Anti-inflammatory Exploration of Sulfonamide Containing Diaryl Pyrazoles with Promising COX-2 Selectivity and Enhanced Gastric Safety Profile

Article abstract of DOI:10.1002/jhet.3118

Novel sulfonamide containing diaryl pyrazoles were synthesized and were subsequently tested for their in vitro cyclooxygenase inhibitory assay. Compounds that showed promising in vitro COX-2 IC₅₀ values and selectivity indices were then evaluat

Full text of DOI:10.1002/jhet.3118

Month 2018
Anti-inflammatory Exploration of Sulfonamide Containing Diaryl
Pyrazoles with Promising COX-2 Selectivity and Enhanced Gastric
Safety Profile
a
b
Laxmikant S. Pavase,a
*
Dhananjay V. Mane, and Kamalkishor G. Baheti
aDepartment of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad 431004, MS, India
bY. B. Chavan College of Pharmacy, Rauza Baugh, Aurangabad 431001, MS, India
*
E-mail: laxmikantpavase@rediffmail.com
Received August 20, 2017
DOI 10.1002/jhet.3118
Published online 00 Month 2018 in Wiley Online Library (wileyonlinelibrary.com).
Novel sulfonamide containing diaryl pyrazoles were synthesized and were subsequently tested for their
in vitro cyclooxygenase inhibitory assay. Compounds that showed promising in vitro COX-2 IC50 values
and selectivity indices were then evaluated for their in vivo anti-inflammatory inhibition assay using standard
carrageenan-induced rat paw edema method. Two promising inhibitors were evaluated for ulcerogenic liabil-
ity. X-ray crystal structure of COX-2 was taken from PDB entry COX-2 (3LN1) having a resolution of
2.80 Å (Angstroms). Structural preparations for docking studies were accomplished using protein prepara-
tion wizard in Maestro 9.0. Compound 10b displayed reasonable COX-2 inhibition (COX-2 IC50 = 0.52 μM)
and COX-2 selectivity index (SI = 10.73) when compared with celecoxib (COX-2 IC50 = 0.78 μM) and
(SI = 9.51). In vivo anti-inflammatory studies demonstrated 64.28% inhibition for 10b in comparison with
the 57.14% for that of celecoxib itself. The results of ulcerogenic liability were also found comparable with
standard celecoxib. Molecular docking studies revealed that all the designed molecules showed good inter-
actions with receptor active site with glide scores in the range ꢀ13.130 to ꢀ10.624.
J. Heterocyclic Chem., 00, 00 (2018).
INTRODUCTION
COX-2 is observed in acute and chronic inflammation
[5,6]. Thus, inhibition of COX-2 accounts for the anti-
inflammatory effects of NSAIDs, whereas interruption of
COX-1 leads to gastrointestinal toxicity ranging from
ulcers to perforation and bleeding [7]. Time-honored non-
selective NSAIDs such as indomethacin, ibuprofen, and
aspirin interact with both forms (COX-1 and COX-2),
accounting for their anti-inflammatory activity in addition
to their pronounced side effects, resulting from the
inhibition of gastroprotective PGs synthesized through
COX-1 pathway [8]. Hence, a number of selective
COX-2 inhibitors such as celecoxib I, rofecoxib II, and
valdecoxib III (coxibs) have been developed and approved
for marketing by virtue of their fewer gastrointestinal side
effects compared with traditional NSAIDs (Fig. 1).
Celecoxib, in the 1,5-diarylpyrazole class of compound,
was the first launched selective COX-2 inhibitor with
Fast and effective relief of pain and inflammation in the
human being is continued to be a major task for the
medicinal chemist. Non-steroidal anti-inflammatory drugs
(NSAIDs) are important therapeutic agents for the
alleviation of pain and inflammation associated with a
number of pathological conditions [1]. NSAIDs bestow
their effect by inhibiting the catalytic activity of
cyclooxygenase (COX), which results in a blockage of
the formation of prostaglandins (PGs) and thromboxane
(TXs) [2,3]. The cyclooxygenase exists as two distinct
isoforms (COX-1 and COX-2) [4]. The maintenance of
physiological functions such as protection of gastric
mucosa, vascular homeostasis, and platelet aggregation is
governed by the constitutively expressed COX-1 isoform
as organization enzyme while the upregulation of the
© 2018 Wiley Periodicals, Inc.

L. S. Pavase, D. V. Mane, and K. G. Baheti
Vol 000
certain
4-(3-hydrazinocarbonyl-5-phenylpyrazol-1-yl)-
benzenesulfonamide derivatives bearing two aryl
moieties at 1- and 5-positions of the pyrazole ring and
carrying different substituent on the 5-amino and
3-hydrazinocarbony residue was synthesized. The
synthesized compounds have a characteristic molecular
pattern and bulk volume to fulfill the pharmacophoric
requirements for better recognition at the COX-2 binding
active site. The newly synthesized analogs were
evaluated for their COX selectivity and their in vivo anti-
inflammatory activity.
Although the synthesis of coxibs is engrained in recent
years, the challenge of exploring the anti-inflammatory
activity with promising COX-2 selectivity and safe
gastric profile is still in need of investigative
accomplishments. In furtherance with our quest
associated with synthesis of safe anti-inflammatory agents
[25], authors here wish to report an investigation about
synthesis, in vitro evaluation of COX selectivity, in vivo
anti-inflammatory (AI) activity, and evaluation of
ulcerogenic liability for two new groups of hydrazide 1,5-
diaryl and amide containing 1,3-diaryl pyrazoles with
promising results.
Figure 1. Representative examples of COX- 2 inhibitors.
excellent selectivity and potent anti-inflammatory activity,
having advantage of not associating with increased
cardiovascular complications [9] but known to have
gastrointestinal side effects [10].
The pyrazole nucleus is known to exhibit wide range of
biological activities such as antimicrobial [11,12], antiviral
[13], antitumor [14], anti-depressant [15], and anti-
inflammatory [8,16–19] activity. Among these, four
functionalized pyrazoles occupy a distinctive position in
medicinal chemistry. The archeology of research
pertaining to the pyrazoles and their anti-inflammatory
activity can be studied at ones by referring recent reviews
[20,21].
The pharmacophoric structural features of the selective
COX-2 inhibitors possess a central heterocyclic five-
member ring system bearing two vicinal aryl moieties,
such as pyrazole (celecoxib), 2(5H) furanone (rofecoxib),
and isoxazole (valdecoxib). The main part of our research
has been devoted to synthetic methods containing the
pyrazole nucleus, as a pharmacophoric moiety for
potential drugs. Also, sulfonamides [22] and hydrazide
with their heterocyclic analogs showed evidence of
diverse biological activities including anticancer [23] and
anti-inflammatory [24] properties. In particular, the
pyrazole nucleus represents a very attractive scaffold to
obtain new molecules endowed with anti-inflammatory
activities [25,26]. On the basis of these considerations,
and in view of the reported COX-2 inhibitory activities of
RESULTS AND DISCUSSION
Chemistry. The present study focuses on the synthesis
of novel amide compounds, N-[5-phenyl-2-(4-
sulfamoylphenyl)-2H-pyrazol-3-yl]-amide (5a–f) and
hydrazide
compounds,
4-[3-(N0-alkyloyl/aryloyl-
hydrazinocarbonyl)-5-phenylpyrazol-1-yl]-
benzenesulfonamide (10a–j).
