2-Hydroxymethyl-4-[5-(4-methoxyphenyl)-3-trifluoromethyl-1H-1-pyrazolyl] -1-benzenesulfonamide (DRF-4367): An orally active COX-2 inhibitor identified through pharmacophoric modulation

Article abstract of DOI:10.1039/b402787f

Analogs of 1,5-diarylpyrazoles with a novel pharmacophore at N¹ were designed, synthesized and evaluated for the in-vitro cyclooxygenase (COX-1/COX-2) inhibitory activity. The variations at/around position-4 of the C-5 phenyl ring in conjunction with a CF₃ and CHF₂ groups at C-3 exhibited a high degree of potency and selectivity index (SI) for COX-2 inhibition. The in-vivo evaluation of these potent compounds with a few earlier ones indicated the 4-OMe-phenyl analog 6 and the 4-NHMe-phenyl analog 9 with a CF₃, and the 4-OEt-phenyl analog 19 with a CHF₂ group at C-3 to possess superior potency than celecoxib. In addition to its impressive anti-inflammatory, antipyretic, analgesic and anti-arthritic properties, compound 6 (DRF-4367) was found to possess an excellent pharmacokinetic profile, gastrointestinal (GI) safety in the long-term arthritis study and COX-2 potency in human whole blood assay. Thus, compound 6 was selected as an orally active anti-inflammatory candidate for pre-clinical evaluation.

Full text of DOI:10.1039/b402787f

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A R T I C L E
2-Hydroxymethyl-4-[5-(4-methoxyphenyl)-3-trifluoromethyl-1H-
1-pyrazolyl]-1-benzenesulfonamide (DRF-4367): an orally active
COX-2 inhibitor identified through pharmacophoric modulation
Sunil Kumar Singh,*a Saibaba Vobbalareddy,a Srinivasa Rao Kalleda,a
Shaikh Abdul Rajjak,a Seshagiri Rao Casturi,b Srinivasa Raju Datla,b Rao N. V. S. Mamidi,b
Ramesh Mullangi,b Ravikanth Bhamidipati,b Rajagopalan Ramanujam,b
Venkateswarlu Akellab and Koteswar Rao Yeleswarapu*a
a
Discovery Chemistry, Discovery Research-Dr. Reddy’s Laboratories Ltd., Bollaram Road,
Miyapur, Hyderabad 500 049, India. E-mail: sunilkumarsingh@drreddys.com;
koteswarraoy@drreddys.com; Fax: +91-40-2304-5438/2304-5007; Tel: +91-40-2304-5439
Discovery Biology, Discovery Research-Dr. Reddy’s Laboratories Ltd., Bollaram Road, Miyapur,
b
Hyderabad 500 049, India
Received 24th February 2004, Accepted 22nd June 2004
First published as an Advance Article on the web 11th August 2004
Analogs of 1,5-diarylpyrazoles with a novel pharmacophore at N1 were designed, synthesized and evaluated for the in-
vitro cyclooxygenase (COX-1/COX-2) inhibitory activity. The variations at/around position-4 of the C-5 phenyl ring in
conjunction with a CF3 and CHF2 groups at C-3 exhibited a high degree of potency and selectivity index (SI) for COX-2
inhibition. The in-vivo evaluation of these potent compounds with a few earlier ones indicated the 4-OMe-phenyl analog 6
and the 4-NHMe-phenyl analog 9 with a CF3, and the 4-OEt-phenyl analog 19 with a CHF2 group at C-3 to possess
superior potency than celecoxib. In addition to its impressive anti-inflammatory, antipyretic, analgesic and anti-arthritic
properties, compound 6 (DRF-4367) was found to possess an excellent pharmacokinetic profile, gastrointestinal (GI)
safety in the long-term arthritis study and COX-2 potency in human whole blood assay. Thus, compound 6 was selected as
an orally active anti-inflammatory candidate for pre-clinical evaluation.
Introduction
The two isoforms of prostaglandin (PG) synthase (cyclooxygenase,
COX) exhibit tissue-dependent expression and regulation.1 The
COX-1, a constitutive enzyme, is primarily expressed in the gastro-
intestinal (GI) tract, and is responsible for the biosynthesis of
PGs required for cytoprotection and platelet aggregation.2 Hence,
interference in its normal function for a long time leads to gastro-
intestinal toxicity such as ulceration, bleeding and perforation.3
On the other hand, the isozyme COX-2, which is induced by the
pro-inflammatory cytokines, viz. tumor necrosis factor- (TNF-),
interleukines, mitogens and endotoxins in the inflammatory cells
at the time of injury, plays a major role in the biosynthesis of PGs
required by inflammatory cells (monocytes and macrophages)
and causes inflammation, pain and fever.4 The conventional non-
steroidal anti-inflammatory drugs (NSAIDs), which are effective in-
hibitors of both COX-1 and COX-2, down regulate the biosynthesis
of both kinds of PGs (cytoprotective and inflammatory) in most
of the tissues and exhibit anti-inflammatory activity with side
effects.5 Thus, the selective inhibition of the isozyme COX-2, spar-
ing COX-1, emerged as a novel approach in designing new anti-
inflammatory agents with greater GI safety, and generated a new
avenue in inflammation research. The clinical proof of this concept
is evident from the launch of celecoxib6 and rofecoxib7 (Fig. 1) for
the treatment of rheumatoid and osteo-arthritis. The more effective
second generation drugs such as valdecoxib8 and etoricoxib9 further
validated this new approach. The recent use of COX-2 inhibitors in
other ailments like cancer10 and Alzheimer’s disease,11 and further
discovery of isozyme COX-3,12 have put forth additional challenges
and opportunities ahead. But, the recent reports have raised some
safety issues over the use of COX-2 inhibitors towards a few vital
organs.13 However, this new way of treating inflammatory disorders
has a great clinical advantage over the conventional NSAIDs, and
still warrants a focused effort to have more efficacious COX-2 in-
hibitors with better patient acceptability.
Fig. 1 COX-2 inhibitors.
sulfonamide (MeSO2NH) group as the pharmacophore, exemplified
by nimesulide;14 and (b) the vicinal diaryl heterocycles having the
4-sulfamoyl (SO2NH2)-phenyl or methanesulfonyl (SO2Me)-
phenyl group as the pharmacophore, exemplified by celecoxib,6
valdecoxib,8 rofecoxib7 and etoricoxib9 (Fig. 1). The latter chemi-
cal class is well explored because of the COX-2 enzyme–ligand
co-crystal structure available for the structure-based drug design.15
The two vicinal phenyl rings of the COX-2 inhibitors orienting
in a rigid cis-stilbene geometry along with the extension of the
4-SO2NH2/SO2Me-phenyl ring towards the hydrophilic region of
the COX-2 secondary pocket became the widely accepted feature
for the selectivity. However, apart from vicinal diaryl carbocycles/
heterocycles,16 other scaffolds have also been reported recently.17
With a view to achieving better COX-2 selectivity from known
scaffolds,18,19 we focused our attention on modulating the vital
4-sulfamoyl (SO2NH2)-phenyl ring of celecoxib.20 During this
exercise, it was observed that a hydroxymethyl group (CH2OH)
introduced adjacent to sulfonamide (SO2NH2) increased the COX-2
selectivity. Our idea of introducing the hydrophilic (CH2OH) group
In contrast to the diverse chemical scaffolds of traditional
NSAIDs, COX-2 inhibitors are broadly represented by two
chemical classes: (a) the diphenyl ethers, having the acidic methane-
2 4 4 2
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 4 4 2 – 2 4 5 0
T h i s j o u r n a l i s © T h e R o y a l S o c i e t y o f C h e m i s t r y 2 0 0 4

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was based on the assumption that this would be preferred by the
hydrophilic pocket of the COX-2 enzyme and lead to better inhibi-
tion. Herein, we report a brief SAR (structure–activity relationship)
varying the substitution pattern at the C-3 and C-5 positions of 1,5-
diarylpyrazole to optimize this new pharmacophore and obtain a
better drug candidate with the minimum of effort. This report† also
covers the comparative animal model study of these potent com-
pounds with a few earlier ones20 which finally led to the identifica-
tion of a better drug candidate than celecoxib.
oral administration) using the carrageenan-induced rat paw edema
model.24 ED50s were calculated for the compounds exhibiting more
than 50% inhibition in the rat paw volume. The potent compounds,
selected based on these data, were further evaluated in different
animal models25–27 of inflammation-related disease after a single
dose pharmacokinetic study at 10 mg kg−1 (po). The gastrointestinal
safety profile of the most potent compound was assessed by the
51Cr excretion test.23b,28 Finally, the COX-2 selectivity of the potent
compound was confirmed by human whole blood assay.29
The results of in-vitro COX-1/COX-2 enzyme inhibition exhib-
ited by new analogs of 1,5-diarylpyrazole are depicted in Table 1
and 2. Few compounds such as 5, 6 and 7 from our earlier report20
have also been included here for the sake of comparison in the in-
vitro as well as in-vivo studies. Since small hydrophobic groups
around position-4 of the C-5 phenyl ring created a favorable envi-
ronment for COX-2 potency and selectivity,20 we planned to design
few novel analogs comprising hitherto unreported groups in conju-
gation with a CF3 group at C-3. We started this study by substituting
small alkyl groups to the amino group of 4-aminophenyl analog 8.
