New celecoxib derivatives as anti-inflammatory agents

Article abstract of DOI:10.1021/jm070821f

A series of 1,5-diarylpyrazoles with a substituted benzenesulfonamide moiety was synthesized and evaluated for cyclooxygenase (COX-1/COX-2) inhibitory activities. Some compounds, for example, (±)-2-[4-(5-p-tolyl-3- trifluoromethyl-pyrazole-1-yl)-benzenesulfonylaminooxy]-propionic acid 16 and its disodium salt 21, had a higher in vivo anti-inflammatory activity compared to celecoxib, despite having no in vitro COX-1 or COX-2 inhibitory activity. Their gastrointestinal side effect profile is essentially more favorable than that of celecoxib.

Full text of DOI:10.1021/jm070821f

142
J. Med. Chem. 2008, 51, 142–147
New Celecoxib Derivatives as Anti-Inflammatory Agents
György Szabó,*,† János Fischer,† Ágnes Kis-Varga,‡ and Klára Gyires§
Medicinal Chemistry Research Laboratory and Department of Pharmacology, Gedeon Richter Plc,
Budapest, Post Office Box 27, H-1475, Hungary, and Department of Pharmacology and Pharmacotherapy, Faculty of Medicine, Semmelweis
UniVersity, Budapest, NagyVárad tér 4, H-1089, Hungary
ReceiVed July 9, 2007
A series of 1,5-diarylpyrazoles with a substituted benzenesulfonamide moiety was synthesized and evaluated
for cyclooxygenase (COX-1/COX-2) inhibitory activities. Some compounds, for example, (()-2-[4-(5-p-
tolyl-3-trifluoromethyl-pyrazole-1-yl)-benzenesulfonylaminooxy]-propionic acid 16 and its disodium salt 21,
had a higher in vivo anti-inflammatory activity compared to celecoxib, despite having no in vitro COX-1 or
COX-2 inhibitory activity. Their gastrointestinal side effect profile is essentially more favorable than that of
celecoxib.
Introduction
The discovery that a key cyclooxygenase enzyme in arachi-
donic acid metabolism exists in two isoforms, namely, COX-
1a and COX-2, the constitutive and inducible forms, has
generated new avenues for drug design. COX-1 is present in
the stomach, intestines, kidneys, and platelets, while COX-2 is
expressed during inflammation1–3 and is also constitutively
expressed in brain, spinal cord,4 kidneys, and pancreatic cellate
cells (PCCs).5 The traditional nonsteroid anti-inflammatory
agents (NSAIDs) inhibit both cyclooxygenase enzymes and
hence downregulate prostaglandin formation in almost all cells
and tissues. This broad inhibitory profile accounts for their anti-
inflammatory activity as well as their pronounced side effects.
It was proposed that a selective inhibitor of COX-2 would be
an attractive approach to the treatment of inflammatory condi-
tions, without concomitant gastric and renal toxicity. Celecoxib
16 and other coxibs, such as rofecoxib 2,7 etoricoxib 3,8 and
Figure 1. COX-2 inhibitors.
valdecoxib 49 (Figure 1), are selective COX-2 inhibitors with
fewer gastrointestinal side effects to traditional NSAIDs.
of Analogue Based Drug Discovery (ABDD)14 to obtain more
effective celecoxib derivatives with a better gastrointestinal side-
effect profile.
However, the long-term use of both traditional NSAIDs and
coxibs have been reported to cause significant cardiovascular
side effects.10 Celecoxib has advantages in this respect, because
it is not associated with an increased incidence of cardiovascular
events compared with placebo and with nonselective NSAIDs.11
There are, however, some characteristics of celecoxib that could
be improved. For example, Celecoxib is not effective in all
patients and has some gastrointestinal side effects. Following
the successful introduction of celecoxib and rofecoxib, subse-
quent coxib research has focused on more selective COX-2
analogues. However, the COX-1/COX-2 selectivities of cele-
coxib and rofecoxib were different, and the VIGOR study12
raised the question of its cardiovascular side effects. The possible
reasons of this phenomenon are described in the study of D.
Mukherjee.13 Celecoxib has a lower COX1/COX2 selectivity,
therefore, we focused our research work on its derivatives.
