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

Article abstract of DOI:10.1016/j.bmc.2008.10.001

A new class of hybrid nitric oxide-releasing anti-inflammatory (AI) ester prodrugs (NONO-coxibs 12a-b) wherein an O²-acetoxymethyl 1-(2-carboxypyrrolidin-1-yl)diazen-1-ium-1,2-diolate (11, O²-acetoxymethyl PROLI/NO) NO-donor moiety was covalently coupled to the bromomethyl group of 5-(4-bromomethylphenyl)-1-(4-aminosulfonylphenyl)-3-trifluoromethyl-1H-pyrazole (9a), and its methanesulfonyl analog (9b), were synthesized. The diazen-1-ium-1,2-diolate compounds 12a-b released a low amount of NO upon incubation with phosphate buffer (PBS) at pH 7.4 (6.1-8.2% range). In comparison, the percentage NO released was significantly higher (76-77% 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 12a-b were incubated in the presence of rat serum. These incubation studies suggest that both NO and the anti-inflammatory 5-(4-hydroxymethylphenyl)-1-(4-aminosulfonylphenyl)-3-trifluoromethyl-1H-pyrazole (10a), and its methanesulfonyl analog (10b), would be released from the parent NONO-coxib 12a or 12b upon in vivo cleavage by non-specific serum esterases. The hydroxymethyl compounds 10a-b were weak inhibitors of the cyclooxygenase-1 (COX-1) and COX-2 isozymes (IC₅₀ = 3.7-10.5 μM range). However, the hydroxymethyl compounds 10a-b and the parent NONO-coxibs 12a-b exhibited good AI activities (ED₅₀ = 76.7-111.6 μmol/kg po range) that were greater than that exhibited by the reference drugs aspirin (ED₅₀ = 710 μmol/kg po) and ibuprofen (ED₅₀ = 327 μmol/kg po), but less than that of celecoxib (ED₅₀ = 30.9 μmol/kg po). These studies indicate hybrid ester AI/NO-donor prodrugs (NONO-coxibs) constitutes a plausible drug design concept targeted toward the development of selective COX-2 inhibitory AI drugs that are devoid of adverse cardiovascular effects.

Full text of DOI:10.1016/j.bmc.2008.10.001

Bioorganic & Medicinal Chemistry 16 (2008) 9694–9698
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-hydroxymethylphenyl)-1-(4-aminosulfonylphenyl)-3-trifluoromethyl-
1H-pyrazole and its methanesulfonyl analog: Synthesis, biological
evaluation and nitric oxide release studies
Khaled R. A. Abdellatif, Morshed Alam Chowdhury, Ying Dong, Carlos Velázquez, Dipankar Das,
*
Mavanur R. Suresh, 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
Article history:
A new class of hybrid nitric oxide-releasing anti-inflammatory (AI) ester prodrugs (NONO-coxibs 12a–b)
wherein an O2-acetoxymethyl 1-(2-carboxypyrrolidin-1-yl)diazen-1-ium-1,2-diolate (11, O2-acetoxy-
methyl PROLI/NO) NO-donor moiety was covalently coupled to the bromomethyl group of 5-(4-bromom-
ethylphenyl)-1-(4-aminosulfonylphenyl)-3-trifluoromethyl-1H-pyrazole (9a), and its methanesulfonyl
analog (9b), were synthesized. The diazen-1-ium-1,2-diolate compounds 12a–b released a low amount
of NO upon incubation with phosphate buffer (PBS) at pH 7.4 (6.1–8.2% range). In comparison, the per-
centage NO released was significantly higher (76–77% of the theoretical maximal release of two mole-
cules of NO/molecule of the parent hybrid ester prodrug) when the diazen-1-ium-1,2-diolate ester
prodrugs 12a–b were incubated in the presence of rat serum. These incubation studies suggest that both
NO and the anti-inflammatory 5-(4-hydroxymethylphenyl)-1-(4-aminosulfonylphenyl)-3-trifluoro-
methyl-1H-pyrazole (10a), and its methanesulfonyl analog (10b), would be released from the parent
NONO-coxib 12a or 12b upon in vivo cleavage by non-specific serum esterases. The hydroxymethyl com-
pounds 10a–b were weak inhibitors of the cyclooxygenase-1 (COX-1) and COX-2 isozymes (IC50 = 3.7–
Received 19 August 2008
Revised 30 September 2008
Accepted 1 October 2008
Available online 5 October 2008
Keywords:
1,5-Diarylpyrazoles
NONO-coxib ester prodrugs
Diazen-1-ium-1,2-diolate nitric oxide donor
Cyclooxygenase inhibition
Anti-inflammatory activity
10.5
exhibited good AI activities (ED50 = 76.7–111.6
by the reference drugs aspirin (ED50 = 710 mol/kg po) and ibuprofen (ED50 = 327
than that of celecoxib (ED50 = 30.9 mol/kg po). These studies indicate hybrid ester AI/NO-donor pro-
l
M range). However, the hydroxymethyl compounds 10a–b and the parent NONO-coxibs 12a–b
mol/kg po range) that were greater than that exhibited
mol/kg po), but less
l
l
l
l
drugs (NONO-coxibs) constitutes a plausible drug design concept targeted toward the development of
selective COX-2 inhibitory AI drugs that are devoid of adverse cardiovascular effects.
Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction
prothrombotic thromboxane A2 (TxA2).2–4 These two adverse bio-
chemical changes in the COX pathway are believed to be responsi-
Selective inhibition of the inducible cyclooxygenase-2 (COX-2)
isozyme in the periphery provided a useful drug design concept
that resulted in the development of effective anti-inflammatory
(AI) drugs that were devoid of adverse gastrointestinal ulcerogen-
city frequently associated with the use of non-steroidal anti-
inflammatory drugs (NSAIDs).1 Unfortunately, some selective
COX-2 inhibitory drugs that include rofecoxib (1) and valdecoxib
(2) alter the natural balance in the COX pathway (see structures
in Fig. 1). In this regard, the amount of the desirable vasodilatory
and anti-aggregatory prostacyclin (PGI2) produced is decreased to-
gether with a simultaneous increase in the level of the undesirable
ble for increased incidences of high blood pressure and myocardial
infarction that ultimately prompted the withdrawal of rofecoxib
(VioxxÒ) and valdecoxib (BextraÒ).5,6 Nitric oxide (NO) exhibits a
number of useful pharmacological actions that include vascular
relaxation (vasodilation), and inhibition of platelet aggregation
and adhesion.7 In an earlier study we reported non-ulcerogenic
NONO-NSAID ester prodrugs of aspirin (4) and indomethacin (5)
having a NO-donor diazen-1-ium-1,2-diolate (NONOate) moiety
that are effectively cleaved by esterases to release the AI drug
and NO.8 Accordingly, attachment of a N-diazen-1-ium-1,2-diolate
moiety offers a potential drug design concept to circumvent the
adverse cardiovascular events associated with the chronic clinical
use of highly selective COX-2 inhibitors. We now describe an
investigation directed toward the design and synthesis of model
* 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.2008.10.001

K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 16 (2008) 9694–9698
9695
Me
O
O
NH2
O
S
Me
NH2
S
S
O
N
O
O
N
O
O
Me
O
N
F3C
Celecoxib (3)
Valdecoxib (2)
Rofecoxib (1)
Cl
O
O
CH3
OR
N
O
O
CH3
OR
N
Me
O
O
4-5, R =
MeO
N
N
O
O
O
O
O
5
4
Figure 1. Chemical structures of the selective cyclooxygenase-2 (COX-2) inhibitors rofecoxib (1), valdecoxib (2) and celecoxib (3), and the second-generation O2-
acetoxymethyl-1-(2-carboxypyrrolidin-1-yl)diazenium-1,2-diolate nitric oxide donor (O2-acetoxymethyl PROLI/NO) prodrug esters of aspirin (4) and indomethacin (5).
hybrid nitric oxide donor N-diazen-1-ium-1,2-diolate ester prodrugs
of 5-(4-hydroxymethylphenyl)-1-(4-methanesulfonylphenyl)-3-tri-
fluoromethyl-1H-pyrazole (12a), and its methanesulfonyl analog
(12b), that may be devoid of adverse cardiovascular effects.
4-aminosulfonylphenylhydrazine hydrochloride (7a), or 4-meth-
ylsulfonylphenylhydrazine hydrochloride (7b), afforded the
respective pyrazole analog 8a (56%) or 8b (44%). Subsequent pho-
tochemical promoted bromination of the tolyl methyl group pres-
ent in compounds 8a–b afforded the respective benzyl bromide
derivative 9a, or 9b, in 46% yields. The bromomethyl compounds
9a–b were converted to the respective hydroxymethyl analogs
10a–b in acceptable yield (54–66%) upon heating under reflux in
an acetone/water solvent system (30:4, v/v) for 110 h. The target
O2-acetoxymethyl PROLI/NO prodrug esters (12a–b) were synthe-
sized in 45% yields by condensation of the bromomethyl com-
pounds 9a–b with O2-acetoxymethyl 1-(2-carboxypyrrolidin-1-
2. Chemistry
The two 5-(4-hydroxymethylphenyl)-1-(4-aminosulfonylphe-
nyl)-3-trifluoromethyl-1H-pyrazole (10a), and its methanesulfonyl
analog (10b), and the two O2-acetoxymethyl PROLI/NO prodrugs
(12a–b), were synthesized using the reaction sequence illustrated
in Scheme 1. Accordingly, reaction of the dione 6 with either
CH2Br
CH3
CH3
NHNH2.HCl
a
b
+
O
N
SO2R
N
SO2R
SO2R
N
N
O
CF3
F3C
F3C
7a; R = NH2
7b; R = CH3
6
8a; R = NH2
8b; R = CH3
9a; R = NH2
9b; R = CH3
d
c
O
N
N
+
O
N
CH3
CH2OH
O
O
O
O
O
N
N
N
CH3
+
O
O
O
C
O
OH
11 (PROLI/NO)
N
N
SO2R
N
SO2R
N
F3C
F3C
10a; R = NH2
10b; R = CH3
12a; R = NH2
12b; R = CH3
Scheme 1. Reagents and conditions: (a) C2H5OH, reflux, 20 h; (b) N-bromosuccinimide, benzoyl peroxide, light from a 100-W sun beam lamp, benzene, 25 °C ? reflux, 3.5–
5 h; (c) acetone, H2O, reflux, 110 h; (d) O2-acetoxymethyl 1-(2-carboxypyrrolidin-1-yl)diazen-1-ium-1,2-diolate (11), Et3N, DMSO, 25 °C, 24 h.