Though 4-amino analog 8, as expected, did not exhibit significant
potency (6.600 M), its 4-methylamino homologue 9, was found
to be highly potent (0.339 M) and selective inhibitor of COX-2
enzyme. The 4-dimethylamino analog 10 was also found to be very
potent (0.411 M) but less selective whereas 4-ethylamino analog
11 remained as selective as 9 with lesser potency (0.744 M). Since
we observed a decreasing trend of potency in the amino analogs with
the increase in size of the alkyl groups, a completely different amino
derivative such as 4-azolanylphenyl analog 17 was synthesized to
see if COX-2 enzyme has some pocket specificity around position-4
of the C-5 phenyl ring. Though this compound did not exhibit COX-
2 potency like compounds 9–11, it was still a selective inhibitor of
COX-2 (37% at 1 M) thereby confirming the presence of a specific
hydrophobic area around position-4 of the phenyl ring for effective
ligand-enzyme interaction. Few 3,4-disubstitutedphenyl derivatives
such as the 3,4-dichloro analog 12 (0.405 M), 3,4-dimethyl analog
13 (0.410 M) and 3-methyl-4-methoxy analog 14 (0.560 M) were
also found to be highly potent. A cyclic prototype of compound 14,
2,3-dihydrobenzo[b]furan-5-yl analog 15 turned out to be the most
potent among the compounds reported here (0.228 M). Similarly,
its carbocyclic prototype 2,3-dihydro-1H-5-indenyl 16 which can
also be visualized as the cyclic prototype of compound 13, turned
out to be highly potent (0.295 M) and selective.
Results and discussion
Chemistry
The synthetic route to various 1,5-diarylpyrazoles 5–28 is depicted
in Scheme 1. Simple coupling of the 3-hydroxymethyl-4-sulfamoyl-
phenyl hydrazine hydrochloride 4 with the appropriate 1-phenyl-
1,3-butanediones 3 in absolute ethanol with under heating afforded
the desired analogs of 1,5-diarylpyrazole in very good yield
(60–85%). Though the regiomeric bias was normally in favor of
the 1,5-diarylpyrazoles, the minor, undesired regiomers were eas-
ily eliminated by triturating the product with a mixture of ethyl
acetate–toluene after column chromatography. The formation of
1,5-diarylpyrazoles was identified by the appearance of a singlet for
the C-4 proton at ca. 6.5 ppm in 1H NMR. The required 1-phenyl-
1,3-butanediones 3 were synthesized by Claisen condensation of the
appropriate acetophenones 1 with ethyl trifluoroacetate and ethyl
difluoroacetate 2. This reaction was carried out under slightly modi-
fied condition using sodium hydride in dry DMF at a temperature
of −5 to 30 °C to afford 1-phenyl-1,3-butanediones 3 in 95–98%
yield. The commercially unavailable acetophenones were prepared
according to the standard literature procedures.6,21 The multi-step
synthesis of the modified 3-hydroxymethyl-4-sulfamoylphenyl
hydrazine hydrochloride 4 will be discussed elsewhere.22 All com-
pounds reported herein were characterized spectroscopically.
To compare the effect of CHF2 and CF3, we synthesized a few
CHF2 analogs of the potent compounds selected from Table 1 and
few of our previously reported monosubstituted analogs.20 The in-
vitro results are shown in Table 2. Normally, the CHF2 analogs were
found to exhibit better potency than CF3 analogs but this observa-
tion was limited only to smaller groups. The 4-chloro analog 20
(0.396 M), 4-bromo analog 21 (0.458 M) and 4-ethoxy analog
19 (0.708 M) showed significantly improved potency over the
parent CF3 analogs.20 But, a reverse trend was observed in the case
of the 4-methylamino analog 23 and 4-dimethylamino analog 24,
which showed a drastic drop in COX-2 potency. Although the 3,4-
dimethyl analog 25 (1.140 M) showed a drop in potency when
compared to its CF3 analog 13, the 3-methyl-4-methoxy analog 26
(0.793 M; SI, 1261) exhibited the greatest selectivity among the
two series (Tables 1 and 2). But, the 2,3-dihydro-1H-5-indenyl ana-
log 28 (0.562 M), a cyclic prototype of 25, displayed an improved
potency whereas the 2,3-dihydrobenzo[b]furan-5-yl analog 27, a
cyclic prototype of 26, was found to be less COX-2 selective (SI,
55). However, the smaller hydrophobic groups, either a mono- or
di-substituted phenyl, e.g. the 3-methoxy analog 18 (1.000 M; SI,
450), and 3-chloro-4-fluoro analog 22 (0.653 M; SI, 636) gener-
ally exhibited very good potency and selectivity.
Scheme 1 Reagents and conditions: a) NaH, DMF, −5 to 30 °C, 4–5 h;
b) dil. HCl; c) absolute ethanol, 50–60 °C, 5–6 h.
Biology
All of the new analogs of 1,5-diarylpyrazole synthesized here were
initially screened at 10 M for their selectivity against COX-1,
obtained from the microsomal fraction of ram seminal vesicles, and
against COX-2 recombinant human enzyme, expressed in Sf-9 cells
infected with baculovirus. The promising compounds were further
tested at lower concentrations. The enzyme activity was measured
by a TMPD method and IC50s were calculated using non-linear
regression analysis of the percent inhibitions.23 Celecoxib and
indomethacin were used as reference standards for COX-2 selec-
tive and non-selective inhibitors respectively. Compounds with a
higher selectivity index (SI), i.e. ratio of IC50s (COX-1/COX-2),
and a higher potency (lower IC50s for COX-2) were taken up for pre-
liminary in-vivo anti-inflammatory screening at 30 mg kg−1 (po, i.e.
Since the importance of the 4-sulfonamide-/methylsulfonyl-
phenyl group in the vicinal diaryl heterocycles is well documented
and the ligand–enzyme binding study has become a common tool
to confirm the rational designs of COX-2 inhibitors,16a we wished
to check whether these novel ligands also bind in the same fash-
ion in the COX-2 pocket. The docking study of compound 6 has
already been reported.20 The binding modes of the most potent
† DRL Publication No. 283C.
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 4 4 2 – 2 4 5 0
2 4 4 3

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Table 1 In-vitro data for 3-trifluoromethyl-1,5-diarylpyrazoles
Table 2 In-vitro data for 3-difluoromethyl-1,5-diarylpyrazoles
IC50/Ma
IC50/Ma
Compound
Ar
COX-1b
COX-2c SId
Compound
Ar
COX-1b
COX-2c
SId
5
6
7
8
9
10
11
12
13
14
4-Me-phenyl
278.0
63.0
59.0
135.0
61.4
50.0
136.5
139.0
108.0
0.760
0.365
0.235
6.600
0.339
0.411
0.744
0.405
0.410
0.560
365
172
251
20
181
121
183
343
263
471
18
19
20
21
22
23
3-OMe-phenyl
4-OEt-phenyl
4-Cl-phenyl
4-Br-phenyl
3-Cl,4-F-phenyl
4-NHMe-phenyl
450.0
>200.0h
243.0
1.000
0.708
0.396
0.458
0.653
91e
450
4-OMe-phenyl
4-SMe-phenyl
4-NH2-phenyl
4-NHMe-phenyl
4-NMe2-phenyl
4-NHEt-phenyl
3,4-Cl2-phenyl
3,4-Me2-phenyl
>282h
614
314.0
686
415.0
636
12e
g
g
1.000
g
24
4-NMe2-phenyl
100e
100e
f,g
49f
3-Me,4-OMe-phenyl 264.0
25
26
3,4-Me2-phenyl
3-Me,4-OMe-phenyl
134.0
1.140
0.793
118
>1000.0h
>1261h
15
16
36.0
0.228
0.295
158
678
g
27
28
36.7
0.663
0.562
55
200.0
186.0
331
17
5e
90e
37f
a Mean of three determinations. b COX-1 (obtained from ram seminal vesi-
cles). c COX-2 (human, expressed in Sf-9-infected cells using baculovirus).
d Selectivity Index (ratio of IC50s, i.e. COX-1/COX-2). e % Inhibition at
10 M concentration (one determination). f % Inhibition at 1 M concentra-
tion (one determination). g Not determined. h > Indicates that precipitation
was observed beyond this concentration, and IC50s may be much higher than
the reported values.
f,g
Celecoxib
Indomethacin
—
—
10.75
0.067
0.076
7.810
141
0.008
a Mean of three determinations. b COX-1 (obtained from ram seminal vesi-
cles). c COX-2 (human, expressed in Sf-9-infected cells using baculovirus).
d Selectivity Index (ratio of IC50s, i.e. COX-1/COX-2). e % Inhibition at
10 M concentration (one determination). f % Inhibition at 1 M concentra-
tion (one determination). g Not determined.
analog 12, 3-methyl-4-methoxy analogs 14 and 26, 2,3-dihydro-
1H-5-indenyl analog 16, 3-methoxy analog 18, 4-bromo analog 21,
3-chloro-4-fluoro analog 22 and 2,3-dihydrobenzo[b]furan-5-yl
analog 15 failed to show the expected efficacy in animal model.
