Synthesis and biological evaluation of some N-arylpyrazoles and pyrazolo[3,4-d]pyridazines as anti-inflammatory agents

Article abstract of DOI:10.1002/ardp.201300193

A series of 3,4-bis-chalcone-N-arylpyrazoles 3a-k was prepared from diacetyl pyrazoles 2a-e. The reaction of 2d and 2e with hydrazine hydrate gave pyrazolo[3,4-d]pyridazine derivatives 4a-b. Furthermore, the reaction of 2a-e with thiosemicarbazide afforde

Full text of DOI:10.1002/ardp.201300193

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–11
1
Full Paper
Synthesis and Biological Evaluation of Some N-Arylpyrazoles
and Pyrazolo[3,4-d]pyridazines as Anti-Inflammatory Agents
Osama I. El-Sabbagh1,2, Samia Mostafa3, Hatem A. Abdel-Aziz4,5, Hany S. Ibrahim6, and
Mahmoud M. Elaasser7
1
Faculty of Pharmacy, Pharmaceutical Chemistry Department, Taif University, Taif, Saudi Arabia
Faculty of Pharmacy, Medicinal Chemistry Department, Zagazig University, Zagazig, Egypt
Faculty of Pharmacy, Pharmaceutical Chemistry Department, Suez Canal University, Ismailia, Egypt
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia
Applied Organic Chemistry Department, National Research Center, Dokki, Cairo, Egypt
Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Egyptian Russian University, Badr City,
2
3
4
5
6
Helwan, Egypt
7
The Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt
A series of 3,4-bis-chalcone-N-arylpyrazoles 3a–k was prepared from diacetyl pyrazoles 2a–e. The
reaction of 2d and 2e with hydrazine hydrate gave pyrazolo[3,4-d]pyridazine derivatives 4a–b.
Furthermore, the reaction of 2a–e with thiosemicarbazide afforded pyrazolo[3,4-d]pyridazine
thiocyanate salts 5a–e. The synthesized compounds were subjected to in vivo anti-inflammatory and
ulcerogenic activity measurements, in addition to determination of their in vitro COX selectivity, to give
a full profile about their anti-inflammatory activities. Compounds 3c, 3f, 3i, and 3e showed significant
anti-inflammatory activity among the synthesized compounds. Moreover, docking studies were
performed to give an explanation for their anti-inflammatory activity through COX selectivity.
Keywords: Anti-inflammatory activity / COX selectivity / Molecular docking / N-Aryl pyrazoles / Pyrazolopyridazine
Received: May 24, 2013; Revised: July 14, 2013; Accepted: July 16, 2013
DOI 10.1002/ardp.201300193
Introduction
N-Arylpyrazoles were found to be a main pharmacophore in
these selective anti-inflammatory agents as celecoxib [6] and
SC-558 [7, 8] (Fig. 1).
Prostaglandins are important biological mediators of
inflammation, originating from the biotransformation of
arachidonic acid catalyzed by cyclooxygenases [1]. The
discovery that cyclooxygenase enzyme in arachidonic acid
metabolism exists in two isoforms, namely, COX-1 and COX-2
(the constitutive and inducible forms), has generated new
avenues for drug design.
It was proposed that a selective inhibitor of COX-2 would be
an attractive approach for the treatment of inflammatory
conditions without concomitant gastric and renal toxicity [2].
Celecoxib and other coxibs, such as rofecoxib [3], etoricoxib [4],
and valdecoxib [5], are selective COX-2 inhibitors with fewer
gastrointestinal side effects than traditional NSAIDs.
Certain chalcone derivatives were reported to possess
analgesic and antipyretic actions [9, 10] in addition to
their marked anti-inflammatory activity [11–14]. Among
the chalcones [15, 16], 2,4-dichloro-40-N-[N0-(400-methyl-
phenylsulfenyl)urenyl]chalcone (Me-UCH9) was found to
exert anti-inflammatory action [15] through dual
inhibition of cyclooxygenase-2 and 5-lipoxygenase activities
(Fig. 1).
This motivated us to design and synthesize new series of bis-
chalcones derivatives containing the N-aryl pyrazole moiety,
which fulfill the pattern of COX-2 inhibition in order to
produce potent anti-inflammatory agents with no or less
gastric ulceration. In addition, the relation between the
variations of substituents on the biological activity will be
studied as well as molecular docking studies will be done after
in vitro studies to analyze their mechanism of action as anti-
inflammatory agents.
Correspondence: Prof. Osama I. El-Sabbagh, Pharmaceutical Chemistry
Department, College of Pharmacy, Taif University, P.O. Box 888, 21974 Al-
Haweiah, Taif, Saudi Arabia.
E-mail: osamaelsabbagh@yahoo.com
Fax: þ966-02-7274299
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2
O. I. El-Sabbagh et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–11
Figure 1. Chemical structures of celecoxib, SC-558, Me-UCH9, and compound 3c.
Results and discussion
ethoxide gave 3,4-diacetyl-5-methyl-N-arylpyrazoles 2a–e [14, 17],
as shown in Scheme 1.
Chemistry
The reaction of the latter pyrazoles, at room temperature,
with different aldehydes in the presence of aqueous sodium
hydroxide afforded the bis-chalcone derivatives 3a–k. The
The reaction of 2-oxo-N0-arylpropanehydrazonoyl chlorides
1a–e with pentan-2,4-dione in ethanol containing sodium
O
N
O
O
O
N
Ar1
Ar1
Cl
Me
Me
Me
H
N
Me
(a)
(b)
N
Ar
Me
N
O
N
1a
−e
Ar
Ar
3a
Ar1
2,4-(Cl)2C6H3
2a−e
−k
1,2
Ar
3
a
b
c
d
e
f
Ar
C6H5
a
b
c
d
e
C6H5
4-MeC6H4
4-ClC6H4
4-FC6H4
4-BrC6H4
4-MeC6H4 2,4-(Cl)2C6H3
4-ClC6H4 4-MeOC6H4
4-ClC6H4 2,4-(Cl)2C6H3
4-ClC6H4 2-furyl
4-FC6H4
4-FC6H4
4-FC6H4
4-MeOC6H4
2,4-(Cl)2C6H3
2-furyl
g
h
i
4-BrC6H4 4-MeOC6H4
4-BrC6H4 2,4-(Cl)2C6H3
4-BrC6H4 2-furyl
j
k
Scheme 1. Synthetic pathway of compounds 2a–e and 3a–k. Reagents and conditions: (a) (CH3CO)2CH2, EtONa/EtOH, RT, stirring 8 h;
(b) 2Ar1–CHO, EtOH, 10% NaOH, RT, stirring 6 h.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–11
New Arylpyrazoles as Anti-Inflammatory Agents
3
N
C
S
H
N
N
O
N
O
N
N
Me
Me
Me
Me
Me
Me
Me
(a)
Me
(b)
N
N
N
N
Me
N
Ar
Ar
Ar
4a, b
2a–e
5a–e
5
a
Ar
4
Ar
C6H5
a
b
4-FC6H4
4-BrC6H4
b
c
d
e
4-MeC6H4
4-ClC6H4
4-FC6H4
4-BrC6H4
Scheme 2. Synthetic pathway of compounds 4a–b and 5a–e. Reagents and conditions: (a) NH2NH2 · H2O, EtOH, reflux 8 h;
(b) NH2NHCSNH2, AcOH/H2O, reflux 4 h.
structures of the bis-chalcone derivatives 3a–k were con-
firmed using IR and 1H NMR whereas their IR spectra revealed
the appearance of a strong absorption band at v ¼ 1655–
1670 cmꢀ1 for the two carbonyl groups, which are in lower
range than that for the corresponding compounds 2a–e.
