Synthesis and anti-inflammatory activity of celecoxib like compounds

Article abstract of DOI:10.3109/14756366.2012.710847

Nine novel 4-[3-(4-Dimethylamino-phenyl)-5-aryl-4,5-dihydro-pyrazol-1-yl]- benzenesulfonamides (2a-i) were synthesized and evaluated for their anti-inflammatory and antiproliferative activities. These compounds (2a-i) showed moderate to strong anti-inflammatory activity in carrageenan rat paw oedema test. Compounds 2b, 2d and 2g showing comparable anti-inflammatory activity to that of reference drug celecoxib were evaluated for their ulcerogenic and analgesic activities. The effect of 2b, 2d and 2g on the content of NO, TNF-α and PGE2 in exudates from rat paw stimulated by carrageenan was also evaluated. The compound 2c showed considerable antitumor activities against all 60 human tumor cell lines with effective GI50 (MG-MID) value of 3.63 μM. It exhibited maximum activity against melanoma (LOX IMVI and SK-MEL-5) cancer cell lines with GI50 value less than 2 μM.

Full text of DOI:10.3109/14756366.2012.710847

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; Early Online: 1–8
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.710847
SHORT COMMUNICATION
Synthesis and anti-inflammatory activity of celecoxib
like compounds
Syed Ovais1, Shafiya Yaseen1, Rafia Bashir1, Pooja Rathore1, Mohammed Samim1,
Surender Singh2, Vinod Nair2, and Kalim Javed1
1Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University), New Delhi, India and
2Department of Pharmacology, All India Institute of Medical Sciences, New Delhi, India
Absract
Nine novel 4-[3-(4-Dimethylamino-phenyl)-5-aryl-4,5-dihydro-pyrazol-1-yl]-benzenesulfonamides (2a-i) were
synthesized and evaluated for their anti-inflammatory and antiproliferative activities. These compounds (2a-i)
showed moderate to strong anti-inflammatory activity in carrageenan rat paw oedema test. Compounds 2b, 2d
and 2g showing comparable anti-inflammatory activity to that of reference drug celecoxib were evaluated for their
ulcerogenic and analgesic activities. The effect of 2b, 2d and 2g on the content of NO, TNF-α and PGE2 in exudates
from rat paw stimulated by carrageenan was also evaluated. The compound 2c showed considerable antitumor
activities against all 60 human tumor cell lines with effective GI50 (MG-MID) value of 3.63 µM. It exhibited maximum
activity against melanoma (LOX IMVI and SK-MEL-5) cancer cell lines with GI50 value less than 2 μM.
Keywords: Pyrazoline, tumor necrosis factor (TNF-α), prostaglandin E2 (PGE2), nitrite (NO), antiproliferative activity
Introduction
structurally novel sulfonamide derivatives have recently
been reported to show substantial antitumor activ-
ity, both in vitro and/or in vivo. Although they have
a common chemical motif of aromatic/heterocyclic
sulfonamide, there are a variety of mechanisms of their
antitumor action, most of them poorly understood at this
moment. Some of these derivatives are currently being
evaluated in clinical trials, and there is much optimism
that they may lead to novel alternative anticancer drugs,
devoid of the side effects of the presently available phar-
macological agents.22 Many selective COX-2 inhibitors
belong to a group of compounds which have a central
tri-substituted planar ring (five-membered heterocyclic
ring) attach to pendent benzene rings and a lipophilic
group. One of the phenyl rings is attached to SO2NH2.
e SO2NH2 pharmacophore is believed to induce COX-2
selectivity.23
Pyrazoline act as useful lead molecules as they show sig-
nificant pharmacological properties and clinical applica-
tions. ese derivatives have been reported to possess
activities like anti-inflammatory,1–4 antidepressant,5
anticancer,5,6 anti-bacterial,5 anti-tubercular,7,8 analge-
sic,7,8 anti-diabetic.9 Some of the pyrazole derivatives like
antipyrine, phenylbutazone, celecoxib etc. have found
application in clinical medicine.
Chlacones bearing dimethylaminophenyl moiety are
reported as potential inhibitors of NO (nitric oxide) and
PGE2 (prostaglandin E2) production in the RAW 264.7
macrophage cell line.10 More over dimethylaminophenyl
bearing pyrazolines have proved to possesses significant
anti-inflammatory2,4 and anti-cancerous activities.11
e sulfonamides constitute an important class of
drugs, with several types possessing a host of biologi-
cal properties including antibacterial,12 anti-carbonic
anhydrase,13,14,15 diuretic,13,16 hypoglycaemic,17 antithy-
roid18 and anti-protease activities.19–21 A large number of
Promoted with the above mentioned findings the
present study aimed at gathering the two bioactive
entities (chlacones containing dimethylaminophenyl
Address for Correspondence: Dr Kalim Javed, Department of Chemistry, Faculty of Science, Jamia Hamdard (Hamdard University),
New Delhi – 110 062, India. Tel: +919873463272. E-mail: kjaved@jamiahamdard.ac.in/kjavedchem@yahoo.co.in
(Received 07 March 2012; revised 06 July 2012; accepted 07 July 2012)
1

2
S. Ovais et al.
moiety and benzenesulfonamide) into one compact
structure and evaluating their biological activities as
anti-inflammatory agent with reduced gastric toxicity.
observed at expected ppms. In 13C spectra of these com-
pounds (2a-i) the chemical shift values of carbon atoms
of pyrazoline ring are C-3 (150.88–151.63 ppm), and C-5
(59.98–62.63 ppm), however the signal of C-4 of pyrazo-
line was found to be merged with solvent (DMSO) peak.