Accordingly, our aim was to synthesize analogues by the method
In our previous work, we prepared an N-hydroxy derivative
of valdecoxib15 (compound 6) that showed marginal in vitro
activity (Table 1), but nevertheless, was more effective than
the parent compound in various animal models of acute and
chronic inflammation.16 Following a similar approach, we
synthesized the N-hydroxy analogue of celecoxib (compound
12; Figure 2), but it proved to be an unstable molecule. In
contrast to 6, which could be isolated as a stable monohydrate
following the elimination of traces of free radicals by recrys-
tallization from aqueous ethanol containing L-ascorbic acid, the
same method was not successful with compound 12. In this
report, we discuss our attempt to prepare stable derivatives of
12. One of its derivatives, 16, and its disodium salt, 21, were
identified as potential analgesic and anti-inflammatory candidates
for oral as well as intravenous administration.17
Synthesis. The key intermediate, 11, required for the synthesis
of compounds 12-22 was prepared according to the procedure
outlined in Scheme 1. Claisen condensation of the commercially
available p-methyl-acetophenone 7 and trifluoroacetic acid ethyl
ester gave diketone 8 in good yield.6 Compound 8 was then
reacted with the commercially available p-hydrazino-benzene-
sulfonic acid 9 in refluxing ethanol to give sulfonic acid
derivative 10 (yield over 80%). This reaction is a regioselective
* To whom correspondence should be addressed. Tel.: +36-1-431-5180.
Fax: +36-1-432-6498. E-mail: gy.szabo@richter.hu.
†
Medicinal Chemistry Research Laboratory, Gedeon Richter Plc.
‡
Department of Pharmacology, Gedeon Richter Plc.
Semmelweis University.
§
aAbbreviations: COX, cyclooxygenase; PCCs, pancreatic cellate cells;
NSAIDs, nonsteroid anti-inflammatory drugs; ABDD, analogue-based drug
discovery; VIGOR, Vioxx Gastrointestinal Outcome Research.
10.1021/jm070821f CCC: $40.75
2008 American Chemical Society
Published on Web 12/12/2007

Celecoxib DeriVatiVes as Anti-Inflammatory Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 143
Scheme 2. Synthesis of the Target Compoundsa
Table 1. Effect of valdecoxib and 6 on human recombinant COX-2 and
ovine COX-1 in Vitro
IC50 ( S.E.M., µM
human
recombinant COX-2
ovine
COX-1
COX-1/COX-2
selectivity
cmpd
4, valdecoxib
6
0.04 ( 0.02
1.1 ( 0.2
157.2
96.2 ( 10.2
3930
88
transformation and the 1,5-diarylpyrazole could be generated
almost exclusively by carrying out the condensation in the
presence of the hydrochloride salt of the phenyl-hydrazine. The
intermediate 10 was converted into its sulfonyl chloride 11 with
phosphorus pentachloride in dry dichloromethane, and the
product could be obtained in high purity after recrystallization
from cyclohexane. The general method employed for the
preparation of the aminooxy-carboxylic acid derivatives 12–18
and its sodium salts 19–22 is illustrated in Scheme 2. Reaction
of 11 with hydroxylamine hydrochloride derivatives in a mixture
of dioxane and water at room temperature provided the
aminooxy derivatives 12–18. Their respective disodium salts
20–22 were prepared in 80–88% yield using 2 equiv of aqueous
NaOH solution in ethanol. From the acetic acid derivative 13,
only the monosodium salt 19 could be isolated in stable
crystalline form using 1 equiv aqueous NaOH solution in
ethanol. As the aminooxy-carboxylic acid moiety of 16 contains
a stereogenic center, the prepared target compound 16 is a
racemate. To investigate the in vivo activity of the pure
enantiomers, the racemic compound was resolved by crystal-
lization using optically active ephedrine in ethanol to furnish
two optically pure compounds 23 and 24 (Scheme 3).
a Reagents and conditions: (a) RONH2 ·HCl, dioxan/H2O, rt, 76-92%;
(b) 2 equiv aq NaOH, ethanol, rt, 80-88%; (c) 1 equiv aq NaOH, ethanol,
rt, 90%.
Scheme 3. Resolvation of 16a
a Reagents and conditions: (a) active ephedrine, ethanol, rt, 60%; (b) 1
N HCl, 60%.
Table 2. Anti-Inflammatory Activity of Compounds at 10 mg/kg p.o.