Though the exact reason was not explored, their poor pharmaco-
kinetic properties and metabolic instability were speculated as the
main cause of this failure. The compounds having CHF2 group at
position-3 of the pyrazole ring in conjugation with small groups at
position-4 of C-5 phenyl ring, even being highly potent in-vitro, did
not show satisfactory in-vivo efficacy except the 4-ethoxy analog 19
which showed ED50 of 4.5 mg kg−1. This could possibly be due to the
decrease in number of the most electronegative fluorine atom which
might have caused a serious deviation from the normal arrangement
in the COX-2 pocket at physiological condition. The compounds
such as 4-methoxy analog 6 (1.9 mg kg−1), 4-methylamino analog
9 (3.5 mg kg−1) and 4-ethoxy analog 19 (4.5 mg kg−1) having better
efficacy than celecoxib in the preliminary screening,24 were selected
for other inflammation-related animal studies.
The single dose comparative pharmacokinetic data for the
active compounds 6, 9 and 19, taking celecoxib as the standard at
10 mg kg−1 (po), is mentioned in Table 5, and the graph is shown
in Fig. 3. While compounds 9 and 19 exhibited almost similar
pharmacokinetic profiles as celecoxib, 6 showed the maximum
drug concentration in blood (AUC0−t, 19.40 g.h.mL−1) with
a better elimination half life (t1/2, 7.01 h) when compared to cele-
coxib (AUC0−t, 9.68 g.h.mL−1; t1/2, 6.87 h). However, the time to
reach the maximum concentration in blood, a criteria for the onset
of activity (Tmax, 3 h), was found to be the same for these com-
pounds like that for celecoxib. Though the overall pharmacokinetic
profile of compound 6 was found to be the best among all, other two
compounds 9 and 19 were also studied in the next animal models
of inflammation.
2,3-dihydrobenzo[b]furan-5-yl analog 15 (0.228 M) and the least
potent 4-amino analog 8 (6.600 M) are shown in Fig. 2(a) and (b)
respectively. In compound 15 [Fig. 2(a)], the two hydrogen atoms
of the sulfonamide form hydrogen bonds with His-90 and Gly-354,
whereas its two oxygen atoms form hydrogen bonds with the back-
bones of Phe-518 and Ile-517. The hydroxymethyl group introduced
adjacent to the sulfonamide plays a crucial role in the ligand–enzyme
interaction and binds in a region formed by Phe-518, Val-349 and
Ile-517. The central pyrazole ring of all the ligands mentioned here
is surrounded by Val-116, Val-349, Leu-359 and Ala-527. The CF3
and CHF2 groups normally bind in a pocket formed by Val-116,
Val-349 and Arg-120. Fluorine atoms of these groups experience
strong electrostatic interactions with the side chain of Arg-120. The
C-5 phenyl ring binds in a large but narrow hydrophobic pocket of
COX-2 formed by Tyr-348, Tyr-385, Trp-387 and Ser-530. The 4-
amino group of compound 8 forms a hydrogen bond with Tyr-385 in
the hydrophobic region, which is an unfavorable phenomenon and
hence exhibits less potency [Fig. 2(b)]. The interaction and strain
energies of a few docked ligands are shown in Table 3. Compound
8 has the highest strain energy and least interaction energy which
supplements its lesser potency, whereas compound 15, with the
highest interaction energy, stands highly potent.
Based on the in-vitro potency criteria described, the potent
compounds of Tables 1 and 2 and a few previously reported ones20
were screened in the carrageenan-induced rat paw edema model at
30 mg kg−1 (po). Many compounds reduced the edema and exhibited
anti-inflammatory activity in this preliminary animal model. Com-
pounds exhibiting ca. 50% inhibition of paw edema were further
tested at 1, 3, 10 and 30 mg kg−1 in six animals per group to calculate
the ED50s. These results are depicted in Table 4. Though many com-
pounds displayed very good potency at 30 mg kg−1, very few were
found to be better than celecoxib based on the ED50 values. The in-
vitro potent compounds like 4-methylsulfanyl analog 7, 3,4-dichloro
The comparative data obtained from different animal models
is depicted in Table 6. The above three compounds were initially
screened at 30 mg kg−1 (po) in the endotoxin-induced pyresis
model for assessing their antipyretic activity.25 Though all three
2 4 4 4
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 4 4 2 – 2 4 5 0

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Table 4 In-vivo data for 1,5-diarylpyrazoles in the carrageenan-induced
rat paw edema model
Compound
% Reduction in paw vol. (30 mg kg−1)a
ED50/mg kg−1 b
5
6
7
8
9
12
14
15
16
18
19
20
21
22
26
63
60
47
56
63
50
52
55
54
61
70
61
42
50
60
53
10.0
1.9
c
25.2
3.5
>30
21.7
22.3
23.5
25.4
4.5
7.9
c
>30
18.5
6.7
Celecoxib
a Results of single experiment carried out taking six animals per group (male
wistar rats) on oral dosing. b The experiment was carried out in eight animals
per group (male wistar rats) on oral dosing of 1, 3, 10 and 30 mg kg−1, and
ED50s are the average of three experiments. c No dose response.
Fig. 3 Oral pharmacokinetics of 1,5-diarylpyrazoles in male wistar rats at
10 mg kg−1 (single dose).
found to be better than celecoxib (2.11 mg kg−1). While compound
19 was found to be equipotent to celecoxib, compound 9 was
relatively less potent. Since a reasonable balance of antipyretic and
analgesic activity is required with the anti-inflammatory properties
in this therapeutic area, it was decided at this stage to study only
compounds 6 and 9 in the arthritis model. In a 21 day adjuvant-in-
duced prophylactic model of arthritis,27 compound 6 (0.11 mg kg−1)
was found to be almost seven fold more potent than celecoxib
(0.70 mg kg−1). Compound 9 (0.62 mg kg−1) was found to have the
same potency as celecoxib in this study. Even in a 30 day adjuvant-
induced curative model of arthritis (treatment commenced on the
fifteenth day after establishing the disease by adjuvant injection),
compound 6 (<0.30 mg kg−1) was found to be almost equipotent to
celecoxib (0.13 mg kg−1). Compound 9 in this study was found to be
less potent (5.20 mg kg−1) than compound 6 and celecoxib.
Fig. 2 Docking of (a) the most active compound 15 and (b) the least active
compound 8 in the active site of murine COX-2 enzyme. Ligand structures
are shown in ball and stick model where carbon atoms are shown in orange.
Hydrogen and backbone atoms are not shown for clarity.
Table 3 Interaction and strain energies of 1,5-diarylpyrazoles
Compound
Interaction energy/kcal mol−1
Strain energy/kcal mol−1
8
11
15
19
20
−35.228
−38.919
−39.257
−39.188
−38.22
4.898
1.044
1.899
1.117
1.789
compounds were active at this dose, compounds 6 and 9 were found
to have ED50s of 4.68 and 3.53 mg kg−1 respectively, were found
to be four times better than celecoxib (15.68 mg kg−1). We again
reasoned that the CHF2 group was behind the failure of compound
19 in the disease condition. Similarly, when these three compounds
were screened in the carrageenan-induced hyperalgesia model to
assess their analgesic activity,26 compound 6 (1.13 mg kg−1) was
Finally, the preliminary gastrointestinal safety of compound
6 was assessed by 51Cr excretion tests both in acute as well as
in chronic models of arthritis using indomethacin and celecoxib
as control drugs.23b,28 This compound was found to be as safe as
celecoxib. The data are presented in Table 7. Compound 6 was also
tested for COX-2 selectivity in human whole blood where it was
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 4 4 2 – 2 4 5 0
2 4 4 5

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Table 5 Oral pharmacokinetic profiles of selected 1,5-diarylpyrazoles in wistar rats at 10 mg kg−1 a
Parameters
6
9
19
Celecoxib
AUC(0–t)b (g.h.mL−1)
AUC(0–∞)b (g.h.mL−1)
Cmaxc (g mL−1)
Tmaxd (h)
19.40 ± 2.99
21.61 ± 1.96
2.55 ± 0.83
2.75 ± 0.50
0.11 ± 0.03
7.01 ± 1.93
9.06 ± 1.44
9.75 ± 3.02
9.68 ± 0.63
13.53 ± 2.88
1.63 ± 0.16
2.80 ± 0.45
0.11 ± 0.03
6.87 ± 2.65
10.48 ± 2.82
1.42 ± 0.21
3.25 ± 1.26
0.26 ± 0.13
3.40 ± 2.07
10.03 ± 2.94
1.21 ± 0.39
2.75 ± 0.50
0.15 ± 0.04
4.81 ± 1.14
Kele (h−1)
t1/2f (h)
aAverage of two experiments, each carried out in the group of five animals on single dosing. bArea under curve. c Peak plasma concentration. d Time taken in
achieving Cmax. e Terminal elimination constant. f Terminal half-life.
