Design and synthesis of some pyrazole derivatives of expected anti-inflammatory and analgesic activities

Article abstract of DOI:10.1007/s00044-011-9606-4

Design and synthesis of some pyrazole derivatives 4-11 of expected anti-inflammatory and analgesic activities. In addition, docking of the tested compounds into cyclooxygenase II using (MOE) was performed to rationalize the obtained biological results and their mechanism of action. The structures of the new compounds were elucidated by spectral and elemental analyses. All the newly synthesized compounds were evaluated for their anti-inflammatory activity using the carrageenan-induced rat paw edema method. Analgesic activity of the target compounds was measured using the p-benzoquinone writhing-induced method, and their ability to induce gastric toxicity was also evaluated. Results showed that the newly synthesized compounds exhibited weak to good activities compared to ibuprofen and celecoxib as reference drugs. Some compounds, such 4a and 11b exhibited significant anti-inflammatory activity with gastric ulcerogenic potential less than that of ibuprofen. Results of the analgesic activity showed that compounds possessing good anti-inflammatory activity showed also good analgesic. Substitution of pyrazole ring with at least one aryl moiety was found to be essential for anti-inflammatory and analgesic activities. Free NH (of pyrazole ring) and/or acidic group (COOH) will improve the anti-inflammatory activity. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9606-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:983–994
DOI 10.1007/s00044-011-9606-4
ORIGINAL RESEARCH
Design and synthesis of some pyrazole derivatives of expected
anti-inflammatory and analgesic activities
•
Nagwa M. Abd-El Gawad Ghaneya S. Hassan
•
Hanan Hanna Georgey
Received: 12 November 2010 / Accepted: 9 February 2011 / Published online: 27 February 2011
Ó Springer Science+Business Media, LLC 2011
Abstract Design and synthesis of some pyrazole deriv-
atives 4–11 of expected anti-inflammatory and analgesic
activities. In addition, docking of the tested compounds
into cyclooxygenase II using (MOE) was performed to
rationalize the obtained biological results and their mech-
anism of action. The structures of the new compounds were
elucidated by spectral and elemental analyses. All the
newly synthesized compounds were evaluated for their
anti-inflammatory activity using the carrageenan-induced
rat paw edema method. Analgesic activity of the target
compounds was measured using the p-benzoquinone
writhing-induced method, and their ability to induce gastric
toxicity was also evaluated. Results showed that the newly
synthesized compounds exhibited weak to good activities
compared to ibuprofen and celecoxib as reference drugs.
Some compounds, such 4a and 11b exhibited significant
anti-inflammatory activity with gastric ulcerogenic poten-
tial less than that of ibuprofen. Results of the analgesic
activity showed that compounds possessing good anti-
inflammatory activity showed also good analgesic. Sub-
stitution of pyrazole ring with at least one aryl moiety was
found to be essential for anti-inflammatory and analgesic
activities. Free NH (of pyrazole ring) and/or acidic group
(COOH) will improve the anti-inflammatory activity.
Introduction
Inflammation is a normal and essential response to any
noxious stimulus, which threatens the host and may
vary from a localized response to a more generalized one
(Fylaktakidou et al., 2004). The inflammatory process is
designed to provide a rapid mechanism by which the
host can respond to the invasion of foreign materials and
return to homeostatic equilibrium. Acute inflammation is
mediated by the release of autacoids such as histamine,
serotonin, bradykinin, prostaglandins, and leukotrienes
(Lacerda et al., 2009). On the other hand, the chronic
inflammatory process involves the release of diverse
mediators, as interleukins, interferon, and tumor necrosis
factor a (TNF-a), a cytokine that plays a major role in this
kind of inflammatory process and whose production is
associated with some inflammatory diseases such as rheu-
matoid arthritis, Crohn’s disease, and others (Lacerda
et al., 2009).
Non-steroidal anti-inflammatory drugs (NSAIDs) are
one of the most useful clinical therapies for the treatment
of pain, fever, and inflammation (Laufer and Luik, 2010).
The major mechanism by which NSAIDs exert their
anti-inflammatory activity is by the inhibition of cycloox-
ygenase-derived prostaglandin synthesis, which is also
responsible for the gastrointestinal (Sostres et al., 2010;
Naesdal and Brown, 2006; Cryer, 2005; Lazzaroni and
Bianchi Porro, 2004; James and Hawkey, 2003), renal
(Schneider et al., 2006; Mounier et al., 2006; Zadrazil,
2006), and hepatic (Yanai et al., 2009) side effects that are
observed mainly in chronic use of NSAIDs. Therefore, the
challenge still exists for the pharmaceutical industry to
develop effective anti-inflammatory agents with enhanced
safety profile. Literature survey revealed that many 2-pyr-
azoline derivatives have been reported to exhibit various
Keywords Pyrazoles ꢀ Anti-inflammatory ꢀ
Analgesic ꢀ MOE
N. M. Abd-El Gawad ꢀ G. S. Hassan ꢀ H. H. Georgey (&)
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Cairo University, 11562 Cairo, Egypt
e-mail: Hanan-Hanna@hotmail.com
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Med Chem Res (2012) 21:983–994
pharmacological activities such as antimicrobial (Kart-
hikeyan et al., 2007; Ramiz et al., 2010), anti-inflammatory
(Barsoum et al., 2006; Shoman et al., 2009; Rathish et al.,
2009; Abbas et al., 2010), and antihypertensive (Turan-
Zitouni et al., 2000). Among the highly marketed COX-2
inhibitors that comprise the pyrazole nucleus, celecoxib is
the one which is treated as a safe anti-inflammatory and
analgesic agent. Some examples of pyrazole derivatives as
NSAIDs are mefobutazone, morazone, famprofazone, and
ramifenazone (Reynold, 1993; Menozzi et al., 2003; Sakya
et al., 2007; Patel et al., 2004).
O
O
Ar
N
O
NH
O
N
R
S
O
R
O
NH
Ar
S
O
a
+
N
NH
R'
4
5
R'
6
O
O
6a R: CH3, R':CH3
6b R: CH3, R': Cl
6c R: C6H5, R': CH3
6d R:C6H5, R':Cl
Ar:
Motivated by the aforementioned findings, it was
designed to synthesize novel series of pyrazole derivatives
that would act as anti-inflammatory agents with reduced
gastric toxicity. The synthesized compounds were tested in
vivo for their anti-inflammatory and analgesic activities as
well as their ulcerogenic liability. In order to rationalize the
pharmacological results obtained and the mechanism of
action, MOE (Molecular Operating Environment, 2005)
docking studies of the synthesized compounds were per-
formed using murine cyclooxygenase-2 co-crystallized
with celecoxib (Protein Data Bank code PDB ID: 6COX)
as a template.
Scheme 2 Reagents and solvents: a toluene
Ar
O
N
S
R
C
N
N
Ar
R
O
NH
+
N
a
N
S
H
Cl
4
7
Cl
8
O
Ar:
8a R: CH3
8b R: C6H5
Result and discussion
O
Chemistry
Scheme 3 Reagents and solvents: a CH3CN, 0°C
The synthesis routes for the preparation of the target
compounds were illustrated in Schemes 1, 2, 3, and 4.
