Synthesis and cyclooxygenase-2 inhibiting property of 1,5-diarylpyrazoles with substituted benzenesulfonamide moiety as pharmacophore: Preparation of sodium salt for injectable formulation

Article abstract of DOI:10.1021/jm020563g

A series of 1,5-diarylpyrazoles having a substituted benzenesulfonamide moiety as pharmacophore was synthesized and evaluated for cyclooxygenase (COX-1/COX-2) inhibitory activities. Through SAR and molecular modeling, it was found that fluorine substituti

Full text of DOI:10.1021/jm020563g

J . Med. Chem. 2003, 46, 3975-3984
3975
Syn th esis a n d Cyclooxygen a se-2 In h ibitin g P r op er ty of 1,5-Dia r ylp yr a zoles w ith
Su bstitu ted Ben zen esu lfon a m id e Moiety a s P h a r m a cop h or e: P r ep a r a tion of
Sod iu m Sa lt for In jecta ble F or m u la tion †
Manojit Pal,*,‡ Manjula Madan,‡ Srinivas Padakanti,‡ Vijaya R. Pattabiraman,‡ Srinivas Kalleda,‡
Akhila Vanguri,§ Ramesh Mullangi,§ N. V. S. Rao Mamidi,§ Seshagiri R. Casturi,§ Alpeshkumar Malde,‡
B. Gopalakrishnan,‡ and Koteswar R. Yeleswarapu*,‡
Discovery-Chemistry and Discovery-Biology, Dr Reddy’s Laboratories Ltd., Bollaram Road, Miyapur, Hyderabad 500050, India
Received December 23, 2002
A series of 1,5-diarylpyrazoles having a substituted benzenesulfonamide moiety as pharma-
cophore was synthesized and evaluated for cyclooxygenase (COX-1/COX-2) inhibitory activities.
Through SAR and molecular modeling, it was found that fluorine substitution on the
benzenesulfonamide moiety along with an electron-donating group at the 4-position of the 5-aryl
ring yielded selectivity as well as potency for COX-2 inhibition in vitro. Among such compounds
3-fluoro-4-[5-(4-methoxyphenyl)-3-trifluoromethyl-1H-1-pyrazolyl]-1-benzenesulfonamide 3 dis-
played interesting pharmacokinetic properties along with antiinflammatory activity in vivo.
Among the sodium salts tested in vivo, 10, the propionyl analogue of 3, showed excellent
antiinflammatory activity and therefore represents a new lead structure for the development
of injectable COX-2 specific inhibitors.
In tr od u ction
is at the 4-position of the phenyl ring. This phenyl
moiety and a lipophilic moiety are attached to the
adjacent positions of the central ring. The central ring
may consist of a carbocycle, heterocycle, or even acyclic
structure, which may adopt favorable geometry. In
contrast to this, a similar type of structural generaliza-
tion is not feasible for nonselective inhibitors of COX
isoforms, e.g. aspirin, indomethacin.1 After the discovery
of the inducible enzyme COX-2 in 1991, the diarylhet-
erocycle I and the methanesulfonanilide II (Figure 1)
were first identified as nonulcerogenic antiinflammatory
agents.4,5 Subsequently, based on rational drug design,
a wide range of diarylheterocycles have been identified
and developed as selective COX-2 inhibitors. As a result,
celecoxib6a and rofecoxib7 (Figure 1) were the first to
enter the market for the treatment of acute pain,
rheumatoid arthritis, and osteoarthritis. They are also
in clinical trials for the treatment of Alzheimer’s disease
and different types of cancer based on the role of COX-2
in the etiopathology.8,9a
The discovery that the key enzyme cyclooxygenase in
the arachidonic acid metabolism exists in two isoforms,
namely, the constitutive cyclooxygenase-1 (COX-1) and
the inducible cyclooxygenase-2 (COX-2), has generated
new avenues for drug design.1 The isoforms differ in
regulation and expression, depending on the tissue.
COX-1 is constitutively expressed in the kidney and the
gastrointestinal tract and is responsible for the biosyn-
thesis of prostaglandins (PGs) involved in the cytopro-
tection of the gastrointestinal tract and platelet aggre-
gation. Thus, interruption of COX-1 activity may lead
to gastrointestinal toxicity such as ulceration, bleeding,
and perforation.2a In contrast, COX-2 is inducible by
proinflammatory molecules such as 1L-1, TNF-R, LPS,
and carrageenan and plays a major role in the biosyn-
thesis of PGs in inflammatory cells (monocytes and
macrophages). COX-2, however, has also been reported
to be produced in the normal kidney.2b The traditional
NSAIDs inhibit both COX-1 and COX-2 and hence down
regulate the PGs in almost all cells and tissues. This
accounts for their antiinflammatory activity as well as
side effects. However, selective inhibition of COX-2 over
COX-1 is beneficial for the treatment of inflammatory
diseases with reduced ulcerogenic side effects.
Recent studies have shown that more than 70% of
patients who undergo surgery experience moderate to
extreme postoperative pain and it is desirable to control
and inhibit the processes that cause pain and inflam-
mation with nonnarcotic drugs. Injectable analgesics are
favored because they have a rapid and reliable onset of
effect without addiction. Ketorolac (Figure 2), the only
injectable nonsteroidal antiinflammatory drug (NSAID)
approved for use in the United States, is a potent
inhibitor of both COX-1 and COX-2. Therefore, its use
is associated with unwanted side effects such as gas-
trointestinal ulceration and bleeding.10 To overcome
these side effects parecoxib sodium11a (Figure 2) has
been developed as a prodrug for the potent and selective
COX-2 inhibitor valdecoxib for parenteral administra-
tion (parecoxib sodium has been marketed in Europe
very recently). Like other COX-2 specific inhibitors, it
The sulfonyl-substituted diarylheterocycle is the most
widely explored pharmacophore for the development
of selective COX-2 inhibitors and is exemplified by
5-bromo-2-(4-fluorophenyl)-3-(4-methylsulfonylphenyl)-
thiophene (I).3 The pharmacophore for this series in-
cludes either a sulfonamide or a methanesulfone, which
† DRF Publication No. 201.
* Corresponding author. Tel: 91-40-3045439. Fax: 91-40-3045438/
3045007. E-mail:
koteswarraoy@drreddys.com.
‡ Discovery-Chemistry.
§ Discovery-Biology.
(M.P.) manojitpal@drreddys.com; (K.R.Y.)
10.1021/jm020563g CCC: $25.00 © 2003 American Chemical Society
Published on Web 08/13/2003

3976 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Pal et al.