Table 6 In-vivo data for selected 1,5-diarylpyrazoles in different animal models
ED50/mg kg−1
Rat adjuvant arthritis
Compound
Paw edemaa
Pyresisb
Hyperalgesiab
Prophylacticc
Curatived
6
9
19
1.80 ± 0.08
3.12 ± 0.28
5.62 ± 1.11
6.65 ± 0.98
4.68 ± 0.82
3.53
>30
15.68
1.13 ± 0.06
7.44 ± 1.27
2.02
0.11
<0.30
0.62
5.20
e
e
Celecoxib
2.11 ± 0.57
0.70
0.13
a The experiment was carried out in six animals per group (male wistar rats) on oral dosing of 1, 3, 10 and 30 mg kg−1, and ED50s are the average of three
experiments. b ED50s are the average of two experiments carried out with six animals per group on oral dosing of 1, 3, 10 and 30 mg kg−1. c Value from 21
days experiment with eight animals per group (see the experimental section). d Value from 30 days experiment carried with eight animals per group (see the
experimental section). e Not determined.
Table 7 51Cr excretion test for compound 6 in wistar rats
51Cr excretion
Table 8 Human whole blood assay of compound 6
IC50/Ma
%
Compound
Dose/mg kg−1
Acute dosinga
Chronic dosingb
Compound
COX-1
COX-2
Ratio (COX-1/COX-2)
Control
6
Celecoxib
Indomethacin
—
0.41 ± 0.01
0.43 ± 0.04
0.31 ± 0.05
1.95 ± 0.36
0.70 ± 0.09
0.64 ± 0.1
0.63 ± 0.07
Died
6 (DRF-4367)
Celecoxib
5.38 ± 1.1
8.4 ± 1.6
0.04 ± 0.007
0.14 ± 0.005
125
62
100
100
100
aAll values are given as the mean ± SE (n = 6).
a Experiments were performed on a group of eight animals for 48 h. The
values are the average of two experiments. b Experiments were performed
on a group of eight animals for 14 days. The values are the average of two
experiments.
cation in the reactions, or were prepared according to the procedure
described in the literature.6,21 Reactions were monitored by thin-
layer chromatography (TLC) on silica gel plates (60 F254; Merck),
visualizing with ultraviolet light or iodine spray. Usually, the flash
column chromatographic purification was performed over 100–200
or 230–400 mesh silica gel using a mixture of ethyl acetate and
petroleum ether. While the standard sample of celecoxib was pre-
pared according to the literature procedure,6 the standard drug indo-
methacin was isolated from capsules bought from the medical stores.
The yields of the products reported here are un-optimized. Melting
points were determined on Buchi melting point B-540 apparatus and
are uncorrected. IR spectra were recorded on a Perkin-Elmer FT-IR
1650 spectrometer. 1H and 13C NMR experiments were respectively
performed at 200 MHz and 50 MHz on a Varian Gemini 200 spectro-
meter and their chemical shifts are reported in  units with respect
to TMS as the internal standard. Mass spectra were recorded on
a HP-5989A spectrometer. Elemental analysis was carried out for
C, H, N using a Perkin-Elmer 2400 series II CHN–O analyzer. All
of the analyses were performed by the Analytical Research group
of Discovery-Research, Dr. Reddy’s Laboratories Ltd. The purity
of the final compounds were determined by HPLC using ‘System 1’
which consisted of a Hichrom RPB (250 mm) column, and mobile
phase 0.01 M KH2PO4/CH3CN (50:50), and ‘System 2’ which
comprised an Intersil ODS 3 V (250 mm) column, and mobile phase
H2O/CH3CN (50:50). Both systems were run at 1.0 mL min−1 with
UV detection at the wavelength of maximum absorption.
found to be more potent (0.04 M) and selective (SI, 125) than the
standard drug celecoxib (0.14 M; SI, 62; Table 8).29 Thus, based
on the results described above, compound 6 was selected as a potent
and safe candidate for further pre-clinical studies.
Conclusion
In this report, we described the design and synthesis of a few
1,5-diarylpyrazoles having a novel pharmacophore at N1. The in-
vitro as well as in-vivo evaluation of their C-3 and C-5 substituted
analogs afforded several potent compounds, viz. 4-OMe-phenyl and
4-NHMe-phenyl analogs 6 and 9, both with CF3, and 4-OEt-phenyl
analog 19 with a CHF2 group at the 3-position. Of these, 6 (DRF-
4367) was found to be superior to celecoxib based on the anti-in-
flammatory, antipyretic, analgesic and anti-arthritic potential.Apart
from its excellent potency, it has exhibited a very high concentration
of the drug in systemic circulation upon oral dosing. This compound
displayed GI safety like celecoxib during the long-term arthritis
study and a far superior COX-2 selectivity in human whole blood
assay. Based on the above data, compound 6 is selected as the orally
active candidate for further pre-clinical evaluation.
Experimental
General procedure for the preparation of 1,5-diarylpyrazoles
Chemistry protocols
Step 1. General procedure for the preparation of aceto-
phenone 1. Representative preparation of 1-(2,3-dihydro-1H-
5-indenyl)-1-ethanone. Acetyl chloride (3.77 mL, 52.86 mmol)
was introduced to a suspension of anhydrous aluminium chloride
Solvents, except LR grade, were distilled before use. Research
chemicals such as acetophenones and halo-esters were either pur-
chased from Lancaster/Aldrich Co. and used without further purifi-
2 4 4 6
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 4 4 2 – 2 4 5 0

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(7.33 g, 55.00 mmol) in dichloromethane (75 mL) under an argon
atmosphere at 0–5 °C. After stirring the reaction mixture for 0.5 h
at this temperature, indan (5.2 mL, 42.30 mmol) was slowly added
over a period of 15 min. After maintaining the reaction mixture at
this temperature for 2 h, it was allowed to stir at room temperature
for the next 3 h and poured over crushed ice. It was extracted with
dichloromethane (3 × 50 mL), and the combined organic layers after
washing with water were dried (anhydrous Na2SO4) and evaporated
to yield an oil which was purified by column chromatography using
1% ethyl acetate–petroleum ether to afford the viscous liquid of the
title compound (5.42 g, 80%). This compound was used in the next
step without further purification. Bp 85 °C (0.5 mm) [lit.21 80 °C
(0.2 mm)]. IR (neat) 2954, 1680, 1608, 1424, 1269 cm−1. 1H NMR
(CDCl3):  7.82 (s, 1H, Ar), 7.76 (d, J = 7.8 Hz, 1H, Ar), 7.29 (d,
J = 8.0 Hz, 1H, Ar), 2.95 (t, J = 7.4 Hz, 4H, 2 × CH2), 2.59 (s, 3H,
COCH3), 2.21–2.05 (m, 2H, CH2). MS (DIP) 160 (M+), 145 (M −
15, 100%)+, 117, 115, 91.
Compound 6. This was prepared from 4-methoxyacetophenone
and ethyl trifluoroacetate following steps 2 and 3. Yield 85%. Mp
188–189 °C. IR (neat) 3311, 1609 cm−1. 1H NMR (CDCl3):  7.97 (d,
J = 8.6 Hz, 1H,Ar), 7.62 (d, J = 1.8 Hz, 1H,Ar), 7.25 (d, J = 8.0 Hz,
1H, Ar), 7.15 (d, J = 8.4 Hz, 2H, Ar), 6.88 (d, J = 8.6 Hz, 2H, Ar),
6.71 (s, 1H, C4H), 5.43 (bs, 2H, SO2NH2), 5.01 (d, J = 3.8 Hz, 2H,
CH2O), 3.83 (s, 3H, OCH3), 2.65 (bs, 1H, OH). 13C NMR (DMSO-
d6):  160.1, 145.1, 142.8, 142.0 (q, J = 35.5 Hz, 1C, C-4), 141.5,
139.9, 130.3 (2C), 128.1, 123.9, 123.3, 121.5 (q, J = 267.4 Hz,
1C, CF3), 120.6, 114.4 (2C), 105.9, 59.3, 55.3. MS (CI method)
428 (M + 1)+, 409, 392, 346. HPLC (System 1) 98.35%. Anal.
(C18H16F3N3O4S) C: calc., 50.58; found, 50.27; H: calc., 3.77; found,
3.92; N: calc., 9.83; found, 9.49.
