Synthesis of novel 1,3,4-trisubstituted pyrazoles as anti-inflammatory and analgesic agents

Article abstract of DOI:10.1016/j.ejmech.2013.03.005

Some novel 1,3,4-trisubstituted pyrazoles were synthesized and screened for their anti-inflammatory and analgesic activities as well as their ulcerogenic liability. They showed anti-inflammatory and analgesic activities with better GIT tolerance than the standard drug phenylbutazone. In addition, IC ₅₀ values for 5e and 8e were recorded. Compound 5e was found to be the most active one as anti-inflammatory and analgesic agent. On the other hand, COX-1/COX-2 isozyme selectivity was also done which showed equal inhibition to both isoforms.

Full text of DOI:10.1016/j.ejmech.2013.03.005

European Journal of Medicinal Chemistry 63 (2013) 645e654
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journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel 1,3,4-trisubstituted pyrazoles as anti-inflammatory
and analgesic agents
*
Fatma A. Ragab, Nagwa M. Abdel Gawad, Hanan H. Georgey , Mona F. Said
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, P.O. Box 11562, Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Some novel 1,3,4-trisubstituted pyrazoles were synthesized and screened for their anti-inflammatory
and analgesic activities as well as their ulcerogenic liability. They showed anti-inflammatory and anal-
gesic activities with better GIT tolerance than the standard drug phenylbutazone. In addition, IC50 values
for 5e and 8e were recorded. Compound 5e was found to be the most active one as anti-inflammatory
and analgesic agent. On the other hand, COX-1/COX-2 isozyme selectivity was also done which
showed equal inhibition to both isoforms.
Received 26 October 2012
Received in revised form
28 February 2013
Accepted 2 March 2013
Available online 14 March 2013
Ó 2013 Elsevier Masson SAS. All rights reserved.
Keywords:
Pyrazole
Schiff’s base
Pyrrole
Anti-inflammatory
Analgesic
COX inhibition
1. Introduction
highly marketed COX-2 inhibitors that comprise the pyrazole nu-
cleus, celecoxib and SC-558 (Fig.1) which belong to diaryl pyrazoles
Design of non-steroidal anti-inflammatory drugs (NSAIDs) with
enhanced safety profile is still a challenge for the pharmaceutical
industry. In spite of the great safety of known NSAIDs over the
steroidal derivatives, they still have some side effects like devel-
opment of peptic and duodenal ulcer in addition to some kidney
and liver malfunctions on long term use [1].
The acceptable mechanism of action of NSAIDs is by the inhi-
bition of the biosynthesis of prostaglandins through inhibition of
the key enzyme known as prostaglandin cyclooxygenase (COX) [2].
COX exists in two isoforms namely COX-1 and COX-2. COX-1 is a
constitutive enzyme and responsible for the production of cyclo-
protective prostaglandin in GIT and proaggregatory thromboxane
in blood platelets, while COX-2 is an inducible enzyme which in-
duces in response to the release of several proinflammatory me-
diators [3e5]. Most NSAIDs inhibit both COX-1 and COX-2 but with
variable degrees of selectivity. Selective COX-2 inhibitors may
eliminate side effects that are associated with NSAIDs due to COX-1
inhibition [6e8]. However, selective COX-2 inhibitors are found to
posses cardiovascular side effects [9].
containing sulfonamide moiety.
Consequently, the present investigation describes the synthesis
of certain 1,3-diaryl pyrazoles, position 1 of the pyrazole ring was
substituted either with benzenesulfonamide or 4-chlorophenyl
group, since these two substituents recorded to exhibit good anti-
inflammatory activity [12e18]. Position 3 carries either p-tolyl or
4-methoxy phenyl substituent. Moreover, position 4 of the pyrazole
ring was substituted with a variety of functionality as azomethine,
chalcone or substituted pyrrole derivatives as it was reported that
these derivatives have a promising effect in potentiating the anti-
inflammatory activity [15,19e25]. All the synthesized compounds
were subjected to anti-inflammatory testing. The ulcerogenic ac-
tivity and acute toxicity profile of the most active compounds and
their selectivity to inhibit COX-1 and COX-2 isozymes by in vitro
COX inhibition assay were determined. In addition, the analgesic
activity of representative examples was also carried out.
2. Results and discussion
Pyrazole derivatives as various coxibs showed potent anti-
inflammatory activity with low GIT toxicity [10,11]. Among the
2.1. Chemistry
The target compounds were synthesized according to the
steps outlined in Schemes 1 and 2. The key intermediates 1-(4-
* Corresponding author. Tel.: þ20 1275600699; fax: þ20 2 23628426.
E-mail address: Hanan-Hanna@hotmail.com (H.H. Georgey).
substituted
phenyl)-2-(1-(4-substituted
phenyl)ethylidene)
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.005

646
F.A. Ragab et al. / European Journal of Medicinal Chemistry 63 (2013) 645e654
F
F
derivatives 3a [28] and the newly synthesized 3b. Reaction of the
F
F
hydrazone derivatives 2c,d with VilsmeiereHaack reagent affor-
ded the corresponding 4-formylpyrazole derivatives with pro-
tected sulfonamido group 3c,d [13,29]. Deprotection was
accomplished using methanolic solution of NaOH with THF as co-
solvent [29]. Bekhit et al. [13] reported that acidic conditions
could be also used for the deprotection of the sulfonamido group,
but the basic conditions reported by Sharma et al. [29] applied
here gave much better yield (Scheme 1).
As shown in Scheme 2, the 4-formylpyrazoles 3a,b and 4a,b
were functionalized by a classical condensation reaction with
substituted anilines in refluxed ethanol with traces of glacial acetic
acid to give the corresponding azomethine derivatives 5ael. The 1H
F
F
Br
N
N
N
N
O
O
S
S
NH2
NH2
O
O
-558
Fig. 1. Structure of COX-2 selective agents.
hydrazines 2aed were synthesized by condensation of the
commercially available 4-chlorophenylhydrazine hydrochloride 1a
or the synthesized sulfamoyl counterpart 1b [26] with 4-
substituted acetophenones. Treatment of the hydrazone de-
rivatives 2a,b with VilsmeiereHaack [27] reagent (DMFePOCl3) in
three equivalents led to corresponding 4-formylpyrazole
NMR spectra of 5ael showed the appearance of a signal at
8.64 ppm corresponding to azomethine proton (N]CH) and the
disappearance of the signal attributed to (CH of aldehyde). Also, 13
d 8.49e
C
NMR spectrum of 5e revealed the disappearance of the signal
corresponding to (HC]O).
R1
O
H
For 2a,b
N
N
R1
NH2 .HCl
HN
O
R1
N
ii
NH
Cl
i
3a,b
R
1a,b
R= Cl
R
R1
1a
O
2a-d
H
1b R= SO2NH2
2a
R= Cl, R1= CH3
2c,d
For
2b R= Cl, R1= OCH3
N
2c R= SO2NH2, R1= CH3
2d R= SO2NH2, R1= OCH3
N
O
N
S
N
O
3a,c
R1= CH3
3b,d R1= OCH3
3c,d
iii
R1
O
H
N
N
O
S
NH2
O
4a,b
4a R1= CH3
4b
R1= OCH3
Scheme 1. Synthesis of the target compounds 1e4: (i) EtOH, glacial acetic, r.t. (ii) POCl3, DMF, reflux. (iii) MeOH, THF, NaOH, r.t.

F.A. Ragab et al. / European Journal of Medicinal Chemistry 63 (2013) 645e654
647
R1
O
R1
H
N
R2
NH2
N
N
N
N
R2
5a
i
R= Cl, R1= CH3, R2= H
5b
R= Cl, R1= CH3, R2= OCH3
R
5c R= Cl, R1= CH3, R2= Cl
R
5d R= Cl, R1= OCH3, R2= H
5e R= Cl, R1= OCH3, R2= OCH3
5f R= Cl, R1= OCH3, R2= Cl
5g R= SO2NH2, R1= CH3, R2= H
3a,b, 4a,b
5a-l
3a R=Cl, R1= CH3
3b R= Cl, R1= OCH3
5h
5i
5j
R= SO2NH2, R1= CH3, R2= OCH3
R= SO2NH2, R1= CH3, R2= Cl
R= SO2NH2, R1= OCH3, R2= H
4a R= SO2NH2, R1= CH3
4b
R= SO2NH2, R1= OCH3
5k R= SO2NH2, R1= OCH3, R2= OCH3
5l R= SO2NH2, R1= OCH3, R2= Cl
ii
R3
R1
Cl
O
Cl
R1
O
O
H
O
N
N
R3
N
N
iii
R
R
7a-h
6a-d
6a R= Cl, R1= CH3
6b R= Cl, R1= OCH3
6c R= SO2NH2, R1= CH3
6d
iv
R= SO2NH2, R1= OCH3
R3
Cl
H
N
R1
7a, 8a
7b, 8b
N
R= Cl, R1= CH3, R3= F
N
R= Cl, R1= CH3, R3= OCH3
7c, 8c R= Cl, R1= OCH3, R3= F
7d, 8d R= Cl, R1= OCH3, R3= OCH3
7e, 8e R=SO2NH2, R1= CH3, R3= F
7f, 8f R= SO2NH2, R1= CH3, R3= OCH3
7g, 8g R= SO2NH2, R1= OCH3, R3= F
R
7h, 8h
R= SO2NH2, R1= OCH3. R3= OCH3
8a-h
Scheme 2. Synthesis of the target compounds 5e8: (i) EtOH, glacial acetic, reflux. (ii) p-chloroacetophenone, NaOH, EtOH, r.t. (iii) DMF, r.t. (iv) NH4-acetate, acetic acid, reflux.