F igu r e 1. Some selective COX-2 Inhibitors.
class of 1,5-diarylpyrazole derivatives14b have been
synthesized, some of which are in preclinical develop-
ment. We now describe the synthesis and structure-
activity relationship (SAR) studies of some 1,5-diaryl-
pyrazole derivatives containing a substituted 1-benzene-
sulfonamide group as a pharmacophore and also explore
the possibility of developing an injectable form for better
management of pain. Our work involves identification
of a potent COX-2 inhibitor followed by the preparation
of corresponding prodrug.
F igu r e 2. Injectable antiinflammatory agents.
Ch em istr y
is believed to reduce pain and inflammation by targeting
the COX-2 enzyme, which is expressed at sites of injury,
including surgical incisions. At therapeutic doses, pare-
coxib sodium does not affect COX-1. However, apart
from parecoxib sodium, there is no other injectable COX-
2-specific inhibitor known or reported to be in any phase
of clinical investigation. Therefore, development of an
alternative, new, injectable analgesic for better manage-
ment of surgical pain with minimal side effects is
desirable. These can also be used in the therapy of
ocular inflammation.9b
Celecoxib as well as other coxibs such as rofecoxib,
valdecoxib, and etoricoxib are considered as selective
COX-2 inhibitors with fewer gastrointestinal side effects
of traditional NSAIDs. However, a recent report has
raised a cautionary flag regarding the use of COX-2
inhibitors in patients at risk for cardiovascular morbid-
ity such as myocardial infarction.12 Thus, despite their
excellent antiinflammatory activities, there is still
further scope and demand for the development of
antiinflammatory agents with improved efficacy and
safety compared to the existing COX-2 inhibitors. Cele-
coxib belongs to the 1,5-diarylpyrazole class of com-
pounds, and a recent study revealed that this class of
compounds could be useful as dual COX-2/5-LO (5-
lipooxygenase) inhibitors for the effective management
of inflammatory diseases.6c Therefore, in pursuance of
our synthesis of various diaryl heterocycles,13 we pre-
ferred 1,5-diarylpyrazoles over others for the develop-
ment of novel class of COX-2 inhibitors.
The benzenesulfonamide group of celecoxib attached
to the nitrogen atom of the pyrazole ring is thought to
be responsible for its high potency and COX-2 selectivity
in different models of inflammation. Extensive SAR
study has been carried out on a large number of
celecoxib analogues, including modification of aryl or
pyrazole moiety.6 However, systematic study of the
attachment of appropriate substituents on the 1-ben-
zenesulfonamide group14 and its effect on COX activity
has not yet been explored. We rationalized that intro-
duction of an appropriate substituent at the 2 or 3
position of the 1-benzenesulfonamide group would re-
inforce the binding of this group with specificity to the
COX-2 pocket. In the light of this hypothesis, a new
The diarylpyrazoles listed in Table 1 were synthesized
according to Schemes 1-3. The arylhydrazine hydro-
chloride 25, a key intermediate for the synthesis of most
of the analogues, was synthesized according to the
procedure outlined in Scheme 1. Thus, chlorosulfonation
of appropriately substituted flourobenzene 21 at low
temperature followed by ammonolysis using aqueous
ammonia in dioxane led to the formation of correspond-
ing sulfonamide 23 in good yield. On treatment with
anhydrous hydrazine in acetonitrile under refluxing
condition, 23 provided 24, which was then converted to
the hydrochloride salt 25.
Synthesis6a,15 of dicarbonyl derivatives 27 was carried
out using (Scheme 2) Claisen condensation of an ap-
propriate acetophenone 26 with either ethyltrifluoro-
acetate or ethyl difluoroacetate in the presence of
sodium hydride as a base in dimethylformamide (DMF).
Reaction of 27 with the hydrochloride salt 25 in ethanol
under refluxing conditions led to the formation of the
expected 1,5-diarylpyrazoles (1-4) as major products
along with trace amounts of other regioisomers. The
latter were removed by chromatography.
Syntheses of sodium salts 10-16 (Table 3) are out-
lined in Scheme 3. Sulfonamides 3 and 4 were acylated
using the required acyl anhydride in the presence of
triethylamine to give the corresponding acylated sul-
fonamides. These were treated with sodium bicarbonate
in methanol to afford the required sodium salts. Other
salts 17-20 were also prepared according to a similar
procedure. 1,5-Diarylpyrazoles were characterized by
the appearance of a peak at ∼6.8 and ∼5.7-4.9 ppm in
the 1H NMR spectra, which were assigned as due to the
C-4 proton signal of the pyrazole ring and -NH of
sulfonamide moiety, respectively. Salts were character-
ized by their high melting points (>240 °C) and the
disappearance of the -NH signal of the acylated sul-
fonamide moiety (R′CONHSO2-), which appeared at
1
∼8.50-12.50 ppm in the H NMR spectra.
Biologica l Assa ys
All the compounds synthesized were tested initially
at 100 µM for selectivity and potency against human
COX-2 (expressed in sf9 insect cells using baculovirus)

1,5-Diarylpyrazoles with Benzenesulfonamide Moiety
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 3977
Ta ble 1. InVitro and in Vivo Data for 1,5-Diarylpyrazoles
a
Human COX-2 (expressed in sf9 insect cells using baculovirus) and COX-1 (ram seminal vesicles) enzyme. Figures in the brackets
indicate concentration in µM. The result is the average of at least three determinations, and the deviation from the mean is <10% of the
b
average value. The carrageenan-induced rat paw edema assay was carried out using six animals (male Wistar rats)/group following
oral dose of the test compound. c nd ) not determined.
Sch em e 1
Sch em e 3
(a) (R′CO)2O, triethylamine, 24 h, rt, quantitative yield; (b)
NaHCO3, MeOH, 20 h, 25 °C, 60-96% yield.
Ta ble 2. H-Bond Interactions of Molecules 1 and 3 with
Residues in the Active Site of COX-2
(a) ClSO3H, 0 °C, 4 h, 74% yield; (b) NH3, dioxane, 0 °C, 6 h,
44-79% yield; (c) NH2NH2, CH3CN, reflux, 6 h, 67-81% yield;
(d) HCl, EtOH, quantitative yield.
amino acid
residue in the
active site
distance
X-Y (Å) for
X-H‚‚‚Y
angle
X-H-Y
(deg)
functional
group
compd
1
Sch em e 2
CF3
SO2
SO2
Arg120 side chain
NH of Phe518
2.69
3.41
3.70
3.36
3.01
2.70
3.28
3.42
2.88
2.83
130.0 ( 0.3
158.4 ( 0.1
137.6 ( 0.1
160.4 ( 0.1
124.0 ( 0.1
137.7 ( 0.1
141.0 ( 0.2
155.4 ( 0.2
151.9 ( 0.1
154.9 ( 0.1
Arg513 side chain
SO2NH2 Gln192 side chain
3-F
CF3
SO2
Arg513 side chain
Arg120 side chain
NH of Phe518
3
SO2NH2 Gln192 side chain
CO of Ser353
OH of Tyr355
(a) NaH, DMF, 0 °C to rt, RCO2Et, 24 h, 12-97% yield; (b) 25,
EtOH, reflux, 44-64% yield.