Dose in Carrageenan-Induced Edema Model in Rats (n ) 6–12
Animals/Group)
Biological Results and Discussion. As an initial screen for
activity, 13–22 were evaluated against human recombinant
COX-1 and COX-2 enzymes,18 but despite exhibiting neither
COX-1 nor COX-2 inhibition at 10 µM concentrations, they
were evaluated for in vivo anti-inflammatory activity at a 10
mg/kg single dose using the carrageenan-induced paw edema
method in rats. Table 2 shows the anti-inflammatory activities
of 13–24 compared to celecoxib as a reference compound in
% inhibition
4 h post dose,
mean ( SEM
% inhibition
6 h post dose,
mean ( SEM
% inhibition
7 h post dose,
mean ( SEM
cmpd
1, celecoxib
24.2 ( 4.3
13.1 ( 7.1
13.3 ( 6.9
23.6 ( 4.3
41.0 ( 6.1**
23.5 ( 4.2*
26.0 ( 5.5*
32.0 ( 3.5**
38.4 ( 3.9**
18.4 ( 4.2
0.9 ( 4.2
18.6 ( 2.3
21.3 ( 5.1
23.1 ( 7.2
12.8 ( 3.6
31.1 ( 4.5*
15.7 ( 3.1
33.2 ( 4.1**
33.9 ( 3.7**
21.8 ( 7.0
15.3 ( 4.3
-9.3 ( 2.8
12.3 ( 4.3
17.1 ( 5.4
20.6 ( 5.9
11.9 ( 3.9
17.9 ( 5.2
7.3 ( 1.9
13
14
15
16
17
19
20
21
23
24
24.7 ( 3.5*
24.9 ( 3.2*
16.2 ( 6.2
6.7 ( 3.1
-6.3 ( 2.4
* p < 0.05. ** p < 0.01 vs control (5% Tween-80% phys. saline),
Tukey’s multiple comparison test.
this model. The aminooxy-acetic acid 13 and its ester derivative
14 showed a weak in vivo activity, while the propionic acid
derivatives (15 and 16) and their disodium salts (20 and 21)
exhibited impressive activity in this model. The best compound,
16, was further tested at 3, 10, and 30 mg/kg in 6-12 animals
per group to calculate the ED30. These results are depicted in
Figure 3. Celecoxib was effective at 4 h after treatment with an
ED30 of 23 mg/kg (0.06 mmol/kg), but its activity practically
disappeared after 6 h. Compound 16 was potent at 4 and 6 h
after treatment with an ED30 of 5.7 mg/kg (0.012 mmol/kg) and
ED30 of 8.4 mg/kg (0.018 mmol/kg), respectively. These results
show that 16 significantly inhibited paw edema at both
investigated time points and exhibited a 4-fold better anti-
inflammatory potency (ED30, 5.7 mg/kg, 0.012 mmol/kg) than
the parent compound celecoxib (ED30, 23 mg/kg, 0.06 mmol/
kg) in this model. Surprisingly, we found that the pure
enantiomers (23 and 24) of 16 were each less active than the
Figure 2. Structure of N-hydroxy-valdecoxib monohydrate and N-
hydroxy-celecoxib.
Scheme 1. Synthesis of the Key Intermediatea
a Reagents and conditions: (a) CF3COOEt, NaOMe/MeOH, reflux, 95%;
(b) EtOH/HCl, reflux, 82%; (c) PCl5, CH2Cl2, reflux, 74%.

144 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Szabó et al.
Table 4. Effect of Celecoxib 1 and 16 on Gastric Mucosal Damage
Induced by Ethanol in Rats (n ) 10)
cmpd
dose, mg/kg p.o.
ulcer index
inhibition%
methylcellulose
1
16
methylcellulose
16
60.8 ( 4.1
49.4 ( 4.5
35.0 ( 4.4*
66.2 ( 1.7
43.0 ( 4.4
15.8 ( 2.8*
97.8 ( 2.8
80.6 ( 2.3
44.4 ( 6.4*
30
30
19
43
15
60
35
76
methylcellulose
1
15
60
18
55
* p < 0.05.