Compound 8. The compound was obtained by the acidic hydro-
lysis of the corresponding acetamide (not reported here), prepared
from N1-(4-acetylphenyl) acetamide and ethyl trifluoroacetate fol-
lowing steps 2 and 3. Yield 65%. Mp 210–212 °C. IR (KBr) 3383,
3251, 1615, 1478 cm−1. 1H NMR (DMSO-d6):  7.85 (d, J = 8.2 Hz,
1H, Ar), 7.75 (s, 1H, Ar), 7.60 (bs, 1H, OH), 7.25 (d, J = 8.2 Hz,
1H, Ar), 6.99 (s, 1H, C4H), 6.93 (d, J = 8.2 Hz, 2H, Ar), 6.51 (d,
J = 8.2 Hz, 2H,Ar), 5.49 (bs, 2H, SO2NH2), 4.90 (s, 2H, CH2O). MS
(CI method) 412 (M+), 395, 331, 303. HPLC (System 1) 96.65%;
(System 2) 97.51%. Anal. (C17H15F3N4O3S) C: calc., 49.51; found,
49.79; H: calc., 3.67; found, 3.88; N: calc., 13.59; found, 13.22.
Step 2. General procedure for the preparation of 1-
phenyl-1,3-butanediones 3. Representative preparation of
1-(2,3-dihydro-1H-5-indenyl)-4,4,4-trifluoro-1,3-butanedione.
1-(2,3-Dihydro-1H-5-indenyl)-1-ethanone (5.0 g, 31.25 mmol),
prepared in step 1, was dissolved in 25 mL of dry DMF under an
argon atmosphere and 60% NaH (1.56 g, 39.06 mmol) was added in
three lots whilst maintaining the temperature between −5 and 0 °C.
After stirring at this temperature for 0.5 h, ethyl trifluoroacetate
(4.64 mL, 39.06 mmol) was injected into the reaction mixture and
was allowed to stir at ambient temperature for 4–5 h. The reaction
mixture was poured into ice water, acidified with 2 M HCl and
extracted with ethyl acetate. The combined organic layers were
washed with water, dried (anhydrous Na2SO4) and evaporated to
obtain a residue which was finally purified by column chromato-
graphy using 2% ethyl acetate–petroleum ether to yield a light
yellow solid of the title compound (5.2 g, 65%) which was used as
such for the next step. Mp 41–42 °C. IR (KBr) 3475, 2958, 1570,
1459 cm−1. 1H NMR (CDCl3):  7.81 (s, 1H,Ar), 7.75 (d, J = 8.0 Hz,
1H, Ar), 7.34 (d, J = 7.6 Hz, 1H, Ar), 6.55 (s, 1H, CH), 2.98 (t,
J = 7.2 Hz, 4H, 2 × CH2), 2.22–2.10 (m, 2H, CH2). MS (DIP) 256
(M+), 228, 187 (100%), 145, 115, 91.
Compound 9. This compound was prepared from 4-methyl-
aminoacetophenone and ethyl trifluoroacetate following steps 2
and 3.Yield 78%. Mp 188–190 °C. IR (KBr) 3414, 3301, 1615 cm−1.
1H NMR (CD3OD):  7.95 (d, J = 8.8 Hz, 1H, Ar), 7.83 (s, 1H, Ar),
7.26 (dd, J = 8.4, 1.8 Hz, 1H, Ar), 7.00 (d, J = 8.8 Hz, 2H, Ar), 6.76
(s, 1H, C4H), 6.53 (d, J = 8.8 Hz, 2H, Ar), 5.00 (s, 2H, CH2O), 2.74
(s, 3H, CH3). 13C NMR (DMSO-d6):  150.6, 146.3, 142.80, 142.4
(q, J = 35.6 Hz, 1C, C-4), 142.1, 139.8, 129.8 (2C), 128.1, 124.0,
123.4, 121.5 (q, J = 267.1 Hz, 1C, CF3), 114.8, 111.6 (2C), 104.7,
59.6, 29.4. MS (CI method) 426 (M+), 408, 345. HPLC (System 1)
99.30%. Anal. (C18H17F3N4O3S) C: calc., 50.70; found, 51.05; H:
calc., 4.02; found, 3.82; N: calc., 13.14; found, 12.89.
Compound 10. Preparation was from 4-dimethylaminoaceto-
phenone and ethyl trifluoroacetate following steps 2 and 3. Yield
75%. Mp 178–180 °C. IR (KBr) 3418, 3318, 1611, 1477 cm−1. 1H
NMR (CDCl3):  7.96 (d, J = 8.6 Hz, 1H, Ar), 7.65 (d, J = 2.0 Hz,
1H, Ar), 7.27–7.23 (m, 1H, Ar), 7.05 (d, J = 8.8 Hz, 2H, Ar), 6.65
(d, J = 8.6 Hz, 2H, Ar), 6.61 (s, 1H, C4H), 5.43 (bs, 2H, SO2NH2),
Step3.Representativepreparationof4-[5-(2,3-dihydro-1H-5-
indenyl)-3-trifluoromethyl-1H-1-pyrazolyl]-2-hydroxymethyl-
1-benzenesulfonamide 16. 3-Hydroxymethyl-4-sulfamoylphenyl
hydrazine hydrochloride 4 (5.08 g, 23.43 mmol) was dissolved in
MeOH (30 mL) under an argon atmosphere and acidified to pH 1–2
using IPA–HCl. The reaction mixture was stirred at room tempera-
ture for 0.5 h and the solvent was completely removed under high
vacuum at 40–50 °C. The solid obtained was dissolved in absolute
alcohol (50 mL) and an ethanolic solution of the above-prepared
1-(2,3-dihydro-1H-5-indenyl)-4,4,4-trifluoro-1,3-butanedione
(5.0 g, 19.53 mmol) was added at room temperature. After heating
at 50–60 °C for 10–12 h under an argon atmosphere, the reaction
mixture was concentrated, stirred with ice-cold water and extracted
with ethyl acetate. The combined organic layers were washed with
water, dried and evaporated. The residue obtained was purified
by column chromatography using 25% ethyl acetate–petroleum
ether to yield a gummy material which upon trituration with ethyl
acetate–toluene afforded the title compound 16 as a colorless solid
5.03 (s, 2H, CH2O), 2.99 (s, 6H, 2 × CH3), 2.65 (bs, 1H, OH). 13
C
NMR (DMSO-d6):  150.5, 146.0, 142.8, 142.6 (q, J = 36.4 Hz,
1C, C-4), 141.9, 139.8, 129.5 (2C), 128.8, 123.9, 123.3, 120.5 (q,
J = 267.4 Hz, 1C, CF3), 115.0, 111.8 (2C), 104.9, 59.4, 39.7 (2C).
MS (CI method) 440 (M+), 421, 358. HPLC (System 1) 96.77%;
(System 2) 97.41%. Anal. (C19H19F3N4O3S) C: calc., 51.81; found,
51.93; H: calc., 4.35; found, 4.66; N: calc., 12.72; found, 12.75.
Compound 11. Preparation was from 4-ethylaminoacetophenone
and ethyl trifluoroacetate following steps 2 and 3. Yield 72%. Mp
171–172 °C. IR (KBr) 3406, 1616, 1451 cm−1. 1H NMR (CDCl3):
 8.00 (d, J = 8.2 Hz, 1H, Ar), 7.67 (s, 1H, Ar), 7.30 (d, J = 8.8 Hz,
1H, Ar), 7.02 (d, J = 8.8 Hz, 2H, Ar), 6.68 (s, 1H, C4H), 6.56 (d,
J = 8.8 Hz, 2H, Ar), 5.54 (bs, 2H, SO2NH2), 5.05 (d, J = 3.8 Hz,
2H, CH2O), 3.18 (q, J = 7.4 Hz, 2H, CH2CH3), 2.65 (bs, 1H, OH),
1.30 (t, J = 6.8 Hz, 3H, CH2CH3). 13C NMR (DMSO-d6):  149.6,
146.2, 142.6, 142.2 (q, J = 36.2 Hz, 1C, C-4), 141.9, 139.6, 129.6
(2C), 127.9, 123.8, 123.2, 121.2 (q, J = 267.1 Hz, 1C, CF3), 114.6,
111.7 (2C), 104.6, 59.4, 37.1, 14.3. MS (CI method) 441 (M + 1)+,
423. HPLC (System 2) 97.80%. Anal. (C19H19F3N4O3S) C: calc.,
51.81; found, 51.79; H: calc., 4.35; found, 4.48; N: calc., 12.72;
found, 12.87.
1
(5.55 g, 65%). Mp 118–120 °C. IR (KBr) 3323, 1602 cm−1. H
NMR (CDCl3):  8.00 (d, J = 8.4 Hz, 1H, Ar), 7.69 (s, 1H, Ar),
7.35–7.25 (m, 2H, Ar), 7.14 (s, 1H, Ar), 6.96 (d, J = 8.0 Hz, 1H,
Ar), 6.74 (s, 1H, C4H), 5.40 (bs, 2H, SO2NH2), 5.00 (s, 2H, CH2O),
3.00–2.80 (m, 4H, 2 × CH2), 2.60 (bs, 1H, OH), 2.20–2.00 (m, 2H,
CH2). 13C NMR (DMSO-d6):  145.7, 145.4, 144.6, 142.7, 142.2
(q, J = 37.3 Hz, 1C, C-4), 141.5, 139.8, 128.0, 126.9, 126.1, 125.8
(q, J = 266.5 Hz, 1C, CF3), 124.7, 124.6, 123.8, 123.2, 106.1,
59.4, 32.3 (2C), 24.9. MS (CI method) 437 (M+), 419, 338. HPLC
(System 1) 96.63%; (System 2) 97.43%. Anal. (C20H18F3N3O3S)
C: calc., 54.92; found, 55.25; H: calc., 4.15; found, 3.91; N: calc.,
9.61; found, 9.75.