SO2NH2
2-F
and COX-1 (ram seminal vesicles) enzymes. On the
basis of their in vitro efficacy, selected compounds were
tested further at lower concentration such as 10 µM
followed by 1 µM. IC50 values for COX-1 and COX-2
were determined16 for selected/promising compounds.
Celecoxib, rofecoxib, and indomethacin were used as
reference compounds for the in vitro assay. Few com-
pounds were selected for further investigation on the
basis of their promising in vitro potencies and were
tested in vivo for antiinflammatory activity at 30 mg/
kg dose in the carrageenan-induced rat paw edema
model.17
Resu lts a n d Discu ssion
Results of in vitro assays of the compounds synthe-
sized are summarized in Table 1. The design of com-
pound 1 was based on the assumption that attachment
of fluorine at the C-2 position of benzenesulfonamide
ring may enhance the interaction of this group with

3978 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Pal et al.
Ta ble 3. In Vitro Data for 1,5-Diarylpyrazoles: IC50 Valuesa
IC50 (µM)
volumeb
(Å3)
selectivity index
(COX-1/COX-2)
compd
COX-1
COX-2
log pb
1
3
4
>170c
13.5 ( 0.13
0.15 ( 0.01
0.034 ( 0.003
0.07 ( 0.005
7.8 ( 0.11
>13
>200
>30c
261 ( 7.0
270 ( 7.0
252 ( 7.0
270 ( 7.0
3.14 ( 0.92
3.81 ( 0.93
3.01 ( 0.86
24.3 ( 0.1
15.33 ( 0.03
0.067 ( 0.001
∼714
celecoxib
indomethacin
∼219
∼0.0085
a
b
The result is the mean value of two determinations, and the deviation from the mean is <10% of the mean value. Calculated using
the ACDLabs program developed by Advanced Chemistry Development Inc. c Precipitation of compound observed beyond this concentration.
COX-2. We have chosen fluorine atom as a substituent
because of its small size, lipophilic nature, and ability
to form a strong H-bond. Thus, the basic skeleton of
celecoxib has been modified in compound 1 with the
introduction of a fluorine atom at the C-2 position of
the benzenesulfonamide moiety along with the replace-
ment of a methyl group by a methoxy group.
3a).18 The phenolic -OH of Tyr355 seems to have an
important role in the hydrogen-bond network of the
active site with the fluorine substituent of the benze-
nesulfonamide moiety, thereby enhancing its interaction
with the COX-2 pocket. A fluorine at C-3 would be in
close proximity to the Tyr355 side chain rather than
when it is at C-2 for better electrostatic interactions.
This is corroborated well by the results of the docking
calculations for the molecules 1 and 3 in the COX-2
binding site. The calculated energy of interaction be-
tween COX-2 and 1 is -34.02 kcal/mol, while that of 3
is -36.06 kcal/mol, suggesting that molecule 3 interacts
better. The salient hydrogen bond interactions (Figure
3b and 3c) and respective distances and angles (Table
2) clearly implicate the positional influence of fluorine
substitution on the aryl sulfonamide moiety. The fluo-
rine substituent at the C-3 position of the aryl sulfona-
mide in molecule 3 shows strong hydrogen-bonding
interaction with -OH of the Tyr355 side chain. However,
in molecule 1, wherein the fluorine is at the C-2 position,
it is interacting weakly with Arg513. The additional
interaction with Tyr355 may be responsible for the
relatively higher potency of molecule 3 vis-a`-vis mol-
ecule 1. To gain further evidence in support of this
hypothesis, the corresponding flouro analogue of cele-
coxib (i.e., 7a , 3-fluoro-4-[5-(4-methylphenyl)-3-trifluo-
romethyl-1H-pyrazolyl]-1-benzenesulfonamide) was pre-
pared and tested in vitro. The flouro analogue 7a was
found to be more potent {IC50 (COX-1) ) 7.13; IC50
(COX-2) ) 0.05} than celecoxib, confirming the favorable
role of fluorine in binding with the COX-2 pocket.
On the basis of in vitro data, a few of these 1,5-
diarylpyrazole derivatives were selected for further
study. The IC50 values for compounds 3 and 4 are given
in Table 3 and both of them were identified as potent
and selective COX-2 inhibitors. They are either compa-
rable or superior to celecoxib in terms of selectivity index
(SI > 200 or 714 vs 219 for celecoxib). Interestingly, the
computed molar volumes of 3 and 4 (Table 3) are
comparable to those of celecoxib and indomethacin,
which accounts for their ability to bind deeply at the
active site of COX pockets. The antiinflammatory activ-
ity of these compounds was tested in the standard rat
model of inflammation and compared with standard
COX-2 inhibitors such as celecoxib or rofecoxib. How-
ever, their in vitro potencies did not translate into in
vivo inhibitory efficacies. In carrageenan-induced rat
paw edema assay, compounds 3 and 4 showed 26% and
33% inhibition, respectively, when compared to 52%
inhibition of compound 1 when dosed orally at 30 mg/
kg (Table 1). The reason for the good in vivo activity of
1 despite its inferior in vitro activity is not clear at the
moment. Since none of these compounds exhibited
improved antiinflammatory activity over celecoxib’s 49%
Compound 1 exhibited COX-2 selectivity with moder-
ate potency in COX-2 inhibition (Tables 1 and 3).
However, it showed good inhibition of edema in the in
vivo rat paw model at a dose of 30 mg/kg. This result
motivated us for further SAR studies in this series.
Replacement of the methoxy substituent on the 5-aryl
group of 1 with a halogen such as fluoro, chloro, or
bromo led to either inactive or COX-1-selective com-
pounds (data not shown). Thus, the methoxy group at
the 4-position of the 5-aryl moiety appeared to be
essential for the COX-2 selectivity and potency in this
series. The fluorine substituent of the benzenesulfona-
mide group was found to be sensitive, and its replace-
ment by other groups led to the alteration of selectivity.
This is exemplified by compound 2, wherein the replace-
ment of fluorine by a methyl group resulted in loss of
selectivity (Table 1). A similar effect of the methyl group
was also observed in diaryl furanones.14d However, the
reason for such observation is not clear at present.