Table 5. Effect of Indomethacin and Celecoxib 1 and 16 on the Healing
of Acetic Acid Induced Chronic Gastric Ulcer in Rats
dose,
change of bodyweight, g
area of
cmpd
mg/kg p.o. (+, increase; -, decrease) ulcer (mm2)
methylcellulose
indomethacin
1
16
+58 ( 3
+2 ( 0.6
+32 ( 4
+43 ( 5
3.0 ( 0,8
39.0 ( 5**
8.2 ( 2.0*
2.4 ( 0.7
2.0
30
30
* p < 0.05. ** p < 0.01.
kg p.o. dose and, thus, a significantly better anti-inflammatory/
analgesic effect compared to celecoxib. In the chronic model,
16 showed 54% reversal, which is comparable with the analgesic
effect of celecoxib. Additionally, 16 was investigated in different
in vivo rat ulcer models to compare its gastrointestinal side
effects relative to celecoxib. In the acid-independent ulcer model
(ethanol induced gastric mucosal damage), acidified ethanol was
used to induce deep hemorrhagic bends on the surface of gastric
mucosa. The ulcer indexes were 60.8, 66.2, and 97.8. Celecoxib
was ineffective in the dose range of 15–30 mg/kg, while at 60
mg/kg, it induced 55% inhibition (p < 0.05). Pretreatment with
16 resulted in 35, 43, and 75% inhibition of mucosal lesions at
doses of 15, 30, and 60 mg/kg (Table 4). Thus, 16 was
significantly more effective than celecoxib in its gastroprotective
effects in this model. In the model of acetic acid induced gastric
mucosal damage, treatment was started 5 days after the
subserosal application of acetic acid. In the control (methyl-
cellulose-treated) group, the ulcerated area was 3 mm3. In-
domethacin increased the ulcerated area by a factor of 10 times
(39 mm2). Celecoxib also increased the size of the ulcer (8.2
mm2). In contrast, 16 reduced the ulcerated area (2.4 mm2; Table
5). In conclusion, healing of the acetic acid induced ulcer, which
is analogous to the human ulcer healing, was delayed signifi-
cantly by celecoxib, while 16 did not influence it.
Figure 3. Anti-inflammatory activity of celecoxib 1 and 16 at 3–30
mg/kg p.o. dose range in carrageenan-induced edema model in rats (n
) 6–12 animals/group).
Table 3. The Analgesic Activity of Celecoxib 1 and 16 in
Carrageenan-Induced Mechanical Hyperalgesia Test in Rats
reversal%
cmpd
dose, mg/kg p.o.
30 min
60 min
120 min
180 min
Acute Model
1
10
30
10
30
0
24
24
0
3
30
0
0
15
35
16
89**
59*
Chronic Model
Conclusion
1
10
30
10
30
24
54*
23
17
34
22
0
34
28
34
0
26
20
19
In this report, we have described the design and synthesis of
new celecoxib derivatives substituted at the benzenesulfonamide
moiety. Although our intention was to prepare stable derivatives
of 12, it is not impossible that these derivatives act as prodrugs.
The investigation of this issue is complicated by the unstable
nature of 12. Summarizing the biological results, it can be seen
that the pharmacological efficacy of 16 in vivo is greater and
of longer duration compared to celecoxib. In the carrageenan-
induced edema test, the anti-inflammatory activity of 16 was
significantly higher than that of celecoxib. In the inflammation-
induced chronic hyperalgesia model, 16 showed significant
analgesic effect and its duration of action was several hours
longer than that of the reference compound. In comparing the
gastrointestinal effects, 16 showed a significantly more favorable
side effect profile than celecoxib. Thus, 16 and its water soluble
16
42
54*
* p < 0.05. ** p < 0.01 vs control (5% Tween-80% phys. saline),
Tukey’s multiple comparison test.
racemate, which can be the result of a synergistic effect. The
investigation with 16 was extended to the carrageenan-induced
acute and chronic hyperalgesia model (Randall-Selitto model)19,20
to monitor its capacity to reverse inflammatory hyperalgesia.
The data are reported in Table 3 compared to those of the
reference compound celecoxib. In both models (acute and
chronic treatment), celecoxib at a 30 mg/kg p.o. dose showed
a partial analgesic effect (24 and 54% reversal). In the acute
model, 16 showed almost complete reversal (90%) at a 30 mg/

Celecoxib DeriVatiVes as Anti-Inflammatory Agents
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1 145
described for 12 and was isolated as a white semisolid. Yield 88%;
mp 224–225 °C (ethanol/water); H NMR (300 MHz, DMSO-d6)
2.32 (s, 3H), 4.44 (s, 2H), 7.19–7.21 (m, 1H), 7.19–7.24 (m, 4H),
7.58–7.64 (m, 2H), 7.91–7.97 (m, 2H), 10.98 (br, 1H), 12.99 (br,
1H); HRMS calcd for C19H16O5N3F3S, 455.107559; found,
455.07573.
disodium salt 21 have been identified as potential analgesic and
anti-inflammatory agents for both oral and parenteral administra-
tion.
