, 2H,
OCH2O), 6.79 (s, 1H, pyrazole H-4), 7.21 (d, J = 8.5 Hz, 2H, benzyl
H-3, H-5), 7.32 (d, J = 8.5 Hz, 2H, benzyl 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 636.01
(M+Na); Anal. Calcd for C25H26F3N5O8Sꢂ1/3H2O: C, 48.47; H, 4.34;
N, 11.30. Found: C, 48.12; H, 4.55; N, 11.68.
The respective acid chloride 14a or 14b (1 mmol), O2-acetoxy-
methyl-1-(2-hydroxymethylpyrrolidin-1-yl)diazen-1-ium-1,2-dio-
late (15, 1 mmol) and triethylamine (1 mmol) were dissolved in
dry THF (10 mL), and the reaction was allowed to proceed at
25 °C for 48 h with stirring. The precipitate (triethylammonium
chloride) was filtered off and the solvent was removed in vacuo.
The residue was dissolved in dichloromethane and washed with
water, the organic layer was dried (Na2SO4), the solvent was evap-
orated under vacuum, and the product was purified by silica gel
column chromatography using EtOAc/hexane (2:1, v/v) as eluent.
Physical and spectral data for 16a–b are listed below.
5.5.2. O2-Acetoxymethyl-1-{N-[2-(2-(4-(1-(4-
aminosulfonylphenyl)-3-trifluoromethyl-1H-pyrazol-5-
yl)phenyl)acetoxy)ethyl]-N-methylamino}diazen-1-ium-1,2-
diolate (13b)
5.7.1. O2-Acetoxymethyl-1-{2-[2-(4-(1-(4-
methanesulfonylphenyl)-3-trifluoromethyl-1H-pyrazol-5-
yl)phenyl)acetoxymethyl]pyrrolidin-1-yl}diazen-1-ium-1,2-
diolate (16a)
Yield, 36%; pale yellow gum; IR (film) 3352, 3259 (NH2), 3024
(C–H aromatic), 2965 (C–H aliphatic), 1734, 1718 (CO2), 1342,
1164 (SO2), 1240, 1100 (N@N–O) cmꢁ1 1H NMR (CDCl3) d 2.13
;
(s, 3H, COCH3), 3.04 (s, 3H, NCH3), 3.65 (t, J = 5.2 Hz, 2H, CH2N),
3.67 (s, 2H, CH2COO, 4.32 (t, J = 5.2 Hz, 2H, CO2CH2), 5.11 (br s,
2H, NH2, exchanges with D2O), 5.76 (s, 2H, OCH2O), 6.77 (s, 1H,
pyrazole H-4), 7.20 (d, J = 8.0 Hz, 2H, benzyl H-3, H-5), 7.30 (d,
J = 8.0 Hz, 2H, benzyl H-2, H-6), 7.47 (dd, J = 7.1, 2.5 Hz, 2H, amin-
osulfonylphenyl H-2, H-6), 7.94 (d, J = 7.1, 2.5 Hz, 2H, aminosulfo-
nylphenyl H-3, H-5); 13C NMR (CDCl3) d 20.8, 40.8, 40.9, 52.5, 61.4,
87.2, 106.5, 121.0, 125.5, 127.5, 127.6, 129.0, 130.0, 135.2, 141.8,
142.1, 143.8, 144.6, 169.6, 170.5; MS 637.08 (M+Na); Anal. Calcd
for C24H25F3N6O8S: C, 46.91; H, 4.10; N, 13.68. Found: C, 47.31;
H, 4.39; N, 13.48.