9696
K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 16 (2008) 9694–9698
yl)diazen-1-ium-1,2-diolate (11) in the presence of triethylamine
in dimethyl sulfoxide.
The percent NO released from the hybrid ester PROLI/NO pro-
drugs (12a–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.12 O2-Substi-
tuted-diazen-1-ium-1,2-diolates are stable compounds that hydro-
lyze slowly even in acidic solution.13 Consistent with these
observations, when compounds 12a–b were incubated for 1.5 h
in PBS at pH 7.4, the percentage of NO released varied from 6.1%
to 8.2% which is indicative of slow NO release. In contrast, the ef-
fect of non-specific esterases present in rat serum on the NO re-
lease properties of compounds 12a–b was substantially higher
(76–77% range). These data indicate the non-specific serum ester-
ases present in rat serum cleave these hybrid prodrug esters more
effectively than PBS at pH 7.4. The hybrid ester prodrugs 12a–b can
not release NO prior to cleavage of the acetoxy moiety present in
the terminal O2-acetoxymethyl- PROLI/NO moiety. Two plausible
pathways for the ester hydrolysis of hybrid PROLI/NO ester pro-
drugs and the subsequent release of acetic acid, formaldehyde,
3. Results and discussion
Three positions on the structure of celecoxib (3) were consid-
ered for attachment of an O2-acetoxymethyl PROLI/NO group via
an ester moiety. 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.9 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.10 The C-
5 para-methylphenyl substituent (benzylic carbon) in celecoxib
provides high selectivity for the COX-2 isozyme. Even though cele-
coxib undergoes sequential metabolic biotransformation (Me ?
CH2OH ? CO2H ? CO2-glucuronide conjugate), it still exhibits po-
tent anti-inflammatory activity.11 We envisaged, from a synthetic
perspective, that a p-CH2Br substituent on the pyrazole C-5 phenyl
ring was a suitable substituent for coupling the O2-acetoxymethyl
PROLI/NO moiety. Accordingly, it was decided based on this struc-
tural information to couple the O2-acetoxymethyl PROLI/NO nitric
oxide donor moiety (11) to a pyrazole ring C-5 p-C6H4–CH2Br
group via an ester moiety to prepare the target NONO-coxib hybrid
ester prodrugs (12a–b).
two molecules of NO, and L-proline was described in an earlier
publication.8 The hybrid ester NO-donor prodrugs 12a–b were de-
signed 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
release formaldehyde to furnish the free diazen-1-ium-1,2-diolate
compound that can subsequently fragment to release two mole-
cules of NO. On the other hand, cleavage of the second ester group
attached to the C-5 position of the pyrazole ring, that releases the
hydroxymethyl compounds 10a–b, can occur either prior to, or
after, NO release has occurred.
In vitro COX enzyme inhibition studies (Table 1) showed that
the
IC50 = 7.1
COX-2 IC50 = 10.5
weaker COX-2 inhibition, than the reference drug celecoxib
(COX-1 IC50 = 7.7 M; COX-2 IC50 = 0.07 M). In contrast to the
hydroxymethyl
M; COX-2 IC50 = 3.7
M) exhibited similar COX-1 inhibition, but
(CH2OH)
compounds
10a
(COX-1
l
lM) and 10b (COX-1 IC50 = 8.6
lM;
l
l
l
The anti-inflammatory (AI) activities exhibited by the 5-(4-
hydroxymethylphenyl)-1-(4-aminosulfonylphenyl)-3-trifluoro-
methyl-1H-pyrazole (10a, R = NH2) and its methanesulfonyl analog
(10b, R = CH3) that would be released upon cleavage of the ester
group attached at the C-5 position of the pyrazole ring, and the hy-
brid PROLI/NO ester prodrugs 12a–b were determined using a car-
rageenan-induced rat foot paw edema model (see data in Table 1).