On the other hand, ClaiseneSchmidt [30,31] condensation be-
tween 3a,b or 4a,b and 4-chloroacetophenone afforded the corre-
sponding chalcones 6aed. 1H NMR spectra of 6aed showed the
appearance of two doublets corresponding to olefinic protons of
spectra showed the appearance of aliphatic protons of (eCH2eCHe)
as a prominent ABX system, with protons Ha, Hb and Hx seen as
doublets of doublets at d 3.23e3.45, 3.43e3.61 and 4.19e4.70 ppm,
respectively. 7aeh were cyclized using ammonium acetate in
acetic acid under PaaleKnorr [33,34] reaction conditions to yield
the corresponding 1-(4-substitutedphenyl)-4-(5-(4-chlorophenyl)-
2-(4-substitutedphenyl)-1H-pyrrol-3-yl)-3-(4-substitutedphenyl)-
1H-pyrazoles 8aeh. Their IR spectra revealed the appearance of a
band at 3383e3417 cmꢀ1 corresponding to (NH) and the disap-
pearance of any band corresponding to (C]O), 1H NMR spectra
showed the appearance of an exchangeable signal of (NH) at
a,b-unsaturated ketone (HC]CHeC]O) at d 7.01e7.89 ppm and the
disappearance of the signal corresponding to (CH of aldehyde). Also
the mass spectra of 6a,b revealed the appearance of their molecular
ion peaks along with isotopes (M þ 2)þ, (M þ 4)þ that indicate the
presence of two chlorine atoms. Addition reaction of 6aed with
anisaldehyde or 4-fluorobenzaldehyde under Stetter [32] reaction
conditions using dimethylformamide in the presence of potassium
cyanide as catalyst afforded compounds 7aeh. Their 1H NMR
d
5.00e5.90 ppm and the disappearance of any doublets of doublets

648
F.A. Ragab et al. / European Journal of Medicinal Chemistry 63 (2013) 645e654
corresponding to the aliphatic protons of (eCH2eCH), also 13C NMR
spectrum of 8d revealed the disappearance of any signal corre-
sponding to (C]O) (Scheme 2).
Table 2
IC50 values of compounds 5e and 8e and the reference drug phenylbutazone.
Cpd. No.
Dose (mmol/kg)
% inhibition
IC50 (mmol/kg)
0.22
Phenylbutazone
0.08
0.16
0.32
0.08
0.16
0.32
0.08
0.16
0.32
20.00
42.10
70.55
37.50
43.00
82.22
14.00
43.60
66.70
2.2. Pharmacological screening
5e
8e
0.20
0.21
2.2.1. Anti-inflammatory activity
All the targeted compounds were evaluated for their anti-
inflammatory activity via carrageenan-induced rat paw
edema method reported by Winter et al. [35] using phenyl-
butazone as reference drug. The results were recorded in
Table 1. IC50 values of the active compounds 5e and 8e and the
reference drug phenylbutazone were calculated and recorded
in Table 2.
Results in Table 1 revealed that all the tested compounds
showed anti-inflammatory activity. After 2 h eleven com-
pounds exhibited better activity than phenylbutazone.
While, after 3 h only five compounds gave better activity than
phenylbutazone.
1-(benzenesulfonamide) derivatives 4a,b showed higher activity
than their 1-(4-chlorophenyl) congeners 3a,b. However, on the
formation of the azomethine derivatives 5ael the case was
different; compounds 5def bearing 4-chlorophenyl pharmaco-
phore exhibited higher activity than 1-(benzenesulfonamide)
analogs 5jel except for benzenesulfonamide azomethine de-
rivatives 5g,i which possessed higher activity than their 4-
chlorophenyl analogs 5a,c.
2.2.2. Structureeactivity relationship
Also, the effect of the nature of substituent at the position-3
of the pyrazole nucleus was studied. This position was sub-
stituted either by p-tolyl or 4-methoxy phenyl moiety; in the
pyrazole carboxyaldehydes 3a,b and 4a,b the 3-(4-methoxy
phenyl) derivatives 3b and 4b exhibited higher activity than
their p-tolyl congeners 3a and 4a. For the 1-(4-chlorophenyl)
derivatives of the azomethine series 5aef, improved activity
was observed by the 3-(4-methoxy phenyl) substituted com-
pounds 5def than their p-tolyl analogs 5aec. On the other
hand, in the benzenesulfonamide azomethine series 5k was the
only derivative which showed improved activity than its p-tolyl
analog 5h.
Moreover, the nature of the aryl substituent attached to the
azomethine group has an observable effect on the activity, as
shown in compounds 5b,e,k bearing 4-electron donating substit-
uent (methoxy group) that exhibited improved activity than their
congeners with 4-electron withdrawing substituent (chloro group)
5c,f,l or with unsubstituted phenyl analogs 5a,d,j. On the contrary,
compound 5i was the only one bearing 4-choloro substituent and
has superior activity than its 4-methoxy or unsubstituted phenyl
congeners 5h,g, respectively.
Chalcones carrying 1-(4-chlorophenyl) pharmacophore 6a,b
exhibited greater activity than their 1-(benzenesulfonamide)
counterparts 6c,d. Also, the 3-(4-methoxyphenyl) derivatives 6b,d
were more active than the 3-(p-tolyl) derivatives 6a,c. While, in the
series of 7aeh, 1-(benzenesulfonamide) compounds 7eeh showed
much greater activity than their 1-(4-chlorophenyl) analogs 7aed.
Rigidification of 7aeh by incorporation of additional pyrrole ring as
shown in compounds 8aeh resulted in improvement of activity
except for compounds 8b,f,g which showed decreased activity
compared with 7b,f,g. Moreover, 4-substitution of phenyl moiety
attached to pyrrole ring by electron withdrawing group as fluoro
group in compounds 8a,c,e,g found to exhibit a fruitful effect on
activity than with electron donating one as methoxy analogs
8b,d,f,h.
Considering the anti-inflammatory activity of the pyrazole
carboxyaldehydes 3a,b and 4a,b, it was noticed that the
Table 1
Anti-inflammatory effect and percentage inhibition of phenylbutazone, and syn-
thesized compounds on carrageenan induced edema of the hind paw in rats (n ¼ 5).
Cpd. NO.
Dose
mmol/kg
Edema (mm) ꢁ SEM
% inhibition
2 h
3 h
2 h
0
3 h
0
Control
Phenylbutazone 0.32
0
3.24 ꢁ 0.39
3.60 ꢁ 0.24
1.38 ꢁ 0.09*** 1.06 ꢁ 0.19*** 57.28 70.55
1.62 ꢁ 0.18*** 1.58 ꢁ 0.12*** 49.90 56.00
1.19 ꢁ 0.11*** 1.53 ꢁ 0.22*** 63.11 57.58
1.35 ꢁ 0.15*** 1.15 ꢁ 0.12*** 58.40 68.17
0.55 ꢁ 0.09*** 0.66 ꢁ 0.11*** 82.96 81.55
1.94 ꢁ 0.04*** 1.82 ꢁ 0.16*** 40.06 49.33
1.23 ꢁ 0.16*** 1.84 ꢁ 0.13*** 62.04 48.89
1.68 ꢁ 0.19*** 1.83 ꢁ 0.26*** 48.27 49.17
1.40 ꢁ 0.09*** 0.99 ꢁ 0.15*** 57.19 72.58
0.99 ꢁ 0.17*** 0.64 ꢁ 0.15*** 69.23 82.22
0.98 ꢁ 0.11*** 0.99 ꢁ 0.17*** 69.60 72.60
1.36 ꢁ 0.20*** 1.54 ꢁ 0.21*** 58.08 57.11
1.66 ꢁ 0.09*** 1.58 ꢁ 0.16*** 48.76 56.19
1.00 ꢁ 0.12*** 0.78 ꢁ 0.14*** 69.10 78.40
3a
3b
4a
4b
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
6a
6b
6c
6d
7a
7b
7c
7d
7e
7f
7g
7h
8a
8b
8c
8d
8e
8f
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
1.99 ꢁ 0.09**
1.61 ꢁ 0.09*** 38.45 55.40
1.11 ꢁ 0.15*** 1.13 ꢁ 0.14*** 65.80 68.50
1.19 ꢁ 0.19*** 1.34 ꢁ 0.22*** 63.04 62.78
1.65 ꢁ 0.17*** 1.39 ꢁ 0.25*** 49.19 61.40
1.50 ꢁ 0.16*** 1.21 ꢁ 0.26*** 53.70 66.39
1.51 ꢁ 0.13*** 1.90 ꢁ 0.13*** 53.33 47.60
1.44 ꢁ 0.29*** 1.58 ꢁ 0.14*** 55.56 55.97
2.40 ꢁ 0.32
2.72 ꢁ 0.17
2.59 ꢁ 0.26
3.47 ꢁ 0.11
2.90 ꢁ 0.29
2.74 ꢁ 0.07
2.24 ꢁ 0.27**
3.25 ꢁ 0.22
25.90 19.44
16.05 23.89
19.80 37.83
0
9.61
1.51 ꢁ 0.06*** 1.58 ꢁ 0.14*** 53.46 56.22
1.49 ꢁ 0.18*** 1.40 ꢁ 0.20*** 53.83 61.11
0.90 ꢁ 0.12*** 1.15 ꢁ 0.30*** 72.22 68.06
2.24 ꢁ 0.24
2.00 ꢁ 0.34*
2.94 ꢁ 0.13
2.79 ꢁ 0.18
2.84 ꢁ 0.29
2.83 ꢁ 0.17
30.80 22.50
38.27 20.97
9.38 21.33
1.51 ꢁ 0.26*** 1.37 ꢁ 0.16*** 53.70 61.89
2.90 ꢁ 0.23
0.79 ꢁ 12***
2.85 ꢁ 0.11
1.19 ꢁ 0.09*** 75.37 66.70
49.69 34.17
10.50 20.72
2.2.3. Analgesic activity
Compounds that exhibited good anti-inflammatory activity and
phenylbutazone were screened for their analgesic activity using the
reported method of p-benzoquinone-induced writhing in mice by
Okun et al. [36] It was observed from the results in Table 3 that, all
the tested compounds showed analgesic activity. Moreover, com-
pound 5e that showed the best anti-inflammatory activity also
found to be the most active one as analgesic.