Moreover, the 1H NMR spectra confirmed the structures of
compounds 3a–k. The two signals of the two COCH3 groups at
d ¼ 2.712 and 2.96 ppm for the starting compounds 2a–e
disappeared while two signals of olefinic protons appeared in
the aromatic region at d ¼ 6.8–8.0 ppm.
The structure of the novel pyrazolo[3,4-d]pyridazin-5-ium
isothiocyanate derivatives was proved using IR and 13C NMR
whereas IR revealed the presence of the absorption band of the
thiocyanate function around 2040 cmꢀ1 in addition to the
absence of absorption band at v ¼ 1720 and 1723 cmꢀ1 due to
carbonyl groups for the starting diacetyl pyrazoles (2a–e).
Moreover, 13C NMR confirmed the structures of compounds
5a–e whereas the two peaks of the two acetyl groups, which
appeared around 27.6–31.70 ppm for the starting diacetyl
pyrazoles (2a–e), were shifted to the range around 18.0 ppm.
The previous results were confirmed by X-ray crystallography
for compound 5b [18].
Furthermore, the reaction of pyrazole derivatives 2d–e with
hydrazine hydrate in ethanol afforded pyrazolo[3,4-d]pyrida-
zine derivatives 4a–b (Scheme 2). The structures of the novel
pyrazolo[3,4-d]pyridazine 4a–b were established using IR and
13C NMR whereas their IR spectra revealed the absence of
absorption band at v ¼ 1720 and 1723 cmꢀ1 due to carbonyl
groups for the starting diacetyl pyrazoles (2d–e). Moreover,
13C NMR confirmed the structures of pyrazolo[3,4-d]pyrida-
zines (4a–b) whereas the two peaks of the two COCH3 groups,
which appeared around d ¼ 27.6–31.7 ppm for the starting
2d–e, were shifted to the range d ¼ 18.0–20.7 ppm.
Biological evaluation
The first target of the biological activity was to examine the
anti-inflammatory activity for all compounds (2a–e, 3a–k,
4a–b and 5a–e) via carrageenan-induced edema method [19].
Moreover, compounds that possessed a marked anti-inflam-
matory activity in rat paw edema test were subjected to
ulcerogenic activity test [20] using celecoxib and indometha-
cin as reference drugs. The second target was to study the
mechanism of action of these compounds through inhibition
of ovine COX-1 and COX-2 using an enzyme immunoassay
(EIA) kit [21].
Our attempt to synthesize bis-thiosemicarbazone deriva-
tives of diacetyl pyrazoles 2a–e gave the pyrazolo[3,4-d]-
pyridazine thiocyanate salts 5a–e. The suggested mechanism
of the latter cyclization is postulated in Fig. 2. Due to the
higher electrophilicity of the acetyl group at position 3 of the
pyrazole moiety, the cyclization process takes place initially
by the formation of its thiosemicarbazone I. The nucleophilic
attack of the other hydrazino nitrogen on carbonyl group at
position 4 afforded the unstable hydroxyl intermediate II,
which rearranged to give intermediate III. The latter non-
isolable intermediate lost an H2O molecule to give structure
IV, which rearranged to afford the final isolable salts 5a–e.
In vivo anti-inflammatory activity
The anti-inflammatory activity for all compounds was
determined via rat paw edema test [19] using celecoxib and
indomethacin as reference drugs. Through analysis of Table 1,
it was generally noted that the diacetyl pyrazoles (2a–e)
showed lower anti-inflammatory activity than their corre-
sponding bis-chalcone derivatives (3a–k). This is clear on
comparing the activity of diacetyl pyrazole (2c), which caused
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

4
O. I. El-Sabbagh et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–11
Figure 2. The proposed mechanism for the synthesis of compounds 5a–e.
reduction of edema by 3.93%, with its corresponding bis-
chalcone derivative (3c), which decreased the inflammation
by 66.21% (Table 1).
ꢁ On the other side, the chalcone 3i exhibited a high
ulcerogenic activity but it is still lesser than that of the
reference drug indomethacin (Table 1).
Moreover, it was observed that the bis-chalcone derivatives
3c, 3f, 3i, and 3e showed the highest anti-inflammatory
activity. The rank order of potency for such compounds is:
indomethacin > 3c > 3f > celecoxib > 3i > 3e.
We observe that compounds 3c, 3f, and 3i had a common
p-methoxyphenyl as a chalcone substituent. From these
results, we could deduce that chloro substituent on N-aryl
pyrazole substituent, as seen in compound 3c, gave the
maximum activity (Table 1).
In vitro cyclooxygenase inhibition assay
Compounds 3c, 3f, 3i, and 3e that had the highest anti-
inflammatory activity in rat paw edema test were studied for
their mechanism of action through inhibition of ovine COX-1
and COX-2 using an EIA kit [21].
The efficacies of the tested compounds were determined as
the concentration causing 50% enzyme inhibition (IC50
;
Furthermore, the bis-chalcone derivatives 3e and 3h
bearing furyl moiety exhibited a moderate activity (Table 1).
Cyclization of the diacetyl pyrazoles (2a–e) showed a lower or
no anti-inflammatory activity as seen in the pyrazolo-
pyridazine derivatives (4a–b, 5a–e).
Table 1). As far as COX-1 inhibitory properties are concerned,
all the tested compounds showed no inhibition of COX-1 up to
100 mM except 3e and 3i, as they possessed little inhibition.
Moreover, a reasonable in vitro COX-2 inhibitory activity was
observed with compounds 3c, 3f, 3e, and 3i. Selectivity index
was a parameter used to diagnose the selectivity between two
isoenzymes. The selectivity indices (COX-1/COX-2) were
calculated and compared with those of the selective COX-2
inhibitor celecoxib.
In the assay system, the IC50 values of celecoxib on COX-1
and COX-2 were determined to be >100 and 0.29 mM,
indicating that celecoxib is a selective COX-2 inhibitor
(SI ¼ 344.82). The results showed that compound 3c showed
potent inhibition against COX-2 (IC50 ¼ 0.30 mM) compared to
the inhibition against COX-1 (IC50 > 100 mM), as listed in
Table 1. Thus, compound 3c gave the highest selectivity index
(SI ¼ 333.33). This indicates that compound 3c, which
Ulcerogenic activity
Compounds 3c, 3f, 3i, and 3e that possessed the highest anti-
inflammatory activity in rat paw edema test were subjected to
ulcerogenic activity test [20]. Celecoxib and indomethacin
were used as reference drugs. The results most likely to be
withdrawn from Table 1 are:
ꢁ The order of ulcerogenic activity is: indomethacin
> 3i > 3e > 3f > 3c > celecoxib.
ꢁ It was also observed that the bis-chalcone 3c has nearly the
same ulcerogenic activity as the selective COX-2 inhibitor
celecoxib.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–11
New Arylpyrazoles as Anti-Inflammatory Agents
5
Table 1. Biological evaluation of the synthesized compounds.