Yield, 69%; pale yellow powder; mp 52–54 °C; ½a D21:0
= ꢁ36.0
ꢃ
(1.0100, CHCl3); IR (film) 3065 (C–H aromatic), 2934 (C–H ali-
phatic), 1755, 1735 (CO2), 1321, 1156 (SO2), 1241, 1098 (N@N–
O) cmꢁ1
;
1H NMR (CDCl3) d 1.80ꢁ2.09 (m, 4H, pyrrolidin-1-yl H-
3, H-4), 2.10 (s, 3H, COCH3), 3.07 (s, 3H, SO2CH3), 3.52–3.73 (m,
2H, pyrrolidin-1-yl H-5), 3.76 (s, 2H, CH2COO), 4.19–4.31 (m, 3H,
pyrrolidin-1-yl H-2, COOCH2), 5.73 (d, J = 7.3 Hz, 1H, –OCHH0OAc),
5.76 (d, J = 7.3 Hz, 1H, OCHH0OAc), 6.77 (s, 1H, pyrazole H-4), 7.23
(d, J = 8.2 Hz, 2H, benzyl H-3, H-5), 7.31 (d, J = 8.2 Hz, 2H, benzyl H-
2, H-6), 7.54 (d, J = 8.5 Hz, 2H, methanesulfonylphenyl H-2, H-6),
7.94 (d, J = 8.5 Hz, 2H, methanesulfonylphenyl H-3, H-5); 13C
NMR (CDCl3) d 20.9, 22.8, 26.9, 40.8, 44.4, 52.6, 59.7, 65.9, 87.3,
106.7, 120.9, 125.7, 127.4, 128.5, 129.0, 130.1, 135.5, 140.0,
143.3, 144.1, 144.7, 169.4, 170.5; MS 662.04 (M+Na); Anal. Calcd
for C27H28F3N5O8S: C, 50.70; H, 4.41; N, 10.95. Found: C, 51.30;
H, 4.64; N, 10.41.
5.6. General method for preparation of acid chlorides (14a–b)
Oxalyl chloride (0.3 mL, 3.4 mmol) was added drop wise to a
solution of the respective acid 11a–b (2.8 mmol) in dry CH2Cl2

5188
K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 17 (2009) 5182–5188
5.7.2. O2-Acetoxymethyl-1-{2-[2-(4-(1-(4-
in vivo carrageenan-induced foot paw edema model reported
previously.27
aminosulfonylphenyl)-3-trifluoromethyl-1H-pyrazol-5-
yl)phenyl)acetoxymethyl]pyrrolidin-1-yl}diazen-1-ium-1,2-
diolate (16b)
Acknowledgements
Yield, 51%; white powder; mp 61–63 °C; ½a D21:0
= ꢁ23.0 (1.0200,
ꢃ
CHCl3); IR (film) 3341, 3271 (NH2), 3065 (C–H aromatic), 2934 (C–
H aliphatic), 1758, 1737 (CO2), 1341, 1168 (SO2), 1239, 1101
We are grateful to the Canadian Institutes of Health Research
(CIHR) (MOP-14712) for financial support of this research.
(N@N–O) cmꢁ1
;
1H NMR (CDCl3) d 1.82ꢁ2.05 (m, 4H, pyrrolidin-
1-yl H-3, H-4), 2.11 (s, 3H, COCH3), 3.52–3.63 (m, 2H, pyrrolidin-
1-yl H-5), 3.68 (s, 2H, CH2COO), 4.17–4.32 (m, 3H, pyrrolidin-1-yl
H-2, CO2CH2), 5.14 (br s, 2H, NH2, exchanges with D2O), 5.73 (d,
J = 7.3 Hz, 1H, -OCHH0OAc), 5.75 (d, J = 7.3 Hz, 1H, OCHH0OAc),
6.77 (s, 1H, pyrazole H-4), 7.19 (d, J = 7.9 Hz, 2H, benzyl H-3, H-
5), 7.29 (d, J = 7.9 Hz, 2H, benzyl H-2, H-6), 7.45 (dd, J = 6.5,
2.5 Hz, 2H, aminosulfonylphenyl H-2, H-6), 7.91(d, J = 6.5, 2.5 Hz,
2H, aminosulfonylphenyl H-3, H-5); 13C NMR (CDCl3) d 20.9,
22.8, 27.9, 40.9, 52.6, 59.7, 65.7, 87.2, 106.5, 121.0, 125.4, 127.5,
127.6, 129.0, 129.7, 135.4, 141.8, 142.1, 143.8, 144.6, 169.7,
170.5; MS 662.92 (M+Na).
References and notes
1. Harris, C. R.; Breyer, D. M. Clin. J. Am. Soc. Nephrol. 2006, 1, 236.
2. Neiderberger, E.; Manderscheid, C.; Geisslinger, G. Biochem. Biophy. Res.
Commun. 2006, 342, 940.