Synthesis of novel amide derivative was prepared as
depicted in Scheme 1. Reaction of sulfanilide 1 with
SnCl2 and NaNO2 in water under cooling condition gave
4-hydrazino-benzenesulfonamide hydrochloride salt 2.
This was transformed into the corresponding amino
Scheme 1. Synthetic route for N-[5-phenyl-2-(4-sulfamoylphenyl)-2H-pyrazol-3-yl]-amide (5a–f). Reagents and conditions: (a) SnCl2, NaNO2, water,
HCl 3 h; (b) ethanol, reflux 4 h; (c) R-acid, N,N-dimethylformamide, EDC.HCl/HOBt, TEA, 25°C, 5 h.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Anti-inflammatory Exploration of Sulfonamide Containing Diaryl Pyrazoles with
Promising COX-2 Selectivity and Enhanced Gastric Safety Profile
1
pyrrole derivative
4
by reaction with 3-oxo-3-
yield. H NMR spectrum of ester intermediate 7 revealed
the presence of triplet signal at δ 1.28 ppm (J = 7 Hz),
and quartet signal at δ 4.32 ppm (J = 7 Hz) corresponds
to ethyl group, and two aromatic protons at δ 8.06 ppm
phenylpropionitrile 3 in ethanol at reflux condition. At
the last step, 4-(5-amino-3-phenylpyrazol-1-yl)-
benzenesulfonamide amide derivatives 5a–e were
obtained in 60–70% yield through coupling of amine
1
(J = 8 Hz) correspond to phenyl ring. Similarly, H NMR
1
intermediate 4 with selected acid side chains. H NMR
spectrum of pyrazole 8 showed shift in triplet signals of
ester intermediate 7 to δ 1.32 ppm (J = 7 Hz) and quartet
signal to δ 4.34 ppm (J = 7 Hz), an aromatic protons
shifted to δ 7.85 ppm (J = 8 Hz), singlet of pyrazolyl
proton was found at δ 7.13 ppm. Hydrazide intermediate
9 was confirmed by the disappeared signal of ester and
by the shift of pyrazolyl proton to at δ 7.05 ppm.
spectrum of 2 revealed the presence of broad singlet
signal at δ 10.43 ppm corresponding to two protons of
hydrazine (─NH2) forming hydrochloride salt. It also
showed singlet proton at δ 8.86 corresponding to
hydrazine (─NH─). ES-MS spectrum showed m/z 188.1
(M + H)+. Intermediate 4 was characterized by the singlet
proton at δ 8.86 ppm corresponding to pyrazole ring and
a broad singlet at δ 5.71 ppm for 5-amino pyrazole,
mutilate for five protons at δ 7.33–7.42 was appeared for
the newly added phenyl ring from the intermediate 3.
ES-MS spectrum showed m/z 315.1 (M + H)+ for the
amine core 4. Novel amide derivatives 5a–f were
prepared by coupling selected acid with the amine core 4.
Substituted aromatic, aliphatic, and heterocyclic aromatic
acids were selected to evaluate the structure activity
relationship among the novel analogue.
The second series of targeted compounds 10a–j were
prepared as outlined in Scheme 2. The starting material,
ethyl-2,4-dioxo-4-phenylbutanoate 7, was prepared from
acetophenone 6 and diethyl oxalate in toluene using
sodium hydride at 0 to 50°C. Cyclization of ester
intermediate 7 to pyrazole 8 was achieved using
(4-sulfamoylphenyl)hydrazine hydrochloride in ethanol
under reflux condition. Reaction of hydrazine hydrate
with the cyclized pyrazole 8 converts its ester group to
corresponding hydrazide intermediate 9 in ethanol with
reflux condition. Coupling hydrazide intermediate 9 with
substituted acid side chains using EDC.HCl and HOBt in
DMF resulted into proposed compounds in 60–70%
Molecular docking studies. Molecular docking studies
were carried out in order to identify the correlations of
novel synthesized compounds with the targeted enzymes
COX-2 to predict its selectivity. Synthesized compounds
5a–f and 10a–j were subjected for docking studies along
with standard celecoxib using crystal structure of COX-2
(3LN1) [26]. An advanced grid-based ligand docking
program, GLIDE (Schrodinger Inc., USA) version 4.5,
was used to estimate binding affinities of docked
compounds. This will approximate the algorithm
energetics to systematic positions, orientations, and
conformations. Elimination of unwanted conformations
was performed by scoring the enzyme pocket via a series
of hierarchical filters. Finally, the conformations were
refined via Monte Carlo sampling of pose conformation
[27,28]. X-ray crystal structure of COX-2 was taken from
PDB entry COX-2 (3LN1) having resolution of 2.80 Å
(Angstroms).
Structural preparations for docking studies were
accomplished using protein preparation wizard in
Maestro 9.0. Protein preparation wizard of Maestro 9.0
accomplished the structural preparation of docking
studies in two steps, viz. preparation and refinement. In a
Scheme 2. Synthetic route for 4-[3-(N0-alkyloyl/aryloyl-hydrazinocarbonyl)-5-phenylpyrazol-1-yl]-benzenesulfonamide (10a–j). Reagents and
conditions: (a) NaH/toluene, N2, 0 to 50°C 4 h, yield 89%; (b) (4-sulfamoylphenyl) hydrazine hydrochloride, EtOH, refluxed for 6 h, yield 84%; (c)
hydrazine hydrate/EtOH, refluxed for 4 h yield 83%; (d) R-acid, EDC.HCl, HOBt, DIPEA, DMF, RT, 4 h, yield 60–70%.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

L. S. Pavase, D. V. Mane, and K. G. Baheti
Vol 000
crystal structure of ligand protein, chemical correctness
was ensured, water molecules were deleted, and
hydrogen atoms were added at missing positions. Bond
order for crystal ligand and protein was adjusted and
minimized up to 0.30
Å
RMSD. Low energy
conformations of ligand using OPLS 2005 force field was
produced from the Ligprep 2.2 module in Maestro 9.0
build panel. Glide provides three different levels of
docking precisions viz. high throughput virtual screening,
standard precision, and extra precision. We carried out
our calculations using extra precision docking mode as
the tool is designed for better refinement in ligands.
Molecular docking studies revealed that all the designed
molecules showed good interactions with receptor active
site with glide scores in the range ꢀ 13.130 to ꢀ10.624.
Molecules 10b and 10g, the most potent COX-2
inhibitors in current series, showed good interactions with
the receptor active site (Fig. 2). Sulfonamide group of
10b showed hydrogen bonding with Leu 338, Ser 339,
Arg 499, and Phe 504. Pyrazole ring of 10b showed pi
stacking with Arg 106. Compound 10g showed similar
interactions with the receptor. All these interactions are
also shown by celecoxib (Fig. 3). Thus, it can be
concluded that these interactions contribute to the
inhibitory activity of these molecules.