Results and discussion
Chemistry
Pharmacology
Anti-inflammatory activity
e target compounds were synthesized according to
steps outlined in scheme 1. e intermediates chalcones
(1a-i) were obtained by Claisen-Schmidt condensation
of 1-[4-(dimethylamino)phenyl]ethanone with appro-
priate aromatic aldehydes in presence of base (NaOH).
Treatment of the intermediate chalcone with 4-hydra-
zinobenzenesulfonamide hydrochloride in refluxing
absolute ethanol yielded corresponding 2-pyrazoline
derivatives (2a-i). e structures of all novel compounds
e anti-inflammatory activity of target compounds (2a-
i) was evaluated by applying carrageenan-induced hind
paw oedema bioassay in rats24 using celecoxib as refer-
encedrugasithassomechemicalstructuralresemblance
with target compounds. e results are summarised
in Table 1. ese compounds (2a-i) showed moderate
to strong anti-inflammatory activity (49.5–75.9% at 3 h
and 42.2–88.8% at 5 h). e anti-inflammatory activity
of 2d and 2g is almost comparable to that exhibited by
standard drug celecoxib at 3 h and 5 h. Compound 2b
showed 86.6% inhibition of paw oedema at 5 h which is
comparable to that of celecoxib (82.2%). Introduction
of methyl or chloro or methoxyl group in 5-phenyl ring
increases the activity. Replacement of 5-phenyl ring with
furyl ring diminishes the activity at 5 h (2a vs. 2h).
was established through spectroscopic (IR, H NMR, 13C
1
NMR, MS) and elemental analysis data. e IR spectra of
pyrazolines (2a-i) showed absorption bands in the region
of 1587–1601cm−1 corresponding to C=N stretching bands
because of ring closure. Also, infrared spectra revealed
NH2 peak at 3309–3413cm−1 and 3216–3305cm−1 and for
SO2NH2 peak at 1311–1334cm−1 and 1146–1155cm−1. In
1
the HNMR spectra of 2-pyrazolines, the three hydrogen
atoms attached to the C-4 and C-5 carbon atoms of the het-
erocyclic ring gave an ABX spin system. Measured chemi-
cal shift and coupling constant values (cf. Experimental
Section) prove the 2-pyrazoline structure. NH proton
of the SO2NH2 group was observed at 6.89–7.03 ppm as
singlet. e protons of methyl and aromatic protons were
Ulcerogenic activity
Ulcerogenic activity of the compounds 2b, 2d and 2g was
also recorded at dose of 0.15 mmole/kg and compared
with reference drug celecoxib25 (0.15 mmole/kg). ese
compounds did not show any ulceration or harmful
effects on the stomach in fasted rats.
1a, 2a; Ar = phenyl 1b,
2b; Ar = 4-chlorophenyl
1c, 2c; Ar = 4-methoxyphenyl
1e, 2e; Ar = 2-chlorophenyl
1g, 2g; Ar = 3,4,5-trimethoxyphenyl
1i, 2i; Ar = anthryl (C-9)
1d, 2d; Ar = 4-methylphenyl
1f, 2f; Ar = 3,4-dimethoxyphenyl
1h, 2h; Ar = furyl (C-2)
Scheme 1. Synthesis of pyrazolines bearing benzenesulfonamide moiety.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and anti-inflammatory activity
3
Table 1. Effect of dimethylamino-phenyl based pyrazolines on carageenan induced hind paw oedema in rats.
Pyrazoline (2a-i)
Increase in paw volume ml SEM after carageenan administrationa
Group
no.
Treatment
(0.05 mmole/kg)
Ar
—
—
3 h
5 h
I
Vehicle
0.416 0.03
0.450 0.10
II
Celecoxib
0.1 0.01** (75.9%)b
0.08 0.02** (82.2%)
III
IV
2a
2b
0.21 0.02** (49.5%)
0.21 0.02** (53.3%)
0.15 0.03** (63.9%)
0.06 0.03** (86.6%)
V
2c
0.15 0.02** (63.9%)
0.20 0.01** (55.5%)
VI
2d
0.10 0.02** (75.9%)
0.08 0.03** (82.2%)
VII
2e
2f
0.20 0.02** (51.9%)
0.16 0.01** (61.5%)
0.15 0.01** (66.6%)
0.10 0.01** (77.7%)
VIII
IX
2g
0.10 0.02** (75.9%)
0.05 0.03** (88.8%)
X
2h
2i
0.21 0.03** (49.5%)
0.18 0.03** (56.7%)
0.26 0.02** (42.2%)
0.18 0.03** (60.0%)
XI
**p < 0.001 compared to control (one-way ANOVA followed by Dunnett’s test).
aValues are presented as mean SEM (n = 6). bValues in parentheses represent percent inhibitions.