Synthesis of the appropriate 1-(2,4-dimethoxyphenyl)alkyl/
aryl ketones 2a and 2b was carried out via the reaction of
1,3-dimethoxybenzene with propionic acid or phenyl acetic
Ar
R
O
O
Ar
O
R
a
O
9a R: CH3
9b R: C6H5
O
9
2
O
O
O
b
COOH
N
O
O
R
a
R
COOC2H5
N
R
N
Ar
2
1
c
N
Ar
10a R: CH3, R': H
10b R: CH3, R': Cl
10c R: C6H5, R': H
10d R: C6H5, R': Cl
b
11a R: CH3
11b R: C6H5
Cl
11
O
R'
O
O
10
R
O
O
O
Ar:
c
O
R
N
NH
Scheme 4 Reagents and solvents: a (COOC2H5)2/NaH/toluene, b
R’C6H4NHNH2ꢀHCl/triethylamine/ethanol, and c KOH/methanol
4
3
2a, 3a, 4a R: CH3
2b, 3b, 4b R: C6H5
acid in polyphosphoric acid (Slotta and Heller, 1930). The
structure of 2b was confirmed by appearance of a singlet
at d 4.29 ppm corresponding to methylene protons.
Scheme 1 Reagents and solvents: a RCH2COOH/PAA, b HCHO/
piperdine/acetic acid, and c NH2NH2ꢀH2O/ethanol
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Med Chem Res (2012) 21:983–994
985
Compounds 3a and 3b were synthesized from 2a and 2b,
respectively, using Mannich reaction/elimination sequence.
1H-NMR spectrum of 3a had two doublets due to geminal
coupling at d 2.93 (J: 7.4 Hz) and 3.04 ppm (J: 7.4 Hz)
corresponding to the methylene protons and that of 3b
appeared at d 4.21 (J: 8.2 Hz) and 4.86 ppm (J: 8.2 Hz).
Moreover, cyclocondensation of 3a and 3b with hydrazine
hydrate yielded the corresponding pyrazoline derivatives
4a and 4b. IR spectra of the prepared pyrazolines 4a and 4b
showed t (C=N) stretching at 1609–1604 cm-1 due to the
ring closure. In addition, a sharp band at 3422–3393 cm-1
one strong absorption band at t 1738–1717 cm-1 corre-
sponding to the carbonyl ester moiety. 1H-NMR also
confirmed the formation of the pyrazole ring from the
disappearance of the characteristic signal for the CH proton
that was observed in 9a and 9b (Scheme 4). Alkaline
hydrolysis of the ester containing compounds 10b and 10d
gave the corresponding free carboxylic acid derivatives
11a,b. IR spectra showed a broad absorption band at t
2950–2830 cm-1 for OH of the carboxylic group. 1H-NMR
confirmed the structures of the pyrazole derivatives 11a,b
from the appearance of an exchangeable proton at d
11.00 ppm corresponding to COOH and disappearance of
the characteristic pattern of the ethyl protons (Scheme 4).
Mass spectra of the newly synthesized compounds were
concomitant with their molecular weight.
1
due to the t (NH) stretching was also observed. H-NMR
spectra recorded for the prepared compounds in CDCl3
clearly supported the proposed structures. Protons of pyr-
azoline ring in compounds 4a and 4b showed a prominent
ABX system, with protons Ha, Hb, and Hx seen as doublets
of doublets at d 2.49–2.80, 3.39–3.81, and 5.00–5.10 ppm
(JHa–Hb: 4.2–4.4, JHa–Hx: 7.4–8.2, JHb–Hx: 7.6 Hz), respec-
tively. On the other hand, Hx of 4a appeared as multiplet as
a result of CH3 substituent (Scheme 1).
Pharmacological evaluation
The experimental tests on animals have been performed in
accordance with the Institutional Ethical Committee
approval, Faculty of Pharmacy, Cairo University.
Coupling of 3,4-disubstituted pyrazolines 4a and 4b
with sulfonylated carbamic acid methyl esters 5a and 5b
(Lange et al., 2004) afforded 6a–d. The structures of the
prepared compounds were confirmed by the appearance of
an additional strong absorption band of (C=O) stretching at
t 1683–1656 cm-1 in addition to absorption bands at t
1380–1327 and 1163–1155 cm-1 that were attributed to
(SO2) stretching. Also, 1H-NMR spectra showed an
increase in the integration of the aromatic protons com-
pared to the parent pyrazolines 4a and 4b and an additional
singlet at d 2.16–2.21 ppm corresponding to the three
protons of the CH3 in compounds 6a and 6c (Scheme 2).
Nucleophilic addition of pyrazoline derivatives 4a and
4b to 4-chlorobenzoyl isothiocyanate 7 (Cho and Shon,
1991) gave the adduct 8a and 8b. IR spectra of 8a and 8b
LD50
LD50 of each compound was determined using Finney’s
method (Finney, 1964). Intraperitoneal injection of the
tested compounds in doses less than 75 mg/kg body weight
failed to kill the mice within 24 h. LD50 celecoxib was
250 mg/kg body weight, and 2b, 3a, 4b, 9a, 9b, 10a, and
10d were 277.5 mg/kg body weight. While of 4a, 6c, 8b,
and 10c were 292.5 mg/kg body weight. LD50 of 6b, 6d,
and 10b was 247.5 mg/kg body weight, and 3b, 6a, 8a,
11a, and 11b were 232.5 mg/kg body weight. As a result,
the chosen dose was 25 mg/kg body weight which is
similar to celecoxib and at the same time it is safe (Buck
and Osweiter, 1976).
revealed the presence of (C=O) at t 1669 and 1656 cm-1
.
1H-NMR of these compounds showed downfield shifting of
the NH proton to d 6.30 and 6.11 ppm (Scheme 3).
Anti-inflammatory activity (Table 1)
Reaction of propan-1-one derivative 2a or phenyl eth-
anone derivative 2b with diethyl oxalate in the presence of
sodium hydride gave 4-aryl-3-substituted-2,4-dioxobuta-
noic acid ethyl esters 9a and 9b. IR spectra of these key
intermediates 9a and 9b showed three strong absorption
bands at t 1665–1612 and 1730–1712 cm-1 (C=O of
All the newly synthesized compounds 2–11 were evalu-
ated for their anti-inflammatory activity using carra-
geenan-induced paw edema described by Winter et al.
(Winter et al., 1962). The tested compounds and reference
drugs ibuprofen and celecoxib were intraperitoneal
injected at a dose level of 25 mg/kg, 30 min before car-
rageenan injection at the right hind paw of albino male
rats, the thickness of both paws was measured at differ-
ent time intervals of 1, 2, 3, and 4 h after carrageenan
injection. Anti-inflammatory activity of the tested com-
pounds and reference drugs was calculated as the per-
centage decrease in edema thickness induced by
carrageenan with the following formula (Alam et al.,
2009).
1
ketones and ester). H-NMR of the 1,3-diketone structures
9a and 9b showed a signal for the CH proton that was
observed as a quartet at nearly d 6.00 ppm in 9a or as a
singlet at d 6.40 ppm in 9b, in addition to the appearance of
ethyl protons at the expected chemical shifts (Scheme 4).