In vivo studies
In vitro studies
Size of the hind paw (mm)a)
Before
treatment
% Edema % Edema
inhibition inhibition
3 h after
6 h after
Ulcer
COX-1 IC50 COX-2 IC50
Compounds with the drug treatment
treatment
(3 h)
(6 h)
indexb)
(mM)c)
(mM)
SId)
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
3k
4a
4b
5a
5b
5c
5d
5e
1.62 ꢃ 0.22
1.78 ꢃ 0.26
1.29 ꢃ 0.16
1.88 ꢃ 0.20
1.90 ꢃ 0.12
1.66 ꢃ 0.10
1.55 ꢃ 0.23
1.55 ꢃ 0.16
1.23 ꢃ 0.12
1.88 ꢃ 0.11
1.33 ꢃ 0.09
1.44 ꢃ 0.18
1.80 ꢃ 0.24
1.80 ꢃ 0.19
1.35 ꢃ 0.28
1.55 ꢃ 0.23
1.55 ꢃ 0.16
1.55 ꢃ 0.13
1.35 ꢃ 0.25
1.65 ꢃ 0.22
1.89 ꢃ 0.23
1.55 ꢃ 0.13
1.30 ꢃ 0.12
1.57 ꢃ 0.14
1.45 ꢃ 0.23
1.90 ꢃ 0.12
4.20 ꢃ 0.16
4.33 ꢃ 0.22
3.86 ꢃ 0.20
4.56 ꢃ 0.22
4.53 ꢃ 0.26
4.32 ꢃ 0.26
3.95 ꢃ 0.11ꢄ
3.98 ꢃ 0.16ꢄ
4.25 ꢃ 0.13
3.98 ꢃ 0.24
4.35 ꢃ 0.19
4.39 ꢃ 0.22
4.10 ꢃ 0.19
4.05 ꢃ 0.12
7.86
8.93
8.21
4.29
6.07
15.71
11.79
3.93
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
11.79
11.07
12.86
10.71
66.21
15.36
27.86
65.21
13.57
16.79
29.93
9.29
1.07
3.21
0.00
8.93
16.79
8.57
1.43
–
–
–
–
–
–
–
5.00
–
–
14.29
55.29
11.79
24.29
53.64
9.29
14.29
35.14
0.00
–
–
–
2.80 ꢃ 0.16ꢄꢄꢄ 2.50 ꢃ 0.11ꢄꢄꢄ
0.90 ꢃ 0.33b
>100
0.30
333.33
3.70 ꢃ 0.16
3.60 ꢃ 0.16
–
4.00 ꢃ 0.11ꢄꢄ 3.90 ꢃ 0.22ꢄꢄ
2.63 ꢃ 0.23ꢄꢄꢄ 2.31 ꢃ 0.20ꢄꢄꢄ
2.1 ꢃ 0.23ab
78
>100
1.5
0.32
–
65.333
312.5
1.1 ꢃ 0.35b
3.98 ꢃ 0.18ꢄ
3.86 ꢃ 0.12ꢄꢄ
–
–
–
4.20 ꢃ 0.13ꢄꢄ 4.13 ꢃ 0.23ꢄꢄ
3.61 ꢃ 0.18ꢄꢄ 3.76 ꢃ 0.22ꢄꢄ
–
–
–
–
2.75 ꢃ 0.25ab
57
–
0.7
–
76
–
4.20 ꢃ 0.16
4.05 ꢃ 0.24
4.31 ꢃ 0.25
4.35 ꢃ 0.12
3.70 ꢃ 0.24
3.80 ꢃ 0.16ꢄ
4.20 ꢃ 0.23
4.23 ꢃ 0.13
3.78 ꢃ 0.12
3.89 ꢃ 0.18
4.32 ꢃ 0.16
4.26 ꢃ 0.23
4.45 ꢃ 0.13
3.90 ꢃ 0.18
3.98 ꢃ 0.13ꢄ
4.45 ꢃ 0.23
4.31 ꢃ 0.23
3.88 ꢃ 0.13
–
–
–
10.71
1.43
0.00
–
–
–
–
–
–
–
–
–
16.07
23.21
17.50
4.29
11.43
50.71
70.13
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
7.86
–
–
>100
–
–
–
Celecoxib
Indomethacin
Control
2.95 ꢃ 0.14ꢄꢄꢄ 2.56 ꢃ 0.23ꢄꢄꢄ
2.85 ꢃ 0.24ꢄꢄꢄ 2.45 ꢃ 0.09ꢄꢄꢄ
64.64
78.45
–
0.83 ꢃ 0.1b
3.17 ꢃ 0.3a
0.25 ꢃ 0.11ab
0.29
–
344.82
–
4.70 ꢃ 0.19
4.90 ꢃ 0.13
–
–
–
a)
For rat paw edema: values represent means ꢃ SEM of six animals for each group. Statistical analysis using one-way ANOVA
(Bonferroni’s multiple comparison test). Significance levels ꢄp < 0.5, ꢄꢄp < 0.01, and ꢄꢄꢄp < 0.001 as compared with the respective
control.
b)
Significance level for ulcer index: ap < 0.05 significantly different from celecoxib and bp < 0.05 significantly different from
indomethacin.
c)
IC50 value is the compound concentration required to produce 50% inhibition of COX-1 or COX-2 for means of two determinations and
deviation from the mean is <10% of the mean value. No inhibition of COX-1 up to 100 mM and precipitation of compounds was observed
beyond this concentration.
d)
Selectivity index (COX-1 IC50/COX-2 IC50).
ꢂ
contains two p-methoxyphenyl groups on the chalcone wings
and p-chloro substituent on N-aryl pyrazole part, gave the
highest activity.
with RMSD value (1.49 A). Compound 3c can form four
hydrogen bonds within the binding pocket (Fig. 3), one of
ꢂ
them with Arg120 (distance ¼ 2.7 A) by the methoxy group
ꢂ
and second one with Arg513 (distance ¼ 2.5 A) by one of the
Molecular docking studies
carbonyl groups of the chalcone wings. The other carbonyl
To investigate potential binding modes of these anti-
inflammatory compounds, a docking study was carried
out using the most potent compound in the series (3c).
Using the reported X-ray structure of COX-2 enzyme co-
crystallized with SC-558 (PDB entry 1CX2) [8], an automated
dock run was conducted using Molsoft ICM-pro software.
Docking simulation of 3c (Fig. 3) shows that this compound
gives a conformational stack energy score (ꢀ55.67 kcal/mol)
group can form two hydrogen bonds ꢂwith Arg513 (distance
ꢂ
¼ 2.49 A) and Hip90 (distance ¼ 2.49 A).
One of the p-methoxyphenyl chalcone wings showed an
interesting interaction within the binding site (Fig. 4).
Actually, it was embedded inside two essential amino
acids for the activity in the binding site, which are
Arg120 and Tyr 335. In addition, methoxy group could
form hydrogen bond with the most important amino
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

6
O. I. El-Sabbagh et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–11
Figure 3. Binding mode of 3c docked in COX-2 active site (COX-2 PDB file: 1CX2) showing four hydrogen bonds with amino acid residues
Arg120, Tyr355, and Hip90 (follow the arrows).
acid essential for activity [22]. Furthermore, p-chlorophenyl Conclusion
group was surrounded by a pocket of different amino acids,
which are Leu531, Ser530, Leu352, Tyr385, Trp387, and
Ala527.
The present study describes the synthesis of some bis-
chalcones-N-arylpyrazoles 3a–k, pyrazolo[3,4-d]pyridazines
Figure 4. Detailed view about the interaction of the one of the p-methoxyphenyl of chalcone wings with important amino acids in the binding
site of COX-2 isozyme (follow the arrow).
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–11
New Arylpyrazoles as Anti-Inflammatory Agents
7
4a–b, and pyrazolo[3,4-d]pyridazine thiocyanate salts 5a–e.
The bis-chalcones N-arylpyrazoles revealed anti-inflammatory
activity through inhibition of COX-2 enzyme. Compound 3c
showed a suitable inhibitory action for this enzyme with good
selectivity index related to the COX-1 enzyme. Further
molecular docking studies have been conducted to investigate
the binding mode of compound 3c with the COX-2 isozyme
binding site.