Figure 3. Docking interactions of celecoxib with COX-2. [Color figure
can be viewed at wileyonlinelibrary.com]
inhibitors for COX-1 isoenzyme (IC50 = 3.01–30.18 μM)
and
exhibited
moderate
COX-2
isoenzyme
(IC50 = 0.52–22.25 μM) with COX-2 selectivity’s in the
range of 0.68–10.31. The acquired data for the two
different diaryl series showed that when the two phenyl
rings attached to the central pyrazole moiety are not
vicinal (5a–e), lower inhibitory activities against both
COX-1 (IC50
=
7.40–30.18 μM) and COX-2
(IC50 = 10.18–22.25 μM).
While when the two phenyl rings attached to the
central heterocyclic pyrazole nucleus are vicinal
(10a–j) comparable inhibitory activities against both
In vitro cyclooxygenase inhibition assay.
The in vitro
COX-1/COX-2 isoenzyme inhibition studies measure the
ability of tested compounds to inhibit ovine COX-1 and
human recombinant COX-2 using an enzyme
immunoassay. The obtained results (Table 1) showed that
all the tested diaryl pyrazole are found to be weak
COX-1 (IC50
=
3.01–10.42 μM) and COX-2
(IC50 = 0.52–3.70 μM) with celecoxib. In this series, we
replaced the trifluoromethyl group of the celecoxib with
hydrazide and attached different alkyl and aryl carboxylic
Figure 2. Interactions of 10b and 10g with the receptor. [Color figure can be viewed at wileyonlinelibrary.com]
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Anti-inflammatory Exploration of Sulfonamide Containing Diaryl Pyrazoles with
Promising COX-2 Selectivity and Enhanced Gastric Safety Profile
Table 1
chains (10d and 10i) also contributed towards the
In vitro anti-inflammatory COX-1 and COX-2 activities of diaryl
sulfonamides.
activities and COX-2 selectivity than the benzoic acid
derivative 10c.
In vivo anti-inflammatory activity. The anti-inflammatory
activities exhibited by selected five compounds (10b, 10c,
10d, 10g, and 10i) using dose 50 mg/kg are listed in
Table 2. The phenyl analog 10c, 2-thienyl analog 10d, and
3-pyridyl analog 10i were found to possess anti-
inflammatory activity (35.71%, 42.85%, and 35.71%
reduction in inflammation after 3 h) less than celecoxib
(57.14% reduction in inflammation after 3 h).
COX-1
COX-2
Compound
R
IC50 (μM)a IC50 (μM)a SIb
5a
5b
5c
5d
5e
10a
10b
phenyl
7.40
18.23
30.18
18.30
24.12
9.75
10.18
12.40
22.25
16.20
15.32
2.08
0.68
1.47
1.35
1.12
1.31
4.68
10.73
3-pyridyl
cyclohexyl
2-thienyl
2-furyl
methyl
3-trifluoromethyl
phenyl
5.58
0.52
While
(3-trifluoromethyl) and 10g (3-chloro), which showed
COX-2 activities in vitro comparable with
substituted
phenyl
derivatives
10b
10c
10d
10e
phenyl
6.51
4.21
10.42
0.82
0.62
2.80
7.93
6.79
3.72
2-thienyl
2,6-dimethoxy
phenyl
cinnamyl
3-chloro phenyl
cyclohexyl
3-pyridyl
2-furyl
celecoxib, showed in vivo activity (edema inhibition %:
64.28 and 60.71%, respectively, after 3 h) higher than
reference drug celecoxib (edema inhibition %: 57.14%
after 3 h).
10f
10g
10h
10i
10j
Celecoxib
Ibuprofen
9.58
7.52
9.23
3.357
5.92
7.42
3.2
3.20
0.90
3.70
0.43
1.62
0.78
1.40
2.99
8.36
2.49
8.30
3.65
9.51
2.28
Ulcerogenic liability.
Compounds 10b and 10g with
potential in vitro COX-2 inhibitory activity and showed
higher in vivo anti-inflammatory activity than celecoxib
were evaluated for their ulcerogenic liability according to
a known method [29]. From the obtained results
(Table 3), compound 10b and 10g were found to be less
ulcerogenic (ulcer index: 6.6 and 8.65, respectively) than
aIC50 values represent concentration of test compound required to produce
50% inhibition; the result is the mean of two values obtained by assay of
enzyme kits obtained from Cayman Chemicals Inc., Ann Arbor, MI, USA.
bSelectivity index (COX-1 IC50/COX-2 IC50).
acid side chains. Within the biologically active series
(10a–j), the trifluoromethyl (10b) and chloro (10g)
analogs were more selective COX-2 inhibitors (selectivity
indices = 10.73 and 8.36, respectively) than unsubstituted
phenyl (10c), 3-pyridyl (10i), and 2-thienyl (10d) analogs
(selectivity indices = 7.93, 8.30, and 6.79, respectively)
in comparison with celecoxib selectivity index = 9.51.
Aliphatic acid side chain compounds (10a and 10h)
were stand inferior both in activity and COX-2 selectivity.
In the active series, it was observed that the aromatic
acid compounds substituted with halogen at third position
(10b and 10g) were most active. Heterocyclic acid side
Table 3
Ulcerogenic liability of compounds.
Average Average no.
Ulcer
Compound
severity
of ulcers
% Incidence indexa
10b
10g
Celecoxib
Ibuprofen
0.5
0.6
1.5
0.5
6.5
5.5
6.5
4.5
8.3
6.6
8.65
5.54
17.06
0.65
0.54
2.26
aStatistical analysis used was one-way ANOVA, followed by Dunnett’s
test, (n = 6).
Table 2
In vivo anti-inflammatory activity employing carrageenan-induced paw edema method in mice.
Change in paw volume in mL after drug treatment ( SEM)
% Inhibition
Compound
0h
1h
2h
3h
1h
2h
3h
Control
Celecoxib
10b
10c
10d
0.38 0.03*
0.27 0.10*
0.34 0.07*
0.37 0.02*
0.30 0.07*
0.33 0.06*
0.35 0.03*
0.48 0.04*
0.33 0.01*
0.40 0.05*
0.44 0.07*
0.37 0.08*
0.38 0.09*
0.42 0.08*
0.57 0.09*
0.36 0.10*
0.42 0.02*
0.50 0.07*
0.41 0.09*
0.42 0.08*
0.47 0.06*
0.66 0.04*
0.39 0.08*
0.44 0.07*
0.55 0.05*
0.46 0.07*
0.44 0.09*
0.52 0.09*
—
40
40
30
30
50
30
—
—
52.63
57.89
31.57
42.10
52.63
36.84
57.14
64.28
35.71
42.85
60.71
35.71
10g
10i
Results of all parameters were expressed as mean standard error of mean for each group. Data were analyzed by one-way ANOVA followed by
Dunnett’s test, (n = 6). Dose levels: Test compounds and celecoxib (50 mg/kg, b. w. p. o.).
*P < 0.05 significant from control.
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L. S. Pavase, D. V. Mane, and K. G. Baheti
Vol 000
the ibuprofen (ulcer index: 17.06) and displayed
comparable ulcerogenic potentials with the celecoxib
(ulcer index: 5.54). Hence, 10b and 10g was found to be
safe gastric profile.
bath. Then the mixture was rapidly added to a cooled
(0°C) solution of tin (II) chloride dehydrate (27.1 g,
120 mmol) in concd HCl (30 mL). The resulting mixture
was stirred at 0°C for 1 h and then warmed to ambient
temperature to stir overnight. The precipitate was
collected by filtration and successively washed with cool
water and Et2O to give the relatively pure compound,
which was used directly without further purification.