1.63 ꢁ 0.22*** 2.37 ꢁ 0.19**
8g
8h
1.43 ꢁ 0.15*** 1.59 ꢁ 0.15*** 55.70 55.90
1.68 ꢁ 0.25*** 2.57 ꢁ 0.28 48.15 28.60
Statistical analysis was carried out by one-way ANOVA test.
*Significance difference from the control value at p < 0.05.
**Significance difference from the control value at p < 0.01.
***Significance difference from the control value at p < 0.001.

F.A. Ragab et al. / European Journal of Medicinal Chemistry 63 (2013) 645e654
649
Table 3
Analgesic activity of phenylbutazone and 17 synthesized compounds in mice.
2.2.6. In vitro COX inhibition assay
Compounds 5e,f, 8e, and indomethacin were evaluated for their
selectivity to inhibit COX-1 and/or COX-2 isozymes using Cayman’s
COX-Activity Assay kit according to the manufacturer’s instruction
[40]. The ratio of percentage inhibition of COX-1/COX-2 was
calculated and recorded in Table 5. Results revealed that the tested
compounds were non-selective as they have almost equal inhibi-
tory activity on both isoforms of COX enzyme.
Cpd. No.
Dose
No. of
No. of
% protection
mmol/kg
animals
protected
animals
Control
Phenylbutazone
0
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
0
2
2
1
1
1
1
4
2
2
2
3
3
3
2
2
2
1
3
0
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
33.33
33.33
16.67
16.67
16.67
16.67
66.67
33.33
33.33
33.33
50
3b
4a
4b
5b
5d
5e
5f
5i
5k
5l
3. Conclusion
From this study, we conclude that 1-(4-chlorophenyl)pyrazole and
pyrazolylbenzenesulfonamide derivatives could be considered to be
useful building blocks for future research to the synthesis of potential
anti-inflammatory and analgesic agents with minimal side effects.
Pharmacological screening and biological evaluation revealed that all
the tested compounds have good anti-inflammatory activity. The
newly synthesized compounds 5e,f, and 8e showed high anti-
inflammatory activity and good analgesic activity. These compounds
were non-selective toward COX-1 or COX-2 but they exhibited better
GIT tolerance than the reference drug phenylbutazone.
7e
7f
50
50
7g
8a
8c
8e
8f
33.33
33.33
33.33
16.67
50
4. Experimental
Table 4
Ulcerogenic effect of phenylbutazone and 9 synthesized compounds in rats.
All chemicals were purchased from VWR International Merck,
Germany or SigmaeAldrich and used without further purification.
Compound (4-chlorophenyl)hydrazine hydrochloride (1a) was
purchased from SigmaeAldrich. Melting Points were carried out by
open capillary tube method using Stuart SMP3 Melting Point
apparatus and they are uncorrected. Elemental Microanalysis was
carried out at the Micro Analytical Center, Cairo University or at The
Regional Center for Mycology and Biotechnology, Al-Azhar Univer-
sity. Infrared spectra were recorded on Shimadzu Infrared spec-
trometer IR Affinity-1 (FTIR-8400S-Kyoto-Japan), and expressed in
wave number (cmꢀ1), using potassium bromide discs. 1H NMR and
13C NMR Spectra were recorded on a Varian Mercury VX-300 MHz,
13C, 70 MHz NMR spectrometer, or Jeol-ECA500, 500 MHz Japan,13C,
125 MHz NMR spectrometer, the spectra were run at 300 MHz or
500 MHz in deuterated chloroform (CDCl3) or dimethylsulfoxide
Cpd. No.
Dose
No. of
% incidence Average no. Average Ulcer
mmol/kg animals divided
by 10
of ulcer
severity index
Control
Phenylbutazone 0.32
4a
4b
5e
5f
0
5
5
5
5
5
5
5
5
5
5
5
0
10
6
8
10
4
4
10
8
8
10
0
0
0
19.0
2.6
2.2
3.2
0.4
0.8
5.4
4.0
3.0
4
1.20
1.00
1.00
1.12
1.00
1.00
1.00
1.00
1.00
1
30.20
9.60
11.20
14.32
5.40
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
0.32
5i
5.80
5k
8a
8c
8e
16.40
13.00
12.00
15.00
2.2.4. Ulcerogenic effect
(DMSO-d6). Chemical shifts were expressed in
d units and were
The ulcerogenic effect of nine active compounds and phenyl-
butazone was evaluated by the reported method of Meshali et al.
[37], and the ulcer index was calculated according to the method of
Robert et al. [38] and recorded in Table 4. It was observed that all
the tested compounds showed lower ulcer indices than phenyl-
butazone. Moreover, concerning the tested Schiff’s bases it was
noticed that the chloro derivatives 5f,i showed less ulcerogenic
effects than their methoxy analogs 5e,k.
related to that of the solvents. As for the proton magnetic resonance,
D2O was carried out for NH and OH exchangeable protons. Mass
Spectra were recorded using Shimadzu Gas Chromatograph Mass
spectrometer-Qp 2010 plus (Japan) or Jeol JMS-AX 500 mass spec-
trometer (Japan). All the reactions were followed by TLC using silica
gel F254 plates (Merck), using chloroform: methanol 9:1 or chlo-
roform as eluting system and were visualized by UV-lamp. Com-
pounds 1b [26], 3a [28], 3c [13,29], 3d [29], 4a [13,29], 4b [29] were
prepared according to reported methods, 2a [28], 2c [13,29] and 2d
[29] were prepared by modified procedure from the reported
method.
2.2.5. Acute toxicity
LD50 of some representative compounds 5e,k,l, and 8e was
determined using Finney’s method [39]. Results revealed that LD50
of compounds 5e,k, and 8e was 3.25 mmol/kg body weight, while
that of compound 5l was 2.44 mmol/kg body weight which indi-
cated a high safety margin of the tested compounds.
4.1. Chemistry
4.1.1. General procedure for the synthesis of compounds (2aed)
A mixture of 1a,b (10 mmol) in glacial acetic acid (1 mL) was
added to a solution of 4-substituted acetophenone (10 mmol) in
ethanol (30 mL). Then, the reaction mixture was stirred at room
temperature for 24 h, concentrated under reduced pressure and
poured onto ice water. The solid formed was collected, washed with
water, dried and recrystallized from ethanol.
Table 5
COX-1/COX-2 percentage inhibition ratio of compounds 5e,
5f, 8e and indomethacin.
Cpd. NO.
COX-1/COX-2
Indomethacin
5e
5f
3.94
1.17
0.95
0.87
4.1.1.1. 1-(4-Chlorophenyl)-2-(1-p-tolylethylidene)hydrazine
Yield 73%; as oil; IR (KBr, cmꢀ1): 3352 (NH), 3074 (CH aromatic),
2916, 2854 (CH aliphatic); 1H NMR (CDCl3, 300 MHz):
2.42 (s, 3H,
(2a).
8e
d

650
F.A. Ragab et al. / European Journal of Medicinal Chemistry 63 (2013) 645e654
CH3), 2.58 (s, 3H, ]CeCH3), 4.73 (s, ex, 1H, NH), 6.77e7.87 (m, 8H,
aromatic H); EIMS, m/z: 260 (M þ 2)þ, 258 (Mþ); Anal. Calcd. For
(aniline, 4-anisidine or 4-chloroaniline) (2 mmol), the reaction
mixture was refluxed for 12e15 h. Then after cooling to room
temperature, the solid formed was collected by filtration, washed
with water and recrystallized from ethanol.
C
15H15ClN2 (258.74): C, 69.63; H, 5.84; N, 10.83. Found: C, 69.74; H,
5.92; N, 10.91.
4.1.1.2. 1-(4-Chlorophenyl)-2-(1-(4-methoxyphenyl)ethylidene)hy-
drazine (2b). Yield 66%; m.p. 128e130 ꢂC; IR (KBr, cmꢀ1): 3356
(NH), 3000 (CH aromatic), 2962, 2935 (CH aliphatic); 1H NMR
4.1.3.1. N-((1-(4-Chlorophenyl)-3-p-tolyl-1H-pyrazol-4-yl)methy-
lene)aniline (5a). Yield 71%; m.p. 197e198 ꢂC; IR (KBr, cmꢀ1): 3030
(CH aromatic), 2920, 2854 (CH aliphatic); 1H NMR (CDCl3,
(CDCl3, 500 MHz):
d
2.55 (s, 3H, ]CeCH3), 3.84 (s, 3H, OCH3), 4.90
300 MHz):
d
2.44 (s, 3H, CH3), 6.68e7.83 (m, 14H, aromatic Hþ CH
(s, ex, 1H, NH), 6.76e7.94 (m, 8H, aromatic H); 13C NMR (CDCl3,
pyrazole), 8.53 (s, 1H, HC]N); EIMS, m/z: 373 (M þ 2)þ, 371 (Mþ);
Anal. Calcd. For C23H18ClN3 (371.86): C, 74.29; H, 4.88; N, 11.30.
Found: C, 74.38; H, 4.97; N, 11.66.
125 MHz):
d 26.4, 55.5, 114.3, 122.4, 124.2, 127.0, 129.1, 130.2, 130.8,
142.4, 159.8, 163.7; EIMS, m/z: 276 (M þ 2)þ, 274 (Mþ); Anal. Calcd.
For C15H15ClN2O (274.74): C, 65.57; H, 5.50; N, 10.20. Found: C,
65.62; H, 5.48; N, 10.47.
4.1.3.2. N-((1-(4-Chlorophenyl)-3-p-tolyl-1H-pyrazol-4-yl)methy-
lene)-4-methoxyaniline (5b). Yield 45%; m.p. 152e153 ꢂC; IR (KBr,
cmꢀ1): 3101, 3059 (CH aromatic), 2920, 2850 (CH aliphatic); 1H
4.1.1.3. 4-(2-(1-p-Tolylethylidene)hydrazinyl)benzenesulfonamide
(2c). Yield 52%; m.p. 226e227 ꢂC; IR (KBr, cmꢀ1): 3363, 3336 (NH2),
3267 (NH), 3050 (CH aromatic), 2950, 2830 (CH aliphatic), 1319,
NMR (CDCl3, 300 MHz): d 2.44 (s, 3H, CH3), 3.83 (s, 3H, OCH3), 6.91e
7.82 (m, 13H, aromatic Hþ CH of pyrazole), 8.51 (s, 1H, HC]N);
EIMS, m/z: 403 (M þ 2)þ, 401 (Mþ); Anal. Calcd. For C24H20ClN3O
(401.88): C, 71.73; H, 5.02; N, 10.46. Found: C, 71.81; H, 5.11; N,
10.73.
1145 (SO2); 1H NMR (CDCl3, 500 MHz):
d 2.26 (s, 3H, CH3), 2.57 (s,
3H, ]CeCH3), 4.65 (s, ex, 1H, NH), 7.25 (s, ex, 2H, NH2), 7.19e7.82
(m, 8H, aromatic H); EIMS, m/z: 303 (Mþ); Anal. Calcd. For
C
15H17N3O2S (303.37): C, 59.38; H, 5.65; N, 13.85. Found: C, 59.57;
H, 5.08; N, 13.46.
4.1.3.3. 4-Chloro-N-((1-(4-chlorophenyl)-3-p-tolyl-1H-pyrazol-4-yl)
methylene)aniline (5c). Yield 55%; m.p. 151e153 ꢂC; IR (KBr, cmꢀ1):
3036 (CH aromatic), 2920, 2850 (CH aliphatic); 1H NMR (CDCl3,
4.1.1.4. 4-(2-(1-(4-Methoxyphenyl)ethylidene)hydrazinyl)benzene-
sulfonamide (2d). Yield 78%; m.p. 230e232 ꢂC; IR (KBr, cmꢀ1):
3353, 3321 (NH2, NH), 3070, 3012 (CH aromatic), 2978 (CH
300 MHz):
d
2.44 (s, 3H, CH3), 6.59e7.84 (m, 13H, aromatic Hþ CH
of pyrazole), 8.51 (s, 1H, HC]N); EIMS, m/z: 409 (M þ 4)þ, 407
(M þ 2)þ, 405 (Mþ); Anal. Calcd. For C23H17Cl2N3 (406.30): C, 67.99;
H, 4.22; N, 10.34. Found: C, 68.08; H, 4.31; N, 10.59.
aliphatic),1319,1145 (SO2); 1H NMR (CDCl3, 500 MHz):
d 2.25 (s, 3H,
CH3), 3.84 (s, 3H, OCH3), 4.65 (s, ex, 1H, NH), 7.26 (s, ex, 2H, NH2),
6.92e7.82 (m, 8H, aromatic H); EIMS, m/z: 319 (Mþ); Anal. Calcd.
For C15H17N3O3S (319.37): C, 56.41; H, 5.37; N, 13.16. Found: C,
56.69; H, 5.58; N, 13.26.
4.1.3.4. N-((1-(4-Chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-
yl)methylene)aniline (5d). Yield 63%; m.p. 159e160 ꢂC; IR (KBr,
cmꢀ1): 3070, 3000 (CH aromatic), 2924, 2904 (CH aliphatic); 1H
4.1.2. General procedure for the synthesis of compounds (3a,b)
A mixture of DMF (2.58 g, 35.30 mmol) and POCl3 (5.40 g,
35.30 mmol) was cooled at 0 ꢂC before being stirred at that tem-
perature. A solution of 2aed (11.76 mmol) in DMF (3 mL) was added
drop wise to the VilsmeiereHaack reagent, warmed to room tem-
perature, and heated at 70e80 ꢂC for 5 h. After cooling to room
temperature, the mixture was basified with cold saturated K2CO3
solution. The precipitate was filtered, washed with water and
crystallized from ethanol.
NMR (CDCl3, 300 MHz): d 3.88 (s, 3H, OCH3), 6.68e7.83 (m, 14H,
aromatic Hþ CH of pyrazole), 8.50 (s, 1H, HC]N); EIMS, m/z: 389
(M þ 2)þ, 387 (Mþ); Anal. Calcd. For C23H18ClN3O (387.86): C, 71.22;
H, 4.68; N, 10.83. Found: C, 71.31; H, 4.77; N, 11.18.
4.1.3.5. N-((1-(4-Chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazol-4-
yl)methylene)-4-methoxyaniline (5e). Yield 60%; m.p. 103e105 ꢂC;
IR (KBr, cmꢀ1): 3070, 3008 (CH aromatic), 2966, 2916 (CH
aliphatic); 1H NMR (CDCl3, 300 MHz):
d
3.82 (s, 3H, OCH3), 3.87 (s,
3H, OCH3), 6.91e7.78 (m, 12H, aromatic H), 8.50 (s, 1H, CH of pyr-
azole), 8.61 (s, 1H, HC]N); 13C NMR (CDCl3, 70 MHz):
55.3, 55.4,
4.1.2.1. 1-(4-Chlorophenyl)-3-p-tolyl-1H-pyrazole-4-carbaldehyde
(3a). Yield 90%; m.p. 132e133 ꢂC; IR (KBr, cmꢀ1): 3050 (CH aro-
matic), 2920 (CH aliphatic), 2870, 2781 (CH aldehyde), 1670 (C]O
d
114.1, 114.8, 116.3, 120.3, 122.0, 124.5, 127.1, 129.5, 129.9, 130.1, 138.0,
144.8, 150.9, 153.8, 158.1, 160.1; EIMS, m/z: 419 (M þ 2)þ, 417 (Mþ);
Anal. Calcd. For C24H20ClN3O2 (417.88): C, 68.98; H, 4.82; N, 10.06.
Found: C, 69.12; H, 4.89; N, 10.38.
aldehyde); 1H NMR (CDCl3, 300 MHz):
d 2.44 (s, 3H, CH3), 7.27e7.77
(m, 8H, aromatic H), 8.51 (s, 1H, CH of pyrazole), 10.05 (s, 1H, CHO);
EIMS, m/z: 298 (M þ 2)þ, 296 (Mþ); Anal. Calcd. For C17H13ClN2O
(296.75): C, 68.81; H, 4.42; N; 9.44. Found: C, 68.94; H, 4.48; N, 9.63.
4.1.3.6. 4-Chloro-N-((1-(4-chlorophenyl)-3-(4-methoxyphenyl)-1H-
pyrazol-4-yl)methylene)aniline (5f). Yield 52%; m.p. 186e188 ꢂC; IR
(KBr, cmꢀ1): 3012 (CH aromatic), 2920, 2850 (CH aliphatic); 1H
4.1.2.2. 1-(4-Chlorophenyl)-3-(4-methoxyphenyl)-1H-pyrazole-4-
carbaldehyde (3b). Yield 92%; m.p. 142e143 ꢂC; IR (KBr, cmꢀ1):
3070 (CH aromatic), 2958, 2931 (CH aliphatic), 2831, 2781 (CH
NMR (CDCl3, 300 MHz):
d 3.88 (s, 3H, OCH3), 7.01e7.90 (m, 13H,
aromatic Hþ CH of pyrazole), 8.49 (s, 1H, HC]N); EIMS, m/z: 421
(Mþ); Anal. Calcd. For C23H17Cl2N3O (422.30): C, 65.41; H, 4.06; N,
9.95. Found: C, 65.38; H, 4.09; N, 10.08.
aldehyde), 1674 (C]O aldehyde); 1H NMR (CDCl3, 300 MHz):
d
3.87
(s, 3H, OCH3), 7.01e7.79 (m, 8H, aromatic H), 8.47 (s, 1H, CH of
pyrazole), 10.03 (s, 1H, CHO); 13C NMR (CDCl3, 70 MHz):
55.3,
d
114.1, 120.7, 122.5, 123.5, 129.7, 130.1, 131.0, 133.4, 137.5, 154.5, 160.6,
4.1.3.7. 4-(4-((Phenylimino)methyl)-3-p-tolyl-1H-pyrazol-1-yl)ben-
zenesulfonamide (5g). Yield 67%; m.p. 152e154 ꢂC; IR (KBr, cmꢀ1):
3321, 3251 (NH2), 3062, 3024 (CH aromatic), 2916 (CH aliphatic),
184.8; EIMS, m/z: 314 (M þ 2)þ, 312 (Mþ); Anal. Calcd. For
C
17H13ClN2O2 (312.75): C, 65.29; H, 4.19; N, 8.96. Found: C, 65.32; H,
4.16; N, 9.33.
1342, 1157 (SO2); 1H NMR (CDCl3, 500 MHz):
d 2.43 (s, 3H, CH3),
6.68e8.03 (m, 13H, aromatic H), 8.06 (s, ex, 2H, NH2), 8.50 (s,1H, CH
of pyrazole), 8.60 (s, 1H, HC]N); EIMS, m/z: 416 (Mþ); Anal. Calcd.
For C23H20N4O2S (416.49): C, 66.33; H, 4.84; N, 13.45. Found: C,
66.52; H, 4.96; N, 13.80.
4.1.3. General procedure for the synthesis of compounds (5ael)
To a hot solution of 3a,b or 4a,b (2 mmol) in ethanol (15 mL)
glacial acetic acid (0.2 mL) was added followed by aromatic amines

F.A. Ragab et al. / European Journal of Medicinal Chemistry 63 (2013) 645e654
651
4.1.3.8. 4-(4-((4-Methoxyphenylimino)methyl)-3-p-tolyl-1H-pyr-
azol-1-yl)benzenesulfonamide (5h). Yield 62%; m.p. 130e132 ꢂC; IR
(KBr, cmꢀ1): 3379, 3298 (NH2), 3062 (CH aromatic), 2920, 2835 (CH
4.1.4.2. 1-(4-Chlorophenyl)-3-(1-(4-chlorophenyl)-3-(4-methoxyph-
enyl)-1H-pyrazol-4-yl)prop-2en-1-one (6b). Yield 89%; m.p. 201e
202 ꢂC; IR (KBr, cmꢀ1): 3066, 3005 (CH aromatic), 2958, 2935
aliphatic),1338,1161 (SO2); 1H NMR (CDCl3, 500 MHz):
d
2.43 (s, 3H,
(CH aliphatic), 1662 (C]O); 1H NMR (CDCl3, 300 MHz):
d 3.88
CH3), 3.82 (s, 3H, OCH3), 6.67e8.03 (m,12H, aromatic H), 8.46 (s,1H,
CH of pyrazole), 8.59 (s,1H, HC]N), 9.16 (s, ex, 2H, NH2); EIMS, m/z:
446 (Mþ); Anal. Calcd. For C24H22N4O3S (446.52): C, 64.56; H, 4.97;
N, 12.55. Found: C, 64.79; H, 5.31; N, 12.70.
(s, 3H, OCH3), 7.01 (d, 1H, CH olefinic, J ¼ 8.4 Hz), 7.60 (d, 1H,
CH olefinic, J ¼ 8.4 Hz) 7.26e7.46, 7.72e7.91 (m, 12H, aromatic H),
8.29 (s, 1H, CH pyrazole); 13C NMR (CDCl3, 70 MHz):
d 55.3, 114.2,
118.3, 120.3, 120.9, 124.4, 126.6, 128.8, 129.6, 129.7, 129.9,
132.6, 135.7, 136.4, 137.8, 145.0, 153.9, 160.2, 188.5; EIMS, m/z:
452 (M þ 4)þ, 450 (M þ 2)þ, 448 (Mþ); Anal. Calcd. For
4.1.3.9. 4-(4-((4-Chlorophenylimino)methyl)-3-p-tolyl-1H-pyrazol-
1-yl)benzenesulfonamide (5i). Yield 61%; m.p. 138e140 ꢂC; IR (KBr,
cmꢀ1): 3360, 3250(NH2), 3012 (CH aromatic), 2924 (CH aliphatic),
C25H18Cl2N2O2 (449.32): C, 66.83; H, 4.04; N, 6.23. Found: C, 66.91;
H, 4.08; N, 6.38.
1342, 1149 (SO2); 1H NMR (CDCl3, 500 MHz):
d 2.38 (s, 3H, CH3),
7.08e8.02 (m, 12H, aromatic H), 8.47 (s, 1H, CH of pyrazole), 8.68 (s,
1H, HC]N), 8.80 (s, ex, 2H, NH2); EIMS, m/z: 450 (Mþ); Anal. Calcd.
For C23H19ClN4O2S (450.94): C, 61.26; H, 4.25; N, 12.42. Found: C,
61.56; H, 4.50; N, 12.08.
4.1.4.3. 4-(4-(3-(4-Chlorophenyl)-3-oxoprop-1-enyl)-3-p-tolyl-1H-
pyrazol-1-yl)benzenesulfonamide (6c). Yield 85%; m.p. 105e107 ꢂC;
IR (KBr, cmꢀ1): 3387, 3263 (NH2), 3086, 3012 (CH aromatic), 2958,
2927 (CH aliphatic), 1678 (C]O), 1384, 1161 (SO2); 1H NMR (CDCl3,
300 MHz):
d
2.37 (s, 3H, CH3), 7.43 (d, 1H, CH olefinic, J ¼ 8.7 Hz),
4.1.3.10. 4-(3-(4-Methoxyphenyl)-4-((phenylimino)methyl)-1H-pyr-
azol-1-yl)benzenesulfonamide (5j). Yield 66%; m.p. 113e115 ꢂC; IR
(KBr, cmꢀ1): 3321, 3251 (NH2), 3012 (CH aromatic), 2924, 2854 (CH
7.89 (d, 1H, CH olefinic, J ¼ 8.1 Hz), 7.17e7.38, 7.80e7.82 (m, 14H,
aromatic Hþ ex, NH2), 8.02 (s, 1H, CH pyrazole); EIMS, m/z: 477
(Mþ); Anal. Calcd. For C25H20ClN3O3S (477.96): C, 62.82; H, 4.22; N,
8.79. Found: C, 63.25; H, 4.43; N, 8.82.
aliphatic),1342,1157 (SO2); 1H NMR (CDCl3, 300 MHz):
d 3.89 (s, 3H,
OCH3), 7.02e8.05 (m, 13H, aromatic H), 8.52 (s, 1H, CH of pyrazole),
8.62 (s, 1H, HC]N), 8.70 (s, ex, 2H, NH2); EIMS m/z: 432 (Mþ); Anal.
Calcd. For C23H20N4O3S (432.49): C, 63.87; H, 4.66; N, 12.95. Found:
C, 63.94; H, 4.71; N, 13.14.
4.1.4.4. 4-(4-(3-(4-Chlorophenyl)-3-oxoprop-1-enyl)-3-(4-methoxyph-
enyl)-1H-pyrazol-1-yl)benzenesulfonamide (6d). Yield 87%; m.p. 171e
173 ꢂC; IR (KBr, cmꢀ1): 3325, 3244 (NH2), 3124 (CH aromatic), 2958,
2927 (CH aliphatic), 1670 (C]O), 1330, 1165 (SO2); 1H NMR (CDCl3,
4.1.3.11. 4-(3-(4-Methoxyphenyl)-4-((4-methoxyphenylimino)
methyl)-1H-pyrazol-1-yl)benzenesulfonamide (5k). Yield 61%; m.p.
124e126 ꢂC; IR (KBr, cmꢀ1): 3379, 3250 (NH2), 3066 (CH aromatic),
2931, 2835 (CH aliphatic), 1342, 1145 (SO2); 1H NMR (CDCl3,
500 MHz):
d
3.82 (s, 3H, OCH3), 6.90e7.87 (m, 16H, aromatic Hþ
olefinic Hþ ex, NH2), 7.98 (s, 1H, CH pyrazole); EIMS, m/z: 492
(M ꢀ 1)þ; Anal. Calcd. For C25H20ClN3O4S (493.96): C, 60.79; H, 4.08;
N, 8.51. Found: C, 61.07; H, 4.21; N, 8.79.
500 MHz):
d 3.82 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 6.93e8.02 (m,
12H, aromatic H), 8.06 (s, ex, 2H, NH2), 8.46 (s, 1H, CH of pyrazole),
4.1.5. General procedure for the synthesis of compounds (7aeh)
Compounds 6aed (3 mmol) were dissolved in the minimum
amount of DMF, then 4-substituted benzaldehyde (3 mmol) was
added to the solutions followed by KCN (0.30 mmol). The reaction
mixture was stirred at (30e35 ꢂC) for 4 h, then poured onto ice
water and neutralized with drops of concentrated hydrochloric
acid; the precipitated solid was filtered washed with water and
crystallized from methanol.
8.57 (s, 1H, HC]N); EIMS, m/z: 462 (Mþ); Anal. Calcd. For
C
24H22N4O4S (462.52): C, 62.32; H, 4.79; N, 12.11. Found: C, 62.42;
H, 4.72; N, 12.38.
4.1.3.12. 4-(4-((4-Chlorophenylimino)methyl)-3-(4-methoxyphenyl)-
1H-pyrazol-1-yl)benzenesulfonamide (5l). Yield 60%; m.p. 118e
119 ꢂC; IR (KBr, cmꢀ1): 3360, 3244 (NH2), 3074 (CH aromatic),
2931, 2835 (CH aliphatic), 1342, 1149 (SO2); 1H NMR (CDCl3,
300 MHz):
d
3.88 (s, 3H, OCH3), 6.60e8.05 (m, 12H, aromatic H),
4.1.5.1. 4-(4-Chlorophenyl)-2-(1-(4-chlorophenyl)-3-p-tolyl-1H-pyr-
azol-4-yl)-1-(4-fluorophenyl)butane-1,4-dione (7a). Yield 78%; m.p.
143e145 ꢂC; IR (KBr, cmꢀ1): 3040 (CH aromatic), 2920 (CH
8.47 (s, 1H, CH of pyrazole), 8.62 (s, 1H, HC]N), 8.80 (s, ex, 2H,
NH2); EIMS, m/z: 466 (Mþ); Anal. Calcd. For C23H19ClN4O3S
(466.93): C, 59.16; H, 4.10; N, 12.00. Found: C, 59.39; H, 4.22; N,
12.31.
aliphatic), 1681 (C]O); 1H NMR (CDCl3, 300 MHz):
d 2.40 (s, 3H,
CH3), 3.26 (dd, 1H, CHeC]O, J ¼ 17.1, 5.1 Hz), 3.45 (dd, 1H, CHeC]
O, J ¼ 17.1, 8.0 Hz), 4.26 (dd, 1H, CH, J ¼ 8.0, 5.1 Hz), 7.20e7.83 (m,
16H, aromatic H), 7.88 (s, 1H, CH pyrazole); EIMS, m/z: 557
(M þ 1)þ; Anal. Calcd. For C32H23Cl2FN2O2 (557.44): C, 68.95; H,
4.16; N, 5.03. Found: C, 69.02; H, 4.14; N, 5.17.
4.1.4. General procedure for the synthesis of compounds (6aed)
To a mixture of 3a,b or 4a,b (10 mmol) and 4-chloroace-
tophenone (1.54 g, 10 mmol) in absolute ethanol (20 mL), sodium
hydroxide (1.20 g, 30 mmol) in water (3 mL) was added. The re-
action mixture was stirred at room temperature for 24 h. Then,
diluted with water and acidified with 10% hydrochloric acid till
pH ¼ 5. The precipitated solid was filtered and crystallized from
methanol.
4.1.5.2. 4-(4-Chlorophenyl)-2-(1-(4-chlorophenyl)-3-p-tolyl-1H-pyr-
azol-4-yl)-1-(4-methoxyphenyl)butan-1,4-dione (7b). Yield 52%;
m.p. 114e116 ꢂC; IR (KBr, cmꢀ1): 3070 (CH aromatic), 2920, 2839
(CH aliphatic), 1678 (C]O); 1H NMR (CDCl3, 300 MHz):
d 2.44 (s,
3H, CH3), 3.25 (dd,1H, CHeC]O, J ¼ 16.8, 6.0 Hz), 3.45 (dd,1H, CHe
C]O, J ¼ 16.8, 8.7 Hz), 3.87 (s, 3H, OCH3), 4.23 (dd, 1H, CH, J ¼ 8.7,
6.0 Hz), 7.00e7.84 (m, 16H, aromatic H), 7.87 (s, 1H, CH pyrazole);
EIMS, m/z: 568 (Mþ); Anal. Calcd. For C33H26Cl2N2O3 (569.47): C,
69.60; H, 4.60; N, 4.92. Found: C, 69.65; H, 4.58; N, 5.12.
4.1.4.1. 1-(4-Chlorophenyl)-3-(1-(4-chlorophenyl)-3-p-tolyl-1H-pyr-
azol-4-yl)prop-2-en-1-one (6a). Yield 79%; m.p. 205e206 ꢂC; IR
(KBr, cmꢀ1): 3066 (CH aromatic), 2916 (CH aliphatic), 1662 (C]O);
1H NMR (CDCl3, 300 MHz):
d 2.44 (s, 3H, CH3), 7.57 (d, 1H, CH
olefinic, J ¼ 8.4 Hz), 7.74 (d, 1H, CH olefinic, J ¼ 9.0 Hz), 7.29e7.49,
7.86e7.92 (m, 12H, aromatic H), 8.32 (s, 1H, CH pyrazole); EIMS, m/
z: 436 (M þ 4)þ, 434 (M þ 2)þ, 432 (Mþ); Anal. Calcd. For
4.1.5.3. 4-(4-Chlorophenyl)-2-(1-(4-chlorophenyl)-3-(4-methoxyph-
enyl)-1H-pyrazol-4-yl)-1-(4-fluorophenyl)butan-1,4-dione
(7c).
C
25H18Cl2N2O (433.32): C, 69.29; H, 4.19; N, 6.46, Found: C, 69.38;
Yield 65%; m.p. 170e171 ꢂC; IR (KBr, cmꢀ1): 3070, 3000 (CH aro-
matic), 2931, 2916 (CH aliphatic), 1681 (C]O); 1H NMR (CDCl3,
H, 4.23; N, 6.69.

652
F.A. Ragab et al. / European Journal of Medicinal Chemistry 63 (2013) 645e654
300 MHz):
d
3.38 (dd, 1H, CHeC]O, J ¼ 17.7, 5.1 Hz), 3.59 (dd, 1H,
4.1.6. General procedure for the synthesis of compounds (8aeh)
Compounds 7aeh (2 mmol) was added to stirred solution of
ammonium acetate (2 mmol) in glacial acetic acid (5 mL). The re-
action mixture was refluxed at 110 ꢂC for 20 h, poured onto ice
water. The precipitate formed was filtered washed with water,
dried and crystallized from ethanol.
CHeC]O, J ¼ 17.7, 8.4 Hz), 3.89 (s, 3H, OCH3), 4.70 (dd, 1H, CH,
J ¼ 8.4, 5.1 Hz), 6.99e7.80 (m, 16H, aromatic H), 8.11 (s, 1H, CH
pyrazole); EIMS, m/z: 572 (Mþ); Anal. Calcd. For C32H23Cl2FN2O3
(573.44): C, 67.02; H, 4.04; Cl, 12.37; N, 4.89. Found: C, 67.05; H,
4.10; Cl, 12.46; N, 4.98.
4.1.5.4. 4-(4-Chlorophenyl)-2-(1-(4-chlorophenyl)-3-(4-methoxyphenyl)-
1H-pyrazol-4-yl)-1-(4-methoxyphenyl)butane-1,4-dione (7d). Yield 46%;
m.p. 108e110 ꢂC; IR (KBr, cmꢀ1): 3070 (CH aromatic), 2924, 2854 (CH
4.1.6.1. 1-(4-Chlorophenyl)-4-(5-(4-chlorophenyl)-2-(4-fluorophenyl)-
1H-pyrrol-3-yl)-3-p-tolyl-1H-pyrazole (8a). Yield 74%; m.p. 132e
134 ꢂC; IR (KBr, cmꢀ1): 3417 (NH), 3059 (CH aromatic), 2916, 2850
aliphatic), 1678 (C]O); 1H NMR (CDCl3, 300 MHz):
d
3.23 (dd, 1H, CHe
(CH aliphatic); 1H NMR (CDCl3, 300 MHz):
d 2.43 (s, 3H, CH3), 5.00 (s,
C]O, J ¼ 16.7, 6.0 Hz), 3.43 (dd, 1H, CHeC]O, J ¼ 16.7, 8.5 Hz), 3.78e
3.98 (m, 6H, 2OCH3), 4.23 (dd, 1H, CH, J ¼ 8.5, 6.0 Hz), 6.88e7.82 (m,
16H, aromatic H), 8.10 (s, 1H, CH pyrazole); 13C NMR (CDCl3, 70 MHz):
ex, 1H, NH), 6.97 (s, 1H, CH of pyrrole), 6.98e8.10 (m, 16H, aromatic
H), 8.25 (s, 1H, CH of pyrazole); EIMS, m/z: 541 (M þ 4)þ, 539
(M þ 2)þ, 537 (Mþ); Anal. Calcd. For C32H22Cl2FN3 (538.44): C, 71.38;
H, 4.12; N, 7.80. Found: C, 71.41; H, 4.15; N, 7.94.