Compound 12. Preparation was from 3,4-dichloroacetophenone
and ethyl trifluoroacetate following steps 2 and 3. Yield 60%. Mp
148–150 °C. IR (KBr) 3454, 3241, 1603 cm−1. 1H NMR (CDCl3):
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 4 4 2 – 2 4 5 0
2 4 4 7

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Compound 19. Preparation was from 4-ethoxyacetophenone
 7.99 (d, J = 8.4 Hz, 1H, Ar), 7.63 (d, J = 2.0 Hz, 1H, Ar),
7.44–7.39 (m, 2H, Ar), 7.22 (dd, J = 8.6, 2.2 Hz, 1H, Ar), 6.93 (dd,
J = 8.0, 2.0 Hz, 1H, Ar), 6.78 (s, 1H, C4H). 5.43 (bs, 2H, SO2NH2),
5.0 (d, J = 3.8 Hz, 2H, CH2O), 2.74 (bs, 1H, OH). MS (CI method)
466 (M+), 447, 430, 384. HPLC (System 1) 96.43%; (System 2)
97.85%. Anal. (C17H12Cl2F3N3O3S) C: calc., 43.79; found, 43.55; H:
calc., 2.59; found, 2.65; N: calc., 9.01; found, 9.25.
and ethyl difluoroacetate following steps 2 and 3. Yield 76%. Mp
1
150–152 °C. IR (KBr) 3453, 3320, 3214, 1607, 1461 cm−1. H
NMR (CDCl3):  7.97 (d, J = 8.5 Hz, 1H, Ar), 7.58 (d, J = 2.0 Hz,
1H, Ar), 7.24–7.10 (m, 4H, Ar), 6.89–6.68 (m, 2H, Ar), 6.66 (t,
J = 54.0 Hz, 1H, CHF2), 5.45 (bs, 2H, SO2NH2), 5.00 (s, 2H,
CH2O), 4.03 (q, J = 7.0 Hz, 2H, CH2CH3), 2.78 (bs, 1H, OH), 1.42
(t, J = 7.0 Hz, 3H, CH2CH3). 13C NMR (DMSO-d6):  159.1, 147.2
(t, J = 28.8 Hz, 1C, C-4), 144.6, 142.6, 141.8, 139.3, 130.2 (2C),
128.0, 123.6, 122.8, 121.0, 114.7 (2C), 111.4 (t, J = 231.4 Hz, 1C,
CHF2), 105.2, 63.3, 59.3, 14.5. MS (CI method) 424 (M + H)+,
406 (100%). HPLC (System 1) 99.89%. Anal. (C19H19F2N3O4S)
C: calc., 53.90; found, 54.12; H: calc., 4.52; found, 4.91; N: calc.,
9.92; found, 9.59.
Compound 13. Preparation was from 3,4-dimethylaceto-
phenone and ethyl trifluoroacetate following steps 2 and 3. Yield
58%. Mp 132–134 °C. IR (KBr) 3522, 1605, 1472 cm−1. 1H NMR
(CDCl3):  7.95 (d, J = 8.4 Hz, 1H, Ar), 7.66 (s, 1H, Ar), 7.25
(d, J = 7.8 Hz, 1H, Ar), 7.16 (m, 2H, Ar), 6.86 (d, J = 7.4 Hz,
1H, Ar), 6.74 (s, 1H, C4H), 5.50 (bs, 2H, SO2NH2), 5.03 (s, 2H,
CH2O), 2.80 (bs, 1H, OH), 2.28 (s, 3H, CH3), 2.24 (s, 3H, CH3).
13C NMR (DMSO-d6):  145.4, 142.8, 142.2 (q, J = 37.5 Hz, 1C,
C-4), 141.5, 140.0, 137.9, 137.1, 129.8 (2C), 128.0, 126.2, 125.8,
123.9, 123.3, 121.5 (q, J = 266.8 Hz, 1C, CF3), 106.1, 59.4, 19.3,
19.2. MS (CI method) 425 (M+), 407, 379, 327. HPLC (System 1)
97.55%; (System 2) 97.45%. Anal. (C19H18F3N3O3S) C: calc.,
53.64; found, 53.48; H: calc., 4.26; found, 4.68; N: calc., 9.88;
found, 10.21.
Compound 20. Preparation was from 4-chloroacetophenone
and ethyl difluoroacetate following steps 2 and 3. Yield 72%. Mp
130–132 °C. IR (KBr) 3329, 1601 cm−1. 1H NMR (CDCl3):  8.00
(d, J = 8.6 Hz, 1H,Ar), 7.59 (s, 1H,Ar), 7.36 (d, J = 8.4 Hz, 2H,Ar),
7.25 (s, 1H, Ar), 7.17 (d, J = 7.0 Hz, 2H, Ar), 6.75 (t, J = 54.8 Hz,
1H, CHF2), 6.74 (s, 1H, C4H), 5.44 (bs, 2H, SO2NH2), 5.01 (s, 2H,
CH2O), 2.80 (bs, 1H, OH). MS (CI method) 413 (M+), 393 (100%),
378, 332. HPLC (System 1) 98.30%; (System 2) 98.50%. Anal.
(C17H14ClF2N3O3S) C: calc., 49.34; found, 49.55; H: calc., 3.41;
found, 3.77; N: calc., 10.15; found, 10.39.
Compound 14. Preparation was from 3-methyl-4-methoxy
acetophenone and ethyl trifluoroacetate following steps 2 and 3.
Yield 80%. Mp 156–158 °C. IR (KBr) 3422, 3317, 1610, 1472 cm−1.
1H NMR (CDCl3):  7.98 (d, J = 8.6 Hz, 1H, Ar), 7.65 (d,
J = 2.2 Hz, 1H, Ar), 7.24 (d, J = 8.4 Hz, 1H, Ar), 7.07 (s, 1H, Ar),
6.90 (d, J = 2.0 Hz, 1H, Ar), 6.76 (d, J = 8.4 Hz, 1H, Ar), 6.70 (s,
1H, C4H), 5.42 (bs, 2H, SO2NH2), 5.00 (s, 2H, CH2O), 3.85 (s, 3H,
OCH3), 2.65 (bs, 1H, OH), 2.19 (s, 3H, CH3). 13C NMR (DMSO-d6):
 158.1, 145.3, 142.6, 142.1 (q, J = 37.6 Hz, 1C, C-4), 141.5, 139.8,
130.9, 127.9, 127.8, 126.3, 123.8, 123.2, 122.2 (q, J = 268.2 Hz,
1C, CF3), 120.1, 110.4, 105.7, 59.2, 55.4, 15.9. MS (CI method)
441 (M+), 422, 343. HPLC (System 1) 99.00%; (System 2) 98.89%.
Anal. (C19H18F3N3O4S) C: calc., 51.70; found, 50.98; H: calc., 4.11;
found, 3.88; N: calc., 9.52; found, 9.01.
Compound 21. Preparation was from 4-bromoacetophenone
and ethyl difluoroacetate following steps 2 and 3. Yield 68%. Mp
1
210–211 °C. IR (KBr) 3373, 1723, 1600, 1451 cm−1. H NMR
(CDCl3):  7.98 (d, J = 8.6 Hz, 1H, Ar), 7.59 (d, J = 1.8 Hz, 1H,
Ar), 7.51 (d, J = 8.2 Hz, 2H, Ar), 7.20 (dd, J = 6.4, 2.0 Hz, 1H,
Ar), 7.05 (d, J = 9.4 Hz, 2H, Ar), 6.76 (t, J = 54.8 Hz, 1H, CHF2),
6.74 (s, 1H, C4H), 5.49 (bs, 2H, SO2NH2), 5.02 (s, 2H, CH2O), 4.25
(bs, 1H, OH). MS (CI method) 460 (M + 2)+, 440, 378. HPLC
(System 1) 98.60%; (System 2) 98.93%. Anal. (C17H14BrF2N3O3S)
C: calc., 44.56; found, 44.80; H: calc., 3.08; found, 3.32; N: calc.,
9.17; found, 9.51.