Change of the position of fluorine from C-2 (1) to C-3
(3) improved in vitro potency with a moderate change
in selectivity. Although the replacement of the 4-meth-
oxyphenyl moiety by other groups such as 4-XC6H4 (X
) fluoro, chloro, or nitro) reduced the in vitro activity
drastically (data not shown), a 4-methylsulfanylphenyl
group (4) resulted in increase of both in vitro potency
and selectivity (Table 1). These observations suggest
that electron-donating groups at the 4-position of the
5-aryl moiety play a crucial role, and being good
electron-donating groups, methoxy and methylsulfanyl
substituents showed a similar effect on COX-2 selectiv-
ity.
The selectivity18 of celecoxib has been shown to result
from the 1-benzenesulfonamide moiety (Figure 3). This
group binds in a COX-2 pocket, which is relatively polar
and is less restricted compared to the COX-1 enzyme.
This could also be the reason for the COX-2 selectivity
of 1, 3, and 4. Since the total volume19 of the COX-2
primary binding site (394 Å3) is about 25% larger than
that of the COX-1 (316 Å3), a fluorine-substituted
benzene sulfonamide moiety should enhance its interac-
tion with the COX-2 pocket significantly. Also it is
evident from the in vitro data that fluorine at the C-3
position of the phenyl ring is more promising than at
C-2. In search of a possible explanation for this observa-
tion we carefully analyzed the nature of interaction of
SC-558 with the active site of COX-2 enzyme (Figure

1,5-Diarylpyrazoles with Benzenesulfonamide Moiety
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 3979
F igu r e 3. (a) Stereoview of SC558-COX-2 complex (6COX crystal structure). The ligand is shown in magenta. (b) Stereoview of
the molecule 1-COX-2 complex. The hydrogen-bonding interactions are shown as red broken lines. The ligand is shown in magenta.
All protein hydrogens are removed for clarity. (c) Stereoview of the molecule 3-COX-2 complex.
inhibition at 30 mg/kg po under similar conditions, it
was felt that poor bioavailability could be one of the
reasons for their lower in vivo potency. The calculated
log p value for compound 3 showed greater similarity
to celecoxib than 4 (Table 3) and was reflected by their
in vivo pharmacokinetic study. Compound 3 displayed
better pharmacokinetics than 4 and was comparable to
celecoxib (see Table 6). However, the pharmacokinetic
profile indicates that there could be absorption rate
limitation for 3 because of its low water solubility, which
could cause prolonged absorption and thereby delayed
convert them into prodrugs that are comparatively more
soluble in water.
Prodrugs11b are usually converted to the parent drug
in plasma, which perhaps would allow them to pass
through the cell membrane easily to reach the target
for further action. The prodrug would also allow a better
absorption of the drug through the gastrointestinal wall,
and after being absorbed it would convert into the
parent compound through hepatic metabolism. Never-
theless, we were also interested in the development of
injectable formulation of a selective COX-2 inhibitor for
parenteral administration. Therefore, we prepared water-
soluble forms of compounds 3 and 4 and their deriva-
T
max. Since diarylheterocycles possess modest aqueous
solubility, the easiest way to address this problem is to

3980 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Ta ble 4. In Vitro and in Vivo Data for 1,5-Diarylpyrazole Salts
Pal et al.
a
b
See footnote a of Table 1. Carrageenan-induced rat paw edema assay was carried out using six animals (male Wistar rats)/group
following oral dose of the test compound.
Ta ble 5. In Vivo Data (Rat Paw Edema Assaya)
respect to the various prodrugs of celecoxib (18-20,
Table 4) when dosed orally at 30 mg/kg under similar
conditions. Maximum inhibition was achieved with the
propionyl analogue 10 (78%). Interestingly, the butyryl
analogue (18) of celecoxib was found to be better than
its propionyl (19) or acetyl (20) congeners. The differ-
ences in their in vivo drug-releasing properties for
different prodrugs of celecoxib, i.e., 18-20, as well as
of compounds 3 and 4 and their derivatives, i.e., 10-
17, could be the reason for such observation. This,
perhaps, provides an explanation for the lower activity
of prodrug 11, taking into account that, like 10, it is
the same type of derivative of the more in vitro potent
4. The ED50 of compound 10 was found to be 6 mg/kg
(Table 5), which is closer to that of celecoxib at 7.9 mg/
kg (The ED50 of the butyryl analogue of celecoxib in
carrgeenan-induced rat paw edema is 6.69 mg/kg.) and
comparable with that of parecoxib sodium (5 mg/kg).11a
The excellent antiinflammatory activity of 10 suggested
that the salt might be protonated followed by the
cleavage of acyl moiety in vivo to deliver the parent
molecule at the target.11 This was further supported by
entry
compd
ED30 (mg/kg)
ED50 (mg/kg)
1
2
3
4
5
1
1.00 ( 0.13
12.70 ( 1.43
6.00 ( 0.21
7.90 ( 0.14
6.69 ( 0.27
5.00b
10
celecoxib (7)
18
nd
nd
nd
nd
parecoxib sodium
a
ED30 and ED50 values were determined using a minimum of
four dose points, six animals (male Wistar rats)/group followed
by oral administration. b Data from ref 11a. c nd ) not determined.
tives, which showed substantial inhibition either in vitro
or in vivo. A water-soluble form was accessible by
N-acylation of the sulfonamide followed by preparation
of its sodium salt, which would serve as possible prodrug
for the parent compound. Thus, a variety of sodium salts
10-17 were prepared according to the procedure de-
scribed above and evaluated in vivo. All these salts were
found to be highly soluble in water and their in vitro
and in vivo activities are summarized in Table 4.
Prodrugs have been reported to be very weak inhibitors
of both COX-1 and COX-2 earlier11a and were found to
be the same in the present case, too. However, all the
analogues exhibited good to excellent inhibition with

1,5-Diarylpyrazoles with Benzenesulfonamide Moiety
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 3981
Ta ble 6. Oral Pharmacokinetics of 1,5-Diarylpyrazoles
parametersa
3
4
celecoxib
rofecoxib
AUC0-t (µg h/mL)
46.45 ( 14.68
3.30 ( 0.98
7.50 (3.32
6.65 ( 1.33
3.21 ( 0.89
1.26 ( 0.50
23.16 ( 1.17
2.47 ( 0.63
3.25 ( 1.26
57.88 ( 4.53
5.61 ( 1.10
3.00 ( 0.00
C
max (µg/mL)
max (h)
T
a
Pharmacokinetic parameters were determined using four animals (male Wistar rats)/group following 100 mg/kg oral dose of the test
compound.
on a Perkin-Elmer 1650 FT-IR spectrometer. UV spectra were
recorded on a Shimadzu UV 2100S UV-vis recording spectro-
photometer. Melting points were determined using a Buchi
melting point B-540 apparatus and are uncorrected. Thermal
analysis data was generated with the help of a Shimadzu DSC-
50 detector. MS spectra were obtained on a HP-5989A mass
spectrometer. Purity was determined by HPLC (AGIL-AUTO)
using the conditions specified in each case: column, mobile
phase (range used), flow rate (ranges used), detection wave-
length, retention times. Microanalyses were performed using
a Perkin-Elmer 2400 C H N S/O analyzer. Elemental data
are within (0.4% of the theoretical values. All yields reported
are unoptimized. Celecoxib was prepared according to the
literature6a procedure. Rofecoxib was prepared according to the
procedure described in WO 9500501. Acetophenones were
either purchased or prepared according to the procedure
described in the literature.