4-Hydrazinylbenzenesulfonamide hydrochloride, white
solid, (6.7 g, yield: 74.1%). 1H NMR (DMSO-d6,
400 MHz) δ 10.40 (br s, 3H, ─NH2.HCl), 8.86 (s, 1H,
Ar─NH─NH2), 7.68 (d, 2H, J = 8 Hz, ─SO2─NH2─),
7.18 (s, 1H, Ar─H), 7.03 (d, 2H, J = 7.6 Hz, Ar─H); ESI-
MS (M ꢀ HCl + H)+: m/z = 188.1.
CONCLUSIONS
Two series containing novel amides (5a–e) and
hydrazide (10a–j) with diaryl pyrazoles were synthesized
and evaluated as selective COX-2 inhibitors, anti-
inflammatory agents, and for the safety gastric profile.
Molecular docking studies revealed that all the designed
molecules showed good interactions with receptor active
site with glide scores in the range ꢀ13.130 to ꢀ10.624.
Biological studies showed that hydrazide analogs were
most selective and potent anti-inflammatory agents as
compared with the amide analogs. Among all the studied
compounds, 10b and 10g showed >60% edema
inhibition. These two compounds were found to exhibit
less ulcerogenic than ibuprofen and showed ulceration
effect comparable with that of celecoxib.
4-(5-Amino-3-phenylpyrazol-1-yl)-benzenesulfonamide
(4). To a clear solution of 3-oxo-3-phenylpropionitrile
(10 g, 68 mmol) in ethanol (100 mL) at 25°C was added
hydrochloride salt of 4-hydrazinylbenzenesulfonamide
(15.4 g, 68 mmol) to form a suspension, which was then
heated to refluxed temperature for 3 h to become a clear
solution, and cooled the reaction mixture to 25°C.
Obtained solid was collected by filtration and dried under
vacuum to yield the desired intermediate 4 as a yellowish
solid, (15 g, yield: 70%); 1H NMR (DMSO-d6,
400 MHz) δ 7.87–7.94 (m, 4H, Ar─H), 7.77–7.79
(m, 2H, Ar─H), 7.33–7.42 (m, 5H, Ar─H), 6.02 (s, 1H,
Pyrazole-H), 5.72 (br s, Ar─NH2─); ESI-MS (M + H)+:
m/z = 315.1.
EXPERIMENTAL
All the raw materials and reagents were procured
commercially and used without further purification. All
the newly synthesized compounds have been
1
characterized by IR, H NMR, 13C NMR, mass spectra,
General procedure for the synthesis of N-[5-phenyl-2-(4-
and elemental analysis. Melting points were determined
on a Veego melting point apparatus model VMP-D
apparatus and are uncorrected. 1H NMR spectra were
recorded on a Varian 400-MHz spectrometer using
CDCl3 and DMSO-d6 as solvent with tetramethylsilane as
an internal standard, and 13C NMR spectra were
measured on a Varian100 MHz spectrometer DMSO-d6
with tetramethylsilane as the internal standard, where J
(coupling constant) values were estimated in hertz (Hz).
Elecronspray ionization-mass spectra were obtained by
using LC-MS/MS Waters (Aquity) TQ detector.
Microanalyses for elemental composition were performed
for C, H, and N using vario MICRO elemental
instrument and were within 0.4% of theoretical values.
Diaryl sulfonamide derivatives were synthesized in three
to four steps via coupling reaction with acids and
hydrazides. All the products were soluble with DMSO
and DMF. The postulated structures of the newly
synthesized compounds (Table 1) were in good
sulfamoylphenyl)-2H-pyrazol-3-yl]-amide (5a–f).
To a
solution of R-acid (32 mmol) in 5-mL DMF was added
EDC.HCl (48 mmol) and 4-(5-amino-3-phenylpyrazol-1-
yl)-benzenesulfonamide (32 mmol) followed by the
addition of triethylamine (96 mmol) and HOBt
(32 mmol) under nitrogen atmosphere. The reaction
mixture was stirred for 6 h and quenched with water
(50 mL), and adjust the pH = 4 of the reaction mixture
with 1N HCl to obtained the solid, which was filtered at
Buchner funnel and washed with diethyl ether then dried
under vacuum to yield desired solid product (5a–e).
N-[5-Phenyl-2-(4-sulfamoylphenyl)-2H-pyrazol-3-yl]-benzamide
(5a). Off-white solid; (1.0 g, yield: 75%); mp = 132–
134°C; 1H NMR (DMSO-d6, 400 MHz) δ 8.11(d, 2H,
J = 8.4 Hz, Ar─H), 7.99 (d, 2H, J = 8.8 Hz, Ar─H), 7.91
(d, 2H, J = 7.2 Hz, Ar─H), 7.62 (d, 2H, J = 7.2 Hz,
Ar─H), 7.50 (t, 2H, J = 8.0 Hz, J = 7.2 Hz, Ar─H), 7.40
(t, 2H, J = 7.6 Hz, Ar─H), 7.33–7.34 (m, 3H, Ar─H),
5.97 (s, 1H, Pyrazole-H), 5.77(br s, 2H, ─SO2─NH2); 13C
NMR (DMSO-d6, 100 MHz) δ 94.7, 120.4 (2 × C), 127.4
(2 × C), 127.6 (2 × C), 127.8 (2 × C), 127.9, 128.8, 128.9
(2 × C), 129.4 (2 × C), 133.1, 134.2, 137.2, 142.9, 147.9,
151.3, 164.8; ES-MS: m/z 420.3 (M + H)+. Anal. Calcd
for C22H18N4O3S; C, 63.14; H, 4.34; N, 13.39; found: C,
63.17; H, 4.33; N, 13.44.
1
agreement with their H NMR, 13C NMR spectral data,
and elemental analysis data.