d
44.3, 50.8, 55.2, 55.5, 114.0, 114.1, 114.2, 114.3, 118.6, 119.8, 119.8, 123.5,
125.5, 128.2, 128.3, 128.5, 128.6, 129.1, 129.2, 129.3, 129.4, 129.5, 130.1,
130.7, 131.0, 134.8, 137.5, 138.2, 151.5, 159.5, 160.3, 197.4; EIMS, m/z: 585
(M þ 1)þ; Anal. Calcd. For C33H26Cl2N2O4 (585.47): C, 67.70; H, 4.48; N,
4.78. Found: C, 67.74; H, 4.53; N, 4.89.
4.1.6.2. 1-(4-Chlorophenyl)-4-(5-(4-chlorophenyl)-2-(4-methoxyphenyl)-
1H-pyrrol-3-yl)-3-p-tolyl-1H-pyrazole (8b). Yield 78%; m.p. 135e137 ꢂC;
IR (KBr, cmꢀ1): 3390 (NH), 3062, 3020 (CH aromatic), 2920, 2850 (CH
aliphatic); 1H NMR (CDCl3, 300 MHz):
d 2.43 (s, 3H, CH3), 3.89 (s, 3H,
4.1.5.5. 4-(4-(4-(4-Chlorophenyl)-1-(4-fluorophenyl)-1,4-dioxobutan-
2-yl)-3-p-tolyl-1H-pyrazol-1-yl)benzenesulfonamide (7e). Yield 87%;
m.p. 112e114 ꢂC; IR (KBr, cmꢀ1): 3340, 3248 (NH2), 3070 (CH aro-
matic), 2924, 2858 (CH aliphatic), 1666 (C]O), 1161, 1338 (SO2); 1H
OCH3), 5.05 (s, ex, 1H, NH), 6.65 (s, 1H, CH of pyrrole), 6.98e7.95 (m,
16H, aromatic H), 8.25 (s, 1H, CH pyrazole); EIMS, m/z: 550 (M þ 1)þ;
Anal. Calcd. For C33H25Cl2N3O (550.47): C, 72.00; H, 4.58; N, 7.63. Found:
C, 71.98; H, 4.59; N, 7.76.
NMR (CDCl3, 500 MHz):
d 2.35 (s, 3H, CH3), 3.22 (dd, 1H, CHeC]O,
J ¼ 16.8, 5.1 Hz), 3.46 (dd, 1H, CHeC]O, J ¼ 16.8, 8.4 Hz), 4.24 (dd,
1H, CH, J ¼ 8.4, 5.1 Hz), 6.85e7.99 (m, 16H, aromatic H), 8.37 (s, ex,
2H, NH2), 8.55 (s, 1H, CH pyrazole); EIMS, m/z: 603 (M þ 1)þ; Anal.
Calcd. For C32H25ClFN3O4S (602.07): C, 63.84; H, 4.19; N, 6.98.
Found: C, 63.93; H, 4.25; N, 7.17.
4.1.6.3. 1-(4-Chlorophenyl)-4-(5-(4-chlorophenyl)-2-(4-fluorophenyl)-
1H-pyrrol-3-yl)-3-(4-methoxyphenyl)-1H-pyrazole (8c). Yield 76%;
m.p. 120e122 ꢂC; IR (KBr, cmꢀ1): 3390 (NH), 3070 (CH aromatic),
2920, 2850 (CH aromatic); 1H NMR (CDCl3, 300 MHz):
d 3.85 (s, 3H,
OCH3), 5.30 (s, ex, 1H, NH), 6.50 (s, 1H, CH of pyrrole), 7.02e8.15 (m,
16H, aromatic H), 8.25 (s, 1H, CH pyrazole); EIMS, m/z: 557 (M þ 4)þ,
555 (M þ 2)þ, 553 (Mþ); Anal. Calcd. For C32H22Cl2FN3O (554.44): C,
69.32; H, 4.00; N, 7.58. Found: C, 69.36; H, 4.05; N, 7.69.
4.1.5.6. 4-(4-(4-(4-Chlorophenyl)-1-(4-methoxyphenyl)-1,4-dioxobutan-
2-yl)-3-p-tolyl-1H-pyrazol-1-yl)benzenesulfonamide (7f). Yield 91%;
m.p.183e186 ꢂC; IR (KBr, cmꢀ1): 3352, 3263 (NH2), 3070 (CH aromatic),
2924 (CH aliphatic), 1674 (C]O), 1338, 1161 (SO2); 1H NMR (CDCl3,
4.1.6.4. 1-(4-Chlorophenyl)-4-(5-(4-chlorophenyl)-2-(4-methoxyphenyl)-
1H-pyrrol-3-yl)-3-(4-methoxyphenyl)-1H-pyrazole (8d). Yield 84%; m.p.
155e157 ꢂC; IR (KBr, cmꢀ1): 3390 (NH), 3066 (CH aromatic), 3920, 2850
300 MHz):
d
2.44(s, 3H, CH3), 3.25 (dd, 1H, CHeC]O, J ¼ 17.1, 5.1 Hz),
3.46 (dd, 1H, CHeC]O, J ¼ 17.1, 7.9 Hz), 3.90 (s, 3H, OCH3), 4.23 (dd, 1H,
CH, J ¼ 7.9, 5.1 Hz), 6.89e7.91 (m, 16H, aromatic H), 8.03 (s, 1H, CH
pyrazole), 8.37 (s, ex, 2H, NH2); EIMS, m/z: 616 (M þ 2)þ, 614 (Mþ);
Anal. Calcd. For C33H28ClN3O5S (614.11): C, 64.54; H, 4.60; N, 6.84.
Found: C, 64.48; H, 4.63; N, 6.98.
(CH aliphatic); 1H NMR (CDCl3, 300 MHz):
d
3.73e3.88 (m, 6H, 2OCH3),
5.05 (s, ex, 1H, NH), 6.55 (s, 1H, CH of pyrrole), 7.01e8.15 (m, 16H, ar-
omatic H), 8.35 (s,1H, CH pyrazole); 13C NMR (CDCl3, 300 MHz):
55.2,
d
110.0, 113.0, 114.1, 120.0, 120.2, 128.1, 128.8, 129.1, 129.3, 129.5, 129.9,
131.3, 130.2, 137.5, 138.0, 160.0; EIMS, m/z: 566 (M þ 1)þ; Anal. Calcd.
For C33H25Cl2N3O2 (566.47): C, 69.97; H, 4.45; N, 7.42. Found: C, 70.02;
H, 4.47; N, 7.58.
4.1.5.7. 4-(4-(4-(4-Chlorophenyl)-1-(4-fluorophenyl)-1,4-dioxobutan-
2-yl)-3-(4-methoxyphenyl)-1H-pyrazol-1-yl)benzenesulfonamide
(7g). Yield 95%; m.p. 123e125 ꢂC; IR (KBr, cmꢀ1): 3340, 3240
(NH2), 3070 (CH aromatic), 2924, 2858 (CH aliphatic), 1678 (C]O),
4.1.6.5. 4-(4-(5-(4-Chlorophenyl)-2-(4-fluorophenyl)-1H-pyrrol-3-
yl)-3-p-tolyl-1H-pyrazol-1-yl)benzenesulfonamide (8e). Yield 78%;
m.p. 185e188 ꢂC; IR (KBr, cmꢀ1): 3402, 3244 (NH2, NH), 3066, 3032
(CH aromatic H), 2920, 2850 (CH aliphatic), 1338, 1157 (SO2); 1H
1338, 1161 (SO2); 1H NMR (DMSO-d6, 500 MHz):
d 3.45 (dd, 1H,
CHeC]O, J ¼ 17.1, 5.4 Hz), 3.61 (dd, 1H, CHeC]O, J ¼ 17.1, 8.0 Hz),
3.88 (s, 3H, OCH3), 4.19 (dd, 1H, CH, J ¼ 8.0, 5.4 Hz), 6.88e7.91,
(m, 16H, aromatic H), 8.69 (s, 1H, CH pyrazole), 9.38 (s, ex, 2H,
NH2); EIMS, m/z: 620 (M þ 2)þ, 618 (Mþ); Anal. Calcd. For
NMR (DMSO-d6, 500 MHz):
d 2.29 (s, 3H, CH3), 7.05 (s, 1H, CH of
pyrrole), 5.90 (s, ex,1H, NH), 7.16 (s, ex, 2H, NH2), 7.27e8.14 (m,16H,
aromatic), 9.02 (s, 1H, CH pyrazole); EIMS, m/z: 582 (M ꢀ 1)þ; Anal.
Calcd. For C32H24ClFN4O2S (583.07): C, 65.92; H, 4.15; N, 9.61.
Found: C, 65.92; H, 4.13; N, 9.83.
C
32H25ClFN3O5S (618.07): C, 62.18; H, 4.08; N, 6.80. Found: C,
62.22; H, 4.15; N, 6.97.
4.1.5.8. 4-(4-(4-(4-Chlorophenyl)-1-(4-methoxyphenyl)-1,4-dioxobutan-
2-yl)-3-(4-methoxyphenyl)-1H-pyrazol-1-yl)benzenesulfonamide (7h).