P r ep a r a t ion of Ar ylh yd r a zin e H yd r och lor id e (25).
Step a : P r ep a r a tion of Ar ylsu lfon yl Ch lor id e (22). To a
cold solution (0 °C) of chlorosulfonic acid (450 mmol) was added
appropriately substituted fluorobenzene 21 (90 mmol) slowly
at 0 °C. The mixture was then stirred for 4 h at 0 °C and
allowed to stand for overnight at the same temperature. The
reaction mixture was poured into crushed ice. The oily layer
that separated was collected and washed with water to give
the desired compound as a liquid.
the pharmacokinetic evaluation of 10 in male rats (100
mg/kg dose), where the plasma levels of 3 (after oral
administration of 3 as such) and 10 were monitored.
Appearance of 3 (HPLC retention time of 10, 3, and 3
released from 10 is 13.4, 9.0, and 9.2 min, respectively)
at the first time point (0.5 h) in plasma suggested that
the conversion of the prodrug into the parent compound
occurred presystemically. Interestingly, this conversion
of prodrug was found to be susceptible to steric influ-
ence, which has been reflected by the complete loss of
in vivo activity in the case of pivolyl analogue 17 (Table
4, prodrug of 3). The significant change in Tmax observed
for 3 (3.00 ( 1.41 h) released from 10 following oral
administration at 100 mg/kg indicated rapid absorption
of 3 in the present form compared to 3 as such (Table
6). Although the other pharmacokinetic parameters of
3 from 10 and 3 alone were comparable even at the
lower dose (10 mg/kg), the higher efficacy of 10 observed
in the inflammation model could be accounted for by a
possible differential distribution of 3, relatively higher
exposure to 3, and the inherent cyclooxygenase activity
of 10. Thus, the sodium salt 10 was identified as a
water-soluble prodrug of 3 with good in vivo potency.
Step b: P r ep a r a tion of Ar ylsu lfon a m id e (23). To a cold
solution of 22 (67 mmol) in dioxane (30 mL) was added 25%
aqueous solution of ammonia (140 mL) with vigorous stirring.
The stirring continued for 6 h at 0 °C and separated solid was
filtered and washed with water (2 × 50 mL) to give the title
compound as a white solid.
Con clu sion
We have synthesized and described 1,5-diarylpyrazole
derivatives having a substituted benzenesulfonamide
moiety as potent inhibitors of COX-2 enzymes. Molec-
ular modeling and SAR studies revealed that fluorine
substitution on the phenyl sulfonamide moiety along
with an electron-donating group at the 4-position of the
5-aryl ring determine the selectivity as well as potency
for COX-2 inhibition. Compound 3 has been identified
as the best molecule in this series and the water-soluble
prodrug 10 has good activity in vivo. Compound 10 is
the first 1,5-diarylpyrazole derivative reported that has
potential to become an investigational COX-2 specific
inhibitor for parenteral administration. The good COX-2
specificity of its parent compound 3 and the in vivo
efficacy of prodrug 10 may provide better nonnarcotic
management of acute to moderate pain compared to the
nonselective ketorolac.
3,4-Diflu or o-1-ben zen esu lfon a m id e (23a ). Yield 44%;
1
mp 90-92 °C; H NMR (CDCl3) δ 7.82-7.74 (m, 1H), 7.39-
7.32 (m, 2H), 5.05 (bs, D2O exchangeable, 2H); IR (KBr, cm-1
)
3364, 3268, 1512; MS (CI, isobutane) 194 (M+1, 100). Anal.
(C6H5F2NO2S) C, H, N.
2,4-Diflu or o-1-ben zen esu lfon a m id e (23b). Yield 53%;
mp 157-158 °C; 1H NMR (CD3OD) δ 7.98-7.87 (m, 1H), 7.24-
7.06 (m, 2H); IR (KBr, cm-1) 3366, 3263; MS (CI, isobutane)
194 (M+1, 100%). Anal. (C6H5F2NO2S) C, H, N.
4-F lu or o-2-m eth yl-1-ben zen esu lfon a m id e (23c). Yield
1
79%; mp 170-171°C; H NMR (DMSO-d6) δ 7.9 (m, 1H), 7.4
(s, 2H, D2O exchangeable), 7.2 (m, 2H), 2.59 (s, 3H). Anal.
(C7H8FNO2S) C, H, N.
Step c: P r ep a r a tion of 4-Hyd r a zin o-1-a r ylsu lfon a m id e
(24). A solution of 23 (2.94 mmol) in acetonitrile (10 mL) was
treated with anhydrous hydrazine (0.6 mL) and the mixture
was refluxed for 6 h. The solvent was then removed under
reduced pressure and the residue was treated with water (10
mL). The separated solid was filtered and washed with cold
water (2 × 50 mL) to give the required product.
Exp er im en ta l Section
Ch em ica l Meth od s. All the solvents used were com-
mercially available and distilled before use. Reactions were
monitored by thin-layer chromatography (TLC) on silica gel
plates (60 F254; Merck), visualizing with ultraviolet light or
iodine spray. Flash chromatography was performed on silica
gel (SRL 230-400 mesh) using distilled petroleum ether, ethyl
3-Flu or o-4-h ydr azin o-1-ben zen esu lfon am ide (24a). Yield
81%; low-melting solid; 1H NMR (CDCl3) δ 7.76-7.64 (m, 2H),
7.38-7.27 (m, 1H); IR (KBr, cm-1) 3340, 1512; MS (CI,
isobutane) 206 (M + 1, 100). Anal. (C6H8FN3O2S) C, H, N.
2-Flu or o-4-h ydr azin o-1-ben zen esu lfon am ide (24b). Yield
73%; low-melting solid; 1H NMR (CD3OD) δ 7.92-7.85 (m, 1H),
6.98-6.75 (m, 2H); IR (KBr, cm-1) 3376, 3272, 1569; MS (CI,
isobutane) 205 (30), 95 (100). Anal. (C6H8FN3O2S) C, H, N.