Effect of 2b, 2d and 2g on TNF-α, nitrite and PGE
production in exudates from rat paw stimulated2by
carrageenan
Analgesic activity
e analgesic activity for the compounds 2b, 2d and
2g was determined by tail immersion26 method at the
dose of 0.05 mmole/kg bw. e result is summarized in
Table 2. e result shows that compound 2d and 2g are
significantly more potent than standard drug celcecoxib.
However compound 2b shows activity almost equal to
standard drug.
Compounds showing good anti-inflammatory activity 2b,
2d and 2g (0.05 mmole/kg) were evaluated for their action
onthepro-inflammatorycytokine(TNF-α),nitriteandPGE2
levels in exudates from rat paw stimulated by carrageenan.
e results are shown in Figure 1a–1c (Supplementary
data) respectively. ese three compounds reduced the
content of NO, TNF-α and PGE2 significantly. Compound
(2g) inhibited the production of TNF-α and PGE2 signifi-
cantly as compared to the celecoxib.
In-vitro anticancer activity
Primary in vitro one-dose (10–5 M) anticancer assay was
performed using full panel of about 60 human tumor
© 2012 Informa UK, Ltd.

4
S. Ovais et al.
cell lines in accordance with the protocol of the Drug
Evaluation Branch, National Cancer Institute (NCI),
Bethesda, and described elsewhere.27–32 e human
tumor cell lines were derived from nine different cancer
types: Leukemia, melanoma, lung, colon, CNS, ovarian,
renal, prostate, and breast cancers. Out of the synthe-
sized compounds (2a-i), two compounds, namely 2b
and 2c were selected by the National Cancer Institute
(NCI). e compounds 2b displayed some sensitivity
towards cell lines K-562 (Leukemia) and PC-3 (prostrate
cancer) (Supplementary Table S1). e compound 2c
(NSC 754832) possessed considerable antiproliferative
activity (Supplementary Table S2) and it was selected for
an advanced assay against a full panel (approximately 60
cell lines) at five concentrations at 10-fold dilution (100,
10, 1, 0.1 and 0.001 μM). A 48 h continuous drug expo-
sure protocol was used and sulforhodamine B (SRB)
protein assay was used to estimate cell growth. Details
of this system and the information which is encoded
by the activity pattern over all cell lines have been pub-
lished.27–29 e anticancer activity of tested compounds
is given by three parameters for each cell line: log GI50
value (GI50 = molar concentration of the compound
that inhibits 50% net cell growth), log TGI value (TGI =
molar concentration of the compound leading to total
inhibition) and log LC50 value (LC50 = molar concentra-
tion of the compound leading to 50% net cell death).
Furthermore, a mean graph midpoint (MG_MID) is
calculated for each of the mentioned parameters, giv-
ing an averaged activity parameter over all cell lines.
For the calculation of the MG_MID, insensitive cell
lines are included with the highest concentration tested.
Selectivity of the compound with respect to one or more
cell lines of the screen is characterized by a high devia-
tion (∆) of the particular cell line parameter compared
to the MG_MID value.
e compound 2c showed considerable antitumor
activities against the entire tested tumor cell lines and
showed effective growth inhibition GI50 (MG-MID) val-
ues of 3.63 µM. e compound 2c exhibited maximum
activity against melanoma (LOX IMVI and SK-MEL-5)
cancer cell lines with a GI50 value of less than 2 μM. It also
displayed good activity against non-small cell lung can-
cer (NCI-H226, NCI-H460, NCI-H522), colon (COLO 205,
HCC-2998, HCT-116, HCT-15), melanoma (LOX IMVI,
SK-MEL-5), renal cancer (A498, RXF 393, SN12C) and
breast (BT-549) cancer cell lines with a GI50 less than 3.0
μM (Supplementary Table S3).
Figure 1. Effect of 2b, 2d and 2g on (a) TNF-α levels; (b) nitrite
levels; (c) PGE2 levels.
Table 2. Analgesic effect of compounds 2b, 2d and 2g by tail
immersion method.
Reaction Timea (Sec)
Treatment
(0.05
mmole/kg) treatmentb
Control 2.59 0.07** 2.87 0.02** 3.01 0.03** 2.98 0.07**
Celecoxib 3.10 0.07** 3.76 0.07** 4.72 0.10** 4.89 0.03**
Before
30 min
60 min
90 min
Conclusions
e present study describes the synthesis of a series
of nine novel 4-[3-(4-Dimethylamino-phenyl)-5-
aryl-4,5-dihydro-pyrazol-1-yl]-benzenesulfonamides
(2a-i) and their evaluation for anti-inflammatory and
anti-poliferative activities. Among these synthesised
compounds three compounds (2b, 2d and 2g) exhib-
ited anti-inflammatory activity comparable with that
2b
2d
2g
2.98 0.02** 4.65 0.01** 5.34 0.10** 4.89 0.07**
3.39 0.02** 5.03 0.03** 5.84 0.02** 5.97 0.06*
3.10 0.01** 5.72 0.03** 6.02 0.02** 6.29 0.04**
*p < 0.05; **p < 0.01; compared to control (one-way ANOVA
followed by Dunnett’s test).
aRepresents time in seconds. bValues are presented as mean SEM
(n = 6).