Compound 15. Preparation was from 2,3-dihydrobenzo[b]furan
and ethyl trifluoroacetate following steps 1–3. Yield 77%. Mp
140–142 °C. IR (KBr) 3337, 1614, 1469 cm−1. 1H NMR (CDCl3):
 8.02 (d, J = 8.2 Hz, 1H, Ar), 7.68 (d, J = 1.8 Hz, 1H, Ar), 7.26
(d, J = 7.0 Hz, 1H, Ar), 7.10 (s, 1H, Ar), 6.97 (d, J = 6.6 Hz, 1H,
Ar), 6.77 (d, J = 8.4 Hz, 1H, Ar), 6.71 (s, 1H, C4H), 5.46 (bs, 2H,
SO2NH2), 5.05 (s, 2H, CH2O), 4.65 (t, J = 8.8 Hz, 2H, OCH2CH2),
3.22 (t, J = 8.4 Hz, 2H, OCH2CH2), 2.77 (bs, 1H, OH). MS (CI
method) 439 (M+), 421, 356. HPLC (System 1) 98.93%. Anal.
(C19H16F3N3O4S) C: calc., 51.94; found, 51.83; H: calc., 3.67; found,
3.79; N: calc., 9.56; found, 9.80.
Compound 22. Preparation was from 3-chloro-4-fluoroaceto-
phenone and ethyl difluoroacetate following steps 2 and 3. Yield
69%. Mp 122–124 °C. IR (KBr) 3349, 1602, 1462, 1424 cm−1. 1H
NMR (CDCl3):  8.03 (d, J = 8.5 Hz, 1H, Ar), 7.61 (s, 1H, Ar),
7.42–7.05 (m, 4H, Ar), 6.77 (s, 1H, C4H), 6.77 (t, J = 53.2 Hz, 1H,
CHF2), 5.05 (d, J = 3.4 Hz, 2H, CH2O). MS (CI method) 431 (M+),
412, 396, 350. HPLC (System 1) 99.23%. Anal. (C17H13ClF3N3O3S)
C: calc., 47.29; found, 46.95; H: calc., 3.03; found, 3.28; N: calc.,
9.73; found, 9.52.
Compound 23. Preparation was from 4-methylaminoaceto-
phenone and ethyl difluoroacetate following steps 2 and 3.
Yield 71%. Mp 149–151 °C. IR 3431, 3323, 1617 cm−1. 1H
NMR (CD3OD + CDCl3):  7.95 (d, J = 8.4 Hz, 1H, Ar), 7.73 (d,
J = 2.4 Hz, 1H, Ar), 7.24 (dd, J = 8.4, 2.4 Hz, 1H, Ar), 7.01 (d,
J = 8.4 Hz, 2H, Ar), 6.64 (t, J = 54.8 Hz, 1H, CHF2), 6.56 (s, 1H,
C4H), 6.52 (d, J = 8.8 Hz, 2H, Ar), 5.00 (s, 2H, CH2O), 2.82 (s,
3H, CH3). 13C NMR (DMSO-d6):  150.4, 146.6 (t, J = 28.8 Hz,
1C, C-4), 145.8, 142.5, 142.3, 139.2, 129.6 (2C), 127.9, 123.6,
122.9, 115.4, 111.5 (t, J = 231.4 Hz, 1C, CHF2), 111.4 (2C), 104.2,
59.4, 29.4. MS (CI method) 408 (M+), 389 (100%), 356. HPLC
(System 1) 98.20%; (System 2) 97.60%. Anal. (C18H18F2N4O3S)
C: calc., 52.94; found, 53.59; H: calc., 4.44; found, 4.75; N: calc.,
13.72; found, 12.95.
Compound 17. Preparation was from 1-phenylazolane and ethyl
trifluoroacetate following steps 1–3. Yield 67%. Mp 186–188 °C.
1
IR (KBr) 3413, 1611, 1450 cm−1. H NMR (CDCl3):  7.96 (d,
J = 8.8 Hz, 1H, Ar), 7.67 (s, 1H, Ar), 7.26 (s, 1H, Ar), 7.02 (d,
J = 8.2 Hz, 2H, Ar), 6.65 (s, 1H, C4H), 6.48 (d, J = 8.2 Hz, 2H,
Ar), 5.45 (bs, 2H, SO2NH2), 5.03 (s, 2H, CH2O), 3.29–3.20 (m, 4H,
2 × NCH2CH2), 2.05–2.00 (m, 4H, 2 × NCH2CH2). MS (CI method)
466 (M+, 100%), 448, 384, 356. HPLC (System 1) 99.75%. Anal.
(C21H21F3N4O3S) C: calc., 54.07; found, 53.88; H: calc., 4.54; found,
4.91; N: calc., 12.01; found, 12.04.
Compound 18. Preparation was from 3-methoxyacetophenone
and ethyl difluoroacetate following steps 2 and 3. Yield 68%. IR
1
(KBr) 3249, 1603, 1481, 1427 cm−1. H NMR (CDCl3):  7.94
Compound 24. Preparation was from 4-dimethylaminoaceto-
(d, J = 8.3 Hz, 1H, Ar), 7.61 (s, 1H, Ar), 7.33–7.18 (m, 2H, Ar),
7.08–6.77 (m, 3H, Ar), 6.77 (t, J = 54.6 Hz, 1H, CHF2), 6.62 (s,
1H, C4H), 5.45 (bs, 2H, SO2NH2), 5.00 (d, J = 3.8 Hz, 2H, CH2O),
3.78 (s, 3H, OCH3), 2.75 (bs, 1H, OH). MS (CI method) 409 (M+),
389, 356, 328. HPLC (System 1) 99.31%. Anal. (C18H17F2N3O4S)
C: calc., 52.81; found, 52.93; H: calc., 4.19; found, 4.50; N: calc.,
10.26; found, 10.53.
phenone and ethyldifluoro acetate following steps 2 and 3. Yield
1
76%. Mp 89–91 °C. IR (KBr) 3460, 3320, 3245, 1618 cm−1. H
NMR (CD3OD):  7.98 (d, J = 6.8 Hz, 1H, Ar), 7.80 (s, 1H, Ar),
7.25 (dd, J = 1.6, 4.8 Hz, 1H, Ar), 7.04 (d, J = 7.8 Hz, 2H, Ar),
6.77 (t, J = 54.8 Hz, 1H, CHF2), 6.75 (d, J = 6.6 Hz, 2H, Ar), 6.62
(s, 1H, C4H), 5.02 (s, 2H, CH2O), 2.95 (s, 6H, 2 × CH3). MS (CI
method) 422 (M+), 405, 340, 312. HPLC (System 1) 98.89%. Anal.
2 4 4 8
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 4 4 2 – 2 4 5 0

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(C19H20F2N4O3S) C: calc., 54.02; found, 53.87; H: calc., 4.77; found,
4.58; N: calc., 13.26; found, 13.52.
In-vitro enzyme assay (ref. 23)
The microsomal fraction of ram seminal vesicles was used as a
source of the COX-1 enzyme, and microsomes from Sf-9 cells
infected with baculovirus expressing human COX-2 cDNA were
used as a source of the COX-2 enzyme in measuring the inhibi-
tory activity by the TMPD method. The assay mixture (1000 L)
contained 100 M Tris pH 8.0, 3 M EDTA, 15 M hematin, 150
units enzyme and 8% DMSO. The mixture was incubated at 25 °C
for 15 min before initiation of enzyme reaction in the presence of
the compound/vehicle. The reaction was initiated by the addition
of 100 M arachidonic acid and 120 M TMPD, and the veloc-
ity of TMPD oxidation over the first 25 s was monitored at 603
nm. The IC50 values were calculated using non-linear regression
analysis of percentage inhibitions.
Compound 25. Preparation was from 3,4-dimethylacetophenone
and ethyl difluoroacetate following steps 2 and 3. Yield 70%. Mp
140–142 °C. IR (KBr) 3403, 1603 cm−1. 1H NMR (CDCl3):  7.93
(d, J = 8.6 Hz, 1H, Ar), 7.60 (s, 1H, Ar), 7.26–7.10 (m, 2H, Ar),
7.05 (s, 1H, Ar), 6.85 (d, J = 7.4 Hz, 1H, Ar), 6.77 (s, 1H, C4H),
6.69 (t, J = 54.8 Hz, 1H, CHF2), 5.55 (s, 2H, SO2NH2), 5.00 (s, 2H,
CH2O), 2.28 (s, 3H, CH3), 2.23 (s, 3H, CH3), 1.92 (bs, 1H, OH). MS
(CI method) 407 (M+), 389, 361. HPLC (System 1) 97.41%. Anal.
(C19H19F2N3O3S) C: calc., 56.01; found, 56.12; H: calc., 4.70; found,
4.89; N: calc., 10.31; found, 10.56.
Compound 26. Preparation was from 3-methyl-4-methoxy-
acetophenone and ethyl difluoroacetate following steps 2 and 3.
Yield 65%. Mp 164–166 °C. IR (Neat) 3452, 3271, 1635, 1605,
In-vivo screening
1
1462 cm−1. H NMR (CDCl3 + DMSO-d6):  7.95 (d, J = 8.4 Hz,
Carrageenan-induced rat paw edema (ref. 24). Male wistar
rats (120–140 g) were fasted for 16 h before starting the experiment.