4-H yd r a zin o-2-m et h yl-1-b en zen esu lfon a m id e (24c).
Yield 67%; low-melting soild; 1H NMR (DMSO-d6) δ 7.6 (m,
1H), 7.4 (m, 2H), 2.57 (s, 3H); IR (KBr, cm-1) 3375, 3270, 1569;
MS (CI, isobutane) 202 (M+, 100). Anal. (C7H11N3O2S) C, H,
N.
1
acetate, dichloromethane, chloroform, and methanol. H and
13C NMR spectra were determined in CDCl3, DMSO-d6,or
MeOH-d4 solution on Varian Gemini 200 MHz spectrometers.
Proton chemical shifts (δ) are relative to tetramethylsilane
(TMS, δ ) 0.00) as internal standard and expressed in ppm.
Spin multiplicities are given as s (singlet), d (doublet), t
(triplet), and m (multiplet) as well as b (broad). Coupling
constants (J ) are given in hertz. Infrared spectra were recorded

3982 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19
Pal et al.
Step d : P r ep a r a tion of Hyd r a zin e Hyd r och lor id e (25).
To a solution of 24 (0.4 g) in ethanol (5 mL) was added
2-propanol (3 mL) saturated with dry hydrochloric acid at 25
°C and the mixture was stirred for 1.5 h at the same
temperature. The solvent was removed under low pressure to
yield the required compound in quantitative yield.
HICHROM RPB, water-acetonitrile (30:70), 1 mL/min, 225
nm, tR 7.7 min. Anal. (C17H13O2N3S2F4) C, H, N.
Gen er a l Meth od for th e P r ep a r a tion of Sod iu m Sa lt
of 1,5-Dia r ylp yr a zoles. Step 1. To a solution of appropriate
pyrazoles (3, 4, or 7, 0.62 mmol) in dichloromethane (12 mL)
was added the required acyl anhydride (1.862 mmol) and
triethylamine (1.862 mmol) and the mixture was stirred for
24 h at room temperature. After completion of the reaction,
solvent was removed under vacuum and the residue was
treated with water followed by extraction with dichlo-
romethane (3 × 50 mL). Combined organic layer was dried
over anhydrous Na2SO4 and concentrated under vacuum. The
isolated residue was purified by column chromatography using
ethyl acetate-petroleum ether to give required acylated
compound.
Step 2. To a solution of acylated compound (0.57 mmol)
obtained above in methanol (15 mL) was added NaHCO3 (0.79
mmol) and the mixture was stirred at room temperature for
20 h. Methanol was removed under vacuum and the residue
was treated with dry diethyl ether (3 × 10 mL). The powder
was collected after filtration and dried under vacuum to give
the required sodium salt of 1,5-diarylpyrazoles.
Gen er a l Met h od for t h e P r ep a r a t ion of 1,5-Dia r yl-
p yr a zoles.6a ,15 Step a : P r ep a r a tion of 1,3-Dik eton es (27).
Gen er a l P r oced u r e. To a solution of acetophenone (26, 2.7
mmol) in dimethylformamide was added a 60% oil suspension
of sodium hydride (3.24 mmol) at 10 °C under nitrogen
atmosphere and the mixture was stirred for 10 min. To this
mixture was added fluorinated ethyl acetate (3.24 mmol) at
the same temperature and stirring was continued for 12 h.
This mixture was then poured into ice-cold hydrochloric acid
solution (100 mL) and was stirred for 10 min. The separated
solid was extracted with ethyl acetate (2 × 50 mL). The
combined organic layer was collected, washed with water (2
× 50 mL), dried over anhydrous Na2SO4, and concentrated
under vacuum. The residue isolated was purified by column
chromatography using 5% ethyl acetate-petroleum ether to
give required compound 27.
Sod iu m
Sa lt
of
N1-P r op ion yl-3-flu or o-4-[5-
Step b: A mixture of hydrazine hydrochloride 25 and 1,3-
diketone 27 in ethanol was heated to reflux with vigorous
stirring under nitrogen atmosphere for 12 h. Ethanol was
removed under low vacuum and the isolated residue was
purified by column chromatography using ethyl acetate-
petroleum ether to give the expected product.
(4-m eth oxyp h en yl)-3-tr iflu or om eth yl-1H-1-p yr a zolyl]-1-
ben zen esu lfon a m id e (10). White powder; yield 82%; Rf 0.1
(40% ethyl acetate-petroleum ether); mp 278-284 °C; 1H
NMR (DMSO-d6) δ 7.65 (d, J ) 7.8 Hz, 2H), 7.58 (s, 2H), 7.18
(d, J ) 4.4 Hz, 2H), 6.93 (d, J ) 8.8 Hz, 2H), 3.74 (s, 3H), 1.92
(q, J ) 7.6 Hz, 2H), 0.88-0.81 (t, J ) 7.3 Hz, 3H); IR (KBr,
cm-1) 1609; UV (MeOH, nm) 245; MS (CI, isobutane) 472 (M+
- 21, 5), 337, 155 (100); HPLC 99.23%, SYMMETRY C18 (150
mm), water-acetonitrile (35:65), 1 mL/min, 245 nm, tR 4.7 min.
Anal. (C20H16F4N3O4SNa) C, H, N.
Sod iu m Sa lt of N1-P r op ion yl-3-flu or o-4-[5-(4-m eth yl-
su lfan ylph en yl)-3-tr iflu or om eth yl-1H-1-pyr azolyl]-1-ben -
zen esu lfon a m id e (11). White powder; yield 96%; Rf 0.1 (40%
ethyl acetate-petroleum ether); mp 309-312 °C; 1H NMR
(DMSO-d6) δ 7.65-7.60 (m, 3H), 7.25-7.23 (m, 5H), 2.5 (s, 3H),
2.0-1.85 (m, 2H), 0.83 (t, J ) 7.3 Hz, 3H); IR (KBr, cm-1) 3426,
2974, 2358, 1610; UV (MeOH, nm) 245; MS (CI, isobutane)
466 (10), 424 (100); HPLC 98%, SYMMETRY C18 (150 mm),
0.01 M potassium dihydrogen phosphate-acetonitrile (35:65),
pH 3.5, 0.8 mL/min, 245 nm, tR 8.4 min. Anal. (C20H16F4N3O3S2-
Na) C, H, N.
Sod iu m Sa lt of N1-P r op ion yl-4-[5-(4-flu or op h en yl)-3-
t r iflu or om e t h yl-1H -1-p yr a zolyl]-3-flu or o-1-b e n ze n e -
su lfon a m id e (12). White powder; yield 60%; Rf 0.1 (40% ethyl
acetate-petroleum ether); mp 245-250 °C; 1H NMR (DMSO-
d6) δ 7.67-7.58 (m, 3H), 7.35-7.24 (m, 5H), 1.98-1.82 (m, 2H),
0.92 (t, J ) 7.3 Hz, 3H); IR (KBr, cm-1) 3502, 1612; UV (MeOH,
nm) 250; MS (CI, isobutane) 460 (M+ - 21, 10), 404, 396, 250
(100); HPLC 99%, INERTSIL ODS 3V (4.6 × 250 mm), 0.01
M potassium dihydrogen phosphate-acetonitrile (30:70), pH
3.5, 1 mL/min, 250 nm, tR 7.0 min. Anal. (C19H13F5N3O3SNa)
C, H, N.
Sod iu m Sa lt of N1-Bu tr yl-3-flu or o-4-[5-(4-m eth oxyp h e-
n yl)-3-tr iflu or om eth yl-1H-1-pyr azolyl]-1-ben zen esu lfon a-
m id e (13). White hygroscopic powder; yield 87%; Rf 0.1 (40%
ethyl acetate-petroleum ether); 1H NMR (DMSO-d6) δ 8.00-
7.72 (m, 3H), 7.11 (d, J ) 8.8 Hz, 2H), 6.84 (d, J ) 8.8 Hz,
2H), 6.74 (s, 1H), 3.81 (s, 3H), 2.24 (t, J ) 7.3 Hz, 2H), 1.68-
1.56 (m, 2H), 0.92 (t, J ) 7.3 Hz, 3H); IR (KBr, cm-1) 1610,
1469; UV (MeOH, nm) 250; MS (CI, isobutane) 486 (M+ - 21,
100); HPLC 97.6%, INERTSIL ODS 3V (4.6 × 250 mm), 0.01M
potassium dihydrogen phosphate-acetonitrile (30:70), pH 3.5,
1 mL/min, 250 nm, tR 8.3 min. Anal. (C21H18F4N3O4SNa) C,
H, N.
2-F lu or o-4-[5-(4-m et h oxyp h en yl)-3-t r iflu or om et h yl-
1H-1-p yr a zolyl]-1-ben zen esu lfon a m id e (1). The title com-
pound was synthesized from 4′-methoxyacetophenone using
the two-step procedure as described above to give a white
powder: yield 44%; Rf 0.50 (30% ethyl acetate-petroleum
ether); mp 124-125 °C; 1H NMR (CDCl3) δ 8.30-8.23 (m, 1H),
7.27-7.17 (m, 3H), 6.87-6.71 (m, 4H), 5.78 (s, D2O exchange-
able, 2H), 3.81 (s, 3H); IR (KBr, cm-1) 3386 (b, s); UV (MeOH,
nm) 255; MS (EI) 415 (M+, 100); HPLC 98.7%, SYMMETRY
SHIELD RP 18 (250 mm), water-acetonitrile (20:80), 1 mL/
min, 255 nm, tR 4.4 min. Anal. (C17H13F4N3O3S) C, H, N.
2-Met h yl-4-[5-(4-m et h oxyp h en yl)-3-t r iflu or om et h yl-
1H-1-p yr a zolyl]-1-ben zen esu lfon a m id e (2). The title com-
pound was synthesized from 4′-methoxyacetophenone using
the two-step procedure as described above to five a white
powder: yield 62%; Rf 0.80 (30% ethyl acetate-petroleum
1
ether); mp 138-140 °C; H NMR (DMSO-d6) δ 7.88-7.81 (m,
2H), 7.50-7.47 (m, 3H), 7.40-7.12 (m, 3H), 6.95 (d, J ) 8.7
Hz, 2H), 3.75 (s, 3H), 2.57 (s, 3H); IR (KBr, cm-1) 3374 (b, s),
2361; UV (MeOH, nm) 250; MS (CI, isobutane) 411 (M+, 38),
323, 303 (100); HPLC 97.37%, SYMMETRY SHIELD RP 18
(150 mm), water-acetonitrile (50:50), 1 mL/min, 210 nm, tR
13.2 min.
3-F lu or o-4-[5-(4-m et h oxyp h en yl)-3-t r iflu or om et h yl-
1H-1-p yr a zolyl]-1-ben zen esu lfon a m id e (3). The title com-
pound was synthesized from 4′-methoxyacetophenone using
the two-step procedure as described above to give white
flakes: yield 64%; Rf 0.2 (30% ethyl acetate-petroleum ether);
1
mp 116-120 °C; H NMR (CDCl3) δ 7.81-7.71 (m, 3H), 7.13
(d, J ) 8.3 Hz, 2H), 6.86 (d, J ) 8.3 Hz, 2H), 6.74 (s, 1H), 4.91
(s, D2O exchangeable, 2H), 3.82 (s, 3H); IR (KBr, cm-1) 3352,
3079, 1612, 1473; UV (MeOH, nm) 255; MS (CI, isobutane)
417 (M + 2, 25), 416 (M + 1, 100); HPLC 99.85%, SYMMETRY
SHIELD RP 18 (250 mm), water-acetonitrile (50:50), 1 mL/
min, 225 nm, tR 20.3 min. Anal. (C17H13O3N3SF4) C, H, N.
3-F lu or o-4-[5-(4-m et h ylsu lfa n ylp h en yl)-3-t r iflu or om -
eth yl-1H-1-p yr a zolyl]-1-ben zen esu lfon a m id e (4). The title
compound was synthesized from 4′-methylsulfanylacetophe-
none using the two-step procedure as described above to give
a white solid: yield 61%; Rf 0.5 (30% ethyl acetate-petroleum
ether); mp 156-159 °C; 1H NMR (CDCl3) δ 7.86-7.66 (m, 3H),
7.18 (d, J ) 8.8 Hz, 2H), 7.10 (d, J ) 8.8 Hz, 2H), 6.78 (s, 1H),
Sod iu m Sa lt of N1-Bu tr yl-3-flu or o-4-[5-(4-m eth ylsu l-
fa n ylp h en yl)-3-tr iflu or om eth yl-1H-1-p yr a zolyl]-1-ben ze-
n esu lfon a m id e (14). White powder; yield 96%; Rf 0.1 (40%
ethyl acetate-petroleum ether); mp 300-304 °C; 1H NMR
(DMSO-d6) δ 7.67-7.59 (m, 3H), 7.25-7.22 (m, 5H), 2.5 (s, 3H),
1.89 (t, J ) 7.3 Hz, 2H), 1.49-1.38 (m, 2H), 0.83 (t, J ) 7.3
Hz, 3H); IR (KBr, cm-1) 3447, 2963, 1605, 1469; UV (MeOH,
4.95 (s, D2O exchangeable, 2H), 2.48 (s, 3H); IR (KBr, cm-1
)
3386, 3249, 1603, 1416; UV (MeOH, nm) 225; MS (CI,
isobutane) 433 (M + 2, 33), 432 (M + 1, 100); HPLC 97.3%,

1,5-Diarylpyrazoles with Benzenesulfonamide Moiety
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 19 3983
nm.) 250; MS (CI, isobutane) 502 (M+ - 21, 2), 438 (100);
HPLC 98.3%, INERTSIL ODS 3V (250 mm), 0.01M potassium
dihydrogen phosphate-acetonitrile (30:70), pH 3.5, 1 mL/min,
250 nm, tR 10.1 min. Anal. (C21H18F4N3O3S2Na) C, H, N.
in two steps: optimization of hydrogen atoms that were added
followed by optimization of active site. The energy-minimized
complexes were analyzed for ligand-receptor interactions in
the active site.