4-Hydrazinobenzenesulfonamide hydrochloride (2). To
a solution of sodium nitrate (3.1 g, 440 mmol) in 12.0-mL
water was added into a mixture of sulphanilamide (6.9 g,
400 mol) in concd HCl (30 mL) over 15 min in ice-water
Journal of Heterocyclic Chemistry
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Month 2018
Anti-inflammatory Exploration of Sulfonamide Containing Diaryl Pyrazoles with
Promising COX-2 Selectivity and Enhanced Gastric Safety Profile
N-[5-Phenyl-2-(4-sulfamoylphenyl)-2H-pyrazol-3-yl]-
nicotinamide (5b). Brownish solid; (0.911 g, yield: 68%);
2-Phenyl-N-[5-phenyl-2-(4-sulfamoylphenyl)-2H-pyrazol-3-yl]-
acetamide (5f). (0.967 g, yield: 70%); mp = 136–138°C;1H
mp = 130–132°C; 1H NMR (DMSO-d6, 400 MHz) δ 9.04
(s, 1H, Py─H), 8.77–8.78 (m, 1H, Py─H), 8.28 (d, 2H,
Py─H, J = 8 Hz, Py–H), 8.11 (d, 2H, Py─H, J = 8.8 Hz,
Ar─H), 7.98 (d, 2H, J = 8.4 Hz, Ar─H), 7.91 (d, 2H,
J = 7.2 Hz, Ar─H), 7.78 (d, 2H, J = 7.2 Hz, Ar─H), 7.72
(d, 2H, J = 8.4 Hz, Ar─H), 5.98 (s,1H, Pyrazole-H); 13C
NMR (DMSO-d6, 100 MHz) δ 94.8, 120.5 (2 × C),
125.2, 127.6 (2 × C), 127.8 (2 × C), 128.8, 129.3
(2 × C), 130.8, 138.1, 142.9, 148.0, 148.3, 151.3, 153.7,
164.8; ES-MS: m/z 420.3 (M + H)+. Anal. Calcd for
C21H17N5O3S; C, 60.13; H, 4.09; N, 16.70; found: C,
NMR (DMSO-d6, 400 MHz): δ 10.14 (s, 1H, ─CO─NH),
7.94 (d, 2H, J = 8.4 Hz, Ar─H), 7.84–7.86 (m, 4H,
Ar─H), 7.78 (d, 2H, J = 8.0 Hz, Ar─H), 7.35–7.46
(m, 6H, Ar─H), 5.90 (s,1H, Pyrazole-H), 3.14 (s, 2H,
Ar─CH2─CO); 13C NMR (DMSO-d6, 125 MHz) δ 42.0,
88.32, 121.9, 125.2, 126.9, 128.0, 128.3, 128.5, 128.8
(3 × C), 128.9, 129.2, 129.3 (2 × C), 133.0, 133.8, 135.6,
143.4, 149.1, 149.1, 151.3, 169; ES-MS: m/z 433.3
(M + H)+. Anal. Calcd for C23H20N4O3S; C, 63.87; H,
4.66; N, 12.95; found: C, 63.83; H, 4.63; N, 12.94.
2,4-Dioxo-4-phenylbutyric acid ethyl ester (7).
To a
60.13; H, 4.06; N, 16.74.
solution of acetophenone 6 (20 g, 166 mmol) in dry
toluene (200 mL) at ꢀ10°C was added sodium hydride
(11.95 g, 498 mmol) and stirred under nitrogen
atmosphere for 1 h. Diethyl oxalate (43.32 g, 249 mmol)
in toluene (100 mL) was added dropwise and allowed
reaction mixture to room temperature. Reaction mixture
then heated to 50°C for 4 h. Cooled reaction mixture to
ꢀ10°C was added with water (100 mL), and crude
product was extracted in toluene. Organic layers were
collected, dried over sodium sulfate, and removed under
vacuum to give crude intermediate, which was purified
by column chromatography (5% EtOAc in Hexane as
eluent) to furnish yellow liquid intermediate 7. (28 g,
Cyclohexanecarboxylic acid [5-phenyl-2-(4-sulfamoylphenyl)-
2H-pyrazol-3-yl]-amide (5c).
Off-white solid; (0.976 g,
yield: 72%); mp = 136–138°C; 1H NMR (DMSO-d6,
400 MHz) δ 7.98–8.01(m, 2H, Ar─H), 7.95–7.99 (m, 2H,
Ar─H), 7.39–7.43 (m, 4H, Ar─H), 7.31–7.34 (m, 2H,
Ar─H), 5.98 (s,1H, Pyrazole-H), 3.00–3.04 (m, 1H,
CH2─CH2─CH2─), 2.23 (br s, 2H, CH2─CH2─CH2─),
1.67–1.77 (m, 4H, ─CH2─CH2─CH2─), 1.15–1.19(m,
4H, ─CH2─CH2─CH2─); 13C NMR (DMSO-d6,
100 MHz) δ 24.8 (2 × C), 27.4, 29.2 (2 × C), 43.1, 94.8,
120.4 (2 × C), 127.5 (2 × C), 127.9 (2 × C), 128.7, 129.3
(2 × C), 133.1, 137.2, 142.7, 148.0, 151.2, 173.2; ES-MS:
m/z 425.3 (M + H)+. Anal. Calcd for C22H24N4O3S; C,
62.24; H, 5.70; N, 13.20; found: C, 62.28; H, 5.67;
1
yield: 76%); H NMR (DMSO-d6, 400 MHz) δ 8.04 (d,
2H, J = 7.2 Hz, Ar─H), 7.93–7.94 (m, 1H, Ar─H), 7.67–
7.70 (m, 1H, Ar─H), 7.54–7.58 (m, 2H, Ar─H), 7.10 (s,
1H, Ar─H), 4.32 (q, 2H, J = 4.4 Hz, ─OCH2─CH3),
1.31 (t, 3H, ─OCH2─CH3,J = 7.2 Hz); ES-MS: m/z
221.1 (M + H)+.
N, 13.24.
Thiophene-2-carboxylic acid [5-phenyl-2-(4-sulfamoylphenyl)-
2H-pyrazol-3-yl]-amide (5d).
Yellowish solid; (0.963 g,
yield: 71%); mp = 142–144°C; 1H NMR (DMSO-d6,
400 MHz) δ 8.11(d, 2H, J = 8.8 Hz, Ar─H), 8.01 (d,
2H, J = 8.8 Hz, Ar─H), 7.96 (d, 2H, J = 8.8 Hz,
Ar─H), 7.71–7.77 (m, 2H, Ar─H), 7.53–7.57 (m, 2H,
Ar─H), 7.31–7.47 (m, 5H, Ar─H), 5.97 (s, 1H,
Pyrazole-H); 13C NMR (DMSO-d6, 100 MHz) δ 94.9,
120.5 (2 × C), 127.4 (2 × C), 127.9 (2 × C), 128.7,
128.9, 129.2 (2 × C), 133.0, 137.2, 137.4, 137.7, 135.9,
142.9, 148.0, 151.3, 161.9; ES-MS: m/z 425.2
(M + H)+. Anal. Calcd for C20H16N4O3S2; C, 56.59; H,
5-Phenyl-1-(4-sulfamoylphenyl)-1H-pyrazole-3-carboxylicacid
ethyl ester (8). To a suspension of (4-sulfamoylphenyl)
hydrazine hydrochloride (25.4 g, 113 mmol) in ethanol
(100 mL) was added 2,4-dioxo-4-phenylbutyric acid ethyl
ester 7 (25 g, 113 mmol) and refluxed the reaction
mixture for 6 h. Reaction mixture becomes clear at the
completion of the reaction under reflux. Cooled the
content to room temperature, precipitated solid was
filtered and washed with chilled ethanol (10 mL) and
dried to obtain white solid as intermediate 8 (30 g, yield:
3.80; N, 13.20; found: C, 56.61; H, 3.83; N, 13.23.
Furan-2-carboxylic acid [5-phenyl-2-(4-sulfamoylphenyl)-
2H-pyrazol-3-yl]-amide (5e).
1
Brownish solid; (0.955 g,
69%); mp = 192–194°C; H NMR (DMSO-d6, 400 MHz)
yield: 73%); mp = 138–140°C; 1H NMR (DMSO-d6,
400 MHz) δ 8.13(d, 2H, J = 8.4 Hz, Ar─H), 7.96 (d, 2H,
J = 8.4 Hz, Ar─H), 7.89 (d, 2H, J = 8.8 Hz, Ar─H),
7.71–7.77 (m, 2H, Ar─H), 7.53–7.57 (m, 2H, Ar─H),
7.31–7.47 (m, 5H, Ar─H), 5.99 (s, 1H, Pyrazole-H); 13C
NMR (DMSO-d6, 100 MHz) δ 94.8, 111.7, 113.4, 120.6
(2 × C), 127.5 (2 × C), 127.9 (2 × C), 128.7, 128.8,
129.2 (2 × C), 133.0, 142.7, 146.1, 147.2, 148.0, 151.3,
161.9; ES-MS: m/z 409.1 (M + H)+. Anal. Calcd for
C20H16N4O4S2; C, 58.81; H, 3.95; N, 13.72; found: C,
58.83; H, 3.97; N, 13.75.