Compounds 10a–b and 12a–b exhibited AI activities (ED50 = 76.7–
hydroxymethyl compounds 10a–b, which are non-selective COX
inhibitors, the PROLI/NO hybrid ester prodrug 12a was a selective
COX-2 inhibitior (COX-1 IC50 > 100 lM; COX-2 IC50 = 4.9 lM).
Table 1
In vitro COX-1/COX-2 enzyme inhibition, in vivo anti-inflammatory activity, and
in vitro nitric oxide release data for the 5-(4-hydroxymethylphenyl)-1-[4-meth-
ane(amino)sulfonylphenyl]-3-trifluoromethyl-1H-pyrazoles (10a–b) and the diazen-
1-ium-1,2-diolate prodrug esters (12a–b)
111.6
l
mol/kg po range) that were greater than that exhibited by
mol/kg po) and ibuprofen
mol/kg po), but less than that of celecoxib
mol/kg po). The relative AI potency profile with re-
the reference drugs aspirin (ED50 = 710
l
a
a
(ED50 = 327
(ED50 = 30.9
l
l
Compound COX-1 IC50
M)
COX-2 IC50
M)
AI activityb ED50
mol/kg)
% NO releasedc
PBSd Serume
(l
(
l
(l
spect to the SO2NH2 and SO2Me substituents was variable with
SO2NH2 (10a) > SO2Me (10b) for the hydroxymethyl compounds,
but SO2Me (12b) > SO2NH2 (12a) for the parent PROLI/NO prodrug
esters. A comparison of AI potency profiles showed that hydroxy-
methyl compound 10a was 1.22-fold more potent than the PRO-
LI/NO prodrug (12a). In contrast, the PROLI/NO prodrug 12b was
1.45-fold more potent than the hydroxymethyl compound 10b.
These AI data provide credence for the drug design concept that
covalent attachment of a PROLI/NO-donor moiety directly to a suit-
ably positioned p-C6H4–CH2Br group in a selective COX-2 inhibitor
offers a potential method to circumvent adverse cardiovascular
effects.
10a
10b
12a
12b
Celecoxib
Aspirin
Ibuprofen
7.1
8.6
>100
NDf
7.7
3.7
10.5
4.9
ND
0.07
2.4
77.1
111.6
94.3
76.7
30.9
710
—
—
8.2
6.1
—
—
—
76.0
77.0
—
0.3
2.9
—
—
—
—
1.1
327
a
The in vitro test compound concentration required to produce 50% inhibition of
M) is the mean of two determinations acquired
COX-1 or COX-2. The result (IC50, l
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
Inhibitory activity in a carrageenan-induced rat paw edema assay. The results
are expressed as the ED50 value (lmol/kg) at 3 h after oral administration (n = 3) of
the test compound.
c
Percent of nitric oxide released based on a theoretical maximum release of
2 mol of nitric oxide/mol of the diazen-1-ium-1,2-diolate test compound (12a–b).
The result is the mean value of three measurements (n = 3) where variation from
the mean% value was 60.5%.
4. Conclusions
A novel type of hybrid ester prodrugs (NONO-coxibs) (12a–b) in
which an O2-acetoxymethyl PROLI/NO nitric oxide donor moiety is
attached to the CH2Br group of 5-(4-bromomethylphenyl)-1-(4-
aminosulfonylphenyl)-3-trifluoromethyl-1H-pyrazole (9a), and its
methanesulfonyl analog (9b) were synthesized for comparative
biological evaluation. COX-1/COX-2 inhibition, NO release, and AI
studies showed that (i) the 5-(4-hydroxymethylphenyl)-1-(4-
d
A solution of the test compound (2.4 mL of a 1.0 ꢀ 10ꢁ2 mM solution in phos-
phate buffer containing a small amount of DMSO at pH 7.4),17 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 phos-
phate buffer containing a small amount of DMSO at pH 7.4 to which 90 lL rat serum
had been added),17 was incubated at 37 °C for 1.5 h.
f
ND = not determined.