Sod iu m Sa lt of N1-Bu tr yl-4-[5-(4-flu or op h en yl)-3-tr i-
flu or om eth yl-1H-1-p yr a zolyl]-3-flu or o-1-ben zen esu lfon a -
m id e (15). White hygroscopic powder; yield 70%; Rf 0.1 (40%
ethyl acetate-petroleum ether); 1H NMR (DMSO-d6) δ 8.02-
7.71 (m, 3H), 7.22-6.99 (m, 4H), 6.79 (s, 1H), 2.24 (t, J ) 7.3
Hz, 2H), 1.68-1.57 (m, 2H), 0.91 (t, J ) 7.3 Hz, 3H); IR (KBr,
cm-1) 1604, 1470; UV (MeOH, nm.) 245; MS (CI, isobutane)
474 (M+ - 21, 100); HPLC 98%, SYMMETRY C18 (4.6 × 150
mm), 0.01 M potassium dihydrogen phosphate-acetonitrile
(40:60), pH 3.5, 1 mL/min, 245 nm, tR 9.7 min. Anal.
(C20H15F5N3O3SNa) C, H, N.
Ack n ow led gm en t. The authors would like to thank
Dr. A. Venkateswarlu, Dr. R. Rajagopalan, and Prof. J .
Iqbal for their constant encouragement and the analyti-
cal research group for excellent support. The authors
also thank the IPM group for literature support, and
Dr. S. K. Singh for providing us compounds 18-20.
Refer en ces
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Sod iu m Sa lt of N1-Acet yl-4-[5-(4-flu or op h en yl)-3-
t r iflu or om e t h yl-1H -1-p yr a zolyl]-3-flu or o-1-b e n ze n e -
su lfon a m id e (16). White powder; yield 60%; Rf 0.1 (40% ethyl
acetate-petroleum ether); mp 242-246 °C; 1H NMR (DMSO-
d6) δ 7.99 (d, J ) 8.3 Hz, 1H), 7.85-7.71 (m, 2H), 7.23-7.01
(m, 4H), 6.79 (s, 1H), 2.09 (s, 3H); HPLC 97.5%, INERTSIL
ODS 3V (4.6 × 250 mm), 0.01 M potassium dihydrogen
phosphate-acetonitrile (30:70), pH 3.5, 1 mL/min, 250 nm, tR
7.0 min. Anal. (C18H11F5N3O3SNa) C, H, N.
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Sod iu m Sa lt of N1-P ivolyl-3-flu or o-4-[5-(4-m et h oxy-
p h e n yl)-3-t r iflu or om e t h yl-1H -1-p yr a zolyl]-1-b e n ze n e
-su lfon a m id e (17). White powder; yield 82%; Rf 0.1 (40% ethyl
1
acetate-petroleum ether); mp >320 °C; H NMR (DMSO-d6)
δ 7.64-7.54 (m, 3H), 7.23-7.18 (m, 3H), 6.92 (d, J ) 8.3 Hz,
2H), 3.74 (s, 3H), 0.96 (s, 9H); HPLC 98%, HICHROM RPB
(250 mm), 0.01 M potassium dihydrogenphosphate-acetoni-
trile (40:60), pH 3.5, 1 mL/min, 245 nm, tR 18.0 min. Anal.
(C22H20F4N3O4SNa) C, H, N.
(5) Futuki, N.; Takahashi, S.; Yokoyama, M.; Arai, I.; Higuchi, S.;
Otomo, S. NS-398, a new antiinflammatory agent, selectively
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Biologica l Assa ys. In Vit r o Bioch em ica l Assa ys.
Sp ectr op h otom etr ic Assa y of COX-1 a n d COX-2. Microso-
mal fraction of ram seminal vesicles were used as a source of
COX-1 enzyme,20 and microsomes from sf-9 cells infected with
baculovirus containing human COX-2 c-DNA were used as a
source of COX-2 enzyme.21 Enzyme activity was measured
using a chromogenic assay based on oxidation of N,N,N′,N′-
tetramethyl-p-phenylenediamine (TMPD) during the reduction
of PGG2 to PGH2 as per the procedure described by Copeland
et al.,22 with the following modifications. The assay mixture
(1000 µL) contained 100 mM Tris pH 8.0, 3 mM EDTA, 15 µM
hematin, 150 units of enzyme, and 8% DMSO. The mixture
was preincubated at 25 °C for 15 min before initiation of
enzymatic reaction in the presence of compound/vehicle. The
reaction was initiated by the addition of 100 µM arachidonic
acid and 120 µM TMPD. The enzyme activity was measured
by estimation of the initial velocity of TMPD oxidation over
the first 25 s of the reaction followed by tracking the increase
in absorbance at 603 nM. The IC50 values were calculated
using nonlinear regression analysis.
(6) (a) Penning, T. D.; Talley, J . J .; Bertenshaw, S. R.; Carter, J .
S.; Collins, P. W.; Doctor, S.; Granto, 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. M.;
Zhang, Y. Y.; Isakson, P. C. Synthesis and Biological Evaluation
of the 1,5-Diarylpyrazole Class of Cyclooxygenase-2 Inhibitors:
Identification of 4-[5-(4-Methylphenyl)-3-(trifluoromethyl)-1H-
pyrazol-1-yl]benzenesulfonamide (SC-58635, Celecoxib) J . Med.
Chem. 1997, 40, 1347-1365. See also (b) Talley, J . J .; Penning,
T. D.; Collins, P. W.; Rogier, D. J ., J r.; Malecha, J . W.; Miyashiro,
J . M.; Bertenshaw, S. R.; Khanna, I. K.; Graneto, M. J .; Rogers,
R. S.; Carter, J . S.; Docter, S. H.; Yu, S. S. US Patent Application
US 5760068 A, 2 J un 1998; Chem. Abstr. 1998, 129, 54367. (c)
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