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and anti-inflammatory activity
5
of celecoxib and had superior gastro-intestinal safety
profile when tested for their ulcerogenic activity. e
effect of 2b, 2d and 2g on the content of NO, TNF-α
and PGE2 in exudates from rat paw stimulated by car-
rageenan was also evaluated. Compound 2g reduced
the production of TNF-α and PGE2 significantly. e
analgesic activity for the compounds 2b, 2d and 2g
was determined by tail immersion method. e com-
pounds 2d and 2g showed more potent analgesic activ-
ity than standard drug celecoxib. However compound
2b shows activity almost equal to standard drug. Two
compounds, namely 2b and 2c were screened for their
antiproliferative activity. e compound 2c showed
considerable antitumor activities against all 60 human
tumor cell lines and showed effective growth inhibition
GI50 (MG-MID) values of 3.63 µM.
4-[3-(4-Dimethylamino-phenyl)-5-phenyl-4,5-dihydro-
pyrazol-1-yl]-benzenesulfonamide (2a)
Recrystalized from MeOH as pale yellow solid, yield 71%,
m.p. 240–241°C, Rf = 0.58 (toluene: ethyl acetate: formic
acid, 5: 4: 1), IR υmax (KBr): 3378 cm−1 & 3272 cm−1 (NH2),
1594 cm−1 (C=N), 1328 cm−1 & 1146 cm−1 (SO2N<); 1H
NMR (300 MHz, DMSO, δ): 2.94 [6H, s, N(CH3)2], 3.20
[1H, dd, J = 12.6 Hz, J = 21.6 Hz, H-4 trans (pyrazoline)],
3.87–3.83 [1H, m, H-4 cis (pyrazoline)], 5.52–5.49 [1H,
m, H-5 (pyrazoline)], 6.72 (2H, d, J = 9.0 Hz, H-3’, H-5’),
6.95 (2H, s, SO2NH2), 6.97 (2H, d, J = 9.0 Hz, H-3’’, H-5’’),
7.31-7.20 (5H, m, H-2, H-3, H-4, H-5, H-6), 7.51 (2H, d,
J = 8.7 Hz H-2’’, H-6’’), 7.58 (2H, d, J = 8.7 Hz, H-2’, H-6’);
13C NMR (75 MHz, DMSO, δ): 43.30 N(CH3)2, 61.91 (C-5
pyrazoline), 151.01 (C-3 pyrazoline). ESI-MS (m/z): 420
[M+], 419 [M+-1], 421[M++1]; CHNS Analysis: Found
(Calculated): C: 65.71 (65.69), H: 5.71 (5.75), N: 13.33
(13.32), S: 7.61 (7.62); Molecular Formula: C23H24N4O2S.
Experimental section
Chemistry
4-[5-(4-Chloro-phenyl)-3-(4-dimethylamino-phenyl)-4,5-
dihydro-pyrazol-1-yl]-benzenesulfonamide (2b)
Melting points were determined by open capillary tubes
and are uncorrected. All the Fourier Transform Infra Red
(FTIR) spectra were recorded on a Brukers Vector 22
spectrophotometer in film; υmax values are given in cm−1.
1H NMR spectra were recorded on a Bruker Spectrospin
DPX 300 MHz/400 MHz spectrometer using deuterated
DMSO as solvent and tetramethyl silane (TMS) as an
internal standard. Chemical shifts are given in δ (ppm)
scale and coupling constants (J values) are expressed
in Hz. Mass spectra (MS) were scanned by ESI Q-TOF
Water. e m/z values of the more intense peaks are
mentioned. 13C NMR spectra was recorded on Bruker
spectrospin DPX 400 MHz using deuterated DMSO
as a solvent and tetramethyl silane (TMS) as internal
standard. Purity of the compounds was checked on TLC
plates (silica gel G) which were visualized by exposing
to iodine vapors. Elemental analysis was carried out on
CHNS Elementar (Vario EL III).
Recrystalized from MeOH as off white solid, yield 61%,
m.p. 228–229oC, Rf = 0.40 (toluene: ethyl acetate: formic
acid, 5: 4: 1), IR υmax (KBr): 3379 cm−1 & 3273 cm−1 (NH2),
1595 cm−1 (C=N), 1325 cm−1 & 1152 cm−1 (SO2N<); 1H
NMR (300 MHz, DMSO, δ): 2.94 [6H, s, N(CH3)2], 3.09
[1H, dd, J = 4.8 Hz, J = 17.7 Hz, H-4 trans (pyrazoline)],
3.85 [1H, dd, J = 12.0 Hz, J = 17.7 Hz, H-4 cis (pyrazoline)],
5.55–5.52 [1H, m, H-5 (pyrazoline)], 6.73 (2H, d, J = 8.7
Hz, H-3’, H-5’), 6.99 (4H, s, SO2NH2 H-3’’, H-5’’), 7.23 (2H,
d, J = 8.1 Hz, H-3, H-5), 7.38 (2H, d, J = 8.4 Hz, H-2, H-6),
7.82–7.54 (4H, m, H-2’, H-6’, H-2’’, H-6’’); 13C NMR (75 MHz,
DMSO, δ): 43.50 N(CH3)2, 61.65 (C-5 pyrazoline), 150.88
(C-3 pyrazoline); ESI-MS (m/z): 454 [M+], 453 [M+-1], 455
[M++1]; CHNS Analysis: Found (Calculated): C: 60.79
(60.72), H: 5.06 (5.10), N: 12.33 (12.31), S: 7.04 (7.05);
Molecular Formula: C23H23ClN4O2S.