Compounds were suspended in 0.25% CMC and administered
orally in a volume of 10 mL kg−1. After 2 h of dosing, 50 L of 1%
-carrageenan, suspended in saline, was injected into the plantar
aponeurosis of the right paw. The paw volume was measured 3 h
before and after the carrageenan injection using a plethysmometer
(Ugo-Basile, Italy). The paw edema was compared with the vehicle
control group, and the percentage inhibition was calculated. ED50s
were calculated using a linear regression plot.
1H, Ar), 7.63 (s, 1H, Ar), 7.18 (d, J = 6.6 Hz, 1H, Ar), 7.07 (s, 1H,
Ar), 6.98 (d, J = 8.8 Hz, 1H, Ar), 6.94 (d, J = 7.2 Hz, 1H, Ar), 6.77
(t, J = 54.8 Hz, 1H, CHF2), 6.64 (s, 1H, C4H), 6.45 (s, 2H, SO2NH2),
4.95 (s, 2H, CH2O), 3.84 (s, 3H, OCH3), 2.38 (bs, 1H, OH), 2.18
(s, 3H, CH3). MS (CI method) 423 (M+), 403. HPLC (System 2)
97.89%. Anal. (C19H19F2N3O4S) C: calc., 53.90; found, 53.81; H:
calc., 4.52; found, 4.83; N: calc., 9.92; found, 10.21.
Compound 27. Preparation was from 2,3-dihydrobenzo[b]furan
and ethyl difluoroacetate following steps 1–3. Yield 59%. Mp
1
76–78 °C. IR (KBr) 3374, 1602 cm−1. H NMR (CDCl3):  7.93
Endotoxin-induced pyresis in rats (ref. 25). Male wistar rats
(150–170 g) were fasted for 16 h before starting the experiment,
and the baseline rectal temperature was recorded with a flexible
temperature probe (YSI series-400) connected to a digital thermo-
meter. At time zero, the rats were injected with 0.36 mg kg−1 of
lipopolysaccharide (LPS, Sigma Chemical Co., St. Louis, USA)
intra-peritoneally, and the rectal temperatures were recorded after 5
and 7 h. The test compounds were administered 5 h after LPS injec-
tion to determine their antipyretic potential. The percentage reversal
of pyrexia was calculated by taking the ratio of the difference in
temperature at 5th and 7th hour and the baseline of the treated and
the control groups.
(d, J = 8.4 Hz, 1H, Ar), 7.59 (d, J = 1.6 Hz, 1H, Ar), 7.20 (d,
J = 6.6 Hz, 1H, Ar), 7.05 (s, 1H, Ar), 6.92 (d, J = 8.0 Hz, 1H, Ar),
6.71 (d, J = 6.6 Hz, 1H, Ar), 6.66 (t, J = 55.4 Hz, 1H, CHF2), 6.47
(s, 1H, C4H), 5.53 (bs, 2H, SO2NH2), 4.99 (s, 2H, CH2O), 4.61 (t,
J = 8.8 Hz, 2H, OCH2CH2), 3.18 (t, J = 8.8 Hz, 2H, OCH2CH2), 3.05
(bs, 1H, OH). MS (CI method) 421 (M+), 403 (100%), 322. HPLC
(System 2) 99.23%. Anal. (C19H17F2N3O4S) C: calc., 54.15; found,
54.22; H: calc., 4.07; found, 3.82; N: calc., 9.97; found, 10.21.
Compound 28. Preparation was from indan and ethyl difluoro-
acetate following steps 1–3. Yield 48%. Mp 99–100 °C. IR (KBr)
3384, 1602 cm−1. 1H NMR (CDCl3):  7.92 (d, J = 7.6 Hz, 1H, Ar),
7.63 (s, 1H,Ar), 7.30–7.05 (m, 3H,Ar), 6.92 (d, J = 7.4 Hz, 1H,Ar),
6.67 (t, J = 54.8 Hz, 1H, CHF2), 6.50 (s, 1H, C4H), 5.50 (bs, 2H,
SO2NH2), 5.00 (s, 2H, CH2O), 3.00–2.82 (m, 4H, 2 × CH2), 2.50
(bs, 1H, OH), 2.20–2.00 (m, 2H, CH2). MS (CI method) 419 (M+),
401 (100%), 320. HPLC (System 2) 97.15%.Anal. (C20H19F2N3O3S)
C: calc., 57.27; found, 57.31; H: calc., 4.57; found, 4.80; N: calc.,
10.02; found, 10.23.
Carrageenan-induced rat paw hyperalgesia (Randal–Selitto
method, ref. 26). Hyperalgesia was induced in the hind paw of male
wistar rats (150–170 g) by intraplantar injection of carrageenan
(2 mg per paw). Test compounds were dosed after 2 h from the
carrageenan injection. The vocalization response to compression
of the carrageenan-injected paw was measured 1 h later by anal-
gesiometer (Ugo-Basile, Italy). For normal response, one group of
animals was given an intraplantar injection of saline. The percent
increase in pain was calculated as difference in threshold in the
treated group versus the control group. ED50s were calculated using
a linear regression plot.
Computational methods
All molecular modeling studies were performed using the SYBYL
program package, version 6.930 on Silicon Graphics Octane 2 work-
station with the IRIX 6.5 operating system. The crystal structure of
murine COX-2 (6COX, 87% homology with human) with SC-558
was used in this study.15 Owing to the position of the NSAID bind-
ing site, only one monomer was considered in the calculation.
Docked molecules were pre-aligned with respect to SC-558 in
the active site of COX-2 using FIT-ATOMS in SYBYL. A manual
docking procedure was adopted where hydrogen atoms were added
to the proteins while all the residues were considered in the neutral
form. The haeme, bound at the peroxidase active site, was removed
since the majority of the molecules exceeded the cutoff radius.
The active site was minimized (5.5 Å around the ligand) using the
MMFF9431 force field. The ligand–enzyme assembly was then
subjected to a molecular dynamics (MD) treatment at a constant
temperature of 300 K for 100 ps with a time step of 1 fs using a
distance-dependent dielectric constant 4r. The resultant minimum
energy structures obtained after molecular dynamics studies were
re-minimized using the MMFF94 force field and were analyzed for
ligand–receptor interactions in the active site.
Rat adjuvant-induced arthritis (ref. 27). Arthritis was induced
by subplantar injection of 0.5 mg of Mycobacterium butyricum in
light mineral oil into the right hind footpad of male wistar rats (140–
160 g). Treatment was carried out in either a therapeutic or pro-
phylactic manner. In the therapeutic treatment, dosing commenced
after 14 days from adjuvant injection, i.e. after establishment of
the disease in culled groups, and continued until the 30th day. Foot
volumes of both injected and contralateral paws were determined on
various days. Radiographs were taken on days 14 and 30 for all the
groups. In the prophylactic treatment, dosing started on day 0 (day
of adjuvant injection) and continued for 21 days. Foot volume, body
weight and food consumption were determined on various days.
Radiographs were taken on the last day of treatment. To assess the
tibiotarsal joint integrity, radiographic scores were assigned based
on soft tissue swelling, bone erosion, periosteal reaction and joint
space destruction on an arbitrary scale of 0–3 by a radiologist who
was blind to the treatment. The ED50s were calculated at the end
O r g . B i o m o l . C h e m . , 2 0 0 4 , 2 , 2 4 4 2 – 2 4 5 0
2 4 4 9

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13 D. Mukherjee, S. E. Nissen and E. J. Topol, J. Am. Med. Assoc., 2001,
of the treatment compared with the control group using a linear
regression plot, based on foot volume of the injected paw.
286, 954–959.
14 R. Xavier, Drugs Today, 1996, 32, 365–384; G. Cignarella, P. Vianello,
F. Berti and G. Rossoni, Eur. J. Med. Chem., 1996, 31, 359–364.
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Stegeman, J. Y. Pak, D. Gildehaus, J. M. Miyashiro, T. D. Penning,
K. Seibert, P. C. Isakson and W. C. Stallings, Nature, 1996, 384,
644–648.
Single dose pharmacokinetic studies. All of the studies were
carried out in male wistar rats obtained from the National Institute
of Nutrition, Hyderabad, India. The animals (200–225 g) were
fasted for 12 h before starting the experiment and had free access
to water throughout. The animals were fed 3 h after drug admin-
istration. The animals were dosed at 10 mg kg−1 (po) as a 0.25%
CMC suspension, and 0.4 mL blood samples were collected into
heparinized microfuge tubes at pre-determined time points from
the retro-orbital plexus. The samples were analyzed by HPLC after
a suitable extraction procedure, and plasma concentration versus
time profiles were generated for the interesting compounds along
with celecoxib. Pharmacokinetic parameters were calculated by
non-compartmental model analysis.
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Acknowledgements
The authors are thankful to Dr K. Anji Reddy and Professorr
J. Iqbal for their constant support and encouragement, and to
Analytical Research group, DRL, for spectral analysis.
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