Yield 50%; m.p. 182e185 ꢂC; IR (KBr, cmꢀ1): 3321, 3244 (NH2),
3124, 3074 (CH aromatic H), 2924, 2835 (CH aliphatic), 1670 (C]
4.1.6.6. 4-(4-(5-(4-Chlorophenyl)-2-(4-methoxyphenyl)-1H-pyrrol-
3-yl)-3-p-tolyl-1H-pyrazol-1-yl)benzenesulfonamide (8f). Yield 85%;
m.p. 204e205 ꢂC; IR (KBr, cmꢀ1): 3383, 3255 (NH2, NH), 3070,
3032 (CH aromatic), 2920, 2850 (CH aliphatic), 1338, 1161 (SO2);
O), 1330, 1165 (SO2); 1H NMR (CDCl3, 300 MHz):
d 3.26 (dd, 1H,
CHeC]O, J ¼ 16.7, 6.0 Hz), 3.46 (dd, 1H, CHeC]O, J ¼ 16.7, 8.4 Hz),
3.71e3.90 (m, 6H, 2OCH3), 4.23 (dd, 1H, CHeC]O, J ¼ 8.4, 6.0 Hz),
6.90e7.86 (m, 16H, aromatic H), 8.03 (s, 1H, CH pyrazole), 8.30 (s,
1H NMR (DMSO-d6, 500 MHz):
d 2.44 (s, 3H, CH3), 3.69 (s, 3H,
OCH3), 5.85 (s, ex, 1H, NH), 6.85 (s, 1H, CH of pyrrole), 7.13e8.05
(m, 16H, aromatic H), 8.14 (s, 1H, CH pyrazole), 9.32 (s, ex, 2H,
NH2); EIMS, m/z: 597 (M þ 2)þ, 595 (Mþ); Anal. Calcd. For
ex, 2H, NH2); EIMS, m/z: 629 (M
33H28ClN3O6S (630.10): C, 62.90; H, 4.48; N, 6.67. Found: C, 62.97;
H, 4.53; N, 6.81.
ꢀ
1)þ; Anal. Calcd. For
C
C33H27ClN4O3S (595.11): C, 66.60; H, 4.57; N, 9.41. Found: C, 66.71;
H, 4.52; N, 9.54.

F.A. Ragab et al. / European Journal of Medicinal Chemistry 63 (2013) 645e654
653
4.1.6.7. 4-(4-(5-(4-Chlorophenyl)-2-(4-fluorophenyl)-1H-pyrrol-3-
yl)-3-(4-methoxyphenyl)-1H-pyrazol-1-yl)benzenesulfonamide (8g).
Yield 58%; m.p. 188e190 ꢂC; IR (KBr, cmꢀ1): 3402, 3240 (NH2, NH),
3070, 3032 (CH aromatic), 2924, 2850 (CH aliphatic), 1338, 1161
Okun et al. [36] Adult male albino mice weighing 20e25 g were used
in this study. Phenylbutazone (reference standard) and the tested
compounds were prepared as suspension in 2% Tween 80. A sensi-
tivity test was carried out one day before drug administration, were
the animals were injected intraperitoneally with 0.2e0.25 mL of
0.02% freshly prepared solution of p-benzoquinone in distilled water.
Animals showing writhing to p-benzoquinone within 30 min were
chosen for studying the analgesic activity. On the next day, mice were
divided into 19 groups, 6 animals each. The control group received
only 2% Tween 80 while the rest of the groups received phenylbu-
tazone and the tested compounds at a dose of 0.32 mmol/kg body
weight. After 1 h, 0.02% solution of p-benzoquinone was adminis-
tered intraperitoneally and the animals were observed for 30 min
after injection of the irritant, the animals showing writhing were
counted in each group. Writhing is known as stretch, torsion to one
side, drawing up of hind leg, retraction of the abdomen, so that the
belly of mouse touches the floor. The mice showing any of the pre-
vious signs are counted as positive responses and this method
depend on the ability of tested compounds to protect the animals
from writhing signs made by p-benzoquinone. The analgesic activity
was evaluated as the percentage protection of tested animals against
irritant p-benzoquinone induced writhing response compared with
the control group according the following equation:
(SO2); 1H NMR (CDCl3, 500 MHz):
d 3.71 (s, 3H, OCH3), 5.24 (s, ex,
1H, NH), 6.93 (s, 1H, CH of pyrrole), 6.94e7.97 (m, 18H, aromatic Hþ
ex, NH2), 8.30 (s, 1H, CH pyrazole); EIMS, m/z: 599 (Mþ); Anal.
Calcd. For C32H24ClFN4O3S (599.07): C, 64.16; H, 4.04; N, 9.35.
Found: C, 64.21; H, 4.09; N, 9.51.
4.1.6.8. 4-(4-(5-(4-Chlorophenyl)-2-(4-methoxyphenyl)-1H-pyrrol-
3-yl)-3-(4-methoxyphenyl)-1H-pyrazol-1-yl)benzenesulfonamide
(8h). Yield 55%; m.p. 197e200 ꢂC; IR (KBr, cmꢀ1): 3387, 3248 (NH2,
NH), 3109, 3078 (CH aromatic), 2920, 2846 (CH aliphatic),1338,1161
(SO2); 1H NMR (DMSO-d6, 500 MHz):
d 3.70 (s, 3H, OCH3), 3.80 (s,
3H, OCH3), 5.11 (s, ex, 1H, NH), 6.88 (s, 1H, CH of pyrrole), 7.00e8.15
(m, 16H, aromatic H), 8.70 (s, 1H, CH pyrazole), 9.22 (s, ex, 2H, NH2);
EIMS, m/z: 613 (M þ 2)þ, 611 (Mþ); Anal. Calcd. For C33H27ClN4O4S
(611.10): C, 64.86; H, 4.45; N, 9.17. Found: C, 64.91; H, 4.42; N, 9.32.
4.2. Pharmacological screening
4.2.1. Anti-inflammatory (in vivo screening)
All the target compounds were evaluated for their anti-
inflammatory activity using carrageenan-induced rat paw edema
method of Winter et al. [35]. The employed technique is based on
the ability of the tested compounds to inhibit the edema produced
in the hind paw of the rat after injection of carrageenan. Male
Wister albino rats (obtained from the animal house of Faculty of
Pharmacy, Cairo University) weighing 120e180 g were used to
investigate the carrageenan-induced rat paw edema. The rats were
kept in animal house under standard conditions of light and tem-
perature with free access to food and water. The animals were
randomly divided into 38 groups of five rats each. The initial hind
paw volume of rats was determined volumetrically by means of
plethysmometer 7150 (UGO Basile, Italy). Phenylbutazone (refer-
ence standard) and the tested compounds suspended in 2% Tween
80 were administered intraperitoneally at a dose of 0.32 mmol/kg
body weight, while the control group received only 2% Tween 80,
1 h before induction of inflammation. The paw edema was induced
by sub-plantar injection of (0.1 mL) of 1% carrageenan solution in
saline (0.9%). Paw edema volume was measured after 2 and 3 h
using the plethysmometer and compared with the initial hind paw
volume of each rat. The difference of average values between
treated and control group is calculated for each time interval and
evaluated statistically. Quantitative variables from normal distri-
bution were expressed as means ꢁ standard error (SEM). The anti-
inflammatory activity was expressed as percentage inhibition of
edema volume in treated animals in comparison with the control
group according to the following equation:
% protection ¼ Number of protected animals
ꢃ 100=total number of animals
4.2.3. Ulcerogenic effect
The ulcerogenic effect of the nine active compounds as anti-
inflammatory agents and phenylbutazone was evaluated by the
reported method of Meshali et al. [37] Adult male albino rats
weighing 120e180 g were used in this study. Animals were fasted
18 h before the drug administration, then divided into 11 groups
each of 5 animals and received the drug orally. The first group
received 2% Tween 80 and kept as control, the second group
received phenylbutazone in a dose of 0.32 mmol/kg body weight
and the rest of the groups were received the tested compounds in
the same dose.
Food was allowed 2 h after administration of the drugs and rats
were received the same dose orally for three successive days. Two
hours after the last dose, rats were sacrificed, the stomach of each
rat were removed, opened along greater curvature and cleaned by
washing with cold saline. The stomach was stretched on a cork-
board using pins and examined with a magnifying lens (10ꢃ) for
the presence of ulcers and erosions. Ulcer index was calculated
according to the method of Robert et al. [38]. The degree of ul-
cerogenic effect was expressed in term of; percentage incidence of
ulcer in each group of animals divided by 10, the average number of
ulcers per stomach and the average severity of ulcers by visual
observation. The ulcer index was expressed as summation value of
the above three values.
% Inhibition ¼ ðVc ꢀ VtÞ100=Vc
where Vc is the mean of edema volume of rat paw after adminis-
tration of carrageenan in the control group, Vt is the mean of edema
volume of rat paw after administration of the tested compounds or
the reference drugs.
The IC50 values of the most active compounds 5e and 8e and
phenylbutazone were applied according to the previous reported
procedure in doses of 0.08, 0.16, and 0.32 mmol/kg body weight.
4.2.4. Acute toxicity
LD50 of some representative compounds was determined using
Finney’s method [39]. Adult male mice weighing 20e25 g were
divided into groups each of six animals. Minimal dose that killed all
animals and the maximal dose that failed to kill any animal were
determined via several increasing intraperitoneal doses. Animals
were kept under observation for 24 h during which any mortality in
each group was recorded.
4.2.2. Analgesic activity
Compounds that exhibited good anti-inflammatory activity and
phenylbutazone were screened for their analgesic activity using the
reported method of p-benzoquinone-induced writhing in mice by
4.2.5. In vitro COX inhibition study
The most active compounds as anti-inflammatory 5e,f, 8e, and
indomethacin were tested for their ability to inhibit COX-1 and/or

654
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Acknowledgments
The authors would like to thank Prof. Dr. Ashraf B. El-Den,
Department of Pharmacoclogy, Faculty of Pharmacy, Ain-Shams
University for carrying out the in vitro screening. Also, thank
Dr. Walaa Wadie, Department of Pharmacology, Faculty of
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