4-[3-(4-Dimethylamino-phenyl)-5-(4-methoxy-phenyl)-4,5-
dihydro-pyrazol-1-yl]-benzenesulfonamide (2c)
General procedure for the synthesis of chalcones (1a-i)
To a cold solution (below 10°C) of acetophenone (0.01
mole) and desired aromatic aldehyde (0.01 mole) in
ethanol (20 mL) was added a chilled solution of sodium
hydroxide (10 mL of 30% solution). e reaction mixture
was covered with a layer of petroleum ether (60–80°C)
and left at room temperature for a period of 12 h. It was
poured on crushed ice (100 mL) and acidified with HCl.
e product obtained was filtered, washed with water
and crystallized from appropriate solvent.
Recrystalized from MeOH as pale yellow solid, yield 67%,
m.p. 185–187oC, Rf = 0.25 (toluene: ethyl acetate: formic
acid, 5: 4: 1), IR υmax (KBr): 3343 cm−1 & 3251 cm−1 (NH2),
1598 cm−1 (C=N), 1329 cm−1 & 1153 cm−1 (SO2N<); 1H
NMR (300 MHz, DMSO, δ): 2.93 [6H, s, N(CH3)2], 3.03
[1H, dd, J = 17.4 Hz, J = 21.6 Hz, H-4 trans (pyrazoline)],
3.78 (3H, s, OCH3), 3.85 [1H, dd, J = 12.0 Hz, J = 17.7 Hz,
H-4 cis (pyrazoline)], 5.46–5.43 [1H, m, H-5 (pyrazoline)],
6.72 (2H, d, J = 8.4 Hz, H-3’, H-5’), 6.85 (2H, d, J = 8.1Hz,
H-3, H-5), 6.95 (2H, s, SO2NH2), 6.97 (2H, d, J = 9.0 Hz,
H-2, H-6), 7.13 (2H, d, J = 8.4 Hz, H-3’’, H-5’’), 7.51 (2H, d, J
General procedure for the preparation of pyrazolines (2a-i)
A solution of appropriate chalcone (1a-i) (0.01 mole)
and 4-hydrazinobenzenesulfonamide hydrochloride
(0.01 mole) in absolute ethanol (20–150 ml) was refluxed
for 12–16 h. e reaction mixture was concentrated to a
small volume and left at room temperature when 2-pyr-
azoline separated out, which was filtered, dried and crys-
tallized from appropriate solvent.
= 8.7 Hz, H-2’’, H-6’’), 7.57 (2H, d, J = 8.4 Hz, H-2’, H-6’); 13
C
NMR (75 MHz, DMSO, δ): 43.80 N(CH3)2, 55.93 (OCH3
at C-3 and C-4), 62.35 (C-5 (yrazoline), 151.41 (C-3 pyr-
azoline); ESI-MS (m/z): 450 [M+], 449 [M+-1], 451 [M++1];
CHNS Analysis: Found (Calculated): C: 64.00 (63.98),
H: 5.77 (5.82), N: 12.44 (12.44), S: 7.11 (7.12); Molecular
Formula: C24H26N4O3S.
© 2012 Informa UK, Ltd.

6
S. Ovais et al.
4-[3-(4-Dimethylamino-phenyl)-5-p-tolyl-4,5-dihydro-
pyrazol-1-yl]-benzenesulfonamide (2d)
4-[3-(4-Dimethylamino-phenyl)-5-(3,4,5-trimethoxy-phenyl)-
4,5-dihydro-pyrazol-1-yl]-benzenesulfonamide (2g)
Recrystalized from MeOH as pale yellow solid, yield 53%,
m.p. 237–239°C, Rf = 0.67 (chloroform: actone: triethyl-
amine, 9: 1: 0.1), IR υmax (KBr): 3413 cm−1 & 3283 cm−1
(NH2), 1593 cm−1 (C=N), 1333 cm−1 & 1155 cm−1 (SO2N<);
1H NMR (300 MHz, DMSO, δ): 2.24 (3H, s, CH3), 2.96 [6H,
s, N(CH3)2], 3.08 [1H,dd, J = 4.8 Hz, J = 17.4 Hz, H-4 trans
(pyrazoline)], 3.88 [1H, dd, J = 12.3 Hz, J = 17.7 Hz,, H-4
cis (pyrazoline)], 5.49 [1H, dd, J = 4.8 Hz, J = 11.7 Hz, H-5
(pyrazoline)], 6.74 (2H, d, J = 9.0 Hz, H-3’, H-5’), 6.98 (2H,
s, SO2NH2), 6.99 (2H, d, J = 8.4 Hz, H-3’’, H-5’’), 7.13 (4H,
s, H-2, H-3, H-5, H-6 of tolyl unit appeared as singlet i.e
accidentally equivalent protons), 7.54 (2H, d, J = 8.7 Hz,
H-2’’, H-6’’), 7.61 (2H, d, J = 9.0Hz, H-2’, H-6’); 13C NMR (75
MHz, DMSO, δ): 20.06 (CH3 at C-4), 43.27 N(CH3)2, 61.70
(C-5 pyrazoline), 150.99 (C-3 pyrazoline); ESI-MS (m/z):
434 [M+], 433 [M+-1], 435 [M++1]; CHNS Analysis: Found
(Calculated): C: 66.35 (66.33), H: 5.99 (6.03), N: 12.90
(12.89), S: 7.37 (7.38); Molecular Formula: C24H26N4O2S.