Condensation of 9a and 9b with hydrazine salts in eth-
anol and triethylamine afforded pyrazol-3-carboxylic acid
ethyl esters 10a–d. The structures of the prepared com-
pounds 10a–d were confirmed by the appearance of only
123

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Med Chem Res (2012) 21:983–994
% of edema inhibition ¼ ðVR ꢁ VLÞ control ꢁ ðVR ꢁ VLÞ treated ꢂ 100
ðVR ꢁ VLÞ control
Table 1 Anti-inflammatory activity of ibuprofen, celecoxib, and the newly synthesized compounds (Carrageenan-induced edema in rats, n = 5)
Compound
Percent inhibition
1 h
2 h
3 h
4 h
Control
Ibuprofen
Celecoxib
2b
0
0
0
0
55.38 1.97***
54.21 2.00***
5.91 2.15
23.66 2.11*
0
63.55 2.41***
61.07 2.36***
11.23 1.87
29.78 2.29*
1.43 2.24
44.45 2.72**
0
66.65 1.85***
63.97 2.37***
15.01 1.74
31.50 2.44**
1.92 1.95
76.10 2.12***
62.75 2.36***
15.92 2.17
32.34 2.13**
2.90 1.67
3a
3b
4a
40.39 2.39**
0
45.08 2.54***
1.80 0.94
44.38 2.53***
1.92 0.94
4b
6a
23.66 1.96*
12.25 2.44
4.90 2.28
24.15 1.97*
0
27.30 1.82*
14.63 2.33
6.33 1.85
27.30 1.97*
0
25.2 2.23*
16.45 2.03
4.83 1.97
25.09 1.93*
18.79 2.33
7.21 1.97
6b
6c
6d
25.69 1.80*
1.94 0.94
23.62 2.07*
1.43 0.66
8a
8b
0
0
1.43 0.95
1.92 0.92
9a
23.66 1.96*
15.05 2.36
4.90 2.43
0
27.20 2.12*
20.02 2.22
4.86 1.56
1.43 2.38
20.51 2.08
12.70 2.17
28.35 2.43*
42.03 2.86**
26.14 2.24*
20.82 2.35
5.81 1.97
24.60 2.08*
21.21 2.35
5.77 2.24
9b
10a
10b
10c
10d
11a
11b
2.22 1.43
2.38 1.66
17.74 2.52
6.89 2.29
24.69 1.96*
36.96 2.20**
20.82 2.36
15.01 2.31
22.75 2.22*
45.53 2.71***
20.23 2.05
15.43 2.16
22.68 2.06*
42.45 2.35***
* Significant at P B 0.05
** Significant at P B 0.01
*** Significant at P B 0.001
where VR represents the mean right paw thickness, VL
represents the mean left paw thickness, (VR - VL) control
represents the mean increase in paw thickness in the
control group of rats, and (VR - VL) treated represents
the mean increase in paw thickness in rats treated with the
tested compounds. The results listed in Table 1 revealed
that some of the synthesized compounds showed moder-
ate to weak anti-inflammatory activity. Compounds 4a
and 11b induced considerable anti-inflammatory activity
relative to ibuprofen, and celecoxib that reached to 67%
after 3 h. Another important observation was the decline
in activity of compounds 6d and 8b upon replacement of
SO2 by C=O. On the other hand, the triaryl carboxylic
acid derivative 11b was found to be twice as active as
diaryl analog 11a.
Analgesic activity (Table 2)
All the synthesized compounds as well as celecoxib were
tested for their analgesic activity using the p-benzoqui-
none-induced writhing test in mice (Collier et al., 1968).
The results showed that compounds possessing good anti-
inflammatory activity induced also good analgesic, except
10b exhibited only analgesic activity (Table 2).
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Med Chem Res (2012) 21:983–994
987
scoring configuration of each of the ligand–enzyme com-
plexes was selected on energetic grounds.
Table 2 Analgesic activity of celecoxib and the new synthesized
compounds in mice (25 mg/kg body weight) using writhing method
(n = 5)
Docking of celecoxib in the active site of murine COX-2
Compound
Percent protection against writhing after (h)
showed hydrogen bonds interactions between the SO2
˚
group and His90 (distances = 2.69 A). Also, hydrogen
1
2
3
4
bonds were observed between Gln192 and Leu352 with
˚
NH2 group (distances = 3.01 and 3.43 A, respectively).
Control
Celecoxib
3a
0
0
0
0
100
20
80
20
40
40
40
80
100
20
60
20
40
40
20
60
80
0
80
0
Ten hydrophobic bonds with Val116, Val349 (distance
˚
3.70 A), Leu352, Leu359, Phe381, Leu384, Try387,
˚
Phe518, and Val523 (distance 3.10 A).
4a
40
0
20
0
6a
Docking of non pyrazole derivatives 2a,b, 3a,b, and
9a,b revealed the appearance of hydrogen bonds between
His90 and the 4-methoxy group of 2b, 3b, 9b, or the car-
bonyl of the ester group of 9a (distances = 3.48, 2.02,
6d
20
20
20
40
0
10b
0
11a
0
˚
1.73, and 3.39 A). Also, hydrogen bonds were detected
11b
20
between Arg120 and 4-methoxy group of 2a and 3a (dis-
˚
tances = 2.68 and 3.37 A). In addition to, the presence of
Ulcerogenic effect (Table 3)
hydrophobic interactions especially with Val523, Leu352
(except 3a), Val349 (except 2b) and Phe518 for 2a, 2b, and
3a.
The ulcerogenic effect of the most active compounds 3a,
4a, 11b, and the reference drugs (ibuprofen, and celecoxib)
was evaluated according to Meshali’s method (Meshali
et al., 1983). The ulcer index was calculated according to
Robert’s method (Robert et al., 1968). The results showed
that compounds 3a, 4a, and 11b exhibited little gastric
ulceration; about 50–60% that of ibuprofen. On the other
hand, these compounds showed higher gastric ulceration
than celecoxib (Table 3).
Concerning 3-(2,4-dimethoxyphenyl)-4-methyl/phenyl-
4,5-dihydro-1H-pyrazole 4a,b, it was found that the
hydrogen bonds were between Arg120, Leu352, and try355
with the 4-methoxy group, NH group, and 4-methoxy
group for 4a or 2-methoxy group for 4b, respectively
(distances = 2.64 and 2.68, 2.51 and 2.44, and 3.44 and
˚
3.09 A for 4a and 4b, respectively). The presence of the
hydrophobic interactions with Val349, Val523 were
observed, and an extra hydrophobic interaction with
Leu352 was found in the case of 4b.
Molecular modeling study
For compounds 6a–d, it was observed the presence of
hydrogen bond between Arg120 and 4-methoxy group of
˚
compound 6d (distances = 2.38 A). On the other hand,
hydrogen bonds were formed between His90 and SO2
Molecular docking of celecoxib and the synthesized com-
pounds were performed to rationalize the obtained bio-
logical results. Besides, molecular docking studies were
helped in understanding the various interactions between
the ligand and enzyme active site. Docking studies of the
inhibitors were performed by MOE (Molecular Operating
Environment, 2005) using murine COX-2 co-crystallized
with celecoxib (PDB ID: 6COX) as a template. We per-
formed 100 docking iterations for each ligand, and the top-
group of 6a, 6b, and 6c or with the carbonyl group of 6d
˚
(distances = 3.17, 2.60, 3.68, and 2.67 A). Also, more
hydrogen bonds were found between Arg513 and 4-meth-
oxy group of them (distances = 2.51, 2.52, 3.07, and
˚
3.07 A for 6a–d). Additional hydrogen bonds were
observed between Leu352 and NH group of 6a, 6b, or
between Ser530 and NH group of 6c. Besides, the hydro-
phobic interactions with Val523, Val349, Phe518, Leu352
(except 6a), Ile517 (for 6a, 6b) and Try387 (for 6c).