δ 7.84 (d, 2H, J = 8.4 Hz, Ar─H), 7.49–7.51 (m, 4H,
Ar─H), 7.38–7.39 (m, 2H, Ar─H), 7.27–7.29 (m, 2H,
Ar─H), 7.13 (s, 1H, Pyrazole-H), 4.34 (q, 2H,
J = 6.8 Hz, ─OCH2─CH3), 1.30 (t, 3H, ─OCH2─CH3,
J = 7.2 Hz); ES-MS: m/z 372.2 (M + H)+.
4-(3-Hydrazinocarbonyl-5-phenylpyrazol-1-yl)-
benzenesulfonamide (9).
To a suspension of ester
intermediate (10 g, 269 mmol) 8 in ethanol (50 mL) was
added hydrazine hydrate (6.72 g, 134 mmol) and refluxed
the reaction mixture for 6 h. Reaction mixture becomes
clear at the completion of the reaction under reflux.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

L. S. Pavase, D. V. Mane, and K. G. Baheti
Vol 000
Cooled the content to room temperature, precipitated solid
was filtered and washed with chilled ethanol (10 mL) and
dried to obtain white solid as hydrazide intermediate 9
4-[3-(N0-Benzoylhydrazinocarbonyl)-5-phenylpyrazol-1-yl]-
benzenesulfonamide (10c). White solid; (0.540 g, yield:
1
83%); mp = 128–130°C; H NMR (DMSO-d6, 400 MHz)
1
δ 10.49 (s, 1H, ─CO─NH─), 10.36 (s, 1H, ─CO─NH─),
7.91(d, 3H, J = 7.2 Hz, Ar─H), 7.86 (d, 2H, J = 8 Hz,
Ar─H), 7.54–7.58 (m, 6H, Ar─H), 7.49 (s, 2H,
Ar─SO2─NH2), 7.40–7.41 (br s, 2H, Ar─H), 7.31–7.32
(br s, 2H, Ar─H), 7.13 (s, 1H, Pyrazole -H), 13C NMR
(DMSO-d6, 100 MHz) δ 108.7, 125.7(3 × C), 126.7
(3 × C), 127.5 (3 × C), 128.5 (2 × C),128.1, 128.8, 128.9
(2 × C), 131.8, 132.5, 141.5, 143.5, 144.5, 146.1, 160.4,
165.7;ES-MS: m/z 462.3 (M + H)+, mp = 128–130°C,
(8 g, yield: 83%); mp = 187–189°C; H NMR (DMSO-
d6, 400 MHz) δ 9.73 (s, 1H, ─NH─NH2), 7.86 (d, 2H,
J = 8.4 Hz, Ar─H), 7.54 (d, 2H, J = 7.6 Hz, Ar─H),
7.38–7.44 (m, 4H, Ar─H), 7.27–7.29 (m, 2H, Ar─H),
7.11 (s, 1H, Pyrazole-H),4.64 (br s, 2H, ─NH─NH2); ES-
MS: m/z 358.1 (M + H)+.
General procedure for the synthesis of 4-[3-(N0-alkyloyl/
aryloyl-hydrazinocarbonyl)-5-phenylpyrazol-1-yl]-
benzenesulfonamide (10a–j). To a clear solution of R-acid
(14 mmol) in dry dimethylformamide (5 mL) was added
EDC.HCl (21 mmol) and 4-(3-hydrazinocarbonyl-5-
phenylpyrazol-1-yl)-benzenesulfonamide (14 mmol),
followed with the addition of HOBt (14 mmol). Reaction
mixture was stirred for 6 h. Water was added (50 mL),
and the product was extracted in EtOAc (2 × 50 mL),
organic layers were washed with NaHCO3 solution in
water, 10% aq HCl, 25% aq NH4Cl solution in water,
brine, and dried over Na2SO4 and removed under
vacuum to obtain crude residue, which was purified by
column chromatography (2% MeOH in Chloroform as
eluent) to furnish title solid product in 60–70% yield
Anal. Calcd for C23H19N5O4S: C, 59.86; H, 4.15; N,
15.18; S, 6.95. Found: C, 59.83; H, 4.17; N, 15.16; S, 6.98.
4-{5-Phenyl-3-[N0-(thiophene-2-carbonyl)-hydrazinocarbonyl]-
pyrazol-1-yl}-benzenesulfonamide (10d).
Brownish solid;
(0.52 g, yield: 79%); mp = 122–124°C; 1H NMR
(DMSO-d6, 400 MHz) δ 10.53 (s, 1H, ─CO─NH─),
10.41 (s, 1H, ─CO─NH─), 7.86–7.9 (m, 5H, Ar─H),
7.56 (d, 2H, J = 8.8 Hz, Ar─H), 7.49 (s, 2H,
Ar─SO2─NH2), 7.41–7.43 (m, 2H, Ar─H), 7.32–7.34
(m, 2H, Ar─H), 7.21–7.23 (m, 1H, Ar─H), 7.13 (s, 1H,
Pyrazole-H), 13C NMR (DMSO-d6, 100 MHz) δ 108.3,
125.4(2 × C), 126.3 (2 × C), 127.5, 128.5 (2 × C), 128.1,
128.8, 128.9 (2 × C), 131.8, 132.5(2 × C), 137.4 (2 × C),
141.5,143.5, 161.5, 163.5; ES-MS: m/z 468.2 (M + H)+.
Anal. Calcd for C21H17N5O4S2: C, 53.95; H, 3.67; N,
(10a–j).
4-[3-(N0-Acetylhydrazinocarbonyl)-5-phenylpyrazol-1-yl]-
benzenesulfonamide (10a).
White solid; (0.52 g, yield:
1
72%); mp = 124–126°C; H NMR (DMSO-d6, 400 MHz)
δ 10.11 (s, 1H, ─CO─NH─), 9.78 (s, 1H, ─CO─NH─),
7.86 (d, 2H, J = 9.2 Hz, Ar─H), 7.54 (d, 2H, J = 9.2 Hz,
Ar─H), 7.48–7.52 (m, 2H, Ar─H), 7.41–7.43 (m, 3H,
Ar─H), 7.29–7.32 (m, 2H, Ar─H), 7.09 (s, 1H,
Pyrazole-H), 2.24 (s, 3H, ─CH3); 13C NMR (DMSO-d6,
100 MHz) δ 25.4, 108.6, 125.5 (3 × C), 126.6 (3 × C),
128.7, 128.6, 129.2, 129.1, 141.5, 143.5, 144.3, 145.8,
162.3, 172.5; ES-MS: m/z 400.3 (M + H)+; Anal. Calcd
for C18H17N5O4S: C, 54.13; H, 4.29; N, 17.53; found: C,
14.98; found: C, 53.93; H, 3.64; N, 14.96.