Recrystalized from MeOH off white solid, yield 51%, m.p.
240–241oC, Rf = 0.45 (petrol: acetone, 7: 3), IR υmax (KBr):
3309 cm−1 & 3216 cm−1 (NH2), 1596 cm−1 (C=N), 1334 cm−1
& 1154 cm−1 (SO2N<); 1H NMR (300 MHz, DMSO, δ): 2.96
[6H, s, N(CH3)2], 3.15 [1H, dd, J = 6.0 Hz, J = 18.0 Hz, H-4
trans (pyrazoline)], 3.61 (3H, s, OCH3 at C-4), 3.68 [6H, s,
2(OCH3) at C-3, C-5], 3.87 [1H, dd, J = 11.1 Hz, J = 16.8 Hz
H-4 cis (pyrazoline)], 5.38 [1H, dd, J = 5.7 Hz, J = 11.1 Hz,
H-5 (pyrazoline)], 6.56 (2H, s, H-2, H-6), 6.74 (2H, d, J =
8.7 Hz, H-3’, H-5’), 6.98 (2H, s, SO2NH2), 7.03 (2H, d, J =
8.4 Hz, H-3’’, H-5’’), 7.57 (2H, d, J = 9.3 Hz, H-2’’, H-6’’), 7.60
(2H, d, J = 9.3 Hz, H-2’, H-6’); 13C NMR (75 MHz, DMSO,
δ): 43.46 N(CH3)2, 55.90 (OCH3 at C-3 and C-5), 60.01
(OCH3 at C-4), 62.63 (C-5 pyrazoline), 151.63 (C-3 pyr-
azoline); ESI-MS (m/z): 510 [M+], 509 [M+-1], 511 [M++1];
CHNS Analysis: Found (Calculated): C: 61.17 (61.16),
H: 5.88 (5.92), N: 10.98 (10.97), S: 6.27 (6.28); Molecular
Formula: C26H30N4O5S.
4-[3-(4-Dimethylamino-phenyl)-5-furan-2-yl-4,5-dihydro-
pyrazol-1-yl]-benzenesulfonamide (2h)
4-[5-(2-Chloro-phenyl)-3-(4-dimethylamino-phenyl)-4,5-
dihydro-pyrazol-1-yl]-benzenesulfonamide (2e)
Recrystalized from MeOH off white solid, yield 47%, m.p.
282–284oC, Rf = 0.68 (chloroform: acetone: triethylamine
9: 1: 0.1), IR υmax (KBr): 3346 cm−1 & 3258 cm−1 (NH2),
1592 cm−1 (C=N), 1325 cm−1 & 1147 cm−1 (SO2N<); 1H
NMR (300 MHz, DMSO, δ): 2.94 [6H, s, N(CH3)2], 3.76
[1H, dd, J = 12.0 Hz, J = 17.4 Hz, H-4 cis (pyrazoline)], 5.66
[1H, dd, J = 4.5 Hz, J= 11.4 Hz, H-5 (pyrazoline)], 6.38 (1H,
d, J = 1.5 HZ, H-3), 6.45 (1H, d, J = 2.4 Hz, H-4), 6.76 (2H, d,
J = 9.0 Hz, H-3’, H-5’), 7.03 (2H, s, SO2NH2), 7.14 (2H, d, J =
9.0 Hz, H-3’’, H-5’’), 7.64-7.57 [5H, m, H-2’, H-5’, H-2’’, H-6’’,
Recrystalized from acetone off white solid, yield 43%,
m.p. 244–245oC, Rf = 0.38 (toluene: ethyl acetate: formic
acid, 5: 4: 1), IR υmax (KBr): 3402 cm−1 & 3305 cm−1 (NH2),
1601 cm−1 (C=N), 1311 cm−1 & 1147 cm−1 (SO2N<); 1H
NMR (300 MHz, DMSO, δ): 2.93 [6H, s, N(CH3)2], 3.10–
3.04 [1H, m, H-4 trans (pyrazoline)], 3.98 [1H, dd, J = 12
Hz, J = 17.4 Hz, H-4 cis (pyrazoline)], 5.71–5.68 [1H, m,
H-5 (pyrazoline)], 6.71 (2H, d, J = 8.1 Hz, H-3’, H-5’), 6.89
(2H, d, J = 8.1 Hz, H-2’, H-6’), 7.59–6.99 (10 H, m, SO2NH2,
H-2’’, H-6’’,H-3’’, H-5’’, H-2, H-3, H-4, H-5); 13C NMR (75
MHz, DMSO, δ): 42.37 N(CH3)2, 59.98 (C-5 pyrazoline),
151.48 (C-3 pyrazoline); ESI-MS (m/z): 454 [M+], 453
[M+-1], 455 [M++1]; CHNS Analysis: Found (Calculated):
C: 60.70 (60.72), H: 5.06 (5.10), N: 12.33 (12.31), S: 7.04
(7.05); Molecular Formula: C23H23ClN4O2S.