K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 16 (2008) 9694–9698
9697
aminosulfonylphenyl)-3-trifluoromethyl-1H-pyrazole (10a), or its
methanesulfonyl analog (10b) that would be released after ester
hydrolysis are weak non-selective inhibitors of the COX-1 and
COX-2 isozymes, (ii) the parent NONO-coxib prodrugs (12a–b)
are relatively stable in phosphate-buffered saline at pH 7 where
NO release is in the 6.1–8.2% range, (iii) the hybrid PROLI/NO ester
prodrugs (12a–b) undergo extensive cleavage of the terminal acet-
oxy group by rat serum esterase(s) that is followed by a significant
release of NO in the 76–77% range, and (iv) the relatively potent AI
activity exhibited by the hydroxymethyl compounds 10a–b
H-3, H-5). This 1H NMR spectral data for 8b is identical to the pre-
viously reported data.10
5.3. 4-[5-(4-Bromomethylphenyl)-3-trifluoromethyl-1H-
pyrazol-1-yl]benzenesulfonamide (9a)
N-Bromosuccinimide (1.32 g, 7.43 mmol) was added to a stirred
solution of 8a (3.02 g, 7.43 mmol) in benzene (100 mL). Benzoyl
peroxide (0.177 g, 0.73 mmol) was added and the reaction mixture
was then irradiated with light from a 100-W sun beam lamp for
3.5 h. After cooling to 25 °C, the reaction mixture was filtered,
the filtrate was washed with water and then brine, the filtrate
was dried (Na2SO4), filtered, and the solvent was removed from
the filtrate in vacuo. The residue was purified by silica gel column
chromatography using a gradient of EtOAc/hexane (1:9, v/v) to
EtOAc/hexane (3:7, v/v) as the eluent to give 9a (1.57 g, 46%) as a
white powder: mp 154–156 °C; IR (film) 3370, 3268 (NH2), 2965
(ED50 = 77.1–111.6 lmol/kg po range) supports the drug design
concept that covalent attachment of the second-generation PRO-
LI/NO-donor moiety directly to a suitably positioned p-C6H4–CH2Br
group present in a selective COX-2 inhibitor offers a rational
approach to circumvent adverse cardiovascular effects.
5. Experimental
(C–H aromatic), 2925 (C–H aliphatic), 1345, 1165 (SO2) cmꢁ1 1H
;
NMR (CDCl3) d 4.50 (s, 2H, CH2Br), 4.92 (br s, 2H, NH2), 6.80 (s,
1H, H-4), 7.22 (d, J = 8.0 Hz, 2H, bromomethylphenyl H-3, H-5),
7.42 (d, J = 8.0 Hz, 2H, bromomethylphenyl H-2, H-6), 7.50 (dd,
J = 6.7, 2.4 Hz, 2H, sulfamoylphenyl H-3, H-5), 7.94 (dd, J = 6.7,
2.4 Hz, 2H, sulfamoylphenyl H-2, H-6); 13C NMR (CDCl3) d 32.2,
106.7, 120.9, 125.5, 127.5, 128.5, 129.1, 129.7, 139.3, 141.6,
142.2, 143.5, 144.4.
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 CDCl3 with TMS as the internal standard.
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. 1-(4-Methylphenyl)-4,4,4-trifluorobutan-1,3-
5.4. 5-(4-Bromomethylphenyl)-1-(4-methanesulfonylphenyl)-
3-trifluoromethyl-1H-pyrazole (9b)
dione (6),9 4-sulfamoylphenylhydrazine hydrochloride (7a)14
,
N-Bromosuccinimide (1.32 g, 7.43 mmol) was added to a stirred
solution of 8b (3.01 g, 7.43 mmol) in benzene (100 mL). Benzoyl
peroxide (0.177 g, 0.73 mmol) was added and the reaction mixture
was irradiated with light from a 100-W sun beam lamp while heat-
ing at reflux for 4.5 h. The product 9b was isolated and purified by
silica gel column chromatography using a procedure similar to that
described for 9a. Product 9b (1.57 g, 46%) was obtained as a yellow
powder: mp 139–142 °C; IR (film) 2965 (C–H aromatic), 2928 (C–H
4-methylsulfonylphenylhydrazine hydrochloride (7b)15 and
O2-acetoxymethyl 1-(2-carboxypyrrolidin-1-yl)diazen-1-ium-1,2-
diolate (11)8 were prepared according to literature procedures.
5.1. 4-[5-(4-Methylphenyl)-3-trifluoromethyl-1H-pyrazol-1-
yl]benzenesulfonamide (8a)
aliphatic), 1318, 1156 (SO2) cmꢁ1 1H NMR (CDCl3) d 3.07 (s, 3H,
;
4-Sulfamoylphenylhydrazine hydrochloride (7a, 0.980 g,
SO2CH3), 4.50 (s, 2H, CH2Br), 6.81 (s, 1H, pyrazole H-4), 7.24 (d,
J = 8.3 Hz, 2H, bromomethylphenyl H-3, H-5), 7.43 (d, J = 8.3 Hz,
2H, bromomethylphenyl H-2, H-6), 7.55 (dd, J = 6.7, 1.7 Hz, 2H,
methanesulfonylphenyl H-2, H-6), 7.97 (dd, J = 6.7, 1.7 Hz, 2H,
methanesulfonylphenyl H-3, H-5); 13C NMR (CDCl3) d 32.1, 44.4,
106.9, 120.9, 125.7, 128.4, 128.6, 129.1, 129.7, 139.4, 140.1,
143.2, 144.3, 144.4.