Thiocarboxamide derivatives 8a,b showed hydrogen
bonds between Ser530 and NH group (distances = 2.98
Table 3 Ulcerogenic effect of celecoxib, ibuprofen, 3a, 4a, and 11b
in rats (25 mg/kg body weight) (n = 5)
Compound Percent incidence
divided by ten
Average no. Average
severity
Ulcer
index
of ulcer
˚
and 2.63 A). Another bond was detected between His90
˚
and 4-methoxy group of 8b (distance = 3.44 A). In addi-
Control
0
6
0
0
0
tion to, the hydrophobic interactions with Val523, Val349,
Leu359, and Leu352.
Celecoxib
1.4
10.0
4.2
2.6
2.4
1.00
0.32
1.04
1.07
1.16
8.40
Ibuprofen 10
22.92
15.24
11.67
11.56
Concerning compounds 10a–d and 11a,b, hydrogen
bonds were appeared between Arg120 and 4-methoxy
group of 10b, 10d, and 11a (distances = 2.92, 2.86 and
3a
10
8
4a
11b
8
˚
2.75 A) and between tyr355 and 4-methoxy group of 10a,
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Med Chem Res (2012) 21:983–994
Fig. 1 3D Docked structure of compound 4a in the active site of
murine COX-2 showed two hydrogen bonds: 4-OCH3 and Arg120
˚
(2.64 A) and pyrazole N and Leu352 (3.44 A). Also, two hydropho-
˚
˚
bic bonds with Val349 and Val523 (3.72 and 3.17 A, respectively)
Fig. 3 Conformational alignment of celecoxib from the crystal
structure of celecoxib-murine COX-2 complex and that of compound
4a from the docking simulation
Fig. 2 3D Docked structure of compound 11b in the active site of
murine COX-2 showed four hydrogen bonds: Arg120, Val523,
˚
Gly526, and Ala527 (2.31, 3.22, 3.64, and 3.50 A). Also, six
hydrophobic bonds: Val349, Leu352, Ile517, Phe518, Val523, and
˚
Leu531 (3.87, 3.87, 3.70, 2.81, 2.71, and 4.32 A, respectively)
10c or 2-methoxy group of 11b (distances = 3.01, 2.78,
˚
and 2.68 A). Also, the same pattern of the hydrophobic
Fig. 4 Conformational alignment of celecoxib from the crystal
structure of celecoxib-murine COX-2 complex and that of compound
11b from the docking simulation
bonds with Val523 and Val349 (Figs. 1, 2).
Conformational alignment of celecoxib from the crystal
structure of celecoxib-murine COX-2 complex and that of
compound 4a from the docking simulation is shown in
Fig. 3. The superposition showed overlapping through
heterocyclic and one aryl regions. While, that of compound
11b is shown in Fig. 4 showed complete overlapping
through heterocyclic and two aryl regions.
well as for their ulcerogenic effect. Results showed that
compounds 3a, 4a, 6a, 6d, 9a, 11a, and 11b exhibited anti-
inflammatory and analgesic activities with the exception of
10b that established only analgesic activity.
SAR: Substitution of the pyrazole ring with at least one
aryl moiety is essential for activity. Moreover, the presence
of acidic center expressed by NH (of pyrazole ring)
‘‘compound 4a’’ or (COOH) ‘‘compound 11b’’ improves
the anti-inflammatory activity. On the other hand, the
ulcerogenic effect of 4a and 11b is slightly higher than that
of celecoxib due to the primary insult effect.
Conclusions
Various substituted pyrazole derivatives were synthesized
and screened for anti-inflammatory, analgesic activities as
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Med Chem Res (2012) 21:983–994
Experimental
989
OCH3), 3.99 (s, 3H, OCH3), 6.48 (s, 1H, ArH-3), 6.56 (d,
1H, ArH-5, J: 8.8 Hz), 7.86 (d, 1H, ArH-6, J: 8.4 Hz). IR
(KBr) cm-1: 3050 (CH Ar), 2934, 2840 (CH aliphatic),
1661 (C=O). MS m/z: 208 [M? ? 2, 1%], 206 [M?, 1%],
165 [C6H3(OCH3)2CO, 100%]. Anal. calcd for C12H14O3
(206.24): C 69.89; H 6.84. Found: C 69.77; H 6.74.
Chemistry
All melting points were determined by the open capillary
method using Gallen Kemp melting point apparatus (MFB-
595-010M) and were uncorrected. Microanalyses were
carried out at the Micro analytical Unit, Faculty of Science,
and Cairo University. IR (KBr) was determined using
Shimadzu Infrared Spectrometer (IR-435) and FT-IR 1650
1-(2,4-Dimethoxyphenyl)-2-phenyl prop-2-en-1-one 3b
Compound 3bwas prepared from 2b as oil (60% yield) by the
1
1
(Perkin Elmer). H-NMR Spectra were carried using Fou-
same procedure as described for 3a. H-NMR (CDCl3) d:
rier transform EM-390, 200 MHz NMR Spectrometer and
Varian Mercury VX-300 MHz NMR Spectrometer. Mass
spectra were carried using Fining SSQ 7000 Gas Chro-
matograph Mass spectrometer and Shimadzu Gas Chro-
matograph Mass spectrometer-QP 1000 EX. Nomenclature
of presented compounds was according to IUPAC system.
Compounds 2a, 5a, and 5b were prepared according to the
literature procedures (Slotta and Heller, 1930; Lange et al.,
2004; Cho and Shon, 1991), respectively.
3.79 (s, 3H, OCH3), 3.82 (s, 3H, OCH3), 4.21 (d, 1H, CH,
methylene, J: 8.2 Hz), 4.86 (d, 1H, CH, methylene, J:
8.2 Hz), 6.12 (s, 1H, ArH-3), 6.30–7.42 (m, 5H, Ar), 7.59 (d,
1H, ArH-5, J: 8.6 Hz), 8.31 (d, 1H, ArH-6, J: 8.6 Hz). IR
(KBr) cm-1: 3030 (CH Ar), 2930, 2840 (CH aliphatic), 1660
(C=O). MS m/z: 268 (M?, 14%), 165 [C6H3(OCH3)2CO,
100%]. Anal. calcd for C17H16O3 (268.31): C 76.10; H 6.01.
Found: C 76.37; H 5.81.
3-(2,4-Dimethoxypheny)-4-methyl-4,5-dihydro-1H-
pyrazole 4a
1-(2,4-Dimethoxyphenyl)-2-phenyl ethanone 2b
A solution of 1,3-dimethoxybenzene 1 (2.76 g, 20 mmol)
and phenylacetic acid (2.72 g, 20 mmol) in polyphosphoric
acid (50 ml) was heated in boiling water bath for 30 min.