H-5 (thio group)]; ESI-MS (m/z): 410 [M+], 411 [M++1]; 13
C
NMR (75 MHz, DMSO, δ): 60.72 (C-5 pyrazoline), 151.53
(C-3 pyrazoline); CHNS Analysis: Found (Calculated):
C: 61.46 (61.44), H: 5.36 (5.40), N: 13.60 (13.65), S: 7.80
(7.81); Molecular Formula: C21H22N4O3S.
4-[5-Anthracen-9-yl-3-(4-dimethylamino-phenyl)-4,5-
dihydro-pyrazol-1-yl]-benzenesulfonamide (2i)
4-[5-(3,4-Dimethoxy-phenyl)-3-(4-dimethylamino-phenyl)-
4,5-dihydro-pyrazol-1-yl]-benzenesulfonamide (2f)
Recrystalized from MeOH yellow solid in 53% yield,
m.p. 316–318°C, Rf = 0.32 (toluene: ethyl acetate: formic
acid, 5: 4: 1), IR υmax (KBr): 3403 cm−1 & 3304 cm−1 (NH2),
1587 cm−1 (C=N), 1332 cm−1 & 1152 cm−1 (SO2N<); 1H
NMR (300 MHz, DMSO, δ): 2.99 [6H, s, N(CH3)2], 3.46
[1H, dd, J = 9.6 Hz, J = 18.3 Hz, H-4 cis (pyrazoline)], 4.25
[1H, dd, J = 13.5 Hz, J = 17.7 Hz,, H-5 (pyrazoline)], 6.89–
6.75 (6H, m, SO2NH2, H-3’, H-5’, H-2’’, H-6’’), 7.31 (2H,
d, J = 8.7 Hz, H-3’’, H-5’’), 7.45–7.35 (2H, m, H-3, H-6),
7.64 (1H, t, H-7/H-2), 7.73 (3H, d, J = 8.4Hz, H-7/H-2,
H-2’, H-6’), 7.99 (1H, d, J = 8.7 Hz, H-5/H-4), 8.10 (1H,
d, J = 8.1 Hz, H-5/H-4), 8.21 (1H, d, J = 8.1 Hz, H-8/H-
1), 8.67 (1H, s, H-9), 8.84 (1H, d, J = 9.0 Hz, H-8/H-1);
ESI-MS (m/z): 520 [M+], 519 [M+-1], 521 [M++1]; CHNS
Analysis: Found (Calculated): C: 71.53 (71.51), H:
5.38 (5.42), N: 10.76 (10.76), S: 6.15 (6.16); Molecular
Formula: C31H28N4O2S.
Recrystalized from MeOH pale yellow solid, yield 58%,
m.p. 161–162oC, Rf = 0.22 (toluene: ethyl acetate: for-
mic acid, 5: 4: 1), IR υmax (KBr): 3344 cm−1 & 3265 cm−1
(NH2), 1599 cm−1 (C=N), 1334 cm−1 & 1151 cm−1 (SO2N<);
1H NMR (300 MHz, DMSO, δ): 2.93 [6H, s, N(CH3)2],
3.05–3.11 [1H, m, H-4 trans (pyrazoline)], 3.58–3.67 [6H,
m, (OCH3)2], 3.82–3.84 [1H, m, H-4 cis (pyrazoline)],
5.41–5.43 [1H, m, H-5 (pyrazoline)], 6.63 (1H, d, J = 9.0
Hz H-5), 6.71–6.85 (2H, d, J = 8.7 Hz, H-3’, H-5’), 6.97–6.85
(6H, m, SO2 NH2, H-2, H-6, H-3’’, H-5’’), 7.52 (2H, d, J =
6.9 Hz, H-2’’, H-6’’), 7.57 (2H, d, J = 8.1 Hz, H-2’, H-6’); 13
C
NMR (75 MHz, DMSO, δ): 43.80 N(CH3)2, 55.93 (OCH3
at C-3 and C-4), 62.35 (C-5 pyrazoline), 151.41 (C-3 pyr-
azoline); ESI-MS (m/z): 480 [M+], 479 [M+-1], 481 [M++1];
CHNS Analysis: Found (Calculated): C: 62.50 (62.48),
H: 5.83 (5.87), N: 11.66 (11.66), S: 6.66 (6.67); Molecular
Formula: C25H28N4O4S.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and anti-inflammatory activity
7
drug administration. At specified time (5 h) after the
subplantar injection of carrageenan, rats were sacri-
ficed and each paw was cut at the level of the calcaneus
bone. Paws were gently centrifuged at 250 g for 20 min.
in order to recover a sample of the oedematous fluid.
e amount of fluid that was recovered from each
paw was then passed through millipore cut-off filter
(10,000 mol wt.) to remove any traces of blood cells
(Millipore, Bedford, MA, USA).