4.4 mmol) was added to
a stirred solution of the dione 6
(0.921 g, 4.0 mmol) in EtOH (50 mL), and the reaction was allowed
to proceed at reflux for 20 h with stirring. After cooling to 25 °C, the
solvent was removed in vacuo. The residue was dissolved in EtOAc
(25 mL), washed with water and then brine, the EtOAc fraction was
dried (MgSO4), filtered, and the solvent was removed in vacuo to
furnish 8a (0.91 g, 56%) as a white powder; mp 157–159 °C (lit.9
mp 157–159 °C); IR (film) 3358, 3263 (NH2), 2979 (C–H aromatic),
2920 (C–H aliphatic), 1339, 1169 (SO2) cmꢁ1 1H NMR (CDCl3) d
;
5.5. 4-[5-(4-Hydroxymethylphenyl)-3-trifluoromethyl-1H-
pyrazol-1-yl]benzenesulfonamide (10a)
2.39 (s, 3H, CH3), 4.92 (br s, 2H, NH2), 6.75 (s, 1H, pyrazole H-4),
7.12 (d, J = 7.9 Hz, 2H, methylphenyl H-3, H-5), 7.19 (d, J = 7.9 Hz,
2H, methylphenyl H-2, H-6), 7.49 (dd, J = 6.8, 1.9 Hz, 2H, sulfa-
moylphenyl H-3, H-5), 7.92 (dd, J = 6.8, 1.9 Hz, 2H, sulfamoylphe-
nyl H-2, H-6).
A solution of the bromomethyl compound 9a (1.16 g, 2.5 mmol)
in acetone (30 mL) and water (4 mL) was refluxed for 110 h. After
removal of the solvents in vacuo, the residue was dissolved in
EtOAc, the EtOAc fraction was dried (MgSO4), and the solvent
was removed in vacuo. The residue was purified by silica gel col-
umn chromatography using EtOAc/hexane (1:1, v/v) as eluent to
give the alcohol 10a as a pale yellow solid (0.536 g, 54%): mp
82–84 °C; IR (film) 3360, 3261 (NH2), 2959 (C–H aromatic), 2919
5.2. 1-(4-Methanesulfonylphenyl)-5-(4-methylphenyl)-3-
trifluoromethyl-1H-pyrazole (8b)
The title compound 8b was synthesized using the same proce-
dure described for the preparation of 8a, by using 4-meth-
ylsulfonylphenylhydrazine hydrochloride (7b) in place of 7a, in
44% as a yellow powder; IR (film) 3025 (C–H aromatic), 2926 (C–
(C–H aliphatic), 1340, 1159 (SO2) cmꢁ1 1H NMR (CDCl3) d 1.75
;
(br s, 1H, OH, exchanges with D2O), 4.76 (s, 2H, CH2), 4.93 (br s,
2H, NH2, exchanges with D2O), 6.79 (s, 1H, pyrazole H-4), 7.24 (d,
J = 7.9 Hz, 2H, hydroxymethylphenyl H-3, H-5), 7.43 (d, J = 7.9 Hz,
2H, hydroxymethylphenyl H-2, H-6), 7.48 (d, J = 8.9 Hz, 2H, sulfa-
moylphenyl H-3, H-5), 7.92 (d, J = 8.9 Hz, 2H, sulfamoylphenyl H-
2, H-6); 13C NMR (CDCl3) d 64.4, 106.5, 120.9, 125.5, 127.4, 127.5,
127.7, 129.0, 141.5, 142.3, 142.4, 144.1, 144.9; MS 398.08 (M+1).
H aliphatic), 1320, 1157 (SO2) cmꢁ1 1H NMR (CDCl3) d 2.40 (s,
;
3H, phenyl CH3), 3.07 (s, 3H, SO2CH3), 6.76 (s, 1H, pyrazole H-4),
7.12 (d, J = 8.1 Hz, 2H, methylphenyl H-3, H-5), 7.20 (d, J = 8.1 Hz,
2H, methylphenyl H-2, H-6), 7.54 (d, J = 7.9 Hz, 2H, methanesulfo-
nylphenyl H-2, H-6), 7.95 (d, J = 7.9 Hz, 2H, methanesulfonylphenyl

9698
K. R. A. Abdellatif et al. / Bioorg. Med. Chem. 16 (2008) 9694–9698
5.6. 5-(4-Hydroxymethylphenyl)-1-(4-methanesulfonylphenyl)-
3-trifluoromethyl-1H-pyrazole (10b)
6.80 (s, 1H, pyrazole H-4), 7.25 (d, J = 8.5 Hz, 2H, benzyl H-3, H-5),
7.38 (d, J = 8.50 Hz, 2H, benzyl H-2, H-6), 7.88 (d, J = 9.2 Hz, 2H,
methanesulfonylphenyl H-2, H-6), 7.97 (d, J = 9.2 Hz, 2H, metha-
nesulfonylphenyl H-3, H-5); MS 626.15 (M+1). Anal. Calcd for
C26H26F3N5O8Sꢃ1/4H2O: C, 49.57; H, 4.24; N, 11.12. Found: C,
49.92; H, 4.64; N, 10.74.