Synthesis of bis-chalcones 3a–k
To a stirred solution of the appropriate pyrazole 2a–e (10 mmol)
and the appropriate aldehyde (20 mmol) in ethanol (30 mL), 10%
aqueous sodium hydroxide (10 mL) was added portionwise at
room temperature for 10 min. The reaction mixture was further
stirred for 6 h. The resulting solid was filtered, washed with water,
dried, and crystallized from EtOH/DMF.
1,10-(5-Methyl-1-phenyl-1H-pyrazole-3,4-diyl)bis(3-(2,4-
dichlorophenyl)prop-2-en-1-one) (3a)
Orange crystals, 65% yield; mp >300°C; IR (KBr) nmax/cmꢀ1 1670,
Experimental
1622 (2C O), 1570 (C N); 1H NMR (pyridine-d5) d ¼ 2.52 (s, 3H,
–
–
–
–
–CH ), 7.21–8.52 (m, 17H, Ar–H þ olefinic –CH ) ppm; 13C NMR
–
–
3
Chemistry
(pyridine-d5) d ¼ 11.77 (–CH3), 123.00, 123.50, 126.06, 126.42,
Melting points (°C, uncorrected) were determined using a Stuart
melting point apparatus. The IR spectra (KBr) were recorded on a
PerkinElmer FT/IR spectrometer. The NMR spectra were recorded
by Varian Gemini-2000 200 MHz FT-NMR spectrometers (Varian,
Inc., Palo Alto, CA). Chemical shifts are reported in parts per
million (d), and coupling constants (J) are expressed in Hertz. TMS
was used as an internal standard, and chemical shifts were
measured in d ppm. 1H and 13C spectra were run at 200 and
50 MHz, respectively. Electron impact mass spectra were
measured on a Varian MAT 311-A (70 eV). Elemental analysis
was carried out for C, H, and N at the Microanalytical Center of
Cairo University. All reagents were purchased from Sigma–
Aldrich Chemical Company.
127.98, 129.40, 129.68, 129.86, 130.11, 131.27, 134.99, 135.49,
–
–
–
–
137.98 (aromatic C), 149.28 (C O), 149.81 (C O) ppm; MS m/z (%)
558 (Mþþ2, 3.31), (556 (Mþ, 8.9), 521 (1.5), 251 (6.3), 170 (42.74),
135 (100). Anal. calcd. for C28H18Cl4N2O2 (556.27): C, 60.46; H,
3.26; N, 5.04. Found: C, 60.51; H, 3.29; N, 5.13.
1,10-(5-Methyl-1-(4-methylphenyl)-1H-pyrazole-3,4-diyl)-
bis-(3-(2,4-dichlorophenyl)prop-2-en-1-one) (3b)
White crystals, 78% yield; mp >300°C; IR (KBr) nmax/cmꢀ1 1695,
1625 (2C O), 1535 (C N); 1H NMR (pyridine-d5) d ¼ 1.82 (s,
–
–
–
–
3H, –CH3), 2.05 (s, 3H, –CH3), 6.65–7.94 (m, 14H, Ar–H þ olefinic
–CH ) ppm; 13C NMR (pyridine-d5) d ¼ 12.30 (–CH3), 21.42 (–CH3),
–
–
123.50, 124.49, 126.41, 128,49, 129.89, 130.86, 131.88, 135.56,
Synthesis of compounds 2a–e
–
–
–
–
136.49, 138.49 (aromatic C), 149.82 (C O), 150.91 (C O) ppm; MS
m/z (%) 572 (Mþþ2, 6.31), 570 (Mþ, 14.76), 536 (62.22), 135 (100).
Anal. calcd. for C29H20Cl4N2O2 (570.29): C, 61.08; H, 3.53; N, 4.91.
Found: C, 61.12; H, 3.51; N, 4.98.
A
mixture of pentan-2,4-dione (1.34 g, 10 mmol) and the
appropriate 2-oxo-N0-arylpropanehydrazonoyl chloride 1a–e
(10 mmol) in ethanolic sodium ethoxide (10 mol in 20 mL ethanol)
was stirred at room temperature for 8 h to afford compounds 2a–
e. Compounds 2a–c are reported and their experimental data are
mentioned in the literature [23, 24].
1,10-(1-(4-Chlorophenyl)-5-methyl-1H-pyrazole-3,4-diyl)-
bis(3-(4-methoxyphenyl)prop-2-en-1-one) (3c)
Pale yellow crystals, 59% yield; mp 198–200°C; IR (KBr) nmax/cmꢀ1
1,10-(1-(4-Fluorophenyl)-5-methyl-1H-pyrazole-3,4-diyl)-
1
–
–
1631, 1643 (2C O), 1598 (C N); H NMR (pyridine-d ) d ¼ 2.52 (s,
–
–
5
diethanone (2d)
3H, –CH3), 3.67 (s, 6H, 2 –OCH3), 6.94–8.18 (m, 16H, Ar–H and
White crystals, 63% yield; mp 155–157°C; IR (KBr) nmax/cmꢀ1
olefinic –CH ) ppm; 13C NMR (pyridine-d5) d ¼ 12.24 (–CH3), 55.84
–
–
1
–
–
1720, 1725 (2C O), 1603 (C N); H NMR (pyridine-d ): d ¼ 2.38 (s,
–
–
5
(2-OCH3), 113.66, 115.21, 121.78, 122.28, 122.78, 126.91, 129.43,
129.72, 133.74, 133.80, 134.22, 134.28, 134.72, 134.79, 141.16,
3H, –CH3), 2.69 (s, 3H, –COCH3), 2.71 (s, 3H, –COCH3), 7.50–7.78
(dd, 4H, J ¼ 7.2 Hz, Ar–H) ppm; 13C NMR (pyridine-d5) d ¼ 11.70
(–CH3), 27.61 (–CH3), 30.13 (–CH3), 116.43, 116.88, 117.25, 127.52,
–
–
–
–
142.92, 148.69, 149.22 (aromatic C), 162.47 (C O), 162.77 (C O)
ppm; MS m/z (%) 514 (Mþþ2, 6.71), 512 (Mþ, 14.49), 391 (92.97),
152 (83.31), 132 (100), 89 (88.93), 76 (61.0). Anal. calcd. for
C30H25ClN2O4 (512.98): C, 70.24; H, 4.91; N, 5.46. Found: C, 70.23;
H, 4.97; N, 5.50.
–
–
–
135.02, 141.69, 143.79 (aromatic C), 149.01 (–C O), 150.43 (–C O)
–
ppm. MS m/z (%) 260 (Mþ, 100), 246 (Mþ, 97.3), 149 (54.88), 136
(91.7), 95 (68.8). Anal. calcd. for C14H13FN2O2 (260.26): C, 64.61; H,
5.03; N, 10.76. Found: C, 64.44; H, 5.02; N, 10.95.
1,10-(1-(4-Chlorophenyl)-5-methyl-1H-pyrazole-3,4-diyl)-
1,10-(1-(4-Bromophenyl)-5-methyl-1H-pyrazole-3,4-diyl)-
bis(3-(2,4-dichlorophenyl)prop-2-en-1-one) (3d)
diethanone (2e)
Pale yellow crystals, 67% yield; mp 247–249°C; IR (KBr) nmax/cmꢀ1
Pale yellow powder, 57% yield; mp 151–153°C; IR (KBr) nmax/cmꢀ1
1
1
–
–
1665, 1654 (2C O), 1604 (C N); H NMR (pyridine-d ) d ¼ 2.54 (s,
–
–
–
–
5
–
–
1723, 1721 (2C O), 1587 (C N); H NMR (pyridine-d ): d ¼ 2.40 (s,
–
–
5
3H, –CH ), 7.20–8.49 (m, 14H, Ar–H þ olefinic –CH ) ppm; 13C
3
3H, –CH3), 2.69 (s, 3H, –COCH3), 2.71 (s, 3H, –COCH3), 7.50–7.78
(dd, 4H, J ¼ 7.2 Hz, Ar–H) ppm; 13C NMR (pyridine-d5) d ¼ 12.03
(–CH3), 27.87 (–CH3), 31.74 (–CH3), 123.29, 124.27, 127.93, 133.18,
NMR (pyridine-d5) d ¼ 12.30 (–CH3), 123.50, 124.50, 126.41, 128.04,
129.95, 130.51, 131.88, 135.47, 136.47, 138.72þ(aromatic C), 149.84
þ
–
–
–
(C O), 150.94 (C O) ppm; MS m/z (%) 593 (M þ2, 2.21), 591 (M ,
–
–
–
–
135.77, 136.26 (aromatic C), 149.64 (–C O), 150.72 (–C O) ppm.