Oedematous levels of pro-inflammatory cytokine
TNF- α were measured by the commercially available kit
(Rat TNF-α ELISA Ready-SET-Go! eBioscience, USA) fol-
lowing manufacturers protocol. e nitrite concentration
in oedematous fluid was measured according to Griess
reaction, and the calculated concentration was taken
as an indicator of NO production. 100 µl of oedematous
fluid was mixed with an equal volume of Griess reagent
(1% sulphanilamide in 5% phosphoric acid and 0.1%
naphthyl ethylenediamine dihydrochloride in water).
e reaction mixture was incubated at room temperature
for 10–15 min. e optical density at 550nm was mea-
sured and calculated against a sodium nitrite standard
curve. e oedematous levels of PGE2 by ELISA based
kit following instruction of the manufacturer (Cayman
chemicals, Ann Arbor, MI, USA).
Pharmacological activity
All the experiments were carried out in albino rats
of Wistar strain (either sex) were procured from
Central Animal House of Jamia Hamdard, New Delhi
(Registration no. 173/CPCSEA). e experiments were
performed in accordance with the guidelines for the
care and use of laboratory animals, laid down by the
Committee for the Purpose of Control and Supervision
of Experiments in Animals (CPCSEA), Ministry of Social
Justice and Empowerment, Govt. of India, Jan. 2000.
Animals described as fasted were deprived of food for at
least 16–24 h but allowed free access to water. CMC (1%
w/v) in distilled water was used as vehicle for dosing
in all the experiments. All treatments were given orally
except carrageenan.
Anti-inflammatory activity
Carrageenan induced hind paw oedema method was
used for evaluating anti-inflammatory activity.24 Fasted
rats were divided into eleven groups of six animals each.
Group I served as control and was given vehicle only
(10 mL/kg), while animals of group II were given cele-
coxib 0.05 mmole/kg suspended in the vehicle and served
as standard. Test compounds (2a-i) (0.05 mmole/kg)
suspended in vehicle (10 mL/kg) were administered to
respective groups. After 30 min, all animals were injected
with 0.1 mL of 1% carageenan solution (prepared in nor-
mal saline) in the subplantar aponeurosis of left hind
paw to induce inflammation and the volume of injected
paw was measured by using plethysmometer immedi-
ately (at 0 h). e paw volume was again measured after
3 h and 5 h. e average paw volume in a group of treated
rats was compared with vehicle (control group) and the
percentage inhibition of oedema was calculated by using
the formula:
Ulcerogenic activity
Acute gastric ulcerogenic effect of the compounds 2b,
2d and 2g were evaluated in Albino Wistar rats.25 Albino
wistar rats (150–220g) were fasted over 24 h and where
randomly allotted into five groups of six animals each.
Group I served as control and was given vehicle 10 ml/kg
(CMC 1% w/v in distilled water) orally. Groups II served
as standard and was administered orally celecoxib (0.15
mmole/kg i.e. three times the dose given in anti-inflam-
matory activity) suspended in vehicle. Group III, IV and V
were administered orally compounds 2b, 2d and 2g (0.15
mmole/kg) suspended in vehicle, respectively. ey were
sacrificed under deep anathesia after 6 h of dosing. eir
stomach were removed and opened through greater cur-
vature for examining lesions or bleedings. Compounds
2b, 2d and 2g showed no ulcers.
Percent inhibition = (1− Vt/Vc) × 100
Where Vt is the mean paw volume of the test drug treated
rats and Vc is the mean paw volume of the control.
e experiment was carried out under normal labo-
ratory conditions. e animals were handled gently to
avoid too much of stress on them which could result in an
increased adrenal output. A mark was made at the lateral
maleous of the left hind paw so that dipping was done to
the same level while measuring the paw volume.
Analgesic activity
Analgesic activity of the compounds 2b, 2d and 2g were
evaluated in Albino Wistar rats.26 e albino rats weigh-
ing 150–200 g were fasted overnight and were made into
five groups of six animals. Group I served as control and
was given vehicle 10ml/kg (CMC 1% w/v in distilled
water) orally. While the animals of group II were given
celecoxib 0.05 mmole/kg suspended in the vehicle (CMC
1% w/v). Rest of three groups were given 2b, 2d and 2g
(0.05 mmole/kg b.w.) suspended in the vehicle (CMC 1%
w/v), respectively. 3–4 cm area of the tail was immersed
in the water bath thermostatically maintained at 55°C.
e withdrawal time of the tail from the hot water (in
seconds) was noted as reaction time, initially as tail flick
latency before the administration of any drug and then
Determination of TNF-α, nitrite and prostaglandin E2
in exudates from rat paw stimulated by carrageenan
Overnight fasted rats (16 h) were divided into five
groups of six animals each. One group of rats, which
served as control was given vehicle (1% CMC in water
in a volume of 10 ml/kg) only. Celecoxib (0.05 mmole/
kg b.w.) and test compounds (0.05 mmole/kg b.w.),
suspended in vehicle (10 ml/kg) were administered
orally to respective groups. ey received a subplantar
injection of carrageenan (0.1 ml of a 1% suspension in
normal saline) into the right hind paw after 30 min of
© 2012 Informa UK, Ltd.

8
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