1
Experimental Section
[4-(5-p-Methylphenyl-3-trifluormethyl-pyrazole-1-yl)-benze-
nesulfonylaminooxy]-acetic Acid Ethyl Ester (14). Compound
14 was obtained from 11 and aminooxy-acetic acid ethyl ester
according to the procedure described for 12 and was isolated as a
Chemistry. Research chemicals were either purchased from
Aldrich Co. or Fluka and used without further purification in the
reactions or were prepared according to procedures described in
the literature. Reactions were monitored by thin-layer chromatog-
raphy (TLC) on silica gel plates (60 F254; Merck) visualizing with
ultraviolet light or iodine. Column chromatography was performed
on Silica Gel 60 (0.043–0.060 mm), Merck. The main reference
compound, Celecoxib was prepared according to the literature
procedure.6 The yields of the products reported here are unopti-
mized. Melting points were determined with a Büchi 535 apparatus
1
white semisolid. Yield 82%; mp 143–144 °C (ethanol); H NMR
(500 MHz, DMSO-d6) 1.22 (t, 3H), 2.32 (s, 3H), 4.16 (q, 2H),
4.53 (s, 2H), 7.19–7.21 (m, 1H), 7.19–7.24 (m, 4H), 7.58–7.64 (m,
2H), 7.91–7.97 (m, 2H), 11.00 (s, 1H); HRMS calcd for
C21H20O5N3F3S, 483.10726; found, 483.10703.
3-[4-(5-p-Methylphenyl-3-trifluoromethyl-pyrazole-1-yl)-ben-
zenesulfonylaminooxy]-propionic Acid (15). Compound 15 was
obtained from 11 and aminooxy-propionic acid according to the
procedure described for 12 and was isolated as a white semisolid.
Yield 92%; mp 176–178 °C (ethanol/water); 1H NMR (300 MHz,
DMSO-d6) 2.32 (s, 3H), 2.52 (t, 2H), 4.09 (t, 2H), 7.19–7.21 (m,
1H), 7.19–7.24 (m, 4H), 7.56–7.63 (m, 2H), 7.88–7.93 (m, 2H),
10.60 (br s, 1H), 12.30 (br s, 1H); HRMS calcd for C20H18F3N3O5S,
469.09192; found, 469.0921.
1
and are uncorrected. H NMR spectra were recorded on a Varian
INOVA-500 spectrometer, operating at 500 MHz. Chemical shifts
(δ) are reported in parts per million downfield from tetramethyl-
silane. Mass spectra were scanned on a Finnigan MAT 95SQ
spectrometer.
4,4,4-Trifluoro-1-(4-methyl-phenyl)-butane-1,3-dione (8). In
a solution of 5.7 g (0.25 mol) of sodium in 80 mL of methanol, 22
mL (0.16 mol) of ethyl trifluoroacetate was added, and afterward
over 30 min, 21 g (0.15 mol) of 4-methyl acetophenone 7 dissolved
in 40 mL of methanol was added. The resulting solution was stirred
for 10 h at 80 °C then evaporated to dryness. The sodium salt
obtained was dissolved in 50 mL of water and acidified with 120
mL of 1 N HCl and extracted three times with 80 mL of ethyl
acetate. The organic layer was dried over MgSO4 and evaporated
in vacuo to give 34.6 g of a brown solid, which was taken forward
without further purification. Yield 95%.
4-(5-p-Methylphenyl-3-trifluoromethyl-pyrazole-1-yl)-benze-
nesulfonic Acid (10). A total of 42 g (0.22 mol) of 9 was added to
a stirred solution of 51.4 g (0.223 mol) of 8 in 450 mL of ethanol
and 74 mL (0.446 mol) of 6 N HCl. The mixture was heated to
reflux and stirred for 8 h. After cooling to room temperature, the
reaction mixture was concentrated in vacuo. The residue was taken
up with ethyl acetate and washed with 100 mL of water and 100
mL of brine, dried over MgSO4, filtered, and evaporated in vacuo
to give an oil that was crystallized from 300 mL of diisopropylether
to give 70.12 g of the pyrazole 10. Yield 82%; mp 258 °C
(decomposition).
4-(5-p-Methylphenyl-3-trifluoromethyl-pyrazole-1-yl)-benze-
nesulfonyl Chloride (11). To a suspension of 43 g (0.11 mol) of
10 in 250 mL of dichloromethane was added 34.2 g (0.168 mol)
of phosphorus pentachloride in portions. To the resulting solution
was added 10 mL of DMF. After stirring at reflux for 8 h, the
mixture was concentrated in vacuo. To the residue was added water
and twice extracted with ethyl acetate, dried over MgSO4, and
evaporated to dryness. The residue could be crystallized from
cyclohexane to yield 34.2 g of product as a white solid. Yield 77%;
mp 97–98 °C (cyclohexane).
N-Hydroxy-4-(5-p-methylphenyl-3-trifluoromethyl-pyrazole-
1-yl)-benzenesulfonamide (12). A total of 1.0 g (15 mmol) of
hydroxylamine hydrochloride was suspended in 10 mL of dioxane
and treated dropwise with a solution of 1.2 g (15 mmol) of sodium
acetate in 5 mL of water. To this solution was added dropwise
over 10 min a solution of 1.5 g (3.7 mmol) of 11 in 20 mL of
CH2Cl2 and allowed to stir for 1 h, which was then concentrated
in vacuo. To the residue was added water and extracted twice with
ethyl acetate. The combined organic layers were washed three times
with water, dried over MgSO4, and evaporated in vacuo to give an
oil, which could be crystallized from ethanol (75%) to give the
pyrazole as a white solid. Yield 76%; mp 203 °C (ethanol/water);
1H NMR (300 MHz, DMSO-d6) 2.32 (s, 3H), 7.18–7.26 (m, 5H),
7.56–7.62 (m, 2H), 7.86–7.92 (m, 2H), 9.71 (d, 1H), 9.74 (d, 1H);
HRMS calcd for C17H14O3N3F3S, 397.07002; found, 397.07025.
[4-(5-p-Methylphenyl-3-trifluormethyl-pyrazole-1-yl)-benze-
nesulfonylaminooxy]-acetic Acid (13). Compound 13 was obtained
from 11 and aminooxy-acetic acid according to the procedure
(()-2-[4-(5-p-Methylphenyl-3-trifluoromethyl-pyrazole-1-yl)-
benzenesulfonylaminooxy]-propionic Acid (16). Compound 16
was obtained from 11 and 2-aminooxy-propionic acid according
to the procedure described for 12 and was isolated as a white
1
semisolid. Yield 85%; mp 187–189 °C (toluene); H NMR (300
MHz, DMSO-d6) δ [ppm] 1.27 (d, 3H), 2.31 (s, 3H), 4.47 (q, 1H),
7.18 (s, 1H), 7.19–7.24 (m, 4H), 7.59– 7.64 (m, 2H), 7.92–7.96
(m, 2H), 10.80 (br s, 1H, NH), 12.94 (br s, 1H, COOH); HRMS
calcd for C20H18O5N3F3S, 469.09063; found, 469.09138.
(()-2-[4-(5-p-Methylphenyl-3-trifluoromethyl-pyrazole-1-yl)-
benzenesulfonylaminooxy]-propionic Acid Ethyl Ester (17).
Compound 17 was obtained from 11 and 2-aminooxy-propionic
acid ethyl ester according to the procedure described for 12 and
was isolated as a white semisolid. Yield 82%; mp 137–138 °C
(cyclohexane); 1H NMR (500 MHz, CDCl3) 1.29 (t, 3H), 1.38 (d,
3H), 2.39 (s, 3H), 4.21 (q, 2H), 4.63 (q, 1H), 6.74 (s, 1H), 7.10–7.13
(m, 2H), 7.17–7.21 (m, 2H), 7.49–7.54 (m, 2H), 7.88–7.92 (m, 2H);
HRMS calcd for C22H22O5N3F3S, 497.12366; found, 497.12268.
2-Methyl-2-[4-(5-p-methylphenyl-3-trifluoromethyl-pyrazole-
1-yl)-benzenesulfonylaminooxy]-propionic Acid (18). Compound
18 was obtained from 11 and 2-aminooxy-2-methyl-propionic acid
according to the procedure described for 12 and was isolated as a
white semisolid. Yield 80%; mp: 94–96 °C (ethanol); 1H NMR
(400 MHz, DMSO-d6) 1.36 (s, 6H), 2.32 (s, 3H), 7.18–7.25 (m,
5H), 7.57–7.63 (m, 2H), 7.90–7.96 (m, 2H), 10.26 (br s, 1H), 12.89
(br s, 1H); HRMS calcd for C21H20F3N3O5S, 483.10737; found,
483.10694.
[4-(5-p-Methylphenyl-3-trifluoromethyl-pyrazole-1-yl)-benze-
nesulfonylaminooxy]-acetic Acid Sodium Salt Trihydrate (19).
To a solution of 0.40 g (0.88 mmol) of 13 in 7 mL of ethanol was
added 0.44 mL (0.88 mmol) of 2 N NaOH, and the mixture was
stirred for 2 h at 0 °C. The precipitated product was collected by
filtration to give a white solid. Yield 90%; mp > 300 °C; 1H NMR
(400 MHz, DMSO-d6) 2.32 (s, 3H), 3.93 (s, 2H), 7.18–7.26 (m,
5H), 7.52–7.58 (m, 2H), 7.89–7.95 (m, 2H); C19H15F3N3
NaO5S·3H2O MW, 531.44.
3-[4-(5-p-Methylphenyl-3-trifluoromethyl-pyrazole-1-yl)-ben-
zenesulfonylaminooxy]-propionic Acid Disodium Salt Mono-
hydrate (20). To a solution of 1.4 g (3 mmol) of 15 in 20 mL of
ethanol was added 1.9 mL (7.5 mmol) of 3.9 N NaOH, and the
mixture was stirred for 2 h at 0 °C. The precipitated product was
collected by filtration to give a white solid. Yield 87%; mp > 300
°C; 1H NMR (400 MHz, DMSO-d6) 1.98 (t, 2H), 2.30 (s, 3H),
3.51 (t, 2H), 7.12–7.14 (m, 1H), 7.15–7.23 (m, 4H), 7.24–7.30 (m,
2H), 7.66–7.71 (m, 2H); C20H18F3N3Na2O6S MW, 531.41.
(()2-[4-(5-p-Methylphenyl-3-trifluoromethyl-pyrazole-1-yl)-
benzenesulfonylaminooxy]-propionic Acid Disodium Salt Mono-

146 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 1
Szabó et al.
hydrate (21). Compound 21 was obtained from 16 according to
the procedure described for 20 and was isolated as a white solid.
Yield 88%; mp > 300 °C; 1H NMR (300 MHz, DMSO-d6) δ [ppm]
0.97 (d, 3H), 2.28 (s, 3H), 3.58 (q, 1H), 7.12 (s, 1H), 7.12–7.21
(m, 4H), 7.26–7.32 (m, 2H), 7.76–7.83 (m, 2H); C20H18F3N3Na2O6S
MW, 532.42.
magnitude and duration of the hyperalgesia. The maximum reduc-
tion in threshold was measured 2–3 h after injection (the pain
threshold of the inflammed paw is 20–30 g, which was decreased
by 65–80% in comparing to the basic value).
Measurement of Gastrointestinal Side Effects. Animals.
Experiments were performed in male Wistar rats weighing 140–160
g. They were housed in wire mesh bottom cages to prevent
coprophagy. The rats were kept on a 12 h light-dark cycle and
under condition of controlled temperature.
Gastric Mucosal Damage Induced by Acidified Ethanol. After
24 h food deprivation, the animals were given orally 0.5 mL of
acidified ethanol (98% ethanol in 200 mmol/l HCl). One hour later,
the animals were killed by overdose of ether and the stomachs were
excised, opened along the greater curvature, rinsed with saline, and
examined for lesions. Total number of mucosal lesions was assessed
in a blinded manner by calculation of lesion index based on a 0–4
scoring system described previously.21 The lesion index was
calculated as the total number of lesions multiplied by the respective
severity factor. The percentual inhibition of mucosal damage was
calculated as follows 100 - [lesion index in treated group lesion
index in control group] × 100.
2-Methyl-2-[4-(5-p-methylphenyl-3-trifluoromethyl-pyrazole-
1-yl)-benzenesulfonylaminooxy]-propionic Acid Disodium Salt
Monohydrate (22). Compound 22 was obtained from 18 according
to the procedure described for 20 and was isolated as a white solid.
1
Yield 80%; mp > 300 °C; H NMR (300 MHz, DMSO-d6) 0.90
(s, 6H), 2.29 (s, 3H), 7.10–7.17 (m, 5H), 7.26–7.32 (m, 2H),
7.68–7.73 (m, 2H); C21H20F3N3Na2O6S MW, 545.42.
(-)-2-[4-(5-p-Methylphenyl-3-trifluoromethyl-pyrazol-1-yl)-
benzenesulfonylaminooxy]-propionic Acid (23). To a stirred
solution of 10 g (21 mmol) of 16 in 50 mL of 97% ethanol was
added 1.76 g (10 mmol) of (-)-ephedrine. The mixture was stirred
at room temperature for 1 day, and the resulting crystals were
filtered off and washed with 97% ethanol to yield 3.38 g (25%) of
product, calculated on the basis of the starting racemic mixture.
The so-obtained crystals were recrystallized twice from 97% ethanol
to yield 2.02 g (60%) of white crystalline material. The diastereomer
salt was suspended in 30 mL of ethyl acetate, and 15 mL of 1 N
hydrochloric acid was added. The organic layer was separated, dried
over MgSO4, filtered, and concentrated in vacuo. The obtained oil
was crystallized from petroleum ether to yield 0.9 g (60%) of the
title compound. Mp 158–160 °C; [R]20D ) -505.3° (c 1, methanol).
(+)-2-[4-(5-p-Methylphenyl-3-trifluoromethyl-pyrazol-1-yl)-
benzenesulfonylaminooxy]-propionic Acid (24). Compound 24
was obtained from 16 according to the procedure described for 23
using (+)-ephedrine as resolving agent and isopropanol as solvent.
Yield 0.62 g, mp 161–163 °C; [R]20D ) +457.6° (c 1, methanol).
Biological Methods. In Vitro Screening. Spectrophotomet-
ric Assay with Human Recombinant COX-2 and Ovine COX-
1. Enzymatic activities of the human recombinant COX-2 and ovine
COX-1 were measured using a spectrophotometric assay based on
the oxidation of TMPD during the reduction of PGG2 to PGH2.
In Vivo Screening. Carrageenan-Induced Edema in Rats.
Edema was induced by subcutaneous injection of 50 µL of 1%
carrageenan (CARR) suspension in the subplantar region of the
right hind paw of male Wistar rats (140–150 g). The injected
carrageenan induced paw inflammation. The edema, that is, the
difference between the pre- and post-treatment volume (in mL) of
the injected hind paw was measured using a water displacement
Plethysmometer (Ugo Basile, type 7150). The treated paw was
immersed up to the tibio-tarsal articulation into the chamber, and
the volume of displaced liquid was determined as the degree
of the inflammation: Degree of inflammation (mL) ) volume after
the CARR treatment (mL) - volume before the CARR treatment
(mL).
Chronic Gastric Ulcer Induced by Acetic AcidsStudy on
Healing Process. The method of Okabe22 was applied. Male Wistar
rats (150–170 g) were used. Laparotomy by a midline, epigastric
incision was made in rats under ether anesthesia. After exteriorizing
the stomach, 0.05 mL/rat of 20% acetic acid was injected into the
submucosal layer of the anterior wall of the glandular stomach.
Care was taken not to puncture any of the superficial, large blood
vessels. After injection, pressure (by hand) was applied over the
inserted needle to prevent leakage of the acetic acid via the puncture
wound and was maintained momentarily after needle withdrawal.
The site of the injection was readily distinguished by the localized
swelling and blanching of the serosa at the injection site. After
surgery, the abdomen was closed and all rats were maintained
conventionally on laboratory chow and water ad libitum. Animals
were sacrificed 12 days later. Then the stomach was opened along
the greater curvature and examined grossly for lesions. Because
ulcers produced by the acetic acid are round or oval, the length
and width were measured, and an ulcer index based upon the
product of length and width was used. The test compounds were
given from the fifth day after the operation for 7 days. A total of
24 hours after the last dose, the animals were sacrificed and the
ulcer index was determined.
Supporting Information Available: Spectroscopic (1H NMR
and HRMS) data for all synthesized compounds, HPLC purity data
for target compounds 12-24, and analytical HPLC trace for
compound 16. This material is available free of charge via the
Internet at http://pubs.acs.org.
Carrageenan Induced Acute Hyperalgesia in Rats (Randall-
Selitto Model). Edema (inflammation) was induced by subcutane-
ous injection of carrageenan (CARR) suspension in the subplantar
region of the right hind paw of male Wistar rats, weighted 140–190
g (n ) 8–12 animal/group). The nociceptive thresholds of the
inflammed hind paw after painful mechanical stimuli were measured
with an Analgesimeter (Ugo Basile, type 37215, Italy). The
apparatus is suitable for the measuring of the extent and the latency
of the pain reaction threshold of the sensitized paw after painful
stimuli. The analgesics elevate the low pain reaction threshold of
the inflammed paw and the degree of its antinociceptive effect is
expressed in reversal%.
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