–
MS m/z (%) 323 (Mþþ2, 28.05) 321 (Mþ, 28.25), 165 (23.86), 154
(23.89). Anal. calcd. for C14H13BrN2O2 (321.17): C, 52.36; H, 4.08; N,
8.72. Found: C, 52.47; H, 3.70; N, 8.70.
6.87), 554 (14.69), 518 (9.55), 483 (6.62), 135 (100). Anal. calcd. for
C28H17Cl5N2O2 (590.71): C, 56.93; H, 2.90; N, 4.74. Found: C, 56.63;
H, 2.80; N, 4.37.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

8
O. I. El-Sabbagh et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–11
1,10-(1-(4-Chlorophenyl)-5-methyl-1H-pyrazole-3,4-diyl)-
131.16, 131.45, 133.45, 133.49, 136.49, 143.01, 144.78, 149.84,
–
–
150.94 (aromatic C), 162.47 (C O), 162.76 (C O) ppm; MS m/z (%)
–
–
bis(3-(furan-2-yl)prop-2-en-1-one) (3e)
558 (Mþ, 15.88), 438 (50.84), 161 (57.08), 133 (100). Anal. calcd. for
C30H25BrN2O4 (557.43): C, 64.64; H, 4.52; N, 5.03. Found: C, 64.2; H,
4.44; N, 4.66.
Pale yellow crystals, 72% yield; mp 194–196°C; IR (KBr) nmax/cmꢀ1
1
–
–
1669, 1657 (2C O), 1600 (C N); H NMR (pyridine-d ) d ¼ 2.48 (s,
–
–
5
3H, –CH ), 6.49–7.99 (m, 14H, Ar–H þ olefinic CH) ppm; 13C NMR
–
–
3
(pyridine-d5) d ¼ 12.24 (–CH3), 113.12, 113.27, 116.26, 117.08,
121.03, 123.01, 123.52, 126.06, 127.39, 128.83, 129.87, 130.33,
134.99, 135.08, 135.49, 145.64, 145.93, 149.36þ(aromatic C), 149.89
1,10-(1-(4-Bromophenyl)-5-methyl-1H-pyrazole-3,4-diyl)-
bis(3-(2,4-dichlorophenyl)prop-2-en-1-one) (3j)
–
–
–
Pale yellow crystals, 59% yield; mp 244–246°C; IR (KBr) nmax/cmꢀ1
(C O), 152.03 (C O) ppm; MS m/z (%) 432 (M , 2.46), 281 (28.18),
–
1
207 (100), 73 (52.93). Anal. calcd. for C24H17ClN2O4 (432.86): C,
66.59; H, 3.96; N, 6.47. Found: C, 66.37; H, 3.45; N, 6.40.
–
–
1678, 1680 (2C O), 1606 (C N); H NMR (pyridine-d ) d ¼ 2.55 (s,
–
–
5
–
–
3H, –CH ), 7.21–8.52 (m, 14H, Ar–H þ olefinic –CH ) ppm; 13C
3
NMR (pyridine-d5) d ¼ 11.77 (–CH3), 122.99, 123.50, 127.78, 128.04,
129.46, 130.30, 131.27, 133.30, 135.01, 135.50þ(aromatic C), 149.34
1,10-(1-(4-Fluorophenyl)-5-methyl-1H-pyrazole-3,4-diyl)-
þ
–
–
–
–
(C O), 149.87 (C O) ppm; MS m/z (%) 637 (M þ2, 10.3), 635 (M ,
bis(3-(4-methoxyphenyl)prop-2-en-1-one) (3f)
10.45), 433 (45), 199 (40.37). Anal. calcd. for C28H17BrCl4N2O2
(635.16): C, 52.95; H, 2.70; N, 4.41. Found: C, 52.66; H, 2.65; N, 4.51.
Creamy white crystals, 70% yield; mp 176–178°C; IR (KBr)
1
nmax/cmꢀ1 1669, 1657 (2C O), 1596 (C N); H NMR (pyridine-d )
–
–
–
–
5
d ¼ 2.51 (s, 3H, –CH3), 3.67 (s, 6H, 2-OCH3), 6.95–8.18 (m, 16H,
1,10-(1-(4-Bromophenyl)-5-methyl-1H-pyrazole-3,4-diyl)-
Ar–H þ olefinic –CH ) ppm; 13C NMR (pyridine-d5) d ¼ 12.24
–
–
bis(3-(furan-2-yl)prop-2-en-1-one) (3k)
(–CH3), 54.07 (2-OCH3), 113.66, 115.21, 121.78, 122.28, 122.78,
126.91, 129.43, 129.72, 133.74, 133.80, 134.22, 134.28, 134.72,
Yellow crystals, 70% yield; mp 195–197°C; IR (KBr) nmax/cmꢀ1
1
–
–
–
1689, 1678 (2C O), 1590 (C N); H NMR (pyridine-d ) d ¼ 2.50 (s,
134.79, 141.16, 142.92, 148.68, 149.22 (aromatic C), 162.47 (C O),
–
–
–
5
3H, –CH ), 6.54–8.17 (m, 14H, Ar–H þ olefinic CH) ppm; 13C NMR
þ
–
–
–
162.76 (C O) ppm; MS m/z (%) 497 (M , 8.35), 376 (64.64), 241
–
3
(pyridine-d5) d ¼ 11.68 (–CH3), 114.93, 116.44, 116.91, 121.63,
(32.9),161 (51.5),136 (100). Anal. calcd. for C30H25FN2O4 (496.53):
C, 72.57; H, 5.07; N, 5.64. Found: C, 72.33; H, 4.90; N, 5.75.
122.99, 123.50, 126.72, 128.16, 128.34, 129.87, 130.66, 130.95,
–
–
135.02, 135.50, 142.42, 144.20 (aromatic C), 149.34 (C O), 152.87
þ
þ
–
–
(C O) ppm; MS m/z (%) 479 (M þ2, 3.38), 477 (M , 3.8), 121 (100).
1,10-(1-(4-Fluorophenyl)-5-methyl-1H-pyrazole-3,4-diyl)-
Anal. calcd. for C24H17FN2O4 (477.31): C, 60.39; H, 3.59; N, 5.87.
Found: C, 60.43; H, 3.62; N, 5.91.
bis(3-(2,4-dichlorophenyl)prop-2-en-1-one) (3g)
Yellow crystals, 55% yield; mp 222–224°C; IR (KBr) nmax/cmꢀ1
1
–
–
1670, 1678 (2C O), 1608 (C N); H NMR (CDCl -d) d ¼ 2.45 (s, 3H,
–
–
3
–
–
Synthesis of compounds 4a–b
–CH ), 7.26–8.26 (m, 14H, Ar–H þ olefinic –CH ) ppm; 13C NMR
3
To compounds 2d–e (2 mmol) in ethanol (10 mL) hydrazine
hydrate (1 mL, 99%) was added and then the reaction mixture was
heated at reflux for 8 h. The precipitated product was filtered off,
washed with ethanol, dried, and finally crystallized from EtOH/
acetic acid.
(CDCl3-d) d ¼ 13.23 (–CH3), 117.69, 118.16, 126.91, 128.81, 128.94,
129.08, 129.96, 130.10, 131.24, 131.45, 131.40, 133.07, 137.36,
–
–
–
138.34, 140.16 (aromatic C), 149.34 (C O), 149.88 (C O) ppm; MS
–
m/z (%) 576 (Mþþ2, 4.06), 574 (Mþ, 12.26), 539 (100), 136 (40.37).
Anal. calcd. for C28H17Cl4FN2O2 (574.26): C, 58.56; H, 2.98; N 4.88.
Found: C, 58.73; H, 2.60; N 4.64.
2-(4-Fluorophenyl)-3,4,7-trimethyl-2H-pyrazolo[3,4-d]-
pyridazine (4a)
1,10-(1-(4-Fluorophenyl)-5-methyl-1H-pyrazole-3,4-diyl)-
Brown crystals, 70% yield; mp 225–227°C; IR (KBr) nmax/cmꢀ1
1
–
–
bis(3-(furan-2-yl)prop-2-en-1-one) (3h)
1608 (C N); H NMR (pyridine-d ) d ¼ 2.55 (s, 3H, –CH ), 2.85 (s, 3H,
5
3
Yellow crystals, 62% yield; mp 166–168°C; IR (KBr) nmax/cmꢀ1
–CH3), 3.01 (s, 3H, –CH3), 7.32–7.62 (dd, 4H, J ¼ 7.5 Hz, Ar–H) ppm;
13C NMR (pyridine-d5) d ¼ 12.61 (–CH3), 18.01 (–CH3), 20.77 (–CH3),
111.89, 122.99, 123.50, 127.26, 134.97, 135.47, 149.34, 149.89
(aromatic C) ppm; MS m/z (%) 256 (Mþ, 78.14), 238 (100), 168 (15.7),
149 (19.34). Anal. calcd. for C14H13FN4 (256.11): C, 65.61; H, 5.11;
N, 21.86. Found: C, 65.45; H, 5.00; N, 21.48.
1
–
–
1669, 1657 (2C O), 1600 (C N); H NMR (pyridine-d ) d ¼ 2.46 (s,
–
–
5
3H, CH ), 6.55–8.1 (m, 14H, Ar–H þ olefinic CH) ppm; 13C NMR
–
–
3
(pyridine-d5) d ¼ 12.24 (–CH3), 113.09, 113.23, 116.20, 116.38,
116.85, 117.02, 121.04, 121.04, 122.99, 123.50, 123.99, 126.06,
128.02, 128.20, 128.73, 130.24, 134.96, 135.46, 135.50, 136.00,
–
–
–
145.60, 145.88, 149.34 (aromatic C), 149.87 (C O), 152.01 (C O)
–
ppm; MS m/z (%) 417 (Mþ, 17.32), 295 (32.9), 136 (100), 121 (76.01).
Anal. calcd. for C24H17FN2O4 (416.40): C, 69.23; H, 4.12; N, 6.73.
Found: C, 69.01; H, 4.1; N, 6.68.
2-(4-Bromophenyl)-3,4,7-trimethyl-2H-pyrazolo[3,4-d]-
pyridazine (4b)
White crystals, 73% yield; mp 268–269°C; IR (KBr) nmax/cmꢀ1 1606
(C N); 1H NMR (pyridine-d5) d ¼ 2.57 (s, 3H, –CH3), 2.85 (s, 3H,
–
–
1,10-(1-(4-Bromophenyl)-5-methyl-1H-pyrazole-3,4-diyl)-
–CH3), 3.00 (s, 3H, –CH3), 7.21–7.82 (dd, 4H, J ¼ 7.5 Hz, Ar–H) ppm;
13C NMR (pyridine-d5) d ¼ 12.50 (–CH3), 18.27 (–CH3), 20.77 (–CH3),
111.89, 122.99, 123.50, 128.33, 132.95, 135.00, 135.50, 149.34,
149.89 (aromatic C) ppm; MS m/z (%) 319 (Mþþ2, 99.59), 317 (Mþ,
100), 237 (24.59), 198 (29.78), 167 (14.24), 154 (43.1). Anal. calcd.
for C14H13BrN4 (317.18): C, 53.01; H, 4.13; N, 17.66. Found: C,
53.23; H, 4.32; N, 17.80.
bis(3-(4-methoxyphenyl)prop-2-en-1-one) (3i)
Pale yellow crystals, 67% yield; mp 182–184°C; IR (KBr) nmax/cmꢀ1
1
–
–
1669, 1657 (2C O), 1600 (C N); H NMR (pyridine-d ) d ¼ 2.51 (s,
–
–
5
3H, –CH3), 3.66 (s, 6H, 2-OCH3), 6.93–8.16 (m, 16H, Ar–H þ olefinic
CH) ppm; 13C NMR (pyridine-d5) d ¼ 12.24 (–CH3), 55.85 (2-OCH3),
–
–
115.43, 122.07, 123.52, 124.50, 127.19, 128.22, 128.52, 128.75,
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–11
New Arylpyrazoles as Anti-Inflammatory Agents
9
Synthesis of compounds 5a–e
–CH3), 2.94 (s, 3H, –CH3), 2.97 (s, 3H, –CH3), 7.72–7.82 (dd, 4H,
J ¼ 7.5 Hz, Ar–H) ppm; 13C NMR (pyridine-d5) d ¼ 12.30 (–CH3),
21.42 (–CH3), 123.28, 124.27, 128.55, 128.49, 129.97, 132.96,
133.31, 135.27, 136.27, 138.49, 149.61, 150.69 (aromatic C) ppm;
MS m/z (%) 378 (Mþþ2, 2.25), 376 (Mþ, 2.39), 318 (15.31), 257 (4.41),
157 (13.25). Anal. calcd. for C15H14BrN5S (376.27): C, 47.88; H, 3.75;
N, 18.61; S, 8.52. Found: C, 47.71; H, 3.50; N, 18.47; S, 8.52.
A mixture of compounds 2a–e (1 mmol) and thiosemicarbazide
(0.18 g, 2 mmol) in aqueous ethanol (1:1, 50 mL) containing a
catalytic amount of acetic acid (0.5 mL) was heated at reflux for
4 h. The precipitated product was filtered off, washed with
ethanol, dried, and finally crystallized from EtOH/acetic acid.
3,4,7-Trimethyl-2-phenyl-2H-pyrazolo[3,4-d]pyridazin-5-
ium isothiocyanate (5a)
Biological evaluation
Animals
Pink crystals, 65% yield; mp. 238–239°C; IR (KBr) nmax/cmꢀ1 2042
1
–
–
(SCN), 1627 (C N); H NMR (pyridine-d ) d ¼ 2.32 (s, 3H, –CH ), 2.64
5
3
Male albino rats weighing 150–200 g were used. All experimental
animals were provided by the Regional Centre of Mycology and
Biotechnology, Al Azhar University, Cairo, Egypt. All animals
were held under standard laboratory conditions in the animal
house (temperature 25°C) with a 12/12 light–dark cycle. Animals
were fed laboratory diet and water ad libitum. All experiments
were carried out using six animals per group. The animal
experiments were performed in accordance with international
guidelines.
(s, 3H, –CH3), 2.98 (s, 3H, –CH3), 7.38–7.67 (dd, 4H, J ¼ 7.45 Hz,
Ar–H) ppm; 13C NMR (pyridine-d5) d ¼ 12.61 (–CH3), 18.01 (–CH3),
20.04 (–CH3), 123.50, 124.50, 128.54, 130.47, 135.49, 136.49,
149.80, 150.87 (aromatic C) ppm; MS m/z (%) 298 (Mþþ1, 3.38), 297
(Mþ, 40.8), 252 (100), 168 (18.57). Anal. calcd. for C15H15N5S
(297.38): C, 60.58; H, 5.08; N, 23.55; S, 10.78. Found: C, 60.77; H,
5.15; N, 23.39; S, 10.66.
3,4,7-Trimethyl-2-(p-tolyl)-2H-pyrazolo[3,4-d]pyridazin-5-
ium isothiocyanate (5b)
Drugs
White crystals, 48% yield; mp. 270–271°C; IR (KBr) nmax/cmꢀ1
Carrageenan (carrageenan kappa-type III) and all other reagents
were purchased from Sigma Chemical Co. (St. Louis, MO, USA).
2041 (SCN), 1610 (C N); 1H NMR (pyridine-d5) d ¼ 2.32 (s, 3H,
–
–
–CH3), 2.69 (s, 3H, –CH3), 2.93 (s, 3H, –CH3), 2.99 (s, 3H, –CH3),
7.34–7.66 (dd, 4H, J ¼ 7.5 Hz, Ar–H) ppm; 13C NMR (pyridine-d5)
d ¼ 12.61 (–CH3), 18.01 (–CH3), 20.04 (–CH3), 20.95 (–CH3), 122.99,
123.50, 126.28, 130.34, 135.02, 135.52, 140.28, 149.31, 149.86
(aromatic C) ppm; MS m/z (%) 311 (Mþ, 25.23), 252 (100), 168
(15.25). Anal. calcd. for C16H17N5S (311.40): C, 61.71; H, 5.50; N,
22.49; S, 10.30. Found: C, 61.26; H, 5.20; N, 22.8; S, 10.69.
In vivo anti-inflammatory activity
The test compounds were evaluated using the in vivo rat
carrageenan-induced foot paw edema model reported previously
[19]. The dose for the reference drugs and the test compounds was
10 mmol/kg, given orally.
Test compounds and celecoxib were suspended in gum acacia
(7%). The diameter of the right paw of each animal was
determined using a micrometer. The control group received
only the corresponding vehicle. Thirty minutes later, paw edema
was induced by subcutaneous injection of 0.1 mL of carrageenan
(0.1%) into the subplantar surface of the right hind paw of all
animals. The paw diameter was measured 3 and 6 h after
carrageenan injection and is recorded in Table 1.
2-(4-Chlorophenyl)-3,4,7-trimethyl-2H-pyrazolo[3,4-d]-
pyridazin-5-ium isothiocyanate (5c)
Pale yellow crystals, 62% yield; mp. 267–268°C; IR (KBr) nmax/cmꢀ1
2043 (SCN), 1612 (C N); 1H NMR (pyridine-d5) d ¼ 2.64 (s, 3H,
–
–
–CH3), 2.92 (s, 3H, –CH3), 2.98 (s, 3H, –CH3), 7.62–7.79 (dd, 4H,
J ¼ 7.5 Hz, Ar–H) ppm; 13C NMR (pyridine-d5) d ¼ 12.61 (–CH3),
18.01 (–CH3), 20.04 (–CH3), 123.50, 124.50, 128.54, 130.47, 135.49,
136.49, 149.80, 150.87 (aromatic C) ppm; MS m/z (%) 333 (Mþþ2,
3.21), 331 (Mþ, 9.67), 272 (100), 168 (12.55). Anal. calcd. for
C15H14ClN5S (331.82): C, 54.29; H, 4.25; N, 21.11; S, 9.66. Found: C,
54.38; H, 4.2; N, 21.06; S, 9.40.
Ulcerogenic activity
The compounds 3c, 3e, 3f, and 3i, which exhibited the highest
anti-inflammatory activity, were investigated for their ulcerogen-
ic activity [20]. Male albino rats weighing 150–200 g were fasted
for 12 h prior to drug administration. Water was supplied ad
libitum. The animals were divided into seven equal groups (each of
six). The first group received 7% gum acacia (suspending vehicle)
orally once a day and was left as a control, whereas the other
groups received the reference drugs and test compounds with a
dose of 100 mmol/kg/day orally. The test compounds were
administered once a day for three successive days. The animals
were killed by an overdose of ether 6 h after the last dose. The
stomachs were removed, opened along the greater curvature, and
examined for ulceration. The number and diameter of discrete
areas of damage in the glandular mucosa were scored (Table 1).
The ulcer score was calculated according to the method of Vijaya
and Mishra [20]: 0.0 – normal (no injury); 0.5 – latent injury; 1.0 –
slight injury (two to three dotted lines); 2.0 – severe injury
(continuous lined injury or five to six dotted injuries); 3.0 – very
severe injury (several continuous lined injuries); 4.0 – widespread
lined injury.
2-(4-Fluorophenyl)-3,4,7-trimethyl-2H-pyrazolo[3,4-d]-
pyridazin-5-ium isothiocyanate (5d)
Creamy white crystals, 55% yield; mp. 270–272°C IR (KBr) nmax
/
1
cmꢀ1 2044 (SCN), 1608 (C N); H NMR (pyridine-d ) d ¼ 2.64 (s, 3H,
–
–
5
–CH3), 2.93 (s, 3H, –CH3), 2.98 (s, 3H, –CH3), 7.36–7.84 (dd, 4H,
J ¼ 7.5 Hz, Ar–H) ppm; 13C NMR (pyridine-d5) d ¼ 12.56 (–CH3),
17.90 (–CH3), 19.89 (–CH3), 116.57, 117.02, 122.99, 123.50, 126.41,
128.63, 128.81, 133.03, 135.53, 149.25, 149.78 (aromatic C) ppm;
MS m/z (%) 315 (Mþ, 3.21), 256 (44.16), 185 (7.6), 136 (29.53). Anal.
calcd. for C15H14FN5S (315.37): C, 57.13; H, 4.47; N, 21.21; S, 10.17.
Found: C, 56.98; H, 4.07; N, 21.31; S, 10.56.
2-(4-Bromophenyl)-3,4,7-trimethyl-2H-pyrazolo[3,4-d]-
pyridazin-5-ium isothiocyanate (5e)
Pale yellow crystals, 72% yield; mp. 275–276°C; IR (KBr) nmax/cmꢀ1
2046 (SCN), 1608 (C N); 1H NMR (pyridine-d5) d ¼ 2.659 (s, 3H,
–
–
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

10
O. I. El-Sabbagh et al.
Arch. Pharm. Chem. Life Sci. 2013, 346, 1–11
Statistics
2 and SC-558; the receptor grid maps were calculated with a grid
ꢂ
spacing of 0.5 A; the grid was defined in such a way that it
included the ligand and key residues in each of the x, y, and z
directions [25]. For the ligands, 2D–3D conversion is followed by
energy minimization using the MMFF option as a force field. The
docking algorithm implemented in ICM-Pro (version 3.3-02a)
optimized the entire ligand in the receptor field, applying a
multistart Monte Carlo minimization procedure in internal
coordinate space.
In order to study the interactions, we used the docking software
ICM-Pro (version 3.3-02a) and the COX-2 enzyme protein data co-
crystallized with SC-558 (PDB entry: 1CX2). After converting the
PDB data into an ICM format, defining the receptor active site, and
generating the receptor grid maps, we were able to calculate the
interaction of our compounds with COX-2.
In anti-inflammatory study, data were expressed as value ꢃ SEM.