3. Hinz, B.; Brune, K. J. Pharmacol. Exp. Ther. 2002, 300, 367.
4. Patel, H. H.; Gross, G. J. J. Mol. Cell Cardiol. 2002, 34, 1.
5. Mukherjee, D. Biochem. Pharmacol. 2002, 63, 817.
6. Scheen, A. J. Rev. Med. Liege 2004, 59, 565.
7. Dogné, J.-M.; Supuran, C. T.; Pratico, D. J. Med. Chem. 2005, 48, 2251.
8. Butler, A. R.; Williams, D. L. H. Chem. Soc. Rev. 1993, 22, 233.
9. Anning, B. P.; Coles, B.; Morton, J.; Wang, H.; Uddin, J.; Morrow, D. J.; Dey, K. S.;
Marnett, J. L.; O’Donell, B. V. Blood 2006, 108, 4059.
10. Abdellatif, K. R. A.; Chowdhury, M. A.; Dong, Y.; Chen, Q.-H.; Knaus, E. E. Bioorg.
Med. Chem. 2008, 16, 3302.
11. Abdellatif, K. R. A.; Chowdhury, M. A.; Dong, Y.; Velázquez, C. A.; Das, D.;
Suresh, M. R.; Knaus, E. E. Bioorg. Med. Chem. 2008, 16, 9694.
12. Abdellatif, K. R. A.; Dong, Y.; Chen, Q.-H.; Chowdhury, M. A.; Knaus, E. E. Bioorg.
Med. Chem. 2007, 15, 6796.
13. Csont, T.; Ferdinandy, P. Pharmacol. Ther. 2005, 105, 57.
14. Fung, H. L.; Bauer, J. A. Cardiovasc. Drugs Ther. 1994, 8, 489.
15. Hu, R.; Siu, C. W.; Lau, E. O.; Wang, W. Q.; Lau, C.-P.; Tse, H.-F. Int. J. Cardiol.
2007, 120, 351.
16. 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.
6. Cyclooxygenase inhibition assays
The ability of the test compounds listed in Table 1 to inhibit
ovine COX-1 and human recombinant COX-2 (IC50 value,
was determined using an enzyme immuno assay (EIA) kit (catalog
no. 560131, Cayman Chemical, Ann Arbor, MI, USA) according to
our previously reported method.25
lM)
7. Nitric oxide release assays
17. Ahlström, M. M.; Ridderström, M.; Zamora, I.; Luthman, K. J. Med. Chem. 2007,
50, 4444.
18. 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.
19. Saavedra, J. E.; Dunams, T. M.; Flippen-Anderson, J. L.; Keefer, L. K. J. Org. Chem.
1992, 57, 6134.
20. Hrabie, J. A.; Klose, J. R.; Wink, D. A.; Keefer, L. K. J. Org. Chem. 1993, 58, 1472.
21. Saavedra, J. E.; Shami, P. J.; Wang, L. Y.; Davies, K. M.; Booth, M. N.; Citro, M. L.;
Keefer, L. K. J. Med. Chem. 2000, 43, 261.
22. Velázquez, C. A.; Praveen Rao, P. N.; Citro, M. L.; Keefer, L. K.; Knaus, E. E. Bioorg.
Med. Chem. 2007, 15, 4767.
23. Velazquez, C.; Knaus, E. E. Bioorg. Med. Chem. 2004, 12, 3831.
24. Velázquez, C.; Chen, Q.-H.; Citro, M. L.; Keefer, L. K.; Knaus, E. E. J. Med. Chem.
2008, 51, 1954.
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 ꢀ 10ꢁ2 mM
solution in phosphate buffer at pH 7.4, or with 2.4 mL of a
1.0 ꢀ 10ꢁ2 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 ( 13a–b, 16a–b, 17–18) using the re-
ported procedures.26
8. In vivo anti-inflammatory assay
25. Rao, P. N. P.; Amini, M.; Li, H.; Habeeb, A.; Knaus, E. E. J. Med. Chem. 2003, 46,
4872.
The test compounds 11a–b, 13a–b, 16a–b and the reference
drugs celecoxib, aspirin and ibuprofen were evaluated using the
26. Velázquez, C.; Vo, D.; Knaus, E. E. Drug Dev. Res. 2003, 60, 204.
27. Winter, C. A.; Risley, E. A.; Nuss, G. W. Proc. Soc. Exp. Biol. Med. 1962, 111, 544.