The title compound 10b was synthesized and the product was
purified using a procedure similar to that described for the prepa-
ration of 10a using the bromomethyl compound 9b in place of 9a.
The product 10b was obtained in 66% yield as a yellow powder: mp
159–160 °C; IR (film) 3592–3196 (O–H), 2962 (C–H aromatic),
6. Cyclooxygenase inhibition assays
2926 (C–H aliphatic), 1319, 1153 (SO2) cmꢁ1 1H NMR (CDCl3) d
;
1.71 (br s, 1H, OH, exchanges with D2O), 3.08 (s, 3H, SO2CH3),
4.77 (s, 2H, CH2), 6.70 (s, 1H, pyrazole H-4), 7.25 (d, J = 7.9 Hz,
2H, hydroxymethylphenyl H-3, H-5), 7.41 (d, J = 7.9 Hz, 2H,
hydroxymethylphenyl H-2, H-6), 7.55 (dd, J = 7.1, 1.9 Hz, 2H, meth-
anesulfonylphenyl H-2, H-6), 7.62 (dd, J = 7.1, 1.9 Hz, 2H, metha-
nesulfonylphenyl H-3, H-5); 13C NMR (CDCl3) d 44.5, 64.5, 106.7,
120.9, 125.7, 127.4, 127.6, 128.5, 129.0, 139.9, 142.6, 143.3,
144.3, 144.9; MS 397.04 (M+1).
The ability of the test compounds listed in Table 1 to inhibit
ovine COX-1 and COX-2 (IC50 value,
lM) was determined using
an enzyme immuno assay (EIA) kit (Catalog No. 560101, Cayman
Chemical, Ann Arbor, MI, USA) according to our previously re-
ported method.16
7. Nitric oxide release assays
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 solu-
tion in phosphate buffer at pH 7.4, or with 2.4 mL of a 1.0 ꢀ 10ꢁ2 mM
5.7. O2-Acetoxymethyl 1-{2-[4-(1-(4-sulfamoylphenyl)-3-trifluo-
romethyl-1H-pyrazol-5-yl)phenylmethoxycarbonyl]pyrrolidin-
1-yl}diazen-1-ium-1,2-diolate (12a)
solution in phosphate buffer at pH 7.4 to which 90 lL rat serum had
beenadded, was determinedby quantification of nitrite producedby
the reaction of nitric oxide with oxygen and water using the Griess
reaction. Nitric oxide release data were acquired for test compounds
(12a–b) using the reported procedures.17
A solution of compound 11 (0.20 g, 0.8 mmol) in DMSO (1 mL)
and triethylamine (0.1 mL, 0.8 mmol) was stirred at 25 °C for
5 min. A solution of compound 9a (0.368 g, 0.8 mmol) in DMSO
(1 mL) was added and the reaction was allowed to proceed for
24 h at 25 °C with stirring. Ethyl acetate (50 mL) was added to di-
lute the reaction mixture, the organic phase was washed with
water (5ꢀ 15 mL), dried (MgSO4), and the solvent was removed
in vacuo. The residue was purified by silica gel column chromatog-
raphy using EtOAc/hexane (1:1, v/v) as eluent to give 12a as a
8. In vivo anti-inflammatory assay
The test compounds 10a–b, 12a–b, and the reference drugs
celecoxib, ibuprofen and aspirin were evaluated using the in vivo
carrageenan-induced foot paw edema model reported
previously.18
white powder (0.226 g, 45%): mp 74–76 °C; ½a D21:0
ꢂ
+35.1 (1.0000,
CHCl3); IR (film) 3377, 3260 (NH2), 2963 (C–H aromatic), 2913
(C–H aliphatic), 1749 (CO2), 1344, 1164 (SO2), 1273, 1135 (N@N–
O) cm1; 1H NMR (CDCl3) d 2.06ꢁ2.13 (m, 3H, pyrrolidin-1-yl H-3,
H-4, H0-4), 2.12 (s, 3H, COCH3), 2.32ꢁ2.39 (m, 1H, pyrrolidin-1-yl
H0-3), 3.68ꢁ3.77 (m, 1H, pyrrolidin-1-yl H-5), 3.83ꢁ3.91 (m, 1H,
pyrrolidin-1-yl H0-5), 4.62 (dd, J = 8.5, 3.8 Hz, 1H, pyrrolidin-1-yl
H-2), 5.15 (d, J = 12.9 Hz, 1H, –CHH0OCO), 5.22 (br s, 2H, NH2, ex-
changes with D2O), 5.29 (d, J = 12.9, 1H, CHH0OCO), 5.68 (s, 2H,
OCH2O), 6.80 (s, 1H, pyrazole H-4), 7.22 (d, J = 8.0 Hz, 2H, benzyl
H-3, H-5), 7.36 (d, J = 8.0 Hz, 2H, benzyl H-2, H-6), 7.40 (d,
J = 6.8 Hz, 2H, sulfamoylphenyl H-2, H-6), 7.94 (d, J = 6.8 Hz, 2H,
sulfamoylphenyl H-3, H-5); MS 627.15 (M+1); 13C NMR (CDCl3) d
20.8, 22.4, 27.8, 50.8, 61.6, 66.2, 87.1, 106.4, 120.9, 125.4, 127.5,
128.4, 128.6, 129.1, 137.0, 141.9, 143.5, 144.3, 144.5, 169.8,
170.9. Anal. Calcd for C25H25F3N6O8Sꢃ1/2H2O: C, 47.25; H, 4.12;
N, 13.12. Found: C, 47.87; H, 4.96; N, 12.54.