After cooling, the red residue was decomposed by pouring
on ice-water and extracted with chloroform. The organic
layer was washed twice with water, dried over Na2SO4, and
concentrated. The residue was crystallized from MeOH to
A solution of 3a (2.06 g, 10 mmol), hydrazine hydrate
(6.0 ml, 120 mmol) in absolute ethanol (50 ml) was refluxed
for 3 h. The mixture was concentrated in vacuum; water was
added to the residue and extracted with chloroform. The
organic layer was twice washed with water, dried over
Na2SO4, and concentrated. The residue was oil (70% yield).
1H-NMR (CDCl3-D2O) d: 1.20 (d, 3H, CH3, J: 4.6 Hz), 2.49
(dd, 1H, CH, methylene, J: 4.2, 7.4 Hz), 3.81 (dd, 1H, CH,
methylene, J: 42, 7.4 Hz), 3.86 (s, 3H, OCH3), 3.91 (s, 3H,
OCH3), 5.00 (m, 1H, CH py), 5.29 (broad, 1H, NH exch.),
6.51 (s, 1H, ArH-3), 6.96 (d, 1H, ArH-5, J: 8.2 Hz), 7.45 (d,
1H, ArH-6, J: 8.2 Hz). IR (KBr) cm-1: 3393 (NH), 3050 (CH
Ar), 2936, 2836 (CH aliphatic), 1609 (C=N). MS m/z: 220
[M?, 14%], 177 [C6H3 (OCH3)2CH=NNH, 100%]. Anal.
calcd for C12H16N2O2 (220.27): C 65.43; H 7.32; N 12.72.
Found: C 65.10; H 7.60; N 12.78.
1
give 2b (60% yield), mp 72–73°C. H-NMR (CDCl3) d:
3.78 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 4.29 (s, 2H, CH2),
6.43 (s, 1H, ArH-3), 6.53 (m, 2H, Ar), 7.30 (m, 3H, Ar),
7.80 (d, 1H, ArH-5, J: 8.8 Hz), 8.12 (d, 1H, ArH-6, J:
8.8 Hz). IR (KBr) cm-1: 3050 (CH Ar), 2964, 2939, 2834
(CH aliphatic), 1668 (C=O). MS m/z: 256 [M?, 4%], 165
[C6H3(OCH3)2CO, 100%]. Anal. calcd for C16H16O3
(256.20): C 74.98; H 6.29. Found: C 74.90; H 6.20.
1-(2,4-Dimethoxyphenyl)-2-methyl prop-2-en-1-one 3a
3-(2,4-Dimethoxypheny)-4-phenyl-4,5-dihydro-1H-
pyrazole 4b
To a solution of 1-(2,4-dimethoxyphenyl) propan-1-one 2a
(1.95 g, 10 mmol) in MeOH (50 ml), piperdine (0.12 ml,
1.21 mmol), acetic acid (0.12 ml, 2.08 mmol), and for-
malin (4 ml: 37% aqueous solution, 5.32 mmol) were
successively added, and the resulting mixture was refluxed
for 4 h. The mixture was concentrated in vacuum. Water
was added to the residue, and the mixture was extracted
with chloroform. The organic layer was separated, washed
with water (39), dried over Na2SO4, filtered, and concen-
trated to give 3a (70% yield) as an oil. 1H-NMR (CDCl3) d:
1.21(s, 3H, CH3), 2.93 (d, 1H, CH, methylene, J: 7.4 Hz),
3.04 (d, 1H, CH, methylene, J: 7.4 Hz), 3.80 (s, 3H,
Compound 4b was prepared from 3b as oil (75% yield)
using the same procedure described for 4a. 1H-NMR
(CDCl3–D2O) d: 2.80 (dd, 1H, CH, methylene, J: 4.4,
8.2 Hz), 3.39 (dd, 1H, CH, methylene, J: 4.4, 8.2 Hz), 3.80
(s, 3H, OCH3), 3.85 (s, 3H, OCH3), 5.10 (d, 1H, CH
J:7.6 Hz), 5.53 (s, 1H, NH exch.), 6.14 (s, 1H, ArH-3),
6.30–7.42 (m, 5H, Ar), 7.61 (d, 1H, ArH-5, J: 8.4 Hz), 8.31
(d, 1H, ArH-6, J: 8.4 Hz). IR (KBr) cm-1: 3422 (NH),
3058 (CH Ar), 2934, 2837 (CH aliphatic), 1604 (C=N). MS
m/z: 282 [M?, 5%], 165 [C6H3(OCH3)2CHNH, 100%].
123

990
Med Chem Res (2012) 21:983–994
Anal. Calcd for C17H18N2O2 (282.34): C 72.32; H 6.43; N
9.92. Found: C 72.27; H 6.22; N 9.97.
ArH-5, J: 8.4 Hz), 6.80 (d, 1H, ArH-6, J: 8.2 Hz),
7.00–7.65 (m, 5H, Ar), 7.77 (d, 2H, Ar, J: 8.0 Hz), 7.95 (d,
2H, Ar, J: 8.0 Hz). IR (KBr) cm-1: 3261 (NH), 3063 (CH
Ar), 2939, 2841 (CH aliphatic), 1660 (C=O), 1605 (C=N),
1343, 1162 (SO2). MS m/z: 479 [M?, 1%], 165 [C6H3
(OCH3)2CHNH, 100%]. Anal. calcd for C25H25N3O5S
(479.55): C 62.62; H 5.25; N 8.76. Found: C 62.62; H 5.25;
N 8.83.
3-(2,4-Dimethoxyphenyl)-N-[(4-methylphenyl)sulfonyl]-4-
methyl-4,5-dihydro-1H-pyrazol-1-carboxamide 6a
To a solution of 4a (2.20 g, 10 mmol) in toluene (50 ml),
5a (2.75 g, 12 mmol) was added, and the resulting mixture
was refluxed for 4 h. After cooling of the solution to room
temperature, the mixture was concentrated in vacuum,
water was added and extracted with chloroform. The
organic layer was washed twice with water, dried over
Na2SO4, and concentrated. The product was obtained as oil
N-[(4-Chlorophenyl)sulfonyl]-3-(2,4-dimethoxyphenyl)-4-
phenyl-4,5-dihydro-1H-pyrazol-1-carboxamide 6d
Compound 6d was prepared from 4b and 5b as oil (76%
1
yield) adopting the same procedure described for 6a. H-
1
(80% yield). H-NMR (CDCl3–D2O) d: 1.21 (d, 3H, CH3,
J: 4.6 Hz), 2.21 (s, 3H, CH3), 3.70 (d, 1H, CH, methylene,
J: 4.4 Hz), 3.80 (d, 1H, CH, methylene, J: 4.4 Hz), 3.85 (s,
3H, OCH3), 3.90 (s, 3H, OCH3), 4.11 (m, 1H, CH py), 5.27
(s, 1H, NH exch.), 6.51 (s, 1H, ArH-3), 7.20 (d, 1H, ArH-5,
J: 8.4 Hz), 7.33 (d, 1H, ArH-6, J: 8.7 Hz), 7.80 (d, 2H, Ar,
NMR (CDCl3–D2O) d: 3.40 (d, 1H, methylene, J: 4.0 Hz),
3.60 (d, 1H, methylene, J: 4.0 Hz), 3.81 (s, 3H, OCH3), 4.04
(s, 3H, OCH3), 4.20 (d, 1H, CH J: 7.6 Hz), 5.20 (s, 1H, NH
exch.), 6.15 (s, 1H, ArH-3), 6.605 (d, 1H, ArH-5, J: 8.2 Hz),
6.85 (d, 1H, ArH-6, J: 8.2 Hz), 7.00–7.70 (m, 5H, Ar), 7.88
(d, 2H, Ar, J: 8.4 Hz), 7.99 (d, 2H, Ar, J: 8.2 Hz). IR (KBr)
cm-1: 3363 (NH), 3090 (CH Ar), 2937, 2842 (CH aliphatic),
1656 (C=O), 1604 (C=N), 1346, 1163 (SO2). MS m/z: 501
[M??2, 1%], 499 [M?, 1%], 165 [C6H3(OCH3)2CHNH,
100%]. Anal. calcd for C24H22ClN3O5S (499.97): C 57.65; H
4.44; N 8.41. Found: C 57.91; H 4.62; N 8.48.
J: 8.4 Hz), 7.90 (d, 2H, Ar, J: 8.1 Hz). IR (KBr) cm-1
:
3328 (NH), 3030 (CH Ar), 2969, 2928 (CH aliphatic), 1683
(C=O), 1610 (C=N), 1327, 1155 (SO2). MS m/z: 417 [M?,
1%], 165 [C6H3(OCH3)2CHNH, 8%], 91 [C7H7, 100%].
Anal. calcd for C20H23N3O5S (417.48): C 57.54; H 5.55; N
10.06. Found: C 57.40; H 5.77; N 9.96.
N-[(4-Chlorophenyl)sulfonyl]-3-(2,4-dimethoxyphenyl)-4-
methyl-4,5-dihydro-1H-pyrazol-1-carboxamide 6b
4-Chlorobenzoylisothiocynate 7
Compound 7 was prepared according to the reported pro-
cedure (Cho and Shon, 1991) from 4-chlorobenzoyl chlo-
ride and ammonium thiocyanate and immediately reacted
with 4.
Compound 6b was prepared from 4a and 5b as white
crystals from methanol, m.p. 225°C (78% yield) using the
same procedure described for 6a. H-NMR (CDCl3–D2O)
1
d: 1.45 (d, 3H, CH3, J: 4.6 Hz), 3.07 (d, 1H, CH, methy-
lene, J: 4.6 Hz), 3.16 (d, 1H, CH, methylene, J: 4.4 Hz),
3.70 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 4.60 (broad, 1H,
CH), 4.78 (broad, 1H, NH exch.), 7.28 (s, 1H, ArH-3), 7.53
(d, 3H, Ar, J: 8.0 Hz), 7.91 (d, 3H, ArH, J: 8.2 Hz). IR
(KBr) cm-1: 3300 (NH), 3030 (CH Ar), 2900, 2800 (CH
aliphatic), 1680 (C=O), 1600 (C=N), 1380, 1160 (SO2).
MS m/z: 423 [M?-CH3, 3%], 165 [C6H3 (OCH3)2CHNH,
48%], 57 [C2H5N2, 100%]. Anal. calcd for C19H20ClN3O5S
(437.90): C 52.11; H 4.60; N 9.65. Found: C 52.18; H 5.20;
N 9.57.
N-(4-Chlorobenzoyl)-3-(2,4-dimethoxyphenyl)-4-methyl-
4,5-dihydro-1H-pyrazol-1-thiocarboxamide 8a
Compound 4a (2.20 g, 10 mmol) was added to a cold (0°C)
solution of 7 (2.37 g, 12 mmol) in anhydrous acetonitrile
(20 ml), and the resulting mixture was stirred at room
temperature for 3 h. The precipitate was removed by fil-
tration and thoroughly washed with acetonitrile. The filtrate
was concentrated in vacuum, and the residue was collected
and further purified by extraction with chloroform. The
organic layer was twice washed with water, dried over
Na2SO4, and concentrated. The product was obtained as oil
(61% yield). 1H-NMR (CDCl3–D2O) d: 1.27 (d, 3H, CH3),
3.20 (d, 1H, methylene, J: 4.2 Hz), 3.40 (d, 1H, methylene,
J: 4.2 Hz), 3.70 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 4.49 (m,
1H, CH), 6.30 (s, 1H, NH exch.), 6.40(s, 1H, ArH-3), 7.26
(d, 1H, ArH-5, J: 8.6 Hz), 7.33 (d, 1H, ArH-6, J: 8.6 Hz),
7.76 (d, 2H, Ar, J: 8.8 Hz), 7.85 (d, 2H, Ar, J: 8.8 Hz). IR
(KBr) cm-1: 3360 (NH), 3050 (CH Ar), 2934, 2878 (CH
aliphatic), 1669 (C=O), 1620 (C=N), 1098 (C=S). MS m/z:
3-(2,4-Dimethoxyphenyl)-N-[(4-methylphenyl)sulfonyl]-4-
phenyl-4,5-dihydro-1H-pyrazol-1-carboxamide 6c
Compound 6c was prepared from 4b and 5a as oil (84%
yield) using the same procedure described for 6a. 1H-NMR
(CDCl3–D2O) d: 2.16 (s, 3H, CH3), 3.60 (d, 1H, methylene,
J: 4.0 Hz), 3.77 (d, 1H, methylene, J: 4.0 Hz), 3.82 (s, 3H,
OCH3), 4.01 (s, 3H, OCH3), 4.20 (d, 1H, CH J:7.6 Hz),
5.30 (s, 1H, NH exch.), 6.20 (s, 1H, ArH-3), 6.60 (d, 1H,
123

Med Chem Res (2012) 21:983–994
991
417 [M?, 2%], 385 [M?-S, 3%], 177 [C6H3(OCH3)2CNNH,
100%]. Anal. calcd for C20H20ClN3O3S (417.91): C 57.48;
H 4.82; N 10.05. Found: C 57.68; H 4.84; N 10.43.
1730, 1665 (3 C=O). MS m/z: 417 [M?, 1%], 165
[C6H3(OCH3)2CHNH, 8%], 91 [C7H7, 100%]. Anal. calcd
for C20H20O6 (356.38): C 67.41; H 5.66. Found: C 67.69; H
5.40.
N-(4-Chlorobenzoyl)-3-(2,4-dimethoxyphenyl)-4-phenyl-
4,5-dihydro-1H-pyrazol-1-thiocarboxamide 8b
5-(2,4-Dimethoxyphenyl)-4-methyl-1-phenyl-1H-pyrazole-
3-carboxylic acid ethyl ester 10a
Compound 8b was prepared from 4b and 7 as oil (73%
yield) by the same procedure as described for 8a. 1H-NMR
(CDCl3–D2O) d: 3.20 (d, 1H, methylene, J: 4.2 Hz), 3.36
(d, 1H, methylene, J: 4.2 Hz), 3.89 (s, 3H, OCH3), 4.04 (s,
3H, OCH3), 4.59 (d, 1H, CH J: 7.5 Hz), 6.11 (s, 1H, NH
exch.), 6.53 (s, 1H, ArH-3), 7.26–7.96 (m, 9H, Ar), 8.05 (d,
1H, Ar, J: 9.0 Hz), 8.35 (d, 1H, Ar, J: 8.6 Hz). IR (KBr)
cm-1: 3368 (NH), 3050 (CH Ar), 2853, 2772 (CH ali-
phatic), 1656 (C=O), 1619 (C=N), 1087 (C=S). MS m/z:
481 [M??2, 35%], 479 [M?, 1%], 281 [M?–C6H4(p-
Cl)CONHCS, 50%], 165 [C6H3(OCH3)2C?NH, 100%].
Anal. calcd for C25H22ClN3O3S (479.98): C 62.56; H 4.62;
N 8.75. Found: C 62.50; H 4.60; N 8.76.
To a solution of 9a (2.94 g, 10 mmol) in absolute ethanol
(50 ml), phenyl hydrazine hydrochloride (1.45 g, 10 mmol)
and triethylamine (1.01 g, 10 mmol) were added. The
reaction mixture was then heated under reflux for 9 h. It was
then cooled, washed with water, and extracted with chlo-
roform (39 20 ml). The organic layer was then separated,
dried over sodium sulfate, and evaporated to give 10a. The
1
product was oil (84% yield). H-NMR (CDCl3) d: 1.39 (t,
3H, CH2CH3, J: 4.0 Hz), 2.96 (s, 3H, CH3), 3.83 (s, 3H,
OCH3), 4.02 (s, 3H, OCH3), 4.20 (q, 2H, CH2CH3), 6.42 (s,
1H, ArH-3), 6.60 (d, 1H, ArH-5, J: 8.4 Hz), 7.10–7.80 (m,
5H, Ar), 8.11 (d, 1H, ArH-6, J: 8.4 Hz). IR (KBr) cm-1
:
3061 (CH Ar), 2935, 2843 (CH aliphatic), 1717 (C=O). MS
m/z: 366 [M?, 2%], 165 [C6H3(OCH3)2CO, 100%]. Anal.
calcd for C21H22N2O4 (366.42): C 68.84; H 6.05; N 7.68.
Found: C 68.65; H 6.24; N 7.59.
3-Methyl-2,4-dioxo-4-(2,4-dimethoxyphenyl) butanoic acid
ethyl ester 9a
To compound 2a (1.94 g, 10 mmol) in toluene (20 ml) was
added sodium hydride (0.48 g, 20 mmol), and the mixture
was stirred for 10 min. Then, diethyl oxalate (3.29 g,
3.0 ml, 15 mmol) was added dropwise, and the mixture
was stirred at reflux (1 h). The mixture was cooled to room
temperature, washed with ether, acidified with acetic acid,
and extracted with chloroform. The organic layer was
washed with water, and concentrated. The product was
1-(4-Chlorophenyl)-5-(2,4-dimethoxyphenyl)-4-methyl-1H-
pyrazole-3-carboxylic acid ethyl ester 10b
Compound 10b was prepared from 9a (2.94 g, 10 mmol),
4-chlorophenyl hydrazine hydrochloride (1.79 g, 10 mmol)
and triethylamine (1.01 g, 10 mmol) following the same
procedure described for 10a. The product was obtained as
oil (80% yield). 1H-NMR (CDCl3) d: 1.29 (t, 3H, CH2CH3,
J: 4.0 Hz), 2.92 (s, 3H, CH3), 3.75 (s, 3H, OCH3), 3.85 (s,
3H, OCH3), 4.34 (q, 2H, CH2CH3), 6.38 (s, 1H, ArH-3),
6.43 (d, 1H, ArH-5, J: 8.2 Hz), 7.40 (d, 1H, ArH-6, J:
8.2 Hz), 7.80 (d, 2H, Ar, J: 8.4 Hz), 8.06 (d, 2H, Ar, J:
8.4 Hz). IR (KBr) cm-1: 3050 (CH Ar), 2933, 2843 (CH
aliphatic), 1720 (C=O). MS m/z: 402 [M??2, 1%], 400
[M?, 1%], 165 [C6H3(OCH3)2CO, 100%]. Anal. calcd for
C21H21ClN2O4 (400.98): C 62.90; H 5.28; N 7.02. Found:
C 63.14; H 5.46; N 7.32.
1
obtained as oil (60% yield). H-NMR (CDCl3) d: 1.35 (t,
3H, CH2CH3, J: 4.2 Hz), 2.35 (d, 3H, COCHCH3, J:
2.2 Hz), 3.79 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 4.30 (q,
2H, CH2CH3), 6.00 (q, 1H, CH), 7.41 (s, 1H, ArH-3), 7.66
(d, 1H, ArH-5, J: 8.0 Hz), 8.08 (d, 1H, ArH-6, J: 8.0 Hz).
IR (KBr) cm-1: 3030 (CH Ar), 2969, 2843 (CH aliphatic),
1712, 1612 (3 C=O). MS m/z: 417 [M?, 1%], 165
[C6H3(OCH3)2CHNH, 8%], 91 [C7H7, 100%]. Anal. calcd.
for C15H18O6 (294.31): C 61.22; H 6.12. Found: C 61.49; H
5.88.
2,4-Dioxo-3-phenyl-4-(2,4-dimethoxyphenyl) butanoic acid
ethyl ester 9b
5-(2,4-Dimethoxyphenyl)-1,4-diphenyl-1H-pyrazole-3-
carboxylic acid ethyl ester 10c
Compound 9b was prepared from 2b as oil (72% yield)
1
following the same procedure described for 9a. H-NMR
It was prepared from 9b (3.56 g, 10 mmol), phenyl
hydrazine hydrochloride (1.45 g, 10 mmol) and triethyl-
amine (1.01 g, 10 mmol) using the same procedure
described for 10a. The product was obtained as oil (74%
yield). 1H-NMR (CDCl3) d: 1.37 (t, 3H, CH2CH3, J:
4.2 Hz), 3.78 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 4.30 (q,
2H, CH2CH3), 6.23 (s, 1H, ArH-3), 6.40 (d, 1H, ArH-5, J:
(CDCl3) d: 1.38 (t, 3H, CH2CH3, J: 7.2 Hz), 3.77 (s, 3H,
OCH3), 3.98 (s, 3H, OCH3), 4.28 (q, 2H, CH2CH3), 6.44 (s,
2H, COCH & ArH-3), 6.54 (d, 1H, ArH-5, J: 8.6 Hz),
7.15–7.60 (m, 5H, Ar), 7.83 (d, 1H, ArH-6, J: 8.4 Hz). IR
(KBr) cm-1: 3060 (CH Ar), 2940, 2838 (CH aliphatic),
123

992
Med Chem Res (2012) 21:983–994
8.2 Hz), 6.80–8.00 (m, 11H, Ar). IR (KBr) cm-1: 3057
(CH Ar), 2927, 2843 (CH aliphatic), 1738 (C=O). MS m/z:
428 [M?, 1%], 165 [C6H3(OCH3)2CO, 100%]. Anal. calcd
for C26H24N2O4 (428.49): C 72.88; H 5.65; N 6.54. Found:
C 72.87; H 5.30; N 6.25.
8.4 Hz), 8.20 (d, 2H, ArH, J: 8.7 Hz), 11.00 (s, 1H, COOH,
exch.). IR (KBr) cm-1: 3060 (CH Ar), 2940–2830 (CH
aliphatic and OH), 1730 (C=O). MS m/z: 436 [M??2,
56%], 434 [M?, 20%], 151 [C6H3(OCH3)2CH2, 100%].