4-{3-[N0-(2,6-Dimethoxybenzoyl)-hydrazinocarbonyl]-5-
phenylpyrazol-1-yl}-benzenesulfonamide (10e). White solid;
(0.51 g, yield: 69%); mp = 132–134°C; 1H NMR (DMSO-
d6, 400 MHz) δ 10.29 (s, 1H, ─CO─NH─), 10.13 (s, 1H,
─CO─NH─), 7.86 (d,3H, J = 9.2 Hz, Ar─H), 7.56 (d,
3H, J = 8.8 Hz, Ar─H), 7.49 (m, 2H, Ar─SO2─NH2),
7.43–7.44 (m, 4H, Ar─H), 7.43–7.44 (m, 4H, Ar─H),
7.31–7.36 (m, 5H, Ar─H), 7.14 (s, 1H, Pyrazole-H), 6.70
(d, 3H, J = 8.8 Hz, Ar─H), 3.77 (s, 6H, Ar─OCH3), 13C
NMR (DMSO-d6, 100 MHz) δ 56.1, 56.3, 105.4,
107.3(2 × C), 108.4, 122.3, 125.7(2 × C), 126.4 (2 × C),
127.8, 128.5 (2 × C),128.1, 128.8, 128.9 (2 × C), 131.8,
132.5, 141.5, 144.5, 146.1, 160.4, 165.7; ES-MS: m/z
522.4 (M + H)+, Anal. Calcd for C25H23N5O6S: C, 57.57;
54.16; H, 4.32; N, 17.50.
4-{5-Phenyl-3-[N0-(3-trifluoromethylbenzoyl)-hydrazinocarbonyl]-
pyrazol-1-yl}-benzenesulfonamide (10b).
Greenish solid;
(0.55 g, yield: 74%); greenish solid; mp = 136–138°C;
1H NMR (DMSO-d6, 400 MHz) δ 10.81 (s, 1H,
─CO─NH─), 10.51 (s, 1H, ─CO─NH─), 8.27(s, 1H,
Ar─H), 8.23 (d, 1H, J = 8 Hz, Ar─H), 7.99 (d, 1H,
J = 7.6 Hz, Ar─H), 7.88 (d, 2H, J = 8.4 Hz, Ar─H), 7.78
(t, 1H, J = 7.6 Hz, Ar─H), 7.57 (d, 2H, J = 8.4 Hz,
Ar─H), 7.50 (s, 2H, Ar─SO2─NH2), 7.32–7.34 (m, 2H,
Ar─H 7.42–7.44 (m, 3H, Ar─H), 7.34–7.35 (m, 2H,
Ar─H), 7.16 (s, 1H, Pyrazole-H), 13C NMR (DMSO-d6,
100 MHz) δ 108.7, 122.5, 125.2 (3 × C), 125.7, 126.7,
128.5, 128.8 (2 × C), 129.5, 129.9 (2 × C), 131.6
(2 × C), 133.3, 141.5, 143.6, 144.6, 146.0, 160.4, 164.3;
H, 4.45; N, 13.43; found: C, 57.54; H, 4.43; N, 13.47.
4-{5-Phenyl-3-[N0-(3-phenylacryloyl)-hydrazinocarbonyl]-
pyrazol-1-yl}-benzenesulfonamide (10f).
White solid;
(0.55 g, yield: 80%);mp = 128–130°C; 1H NMR
(DMSO-d6, 400 MHz) δ 10.38 (s, 1H, ─CO─NH─),
10.24 (s, 1H, ─CO─NH─), 7.89 (d, 2H, J = 8 Hz,
Ar─H), 7.71 (br s, 2H, Ar─H), 7.49–7.64 (m, 5H,
Ar─H), 7.49 (s,1H, Ar─SO2─NH2), 7.41–7.45 (m, 2H,
Ar─H), 7.32–7.36 (m, 2H, Ar─H), 7.14 (s, 1H,
Pyrazole-H), 6.77 (s, 1H, ─CO─CH═CH─), 6.73 (s, 1H,
─CH═CH─Ar); 13C NMR (DMSO-d6, 100 MHz) δ
108.5, 119.1, 124.9 (2 × C), 125.3, 126.5 (2 × C), 126.7
(2 × C), 127.3 (2 × C), 128.4 (2 × C), 128.1, 128.8,
ES-MS: m/z 528.5 (M
+
H)+; Anal. Calcd for
C24H18F3N5O4S: C, 54.44; H, 3.43; N, 13.23; found: C,
54.47; H, 3.45; N, 13.25.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2018
Anti-inflammatory Exploration of Sulfonamide Containing Diaryl Pyrazoles with
Promising COX-2 Selectivity and Enhanced Gastric Safety Profile
128.9 (2 × C), 131.8, 132.5, 141.5, 142.8, 144.5, 146.1,
160.4, 165.7; ES-MS: m/z 488.2 (M + H)+; Anal. Calcd
for C25H21N5O4S: C, 61.59; H, 4.34; N, 14.36; found:
(DMSO-d6, 100 MHz) δ 108.5, 113.4, 113.7, 126.3
(2 × C), 127.4, 127.6 (2 × C), 128.1, 128.8, 128.9
(2 × C), 131.7, 133.5(2 × C), 136.5, 137.4 (2 × C),
141.5, 161.5, 163.5; ES-MS: m/z 451.2 (M + H)+, Anal.
Calcd for C21H17N5O5S: C, 55.87; H, 3.80; N, 15.51.
Found: C, 55.90; H, 3.84; N, 15.53.
C, 61.58; H, 4.37; N, 14.39.
4-{3-[N0-(3-Chlorobenzoyl)-hydrazinocarbonyl]-5-phenylpyrazol-
1-yl}-benzenesulfonamide (10g). Yellowish solid; (0.53 g,
yield: 76%); mp = 125–127°C; 1H NMR (DMSO-d6,
400 MHz) δ 10.89 (s, 1H), 10.42 (s, 1H), 7.88 (d, 2H,
J = 8.4 Hz, Ar─H), 7.51–7.57 (m, 5H, Ar─H), 7.46
(s, 2H, Ar─SO2─NH2), 7.42–7.45 (m, 4H, Ar─H), 7.32–
7.34 (m, 2H, Ar─H), 7.14 (s, 1H, Pyrazole-H), 13C NMR
(DMSO-d6, 100 MHz) δ 108.5, 121.8, 125.2 (3 × C),
125.7, 127.3, 128.7, 128.5 (2 × C), 129.5, 131.2 (2 × C),
131.6 (2 × C), 133.4, 142.0, 143.6, 144.6, 146.2, 160.1,
164.2; ES-MS: m/z 496.3 (M + H)+; Anal. Calcd for
C23H18ClN5O4S: C, 55.70; H, 3.66; N, 14.12; found: C,
Determination of anti-inflammatory activity.
All the
prepared target compounds 5a–e and 10a–j were
screened for their anti-inflammatory activities against
cyclooxygenase-1 and cyclooxygenase-2 at Deshpande
Laboratories, Bhopal, M.P. Inhibition by test compounds
5a–e and 10a–j of ovine COX-1 and human
recombinant COX-2 [IC50 values (μM)] was assessed
using a COX Fluorescent Inhibitor Screening Assay Kit
(catalog number 700100, Cayman Chemical, Ann Arbor,
MI, USA).