Acknowledgements
We are grateful to the Canadian Institutes of Health Research
(CIHR) (MOP-14712) for financial support of this research.
References and notes
1. Thomsen, R. W.; Riis, A.; Munk, E. M.; Norgaard, M.; Christensen, S.; Sorensen,
H. T. Am. J. Gastroenterol. 2006, 101, 2704.
2. Hinz, B.; Brune, K. J. Pharmacol. Exp. Ther. 2002, 300, 367.
3. Patel, H. H.; Gross, G. J. J. Mol. Cell. Cardiol. 2002, 34, 1.
4. Mukherjee, D. Biochem. Pharmacol. 2002, 63, 817.
5. Scheen, A. J. Rev. Med. Liege 2004, 59, 565.
6. Dogné, J.-M.; Supuran, C. T.; Pratico, D. J. Med. Chem. 2005, 48, 2251.
7. Butler, A. R.; Williams, D. L. H. Chem. Soc. Rev. 1993, 22, 233.
8. Velázquez, C. A.; Chen, Q.-H.; Citro, M. L.; Keefer, L. K.; Knaus, E. E. J. Med. Chem.
2008, 51, 1954.
9. 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.
5.8. O2-Acetoxymethyl 1-{2-[4-(1-(4-methanesulfonylphenyl)-
3-trifluoromethyl-1H-pyrazol-5-yl)phenylmethoxycarbonyl]-
pyrrolidin-1-yl}diazen-1-ium-1,2-diolate (12b)
10. Ahlström, M. M.; Ridderström, M.; Zamora, I.; Luthman, K. J. Med. Chem. 2007,
50, 4444.
The title compound 12b was synthesized using the same proce-
dure described for the preparation of 12a, except that 9b was used
in place of 9a. Product 12b was obtained in 45% yield as a yellow
11. Borne, R.; Levi, M.; Wilson, N., 6th ed.. In Foye’s Principles of Medicinal
Chemistry; Lemke, T. L., Williams, D. A., Roche, V. F., Zito, S. W., Eds.; Lippincott
Williams and Wilkins: Philadelphia, 2008; p 987 (Chapter 36).
12. Saavedra, J. E.; Dunams, T. M.; Flippen-Anderson, J. L.; Keefer, L. K. J. Org. Chem.
1992, 57, 6134.
13. Hrabie, J. A.; Klose, J. R. J. Org. Chem. 1993, 58, 1472.
14. Soliman, R. J. Med. Chem. 1979, 22, 321.
15. 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.
16. Rao, P. N. P.; Amini, M.; Li, H.; Habeeb, A.; Knaus, E. E. J. Med. Chem. 2003, 46,
4872.
17. Velázquez, C.; Vo, D.; Knaus, E. E. Drug Dev. Res. 2003, 60, 204.
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544.
solid: mp 66–68 °C; ½a 2D1:0
ꢂ
+1.74 (1.0000, CHCl3); IR (film) 2962
(C–H aromatic), 2927 (C–H aliphatic), 1755 (CO2), 1344, 1159
(SO2), 1238, 1102 (N@N–O) cm1; 1H NMR (CDCl3) d 2.07ꢁ2.16
(m, 3H, pyrrolidin-1-yl H-3, H-4, H0-4), 2.14 (s, 3H, COCH3),
2.31ꢁ2.42 (m, 1H, pyrrolidin-1-yl H0-3), 3.09 (s, 3H, SO2CH3),
3.73ꢁ3.79 (m, 1H, pyrrolidin-1-yl H-5), 3.89ꢁ3.95 (m, 1H, pyrroli-
din-1-yl H0-5), 4.64 (dd, J = 9.1, 3.8 Hz, 1H, pyrrolidin-1-yl H-2),
5.17 (d, J = 12.8 Hz, 1H, –CHH0OCO), 5.28 (d, J = 12.8, 1H, CHH0OCO),
5.71 (d, J = 7.4 Hz, 1H, –OCHH0O–), 5.74 (d, J = 7.4, 1H, –OCHH0O–),