Anal. calcd for C24H19ClN2O4 (434.88): C 66.29; H 4.40;
N 6.44. Found: C 66.41; H 4.79; N 6.07.
1-(4-Chlorophenyl)-5-(2,4-dimethoxyphenyl)-4-phenyl-1H-
pyrazole-3-carboxylic acid ethyl ester 10d
Pharmacological evaluation
It was prepared from 9b (3.56 g, 10 mmol), 4-chlorophenyl
hydrazine hydrochloride (1.79 g, 10 mmol) and triethyl-
amine (1.01 g, 10 mmol) following the same procedure
described for 10a. The product was obtained as oil (88%
yield). 1H-NMR (CDCl3) d: 1.26 (t, 3H, CH2CH3, J:
4.0 Hz), 3.79 (s, 3H, OCH3), 4.00 (s, 3H, OCH3), 4.28 (q,
2H, CH2CH3), 6.30 (s, 1H, ArH-3), 6.45 (d, 1H, ArH-5, J:
8.2 Hz), 6.65 (d, 1H, ArH-6, J: 8.2 Hz), 7.00–7.60 (m, 5H,
Ar), 7.80 (d, 2H, Ar, J: 8.6 Hz), 8.10 (d, 2H, Ar, 8.6 Hz).
IR (KBr) cm-1: 3059 (CH Ar), 2937, 2839 (CH aliphatic),
1728 (C=O). MS m/z: 464 [M??2, 1%], 462 [M?, 1%],
165 [C6H3(OCH3)2CO, 100%]. Anal. calcd for
C26H23ClN2O4 (462.93): C 67.46; H 5.01; N 6.05. Found:
C 67.44; H 5.21; N 5.83.
The experimental tests on animals have been performed in
accordance with the Institutional Ethical Committee
approval, Faculty of Pharmacy, Cairo University.
LD50, anti-inflammatory, and analgesic activities of all
the newly synthesized compounds were performed. The
tested compounds were dissolved in dimethyl sulfoxide and
water.
Determination of LD50
LD50 determined using Finney’s method (Finney, 1964).
For this purpose, albino mice (25–30 g) were divided into
groups each of five animals. Preliminary experiments were
done for each compound to determine the minimal dose
that kills all mice and the maximal dose that fails to kill any
animal. Several increasing intraperitoneal doses were given
in between these doses. Animals were kept under obser-
vation for 24 h during which symptoms of toxicity and rate
of mortality in each group were recorded.
1-(4-Chlorophenyl)-5-(2,4-dimethoxyphenyl)-4-methyl-1H-
pyrazole-3-carboxylic acid 11a
Compound 10b (2.00 g, 5 mmol) was dissolved in meth-
anol (20 ml) and to this a solution of KOH (0.56 g,
10 mmol) in methanol (5 ml) was added. The reaction
mixture was heated to reflux for 3 h, cooled, and poured
into ice-water. It was acidified with acetic acid and
extracted with chloroform. The organic layer was washed
with water, dried over Na2SO4, and concentrated. The
residue was obtained as oil (51% yield). 1H-NMR (CDCl3–
D2O) d: 3.08 (s, 3H, CH3), 3.70 (s, 3H, OCH3), 3.85 (s, 3H,
OCH3), 6.23 (s, 1H, ArH-3), 6.50 (d, 1H, ArH-5, J:
8.0 Hz), 7.10 (d, 1H, ArH-6, J: 8.0 Hz), 7.35 (d, 2H, Ar, J:
8.6 Hz), 7.78 (d, 2H, Ar, J: 8.6 Hz), 11.00 (s, 1H, COOH,
exch.). IR (KBr) cm-1: 3030 (CH Ar), 2950–2850 (CH
aliphatic and OH), 1720 (C=O). MS m/z: 372 [M?, 8%],
165 [C6H3(OCH3)2CO, 100%]. Anal. calcd for
C19H17ClN2O4 (372.81): C 61.21; H 4.60; N 7.51. Found:
C 61.35; H 4.80; N 7.57.
Anti-inflammatory activity (in vivo screening,
carrageenan-induced edema in rats)
The anti-inflammatory activity of the newly synthesized
compounds was evaluated according to the method
described by Winter et al. (Winter et al., 1962). One-hun-
dred and five rats of both sexes weighing 150–180 g were
divided into 21 groups. The thickness of the left hind paw of
each rat was measured in millimeter using a vernier caliber.
The first group was kept as a control, while the second was
intraperitoneal injected with celecoxib as a reference in a
dose of 25 mg/kg body weight. Other groups were intra-
peritoneal injected with the tested compounds in a dose
25 mg/kg body weight which nearly equal to 1/10 their
LD50. After 30 min, inflammation was induced by subcu-
taneous injection of 50 ll of 1% carrageenan in a normal
saline into the left hind paw. Degree of inflammation (mm)
is measured as the difference between thickness after car-
rageenan treatment and thickness before carrageenan
treatment. The paw thickness was measured hourly for a
period of 4 h. The anti-inflammatory efficacy of the tested
compounds was assessed by comparing the magnitude of
the paw swelling in the treated animals with that of the
1-(4-Chlorophenyl)-5-(2,4-dimethoxyphenyl)-4-phenyl-1H-
pyrazole-3-carboxylic acid 11b
It was prepared from 10d as oil (57% yield) by using the
same procedure described for 11a. 1H-NMR (CDCl3–D2O)
d: 3.83 (s, 3H, OCH3), 4.27 (s, 3H, OCH3), 6.35 (s, 1H,
ArH-3), 6.50 (d, 1H, ArH-5, J: 8 Hz), 6.62 (d, 1H, ArH-6,
J: 8.2 Hz), 7.60–7.78 (m, 5H, Ar), 7.88 (d, 2H, Ar, J:
123

Med Chem Res (2012) 21:983–994
993
control then calculates percent inhibition with the following
divided by ten, the average number of ulcers per stomach,
and the average severity of ulcers by visual observation.
formula (Alam et al., 2009).
% of edema inhibition ¼ ðVR ꢁ VLÞ control ꢁ ðVR ꢁ VLÞ treated ꢂ 100
ðVR ꢁ VLÞ control
Analgesic activity
The ulcer index is the value that result from the sum of the
above three values.
The analgesic activity of the tested compounds was eval-
uated using the writhing method (Collier et al., 1968). One-
hundred and five mice of both sexes weighing 25–30 g
were divided into 21 groups. The first group was kept as a
control, while the second was intraperitoneal injected with
celecoxib as standard drug in a dose of 25 mg/kg body
weight. Other groups were intraperitoneal injected with the
tested compounds in a dose 25 mg/kg body weight. After
30 min, each mouse was intraperitoneal injected with
0.25 ml of p-benzoquinone aqueous solution (0.1 mg/ml).
Thereafter, mice in all groups were observed for writhing
hourly for 4 h. Animals devoid of writhing in each group
were counted, and the analgesic potency of the tested
compounds was determined as percent protection against
writhing.
Acknowledgment The authors wish to offer their deep gratitude to
Prof. Dr. Gamal. A. Soliman, Department of Pharmacology, Faculty
of Veterinary Medicine, Cairo University for carrying out the phar-
macological evaluation.
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