Results of carrageenan-induced paw edema experiments were
also expressed as percentage of change from control (pre-drug)
values. Differences between vehicle control and treatment groups
were tested using one-way ANOVA followed by multiple
comparisons using Bonferroni’s test. In ulcerogenic study, data
were presented as mean ꢃ SE and were subjected to one-way
ANOVA, followed by multiple comparisons using Bonferroni’s
test.
In vitro cyclooxygenase inhibition studies
The selected compounds were tested for their ability to inhibit
COX-1 and COX-2 using a COX-(ovine) inhibitor screening kit
(Catalog No. 560101, Cayman Chemical, Ann Arbor, MI) according
to the manufacturer’s instructions. Cyclooxygenase catalyzes the
first step in the biosynthesis of arachidonic acid (AA) to PGH2.
PGF2a, produced from PGH by reduction with stannous chloride,
is measured by EIA (ACE competitive EIA). Stock solutions of test
compounds were dissolved in a minimum volume of DMSO.
Briefly, to a series of supplied reaction buffer solutions (960 mL,
0.1 M Tris–HCl pH 8.0 containing 5 mM EDTA and 2 mM phenol)
with either COX-1 or COX-2 (10 mL) enzyme in the presence of
heme (10 mL) was added 10 mL of various concentrations of test
drug solutions (0.001, 0.01, 0.1, 1, 10, 100, and 500 mM in a final
volume of 1 mL). These solutions were incubated for a period of
2 min at 37°C, after which 10 mL of arachidonic acid (100 mM) was
added, and the COX reaction was stopped by the addition of 50 mL
of 1 M HCl after 2 min. PGF2a, produced from PGH by reduction
with stannous chloride, was measured by EIA. This assay is based
on the competition between PGs and a PG-acetylcholinesterase
conjugate (PG tracer) for a limited amount of PG antiserum. The
amount of PG tracer that is able to bind to the PG antiserum is
inversely proportional to the concentration of PGs in the wells
since the concentration of the PG tracer is held constant while the
concentration of PGs varies. This antibody PG complex binds to a
mouse anti-rabbit monoclonal antibody that had been previously
attached to the well. The plate is washed to remove any unbound
reagents and then Ellman’s reagent, which contains the substrate
to acetylcholinesterase, is added to the well. The product of this
enzymatic reaction produces a distinct yellow color that absorbs
at 405 nm. The intensity of this color, determined spectrophoto-
metrically, is proportional to the amount of PG tracer bound to
the well, which is inversely proportional to the amount of PGs
present in the well during incubation: absorbance [bound PG
tracer] 1/PGs. Percent inhibition was calculated by comparison of
compound treated to various control incubations. The concen-
tration of the test compound causing 50% inhibition (IC50, mM)
was calculated from the concentration inhibition response curve
(duplicate determinations).
The authors extend their appreciation to The Regional Centre for
Mycology and Biotechnology, Al Azhar University, Cairo, Egypt for
providing the facilities to perform the biological evaluation.
The authors have declared no conflict of interest.
References
[1] I. Tabas, C. K. Glass, Science 2013, 339, 166–172.
[2] M. M. Hassan, S. A. Khan, A. H. Shaikat, M. E. Hossain,
M. A. Hoque, M. H. Ullah, S. Islam, Vet. World 2013, 6,
68–71.
[3] M. J. Langman, D. M. Jensen, D. J. Watson, S. E. Harper, P.-L.
Zhao, H. Quan, J. A. Bolognese, T. J. Simon, JAMA 1999, 282,
1929–1933.
[4] D. J. Cochrane, B. Jarvis, G. M. Keating, Drugs 2002, 62, 2637–
2651.
[5] A. Di Fiore, C. Pedone, K. D’Ambrosio, A. Scozzafava, G. De
Simone, C. T. Supuran, Bioorg. Med. Chem. Lett. 2006, 16, 437–
442.
[6] S. Dadiboyena, A. T. Hamme Ii, Curr. Org. Chem. 2012, 16,
1390–1407.
[7] R. J. Bing, M. Lomnicka, J. Am. Coll. Cardiol. 2002, 39, 521–
522.
[8] R. G. Kurumbail, A. M. Stevens, J. K. Gierse, J. J. McDonald, R.
A. Stegeman, J. Y. Pak, D. Gildehaus, J. M. iyashiro, T. D.
Penning, K. Seibert, P. C. Isakson, W. C. Stallings, Nature 1996,
384, 644–648.
[9] Y. K. Rao, S.-H. Fang, Y.-M. Tzeng, Biorg. Med. Chem. 2009, 17,
7909–7914.
[10] F. Herencia, M. L. Ferrándiz, A. Ubeda, I. Guillén, J. N.
Dominguez, J. E. Charris, G. M. Lobo, M. J. Alcaraz, Free Radic.
Biol. Med. 2001, 30, 43–50.
Molecular docking methodology
Docking experiments were performed using ICM-Pro software
(version 3.3-02a) by Molsoft. The coordinates for the X-ray crystal
structure of the enzymes COX-2 were obtained from the RCSB
Protein Data Base (COX-2 PDB file: 1CX2) [8]. The structure data
were revised by adding the missing aromatic p systems and ionic
attributes to the amino acids in the binding pocket and by adding
hydrogen to all amino acids. The revised protein data were
converted into the ICM format; the binding pockets were defined
by searching the neighbors of the co-crystallized structure of COX-
[11] O. I. El-Sabbagh, S. M. Ibrahim, M. M. Baraka, H. Kothayer,
Arch. Pharm. 2010, 343, 274–281.
[12] A. Araico, M. C. Terencio, M. J. Alcaraz, J. N. Domínguez, C.
León, M. L. Ferrándiz, Life Sci. 2006, 78, 2911–2918.
[13] M. Ismail, J. Lehmann, D. Abou El Ella, A. Albohy, K. Abouzid,
Med. Chem. Res. 2009, 18, 725–744.
[14] H. A. Abdel-Aziz, K. A. Al-Rashood, K. E. H. ElTahir, H. S.
Ibrahim, J. Chin. Chem. Soc. 2011, 58, 863–868.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2013, 346, 1–11
New Arylpyrazoles as Anti-Inflammatory Agents
11
[15] A. Araico, M. C. Terencio, M. J. Alcaraz, J. N. Domínguez, C.
[20] K. S. G. Vijaya, D. N. Mishra, Methods Find. Exp. Clin. Pharmacol.
2006, 28, 419–422.
León, M. L. Ferrándiz, Life Sci. 2007, 80, 2108–2117.
[16] G. S. B. Viana, M. A. M. Bandeira, F. J. A. Matos, Phytomedicine
2003, 10, 189–195.
[21] K. M. Amin, M. M. Kamel, M. M. Anwar, M. Khedr, Y. M. Syam,
Eur. J. Med. Chem. 2010, 45, 2117–2131.
[17] E. Nassar, H. A. Abdel-Aziz, H. S. Ibrahim, A. M. Mansour, Sci.
Pharm. 2011, 79, 507–524.
[22] O. Llorens, J. J. Perez, A. Palomer, D. Mauleon, J. Mol. Graph.
Modell. 2002, 20, 359–371.
[18] H. A. Abdel-Aziz, A. Bari, S. W. Ng, Acta Crystallogr. Sect. E 2010,
66, o3344.
[23] R. S. Tewari, P. Parihar, Indian J. Chem. Sect. B 1980, 19, 217–
218.
[19] C. A. Winter, E. A. Risley, G. W. Nuss, Proc. Soc. Exp. Biol. Med.
1962, 111, 544–547.
[24] A. A. Fahmi, Int. J. Chem. 1995, 6, 1–4.
[25] V. Maiorov, R. Abagyan, Fold. Des. 1998, 3, 259–269.
ß 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com