In vitro cyclooxygenase inhibition assay. Stock solutions
of test compounds were prepared in a minimum volume
of DMSO. Briefly, to a series of supplied reaction buffer
solutions (150 mL, 100mM Trise-HCl, pH 8.0) with
either COX-1 or COX-2 (10 mL) enzyme in the
presence of Heme (10 mL) and fluorometric substrate
(10 mL) were added 10 mL of various concentrations of
the test compound solutions (final between 0.01 and
100mM). The reactions were initiated by quickly adding
10 mL of arachidonic acid solution and then incubated
for 2 min at room temperature. Fluorescence of resorufin
that is produced by the reaction between PGG2 and the
55.68; H, 3.67; N, 14.10.
4-[3-(N0-Cyclohexanecarbonyl-hydrazinocarbonyl)-5-
phenylpyrazol-1-yl]-benzenesulfonamide (10h). White solid;
(0.50 g, yield: 76%); mp = 134–136°C; 1H NMR (DMSO-
d6, 400 MHz) δ 10.11 (s, 1H, ─CO─NH─), 9.78 (s, 1H,
─CO─NH─), 7.86 (d, 2H, J = 9.2 Hz, Ar─H), 7.54 (d,
3H, J = 9.2 Hz, Ar─H), 7.48 (s, 1H, Ar─SO2─NH2),
7.41–7.43 (m, 3H, Ar─H), 7.29–7.32 (m, 2H, Ar─H),
7.09 (s, 1H, Pyrazole-H), 2.24 (m, 1H, ─CH2─CH2─),
1.62–1.90 (m, 5H, ─CH2─CH2─), 1.15–1.41 (m, 5H,
─CH2─CH2─),13C NMR (DMSO-d6, 100 MHz)
δ
25.1(2 × C), 25.4, 29.0 (2 × C), 42.0, 108.6, 125.5
(3 × C), 126.6 (3 × C), 128.7, 128.8, 129.0, 129.1, 141.5,
143.5, 144.4, 146.1, 160.0, 174.5; ES-MS: m/z 468.3
(M + H)+; Anal. Calcd for C23H25N5O4S: C, 59.09; H,
fluorometric
substrate,
ADHP
(10-acetyl-3,7-
dihydroxyphenoxazine) was read with an excitation
wavelength of 535 nm and an emission wavelength of
590 nm. The intensity of this fluorescence is
proportional to the amount of resorufin, which is
proportional to the amount of PGG2 present in the well
during the incubation. Percent inhibition was calculated
by comparison from the 100% initial activity sample
value (no inhibitor). The concentration of the test
compound causing 50% inhibition of COX-1 and COX-
2 (IC50, mM) was calculated from the concentration-
inhibition response curve (triplicate determinations);
results were tabulated in Table 1.
5.39; N, 14.98; found: C, 59.06; H, 4.37; N, 14.99.
4-{5-Phenyl-3-[N0-(pyridine-3-carbonyl)-hydrazinocarbonyl]-
pyrazol-1-yl}-benzenesulfonamide (10i).
Yellowish solid
(0.49 g, yield: 75%); mp = 148–150°C;1H NMR
(DMSO-d6, 400 MHz) δ 10.49 (s, 1H, ─CO─NH─),
10.36 (s, 1H, ─CO─NH─), 9.08(s, 1H, Ar─H), 8.58–
8.659 (m, 2H, Ar─H) 7.99 (d, 2H, J = 8 Hz, Ar─H),
7.84 (d, 2H, J = 8 Hz, Ar─H), 7.54–7.58 (m, 2H, Ar─H),
7.49 (s, 2H, Ar─SO2─NH2), 7.42–7.44 (m, 3H, Ar─H),
7.31–7.32 (br s, 2H, Ar─H), 7.13 (s, 1H, Pyrazole-H),
13C NMR (DMSO-d6, 100 MHz) δ 108.7, 125.7(2 × C),
126.7 (2 × C), 127.5 (2 × C), 128.5 (2 × C),128.1, 128.7,
128.9 (2 × C), 131.8, 132.5, 141.5, 143.5, 144.5, 148.3,
153.7, 161.3, 165.6; ES-MS: m/z 463.3 (M + H)+, Anal.
Calcd for C22H18N6O4S: C, 57.13; H, 3.92; N, 18.17; O,
In vivo anti-inflammatory activity.
Compounds with
better in vitro anti-inflammatory activities (IC50) were
evaluated further for in vivo anti-inflammatory activity.
Five compounds (10b, 10c, 10d, 10g, and 10i) were
studied using carrageenan-induced rat paw edema method
at a dose of 50 mg/kg. The sub planter region of right
hind paw of each rat was injected with a freshly prepared
aqueous suspension of carrageenan (1.0% w/v, 0.1 mL).
Out of the two groups of animals, one group was kept as
control, and other were pretreated with test drugs and
standard drug 1 h before the carrageenan treatment.
Results are reported in Table 2. Digital plethysmometer
(UGO Basil, Italy) was used to measure the paw volume
13.84; found: C, 57.15; H, 3.95; N, 18.16.
4-{3-[N0-(Furan-2-carbonyl)-hydrazinocarbonyl]-5-phenylpyrazol-
1-yl}-benzenesulfonamide (10j).
Brownish solid (0.54 g,
yield: 85%); mp = 138–140°C; 1H NMR (DMSO-d6,
400 MHz) δ 10.54 (s, 1H, ─CO─NH─), 10.44 (s, 1H,
─CO─NH─), 7.88–7.92 (m, 4H, Ar─H), 7.58 (d, 2H,
J = 8.0 Hz, Ar─H), 7.49 (s, 2H, Ar─SO2─NH2), 7.44–
7.46 (m, 3H, Ar─H), 7.34–7.37 (m, 2H, Ar─H), 7.24–
7.26 (m, 1H, Ar─H), 7.14 (s, 1H, Pyrazole-H), 13C NMR
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

L. S. Pavase, D. V. Mane, and K. G. Baheti
Vol 000
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ðVt ꢀ VcÞcontrol ꢀ ðVt ꢀ VcÞtested compound
%inhibition ¼
ꢁ 100
ðVt ꢀ VcÞ
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10g were studied further for ulcerogenicity index.
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Acknowledgments. The authors are grateful to Deshpande
Laboratories, Bhopal, Madhya Pradesh, India, for their help in
evaluation of anti-inflammatory activity, Principal, R.C. Patel In-
stitute of Pharmaceutical Education and Research, Shirpur, (MS),
India, for providing advanced molecular docking program Glide
(Schrodinger Inc., USA), Padmashree Mrs. Fatima Rafiq Zakaria,
Chairman, Maulana Azad Educational Trust, Aurangabad and All
India Council for Technical Education, (AICTE) New Delhi, for
providing the facilities and financial assistance towards purchase
of docking software, Schrodinger Maestro-9.0 with GLIDE
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SUPPORTING INFORMATION
[5] Al-Hourani, B. J.; Sharma, S. K.; Mane, J. Y.; Tuszynski, J.;
Baracos, V.; Kniess, T.; Suresh, M.; Pietzsch, J.; Wuest, F. Bioor Med
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Additional Supporting Information may be found online
in the supporting information tab for this article.
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet