Synthesis and biological evaluation of new pyrazolone-pyridazine conjugates as anti-inflammatory and analgesic agents

Article abstract of DOI:10.1016/j.bmc.2014.02.042

A new series of pyrazolone-pyridazine conjugates 3 and 4a-l were synthesized and characterized by spectroscopic means and elemental analyses. All compounds were tested in vivo for their anti-inflammatory and analgesic properties against diclofenac, as reference compound. The synthesized compounds were also evaluated for their ability to inhibit the production of certain inflammatory cytokines such as TNF-α and IL-6 in serum samples. The ulcerogenic potential of the synthesized compounds was also determined. IC ₅₀ values for inhibition of COX-1 and COX-2 enzymes were investigated in vitro for the most active candidates. Molecular docking was performed on the active site of COX-2 to predict their mode of binding to the amino acids. Among the synthesized derivatives, compounds 4c and 4e showed good analgesic and anti-inflammatory activities with lower ulcer index than the reference drug.

Full text of DOI:10.1016/j.bmc.2014.02.042

Bioorganic & Medicinal Chemistry 22 (2014) 2080–2089
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of new pyrazolone–pyridazine
conjugates as anti-inflammatory and analgesic agents
Nadia Abdalla Khalil a, Eman Mohamed Ahmed a, Khaled Omar Mohamed a, Yassin Mohammed Nissan b,
,
⇑
Sawsan Abo-Bakr Zaitone c
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Cairo University, Kasr Elini st., Cairo 11562, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562, Egypt
c Department of Pharmacology & Toxicology, Faculty of Pharmacy, Suez Canal University, Ismailia 41522, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
A new series of pyrazolone–pyridazine conjugates 3 and 4a–l were synthesized and characterized by
spectroscopic means and elemental analyses. All compounds were tested in vivo for their anti-inflamma-
tory and analgesic properties against diclofenac, as reference compound. The synthesized compounds
were also evaluated for their ability to inhibit the production of certain inflammatory cytokines such
Received 17 December 2013
Revised 11 February 2014
Accepted 19 February 2014
Available online 3 March 2014
as TNF-a and IL-6 in serum samples. The ulcerogenic potential of the synthesized compounds was also
determined. IC50 values for inhibition of COX-1 and COX-2 enzymes were investigated in vitro for the
most active candidates. Molecular docking was performed on the active site of COX-2 to predict their
mode of binding to the amino acids. Among the synthesized derivatives, compounds 4c and 4e showed
good analgesic and anti-inflammatory activities with lower ulcer index than the reference drug.
Ó 2014 Elsevier Ltd. All rights reserved.
Keywords:
Pyrazolone
Pyridazine
Analgesic
Anti-inflammatory
1. Introduction
possess an adverse ulcerogenic effect.9,10 Chronic use of NSAIDs
may induce GI haemorrhage, ulceration, perforation as well as
Non-steroidal anti-inflammatory drugs (NSAIDs) are a class of
compounds used for the treatment of pain, fever and inflammation,
particularly arthritis.1,2 The pharmacological activity of NSAIDs is
related to the suppression of prostaglandin biosynthesis from ara-
chidonic acid by inhibiting cyclooxygenases (COXs), which exist in
three distinct isoforms: COX-1, COX-2 and COX-3.3–5 The COX-1 is
a constitutive enzyme and is responsible for the production of
cytoprotective prostaglandins in the gastrointestinal (GI) tract,
thrombogenesis and homeostasis, while COX-2 is an inducible en-
zyme, which is induced in response to the release of several pro-
inflammatory mediators such as endotoxins, mitogens or cytokines
nephrotoxicity, which greatly limit their therapeutic useful-
ness.11,12 Therefore, drugs that selectively inhibit COX-2 isoform
(coxibs) have been marketed as a new generation NSAIDs.13 How-
ever, prospective examination of coxibs has revealed unexpected
cardiovascular adverse effects such as myocardial infarction.14
COX-2 selective inhibition also will lead to depletion of PGI2 and
further increase in thromboxane formation which may cause
atherosclerosis.15,16 Several clinical studies showed an increased
cardiovascular risks in treated patients with coxibs compared to
others treated with traditional NSAIDs by five folds.17,18 On the
other hand, coxibs showed similar renal side effects to that exhib-
ited by traditional NSAIDs as they reduce acutely medullary blood
flow, sodium excretion and urine volume.19 Therefore, there exists
an unmet medical need to develop novel gastrointestinal-sparing
NSAIDs with an improved safety profile.
Since the development of antipyrine, the first pyrazoline deriv-
ative used in the management of pain, inflammation and fever,
great attention has been focused on pyrazoline derivatives as a
potent class of anti-inflammatory, analgesic and antipyretic
agents,20–23, (Fig. 1). However, some potent analgesic and anti-
inflammatory pyrazoline derivatives including dipyrone and phe-
nylbutazone possess GI side effects that limit the clinical use of
these drugs.24
including tumor necrosis factor
a (TNFa) and interleukins (ILs)
such as IL-6 and IL-1.6,7 Further, COX-3 which is considered as a
variant of COX-1, is present in central nervous system and has been
proposed to be another target for anti-inflammatory agents.8 How-
ever, the beneficial effects of NSAIDs are usually balanced by their
side effects especially on the GI system due to inhibition of COX-1
isoform. Many NSAIDs interact with both COX-1 and COX-2 iso-
forms and non-selectively inhibit their enzymatic activity, leading
to reduction of prostaglandins PGE2 and PGI2 production, which
⇑
Corresponding author. Tel.: +20 1000848844.
E-mail address: yassin.nissan@hotmail.com (Y.M. Nissan).
http://dx.doi.org/10.1016/j.bmc.2014.02.042
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

N. A. Khalil et al. / Bioorg. Med. Chem. 22 (2014) 2080–2089
2081
survey showed that pyridazine and pyrazole cores were often used
as versatile scaffolds to develop new compounds endowed with
interesting pharmacological properties against cyclooxygenase
and lipoxygenase pathways.28–30
Encouraged with the well-documented anti-inflammatory
properties associated with these heterocyclic cores, herein we re-
port the synthesis of new pyrazolone derivatives in combination
with pyridazine scaffold. The analgesic and anti-inflammatory
activities were investigated for the title compounds utilizing the
acetic acid-induced writhing test and the carrageenan-induced
hind paw edema test, respectively. All the compounds were also
evaluated for the irritative and ulcerogenic action on gastric
mucosa.
O
O
O
O
N
N
N
N
N
N
NH
Antipyrine
Remifenazone
Phenylbutazone
NH
NH
XR
N
N
N
N
N
N
N
H
O
O
2. Results and discussion
2.1. Chemistry
4a-l
Famprofazone
Figure 1. Some reported pyrazolone-containing anti-inflammatory agents and the
newly synthesized compounds.
The synthetic route to the target compounds 3 and 4a–l is out-
lined in Scheme 1. Briefly, 3-(2-aminophenylamino)-1H-pyrazol-
5(4H)-one 1, was prepared according to a procedure previously
described.31 Reaction of 1 with an equimolar amount of the
commercially available 3,6-dichloropyridazine was achieved via
On the other hand, a large number of pyridazine and pyridazi-
none derivatives have been reported as analgesic and anti-inflam-
matory agents without gastrointestinal side effect.25–27 Literature
NH2
NH
NH
Cl
a
N
N
Cl
NH
Cl
N
N
N
N
N
H
N
H
O
O
1
2
b
c
NH
OC2H5
N
N
NH
N
NH
NH
XR
N
N
N
O
H
N
3
N
H
O
4a-l
CH3
XR=
4a=
HN
HN
4g=
4h=
4i=
N
NCH3
OCH3
NO2
N
4b=
4c=
4d=
4e=
4f=
N
O
HN
Cl
HN
SO2NH2
HN
4j=
4k=
H2NOC
Cl
HN
HN
HN
F
H2NOC
O
4l=
Scheme 1. Reagents and conditions: (a) isopropanol/K2CO3/reflux 4 h; (b) sodium ethoxide/ethanol, reflux 30 min; (c) amine/n-butanol/reflux 6 h.

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N. A. Khalil et al. / Bioorg. Med. Chem. 22 (2014) 2080–2089
nucleophilic displacement of chlorine to give 2, which is the key
intermediate for the production of the title compounds. The struc-
ture of compound 2 was confirmed by elemental and spectral anal-
yses. IR spectrum for compound 2 revealed a band at 2954 cmꢀ1
corresponding to aliphatic CH of the pyrazoline ring and a band
at 1643 cmꢀ1 corresponding to (C@O) group. 1H NMR spectrum
for compound 2 showed a singlet signal at d 2.58 ppm correspond-
ing to CH2 of pyrazoline ring and an increase in aromatic proton
multiplet signals at d 7.11–7.50 ppm. The latter was further re-
acted with sodium ethoxide to give the corresponding O-ethyl
derivative 3, which showed the appearance of a triplet and a quar-
tet signals in the 1H NMR spectrum at d 1.23 ppm and 3.87 ppm,
assignable to CH3 and CH2 protons, respectively of –OCH2CH3
function.
On the other hand, reaction of 2 with a number of primary and
secondary amines afforded the title compounds 4a–l. Analytical
and spectral data (IR, 1H NMR, 13C NMR and mass spectra) of the
synthesized compounds were in full agreement with the proposed
structures.
2.2. Molecular docking
In 2003, it was reported that a series of anti-inflammatory drugs
inhibits PGE2 synthesis by interacting with Tyr 385 and Ser 530 in
the active site of COX-2 enzyme and that compounds which inter-
act with such amino acids exhibit anti-inflammatory activity.32
Moreover, interactions with Arg 120 may also play a role in the
inhibitory effect.32 Tyr-385 was found to be essential for the cyclo-
oxygenase activity of PGH synthase and that nitration of this resi-
due could be prevented by indomethacin. We conclude that Tyr
385 is at or near the cyclooxygenase active site of PGH synthase
and the tyrosine residue could be involved in the first step of the
cyclooxygenase reaction, which is the abstraction of the 13-proS
hydrogen from arachidonate.33 Acetylation of Ser 530 of sheep
prostaglandin endoperoxide (PGG/H) synthase by aspirin causes
irreversible inactivation of cyclooxygenase enzyme. Thus, Ser 530
does lie in a highly conserved region, probably involved in cycloox-
ygenase catalysis.34 Arg 120 is located near the mouth of the
hydrophobic channel that forms the cyclooxygenase active site of
prostaglandin endoperoxide H synthases COX-1 and COX-2. Arg
120 forms an ionic bond with the carboxylate group of arachido-
nate and this interaction is an important contributor to the overall
strength of arachidonate binding to COX-1.35
Figure 2. Diclofenac 2D interactions with active site of COX-2.
Molecular docking of all synthesized compounds was per-
formed on the active site of COX-2 enzyme. To perform accurate
validation of the docking protocol, docking of the co-crystallized li-
gand (Diclofenac) should be carried out to study the scoring energy
(S), root mean standard deviation (rmsd) and amino acid interac-
tions. Docking was performed using London dG force and refine-
ment of the results was done using Force field energy. Diclofenac
is fitted in the active site pocket with S = ꢀ11.9408 kcal/mol and
rsmd = 0.3682. Diclofenac interacts with the two amino acids Tyr
385 and Ser 530 by three hydrogen bonds, the two hydrogen bonds
lengths with Ser 530 were 2.7 Å and 2.9 Å while that with Tyr 385
was 2.7 Å as displayed in (Figs. 2 and 3). The dichlorophenyl group
forms van der Waals interactions with Val-349, Ala-527, Leu-531,
and Val-523. Following the aforementioned docking protocol, all
the synthesized compounds were docked on the active site of
COX-2 enzyme. Docking scores, amino acids interactions were
summarized in Table 1.
Figure 3. Diclofenac 2D interactions with active site of COX-2.
exhibited interaction with three amino acids of the active site of
COX-2. The 2D interactions of the compounds 4c and 4d were dis-
played in Figures 4 and 5, respectively.
In light of good docking scores as well as good amino acid inter-
actions displayed by all synthesized compounds, especially 4b–e, it
could be concluded that these compounds might possess promis-
ing anti-inflammatory activity.
2.3. Biological screening
2.3.1. Analgesic activity
All synthesized compounds were fit on the active site of COX-2
enzyme with energy scores range ꢀ11.3566 to ꢀ12.9831 kcal/mol.
Compound 4c showed the best docking score of ꢀ12.9831 kcal/
mol. Compounds 2, 3, 4a and 4f showed interaction with one ami-
no acid of the active site of COX-2, while compounds 4b and 4e
interacted with two amino acids. Further, compounds 4c and 4d
In the present study, the test compounds showed variable de-
grees of percentage inhibition against acetic acid induced writhing.
Diclofenac sodium induced higher degree of protection against
writhing (83.3 3.5%) compared to the tested compounds. Com-
pounds 4b, 4c, 4d and 4e produced significant inhibition of
55.6 5.1, 63.7 5.4, 57.2 4.3 and 68.3 5.3%, respectively

N. A. Khalil et al. / Bioorg. Med. Chem. 22 (2014) 2080–2089
2083
Table 1
4d and 4e significantly reduced its serum level compared to the
control group. Similarly, compounds 4b, 4c, 4d and 4e showed sig-
nificant reduction in serum IL-6 level compared to vehicle treated
mice, (Table 2).
Binding scores and amino acid interactions of the docked compounds on the active
site of COX-2 enzyme
Compound No. S kcal/mol Amino acid interactions Interacting groups
2
3
4a
4b
ꢀ11.3973
ꢀ11.3566
ꢀ11.4265
ꢀ12.2006
Ser 530a
Ser 530a
Ser 530a
Arg 120a
Ser 530a
Arg 120b
Ser 119a
Tyr 355a
Arg 120a
Ser 530a
Tyr 385a
Tyr 385a
Ser 530a
Ser 530a
Arg 120a
Arg 120a
Tyr 355a
Arg 120a
Arg 120a
Arg 120a
NH
C@O (H2O)
C@O
2.3.3. In vitro COX-1 and COX-2 inhibition assay
In an attempt to explore the mechanism of anti-inflammatory
effect produced by the newly synthesized compounds, in vitro
assay for COX-1 and COX-2 enzymes inhibition was conducted.
The most active compounds 4b, 4c, 4d, 4e were screened adopt-
ing enzyme-immuno assay (EIA) kit (Cayman Chemical Com-
pany, USA). The COX-1 and COX-2 inhibitory activity of the
compounds was evaluated following a method described by Gau-
tam et al.36 IC50 values for the most active compounds were
determined using celecoxib as the reference drug. Furthermore,
selectivity index for COX-1/ COX-2 inhibition was calculated
for each compound. IC50 values for COX-1 and COX-2 inhibition
as well as the selectivity index were listed in Table 3. From the
screening results, it was obvious that compounds 4b, 4c, 4d and
4e showed good inhibitory activity on both COX-1 and COX-2
N pyrazoline
C@O (H2O)
Pyridazine
NH
N pyridazine
N pyridazine
C@O (H2O)
NH
4c
4d
4e
ꢀ12.9831
ꢀ12.7081
ꢀ12.7081
C@O
C@O(H2O)
C@O(H2O)
N pyrazoline
N pyrazoline
N pyrazoline
N pyrazoline
N pyrazoline
N pyrazoline
4f
4g
4h
4i
4j
4k
4l
ꢀ11.3094
ꢀ11.4281
ꢀ11.4730
ꢀ12.0538
ꢀ12.1480
ꢀ11.6912
ꢀ11.4787
enzymes with IC50 values range 2.86–4.45
0.39–0.84 M for COX-2. Among the tested compounds, 4c dem-
onstrated the highest activity with IC50 value of 2.86 M for
COX-1 and 0.39 M for COX-2, and exerting the highest selectiv-
lM for COX-1 and
a
l
H-bond.
Arene-cation.
b
l
l
ity index (SI) of 7.36.
compared to control group at P < 0.05, (Table 2). Dose response
curves representing the degree of protection at different doses,
against acetic acid induced writhing exhibited by the most active
test compounds and diclofenac sodium, are shown in Figure 6.
2.3.4. Acute ulcerogenesis
Ulcerogenicity studies revealed that the most active compounds
4b, 4c, 4d and 4e, showed high ulcerogenic indices compared to
the vehicle group. However, the indices registered with com-
pounds 4b and 4e were significantly lower than diclofenac sodium.
Further, the scores assigned for the histologic sections of mice gas-
tric mucosa treated with compounds 4b, 4c, 4d and 4e revealed
that all the four compounds produced significantly higher histo-
logic scores compared to the vehicle group. Compound 4d exhib-
ited histologic score almost similar to that showed with mice
treated with diclofenac sodium (Fig. 7).
2.3.2. Anti-inflammatory activity
Screening the anti-inflammatory activity for the selected
compounds that demonstrated the most promising results in the
writhing test 4b, 4c, 4d and 4e, revealed that these compounds ex-
ert good to moderate anti-inflammatory effect ranging from
62.3 5.8% to 35.4 2.8%, (Table 2). Furthermore, determination
of serum TNF-
a level highlighted that the compounds 4a, 4b, 4c,
Figure 4. Compound 4c on the active site of COX-2 enzyme.

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N. A. Khalil et al. / Bioorg. Med. Chem. 22 (2014) 2080–2089
Figure 5. Compound 4d on the active site of COX-2 enzyme.
Table 2
Percentage protection against writhing and percentage inhibition of carrageenan-induced paw edema method Effect of diclofenac sodium and the test compounds on serum TNF-
and IL-6 levels in mice tested by carrageenan-induced paw edema method
a
Compound
Analgesic activity (% protection)a
Anti-inflammatory activity (% inhibition)a
TNF-a
(ng/ml)b
IL-6 (ng/ml)b
Vehicle
Diclofenac sodium
2
3
4a
4b
4c
4d
4e
4f
4g
4h
4i
0
0
15.48 0.31
5.01 0.73⁄
13.2 1.2
12.4 1.1
9.34 0.83⁄
7.5 0.54⁄
7.2 0.43⁄
6.4 0.48⁄
7.12 0.57⁄
11.2 1.3
12.4 1.2
14.4 1.3
13.5 1.2
16.7 1.5
12.5 0.94
11.98 1.3
23.8 2.5
7.6 0.35⁄
21.4 1.8
22.4 2.5
19.5 1.6
16.5 1.3⁄
11.8 0.87⁄
10.65 0.74⁄
9.76 0.58⁄
17.3 1.2
18.4 1.4
25.7 1.2
18.3 1.2
21.43 1.5
22.3 1.9
22.6 2.1
83.3 3.5⁄
33.4 2.8⁄
18.2 1.6
41.2 3.8⁄
55.6 5.1⁄⁄
63.7 5.4⁄
57.2 4.3⁄
68.3 5.3⁄
36.1 2.9⁄
35.32 3.2⁄
19.43 1.2
13.2 1.1
11.3 1.3
13.2 1.2
14.2 1.2
61.5 5.14⁄
11.2 1.1
10.2 0.92
35.3 3.2⁄
59.4 5.3⁄
62.3 5.8⁄
54.3 4.7⁄
35.4 2.8
11.2 2.3
12.4 1.6
7.3 0.5
5.4 0.87
15.4 1.5
18.2 2.1
9.3 2.4
4j
4k
4l
Bold values indicate the most active compound in each assay.
a
The vehicle group was pretreated with 1% administered orally carboxymethylcellulose solution. The test compounds were (25 mg/kg) thirty minutes prior to testing. Data
are presented as mean SEM and analyzed using one-way ANOVA followed by Bonferroni’s post hoc test. ⁄P < 0.05 compared to the vehicle group.
b
All compounds were administered to mice (25 mg/kg, p.o.) thirty minutes prior to testing. TNF-a: tumor necrosis factor-a, IL-6: interlukin-6. Data are presented as
mean SEM and analyzed using one-way ANOVA followed by Bonferroni’s post hoc test for multiple comparisons.⁄P < 0.05 compared to the vehicle group.
Based on these biological results, It was clear that compound 2
showed a weak anti-inflammatory effect of 11.2% rat paw edema
inhibition and a moderate analgesic effect of 33.4% protection in
writhing test. Both activities were decreased with the ethoxy
derivative 3 to 10.2% and 18.2% for rat paw edema inhibition assay
and writhing test, respectively. On the other hand, when the eth-
oxy substituent in compound 3 was substituted by the N-methyl-
piperazine scaffold, compound 4a, the anti-inflammatory and
analgesic activities were markedly raised to more than three folds
(35.3% and 41.2% respectively). Moreover, the results showed that
other aliphatic amines such as piperidine and morphline, com-
pounds 4b and 4c respectively, also 2- and 4-chloro substituted
aromatic amines, compounds 4d and 4e respectively exhibited a
remarkable activity. However, replacement of the ethoxy group
in compound 3 by certain aromatic amines such as 2-flouroaniline
4f, 3-methylaniline 4g, 4-methoxyaniline 4h, 3-nitoaniline 4i, or
amides including sulfanilamide 4j, 2-aminobenazmide 4k and 2-
hydroxybenzamide 4l, did not improve the activity. Among all
tested compounds, 4c was found to be the most active compound
with 62.3% inhibition in rat paw edema test and 55.6% protection
in writhing test. Compounds 4b, 4d and 4e showed 59.4%, 54.3%
and 35.4%, respectively in rat paw edema test and 55.6%, 57.2%
and 68.3%, respectively in writhing test. Regarding ulcergenicity,
compounds 4b and 4e showed lower ulcerogenic effect than the
reference drug diclofenac. Moreover, 4e demonstrated the least
ulcerogenic potential among all tested compounds. Compounds
4c and 4d were the most active compounds regarding their effect
on the inhibition of TNF-a and IL-6 production in serum. Addition-
ally, compound 4c was the most potent inhibitor of COX-1 and
COX-2 with selectivity index (SI) of 7.3. Also compound 4c showed
the best docking score of -12.9831 kcal/mol. and three amino acids
interaction with the active site of COX-2 enzyme in the molecular
docking study.
These results can lead us to some conclusions about structure
activity relationship of the newly synthesized compounds that
can be summarizes as: neither substitution on pyridazine ring with

N. A. Khalil et al. / Bioorg. Med. Chem. 22 (2014) 2080–2089
2085
a
c
b
d
e
Figure 6. Dose response curves for the percentage protection calculated after administration diclofenac sodium and the most active test compounds (4b, 4c, 4d and 4e) in the
writhing test. 0.1% acetic acid solution was injected intraperitoneally to induce writhing in mice. The different compounds were tested at 10, 15, 20, 25 and 30 mg/kg (p.o.) in
male albino mice. Data are presented as mean SEM.
Table 3
derivative 4d and when chloro substitution to para position activ-
ity has remarkably decreased. Also substititutions with more elec-
COX-1 and COX-2 inhibition IC50 and selectivity index (SI)
Compound
IC50
(
lM)
Selectivity index (SI)
tron withdrawing groups like flouro, methoxy, nitro, sulfamoyl or
carboxamide substituents and electron donating group like methyl
on ortho, meta or para positions led to major decrease in activity
4e–k. The biological evaluation of ortho carboxamido ether deriv-
ative 4l showed also decreased both anti-inflammatory and anal-
gesic activities.
COX-1
COX-2
4b
4c
4d
4e
3.15
2.86
4.02
4.46
>30
0.50
0.39
0.72
0.84
0.22
6.31
7.36
5.51
5.30
>200
Celecoxib
Molecular docking study revealed that all the newly synthe-
sized compounds have interactions with the amino acids of the ac-
tive site of COX-2 enzyme. However these interactions fall into
three categories according to the number of interacted amino acids
which can be summarized as: The first category of compounds
interacted with three amino acids on the active site. These com-
pounds are 4c and 4d which showed very good anti-inflammatory
activity. The second category interacts with two amino acids of the
active site as presented by compound 4b and 4e with also good
biological activity less than or approximate to the activity of the
first category. The third category interacts with only one amino
acid of the active site with much less activity as presented by com-
pounds 2, 3 and 4f–l.
electron withdrawing chloro 2 nor the more bulky ethoxy substitu-
ent 3 showed good anti-inflammatory or analgesic activity. On the
other hand, substitution with bulky aliphatic secondary amines
4a–c showed very good anti-inflammatory and analgesic activity
with the highest activity attributed to the morpholine derivative
4c decreased with the more lipophilic and more electron with-
drawing piperidine derivative 4b and much falling with the lipo-
philic and bulkier methyl piperazine derivative 4a. The last series
of substitutions comprising aromatic amine substituents 4d–k
showed that the most active compound was the ortho chloro

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N. A. Khalil et al. / Bioorg. Med. Chem. 22 (2014) 2080–2089
with UV fluorescent silica gel Merck 60F 254 using acetone/ben-
zene (1:9) and were visualized using UV lamp.
All chemicals were obtained from Aldrich, Fluka, or Merck
chemicals. The starting material 3-(2-aminophenylamino)-1H-pyr-
azol-5(4H)-one (1) was prepared as reported.31
a
b
*
3
2
1
*
*
#
*
#
*
4.1.1. 3-[2-(6-Chloropyridazin-3-ylamino)phenylamino]-1H-
pyrazol-5(4H)-one (2)
A mixture of 1 (3.02 g, 0.01 mol), 3,6-dichloropyridazine (1.48 g,
0.01 mol) and anhydrous K2CO3 (1.96 g, 0.02 mol) in isopropanol
(30 mL) was heated under reflux for 4 h. The reaction mixture
was concentrated under reduced pressure to half its volume. The
precipitated crystalline solid was filtered, washed with water,
and recrystallized from ethanol to give 2.
0
*
15
10
5
*
*
*
*
Yield: 80%; mp 151–152 °C; IR (KBr) (cmꢀ1): 3350–3260 (NH),
3182–3055 (aromatic CH), 2954 (aliphatic CH), 1643 (C@O); 1H
NMR (300 MHz, DMSO-d6) d ppm: 2.58 (s, 2H, CH2 pyrazole),
7.11–7.50 (m, 6H, aromatic CH), 8.60 (s, 1H, NH, D2O exchangeable),
9.60 (s, 1H, NH, D2O exchangeable), 10.41 (s, 1H, NH, D2O exchange-
able); 13C NMR (100.63 MHz, DMSO-d6) d ppm: 35.06(1), 115.07(3),
121.86(4), 139.06(1), 149.72(3), 167.24(1); EIMS (% rel abundance):
304 [M+2] (27.54), 302 [M+] (25.42). Anal. Calcd for C13H11ClN6O: C,
51.58; H, 3.66; N, 27.76; Found C, 51.50; H, 3.62; N, 27.70.
0
4.1.2. 3-[2-(6-Ethoxypyridazin-3-ylamino)phenylamino]-1H-
pyrazol-5(4H)-one (3)
Figure 7. Ulcerogenic index after macroscopic examination of the gastric mucosa
and histology score for histologic sections stained with hematoxylin and eosin. The
test compounds were administered orally (50 mg/kg) to fasting mice. Data are
presented as mean SEM and analyzed using one-way ANOVA followed by
Bonferroni’s post hoc test. ⁄P < 0.05 compared to the vehicle, #P < 0.05 compared
to diclofenac sodium.
An appropriate 2 (0.312 g, 0.001 mol) in absolute ethanol
(25 mL) containing metallic sodium (0.46 g, 0.02 mol) was heated
under reflux for 0.5 h. The reaction mixture was concentrated un-
der reduced pressure, poured into ice cold water then acidified by
dilute hydrochloric acid. The precipitated crystalline solid was fil-
tered, washed with water, and recrystallized from ethanol.
Yield: 66%; mp 259–260 °C; IR (KBr) (cmꢀ1): 3380–3255 (NH),
3187–3032 (aromatic CH), 2924–2854 (aliphatic CH), 1697
(C@O); 1H NMR (300 MHz, DMSO-d6) d ppm: 1.23 (t, 3H,CH3),
2.51 (s, 2H, CH2 pyrazole), 3.87 (q, 2H, CH2), 7.10–7.50 (m, 6H, aro-
matic CH), 9.40 (s, 1H, NH, D2O exchangeable), 10.20 (s, 1H, NH,
D2O exchangeable), 12.21 (s, 1H, NH, D2O exchangeable); 13C
NMR (100.63 MHz, DMSO-d6) d ppm: 21.53(1), 34.63(1), 49(1),
115.09(2), 122.21(4), 138.54(1), 149.00(2), 168.46(2), 172.49(1);
EIMS (% rel abundance): 314 [M+2] (4.99), 313 [M+1] (6.82), 312
[M](4.72). Anal. Calcd for C15H16N6O2: C, 57.68; H, 5.16; N, 26.91;
Found C, 57.62; H, 5.10; N, 26.88.
3. Conclusion
In light of biological results, we can conclude that compounds
4b and 4c can be considered as good candidates for the search of
novel analgesic and anti-inflammatory agents urged by their
in vivo and in vitro activities and lower ulcerogenic effect com-
pared to diclofenac as the reference drug. Further, compound 4c
showed good activity as COX-1 and COX-2 inhibitor in vitro with
a selectivity index of 7.3. It also exhibited good inhibition effect
on the serum production of TNF-a and IL-6. Compound 4c showed
good docking score and three amino acids interaction with the ac-
tive site of COX-2 enzyme.
4.1.3. General procedure for 4a–l
A mixture of 2 (0.312 g, 0.01 mol), anhydrous K2CO3 (1.96 g,
0.02 mol) and the appropriate substituted amine (0.01 mol) in n-
butanol (30 mL) was heated under reflux for 4 h. The reaction mix-
ture was concentrated under reduced pressure to half its volume.
After cooling the precipitated crystalline solid was filtered, washed
with water and recrystallized from ethanol.
4. Experimental
4.1. Chemistry
All chemicals and reagents were obtained from Aldrich (Sigma–
Aldrich, St. Louis, MO, USA), and were used without further purifi-
cation. Reactions were monitored by TLC, performed on silica gel
glass plates containing 60 GF-254, and visualization on TLC was
achieved by UV light or iodine indicator. IR spectra were deter-
mined on Shimadzu IR 435 spectrophotometer (KBr, cmꢀ1). 1H
NMR spectra were carried out using a Mercury 300-BB 300 MHz
using TMS as internal standard. 13C NMR spectra were carried
out using a Mercury 300-BB 75 MHz using TMS as internal stan-
dard. Chemical shifts (d) are recorded in ppm on d scale, Micro ana-
lytical Center, Cairo University, Egypt. Mass spectra were recorded
on Shimadzu Qp-2010 plus Spectrometer, Micro analytical Center,
Cairo University, Egypt. Elemental analyses were carried out at the
Micro analytical Center, Cairo University, Egypt. Melting points
were determined with Stuart apparatus and are uncorrected. Pro-
gress of the reactions was monitored using TLC sheets precoated
4.1.3.1. 3-{2-[6-(4-Methylpiperazin-1-ylamino)pyridazin-3-yla-
mino]phenylamino}-1H-pyrazol-5(4H)-one
(4a).
Yield:
70%; mp 195–196 °C; IR (KBr) (cmꢀ1): 3390–3270 (NH), 3186–
3062 (aromatic CH), 2978–2870 (aliphatic CH), 1651 (C@O); 1H
NMR (300 MHz, DMSO-d6) d ppm: 2.49 (s, 2H, CH2 pyrazole),
2.50 (s, 3H, CH3), 3.29–3.31 (m, 4H, piperazine), 3.64–3.67 (m,
4H, piperazine), 7.10–7.48 (m, 6H, aromatic CH), 8.60 (s, 1H, NH,
D2O exchangeable), 9.15 (s, 1H, NH, D2O exchangeable), 12.21 (s,
1H, NH, D2O exchangeable); 13C NMR (100.63 MHz, DMSO-d6) d
ppm: 35.10(3), 55.00(2), 117.00(1), 118.00(1), 119.00(1),
121.76(4), 127.00(1), 140.00(1), 149.74(2), 167.23(2); EIMS (% rel
abundance): 368 [M+2] (24.34), 367 [M+1] (28.76), 366 [M+]
(32.74). Anal. Calcd for C18H22N8O: C, 59.00; H, 6.05; N, 30.58;
Found C, 58.95; H, 6.01; N, 30.52.

N. A. Khalil et al. / Bioorg. Med. Chem. 22 (2014) 2080–2089
2087
4.1.3.2. 3-{2-[6-(Piperidin-1-yaminol)pyridazin-3-ylamino]phe-
nylamino}-1H-pyrazol-5(4H)-one (4b). Yield: 65%; mp 210–
4.1.3.7.
3-{2-[6-(4-Methoxyanilino)pyridazin-3-ylamino]phe-
Yield: 55%; mp
nylamino}-1H-pyrazol-5(4H)-one (4g).
211 °C; IR (KBr) (cmꢀ1): 3350–3200 (NH), 3186–3055, (aromatic
CH), 2958–2866 (aliphatic CH), 1651 (C@O); 1H NMR (300 MHz,
DMSO-d6) d ppm: 2.48 (s, 2H, CH2 pyrazole), 2.50–2.51 (m, 6H,
3CH2 piperidine, J@1.8 Hz), 3.29–3.30 (m, 4H, 2CH2 piperidine,
J@1.8 Hz), 7.10–7.47 (m, 6H, aromatic CH), 8.60 (s, 1H, NH, D2O
exchangeable), 9.40 (s, 1H, NH, D2O exchangeable), 12.2 (s, 1H,
NH, D2O exchangeable); 13C NMR (100.63 MHz, DMSO-d6) d ppm:
35.11(3), 40.61(3), 121.77(3), 149.74(4), 167.24(4), 172.52(1);
EIMS (% rel abundance): 351 [M+] (21.31). Anal. Calcd for
206–207 °C; IR (KBr) (cmꢀ1): 3354–3247 (NH), 3182–3059 (aro-
matic CH), 2962–2858 (aliphatic CH), 1651 (C@O); 1H NMR
(300 MHz, DMSO-d6) d ppm: 2.43 (s, 2H, CH2 pyrazole), 4.20 (s,
3H, CH3O), 7.01–7.49 (m, 10H, aromatic CH), 8.61 (s, 1H, NH, D2O
exchangeable), 9.36 (s, 2H, 2NH, D2O exchangeable), 12.20 (s, 1H,
NH, D2O exchangeable); 13C NMR (100.63 MHz, DMSO-d6) d ppm:
24.00(1), 35.08(1), 114.01(1), 115.02(2), 118.00(1), 121.80(6),
128.79(1), 134.50(1), 139.23(1), 149.73(2), 167.22(2), 172.43(1);
EIMS (% rel abundance): 390 [M+1] (19.78),389 [M+] (24.18). Anal.
Calcd for C20H19N7O2: C, 61.69; H, 4.92; N, 25.18; Found: C, 62.74;
H, 4.93; N, 25.23.
C18H21N7O: C, 61.52; H, 6.02; N, 27.90; Found C, 61.40; H, 6.08;
N, 27.83.
4.1.3.3. 3-{2-[6-(Morpholinoamino)pyridazin-3-ylaminol]phe-
4.1.3.8. 3-{2-[6-(3-Methylanilino)pyridazin-3-ylamino]phenyla-
nylamino}-1H-pyrazol-5(4H)-one (4c).
Yield: 75%; mp
mino}-1H-pyrazol-5(4H)-one (4h).
Yield: 70%; mp 199–
201–202 °C; IR (KBr) (cmꢀ1): 3300–3250 (NH), 3182–3055 (aro-
matic CH), 2958–2920 (aliphatic CH), 1647 (C@O); 1H NMR
(300 MHz, DMSO-d6) d ppm: 2.48 (s, 2H, CH2 pyrazole), 2.50–
2.51 (m, 4H, 2CH2 morpholine), 3.20–3.29 (m, 4H, 2CH2 morpho-
line), 7.10–7.49 (m, 6H, aromatic CH), 8.61 (s, 1H, NH, D2O
exchangeable), 9.35 (s, 1H, NH, D2O exchangeable), 10.20 (s, 1H,
NH, D2O exchangeable); 13C NMR (100.63 MHz, DMSO-d6) d ppm:
34.84(1),115(3),121.83(6),139.11(4),149.74(4),167.21(1);EIMS (%
rel abundance): 354 [M+1] (28.57), 353 [M+] (38.7). Anal. Calcd
for C17H19N7O2: C, 57.78; H, 5.42; N, 27.75; Found C, 57.76; H,
5.38; N, 27.71.
200 °C; IR (KBr) (cmꢀ1): 3356–3247 (NH), 3188–3057 (aromatic
CH), 2962–2855 (aliphatic CH), 1656 (C@O); 1H NMR (300 MHz,
DMSO-d6) d ppm: 2.48 (s, 2H, CH2 pyrazole), 2.51 (s, 3H, CH3),
7.09–7.49 (m, 10H, aromatic CH), 8.60 (s, 1H, NH, D2O exchange-
able), 10.10 (s, 2H, 2NH, D2O exchangeable), 10.21 (s, 1H, NH,
D2O exchangeable); 13C NMR (100.63 MHz, DMSO-d6) d ppm:
35.10(1), 55.11(1), 115.00(1), 118.24(1), 121.77(6), 123.00(1),
127.79(1), 131.50(1), 142.00(1), 149.74(3), 167.24(2), 172.00(1);
EIMS (% rel abundance): 374 [M+1] (17.13), 373 [M+] (25.55). Anal.
Calcd for C20H19N7O: C, 64.33; H, 5.13; N, 26.26; Found C, 64.28; H,
5.09; N, 26.21.
4.1.3.4. 3-{2-[6-(2-Chloroanilino)pyridazin-3-ylamino]phenyla-
4.1.3.9. 3-{2-[6-(3-Nitroanilino)pyridazin-3-ylamino]phenyla-
mino}-1H-pyrazol-5(4H)-one (4d).
Yield: 65%; mp 173–
mino}-1H-pyrazol-5(4H)-one (4i).
Yield: 68%; mp 195–
174 °C; IR(KBr) (cmꢀ1): 3370–3280 (NH), 3186–3035 (aromatic
CH), 2978–2866 (aliphatic CH), 1651 (C@O); 1H NMR (300 MHz,
DMSO-d6) d ppm: 2.49 (s, 2H, CH2 pyrazole), 7.15–7.50 (m, 10H,
aromatic CH), 8.60 (s, 1H, NH, D2O exchangeable), 10.20 (s, 2H,
2NH, D2O exchangeable), 10.40 (s, 1H, NH, D2O exchangeable);
13C NMR (100.63 MHz, DMSO-d6) d ppm: 34.95(1), 112.89(4),
196 °C; IR (KBr) (cmꢀ1): 3310–3250 (NH), 3186–3059 (aromatic
CH), 2958–2870 (aliphatic CH), 1651 (C@O); 1H NMR (300 MHz,
DMSO-d6) d ppm: 2.49 (s, 2H, CH2 pyrazole), 7.10–7.49 (m, 10H,
CH aromatic), 8.61 (s, 1H, NH, D2O exchangeable), 9.40 (s, 2H,
2NH, D2O exchangeable), 10.30 (s, 1H, NH, D2O exchangeable);
13C NMR (100.63 MHz, DMSO-d6) d ppm: 35.09(1), 115.91(1),
119.07(1), 121.79(6), 124.80(1), 134.50(1), 136.00(1), 147.22(1),
149.74(3), 167.23(2), 172.00(1); EIMS (% rel abundance): 405
[M+1] (18.15),404 [M+] (13.13); Anal. Calcd for C19H16N8O3: C,
56.43; H, 3.99; N, 27.71; Found: C, 56.40; H, 4.02; N, 27.67.
115.07(2),
121.83(2),
127.89(2),
128.79(4),
130.50(1),
149.21(1),152.38(1),167.38(1); EIMS (% rel abundance): 395
[M+2] (10.87), 393 [M+] (6.51). Anal. Calcd for C19H16ClN7O: C,
57.94; H, 4.09; N, 24.90; Found: C, 57.98; H, 4.12; N, 24.85.
4.1.3.5. 3-{2-[6-(4-Chloroanilino)pyridazin-3-ylamino]phenyla-
4.1.3.10.
no)phenylamino)pyridazin-3-ylamino}benzenesulfonamide
(4j).
Yield: 75%; mp 196–198 °C; IR(KBr) (cmꢀ1): 3475–3275
4-{6-[2-(5-Oxo-4,5-dihydro-1H-pyrazol-3-ylami-
mino}-1H-pyrazol-5(4H)-one (4e).
Yield: 60%; mp 169–
170 °C; IR (KBr) (cmꢀ1): 3375–3282 (NH), 3186–3040 (aromatic
CH), 2980–2868 (aliphatic CH), 1655 (C@O); 1H NMR (300 MHz,
DMSO-d6) d ppm: 2.49 (s, 2H, CH2 pyrazole), 6.57–7.49 (m, 10H,
aromatic CH), 8.60 (s, 1H, NH, D2O exchangeable), 10.10 (s, 2H,
2NH, D2O exchangeable), 10.30 (s, 1H, NH, D2O exchangeable);
13C NMR (100.63 MHz, DMSO-d6) d ppm: 34.95(1), 112.91(3),
115.07(1), 121.72(2), 127.89(4), 128.79(1), 130.50(2), 149.74(1),
157.12(2), 167.24(1), 172.13(1); EIMS (% rel abundance): 395
[M+2] (15.20), 393 [M+] (15.79). Anal. Calcd for C19H16ClN7O: C,
57.94; H, 4.09; N, 24.90; Found: C, 57.89; H, 4.06; N, 24.95.
(NH), 3113–3055 (aromatic CH), 2962–2866 (aliphatic CH), 1674
(C@O); 1H NMR (300 MHz, DMSO-d6) d ppm: 2.49 (s, 2H, CH2 pyr-
azole), 7.10–7.49 (m, 10H, CH aromatic), 7.92 (2H, NH2, D2O
exchangeable), 8.60 (s, 1H, NH, D2O exchangeable), 9.60 (s, 2H,
2NH, D2O exchangeable), 10.20 (s, 1H,NH, D2O exchangeable);
13C NMR (100.63 MHz, DMSO-d6) d ppm: 35.09(1), 112.91(1),
116.00(1), 118.17(1), 121.79(6), 128.80(1), 129.21(1), 134.50(1),
142.00(1), 149.73(2), 167.22(2), 172.00(1); EIMS (% rel abun-
dance): 440 [M+2] (0.15), 439 [M+1] (0.17), 438[M] (0.21). Anal.
Calcd for C19H18N8O3S: C, 52.05; H, 4.14; N, 25.56; Found: C,
52.10; H, 4.18; N, 25.55.
4.1.3.6. 3-{2-[6-(2-Fluoroanilino)pyridazin-3-ylamino]phenyla-
mino}-1H-pyrazol-5(4H)-one (4f).
Yield: 68%; mp 192–
193 °C; IR (KBr) (cmꢀ1): 3350–3240 (NH), 3186–3059 (aromatic
CH), 2962–2850 (aliphatic CH), 1651 (C@O); 1H NMR (300 MHz,
DMSO-d6) d ppm: 2.48 (s, 2H, CH2 pyrazole), 7.09–7.51 (m, 10H,
aromatic CH), 8.61 (s, 1H, NH, D2O exchangeable), 9.40 (s, 2H,
2NH, D2O exchangeable), 10.21 (s, 1H, NH, D2O exchangeable);
13C NMR (100.63 MHz, DMSO-d6) d ppm: 34.09(1), 108.91(1),
113.17(1), 118.34(1), 121.79(6), 125.89(1), 127.88(1), 130.50(1),
146.11(1), 149.73(2), 167.22(2), 172.33(1); EIMS (% rel abun-
dance): 377 [M+] (12.98); Anal. Calcd for C19H16FN7O: C, 60.47;
H, 4.27; N, 25.98; Found: C, 60.44; H, 4.23; N, 25.93.
4.1.3.11.
2-{6-[2-(5-Oxo-4,5-dihydro-1H-pyrazol-3-ylamino)
Yield:
phenylamino]pyridazin-3-ylamino}benzamide (4k).
75%; mp 187–188 °C; IR (KBr) (cmꢀ1): 3478–3272 (NH), 3119–
3055 (aromatic CH), 2962–2868 (aliphatic CH), 1670 (C@O); 1H
NMR (300 MHz, DMSO-d6) d ppm: 2.50 (s, 2H, CH2 pyrazole),
7.10–7.52 (m, 10H, CH aromatic), 7.90 (s, 2H, NH2, NH2, D2O
exchangeable), 8.61 (s, 1H, NH, D2O exchangeable), 9.45 (s, 2H,
2NH, D2O exchangeable), 10.20 (s, 1H, NH, D2O exchangeable);
13C NMR (100.63 MHz, DMSO-d6) d ppm: 32.46(1), 114.77(1),
119.61(1), 122.21(1), 124.57(1), 125.43(1), 127.47(1), 127.70(3),

2088
N. A. Khalil et al. / Bioorg. Med. Chem. 22 (2014) 2080–2089
129.26(1), 134.46(1), 135.68(1), 138.03(1), 144.71(1), 145.94(1),
147.34(1), 157.60(1), 160.72(1), 171.23(1); EIMS (% rel abun-
dance): 404 [M+2] (9.84), 403 [M+1] (9.84), 402[M] (11.64). Anal.
Calcd for C20H18N8O2: C, 59.69; H, 4.51; N, 27.85; Found: C,
59.73; H, 4.57; N, 27.82.
4.3.3. Analgesic activity
Analgesic activity was tested using acetic acid-induced writhing
method.37 Mice were habituated for 30 min individually in the
testing cage prior to acetic acid injection. Screening of the analgesic
activity was performed after administration of the test compounds
(25 mg/kg, p.o.) and compared to an equal dose of diclofenac so-
dium as the reference control. Control mice received oral doses
of 1% CMC suspension. Thirty minutes after drug administration,
acetic acid solution (0.1 mL, 1%, ip) was given to each mouse to in-
duce writhing.
The total number of abdominal contractions (writhing move-
ments) was counted for the next 15 min, starting with the 5th min-
ute after acetic acid injection. The analgesic activity was expressed
as follows:
4.1.3.12.
2-{6-[2-(5-Oxo-4,5-dihydro-1H-pyrazol-3-ylamino)
(4l). Yield:
phenylamino]pyridazin-3-yloxy}benzamide
70%; mp 171–172 °C; IR (KBr) (cmꢀ1): 3470–3278 (NH), 3115–
3058 (aromatic CH), 2962–2869 (aliphatic CH), 1674 (C@O); 1H
NMR (300 MHz, DMSO-d6) d ppm: 2.49 (s, 2H, CH2 pyrazole),
7.10–7.85 (m, 10H, CH aromatic), 8.21 (2H, NH2, D2O exchange-
able), 8.52 (s,1H, NH, D2O exchange), 9.80 (s,1H,NH, D2O exchange-
able), 10.80 (s, 1H, NH, D2O exchangeable); 13C NMR (100.63 MHz,
DMSO-d6)
d
ppm: 32.57(1), 119.63(1), 127.08(3), 127.24(2),
% Analgesic activity = {(n–n0)/n} ꢁ 100, where n = mean number
of writhes of control group and n0 = mean number of writhes of test
group. The test compounds with percentage protection greater
than 50%, a dose response curve was plotted using the data taken
from different five doses (10, 15, 20, 25 and 30 mg/kg).
129.27(2), 134.54(2), 138.04(2), 144.73(2), 147.10(2), 157.53(2),
170.11(1); EIMS (% rel abundance): 403[M] (16.90). Anal. Calcd
for C20H17N7O3: C, 59.55; H, 4.25; N, 24.3; Found: C, 59.51; H,
4.20; N, 24.28.
4.2. Molecular docking
4.3.4. Anti-inflammatory activity
The anti-inflammatory activity was tested using carrageenan-
induced hind paw edema method.38 Animals were divided in
groups of six each. One group of mice was kept as control and
the mice of other groups were pre-treated with the test com-
pounds suspended in 1% CMC aqueous solution, given orally thirty
minutes before carrageenan injection. Carrageenan solution was
injected into the subplantar tissue of the right hind paw of each
mouse, whereas control mice were injected with saline. The paw
volume was measured before and four hours after injection of car-
rageenan. Mean values of the treated groups were compared with
the mean value of the control group. The percentage inhibition of
inflammation was calculated by applying the following formula:
Anti-inflammatory activity (% inhibition) = (Vc ꢀ Vt)/Vc ꢁ 100,
where Vc = edema volume in control group, Vt = edema volume in
groups treated with the test compounds.
Thereafter, mice were sacrificed by decapitation under anesthe-
sia using a mixture of ketamine (50 mg/kg, ip) and xylazine
(10 mg/kg, ip). A laparotomy was performed, and blood samples
were withdrawn from the heart. Thirty minutes after collection,
blood samples were centrifuged at 2000g for 15 min. Serum sam-
ples were separated, collected and stored at ꢀ80 °C until used for
enzyme linked immunosorbent assay (ELISA). ELISA kits for TNF-
All the molecular modeling studies were carried out on an Intel
Pentium 1.6 GHz processor, 512 MB memory with Windows XP
operating system using Molecular Operating Environment (MOE,
10.2008) software. All the minimizations were performed with
MOE until a RMSD gradient of 0.05 kcal molꢀ1 Åꢀ1 with MMFF94X
forcefield and the partial charges were automatically calculated.
The X-ray crystallographic structure of COX-2 enzyme with its
co-crystallized ligand (Diclofenac) in the file (PDB ID: 1PXX) was
obtained from the protein data bank. The enzyme was prepared
for docking studies where: (i) Ligand molecule was removed from
the enzyme active site. (ii) Hydrogen atoms were added to the
structure with their standard geometry. (iii) MOE Alpha Site Finder
was used for the active sites search in the enzyme structure and
dummy atoms were created from the obtained alpha spheres.
(iv) The obtained model was then used in predicting the ligand en-
zyme interactions at the active site.
4.3. Pharmacological activity
4.3.1. Animals
Male healthy Swiss albino mice with a weight of 25–32 g were
used in the current experiment. Mice were purchased from the
Modern Veterinary Office for Laboratory Animals (Cairo, Egypt).
Animals were allowed to habituate for one week before starting
the experiment then divided into different experimental groups,
6 mice each with food and water ad libitum. All experiments were
conducted between 16:00 and 18:00 h to ensure animal activity
and to eliminate circadian influence on animal behavior. All exper-
imental protocols were approved by the Institutional Animal Care
and Use Committee at the Faculty of Pharmacy, Suez Canal Univer-
sity, Ismailia, Egypt.
a
and IL-2 (Glory Science Co., Ltd, Del Rio, TX, USA) were used
for determination of tissue levels of these markers. The assays were
carried out following the instructions of the manufacturer using an
automated ELISA reader (Europe S.A., Belgium). The most biologi-
cally active compounds were further screened for ulcerogenic
potential.
4.3.5. Acute ulcerogenesis
Compounds that showed analgesic and anti-inflammatory
activity were tested for acute ulcerogeneic potential.39 Food pellets
were removed 24 h before administration of the test compounds.
Ulcerogenicity was evaluated after oral administration of the test
compound or diclofenac at a dose of 50 mg/kg. Control mice were
treated orally with the vehicle (1% CMC suspension). Mice were
treated with the test compounds, then fed with a normal palatable
diet for 18 h. Mice were then anesthetized with a mixture of keta-
mine (50 mg/kg, ip)/xylazine (10 mg/kg, ip) and sacrificed by
decapitation. The stomach was removed, opened along the greater
curvature then washed with distilled water and cleaned gently in
saline. The gastric mucosa of the mice were examined by means
of a magnifying glass. For each stomach, the severity of mucosal
damage was assessed by measuring ulcerogenic index that is,
severity of drug to cause mucosal damage, which is the difference
4.3.2. Preparation of the tested compounds and the standard
drug
The test compounds and diclofenac sodium were suspended in
a 1% sodium carboxymethylcellulose (CMC) aqueous solution.
Therapies were given to mice by oral gavage in a volume equals
5 mL/kg. Control mice received appropriate volumes of the dosing
vehicle (5 mL/kg). Diclofenac sodium (25 mg/kg) was used as a ref-
erence analgesic anti-inflammatory drug. Glacial acetic acid was
purchased from ADWIC Company (Cairo, Egypt) and used to pre-
pare 1% acetic acid solution. Carrageenan (Sigma–AldrichÒ, St.
Louis, Missouri, USA) was freshly prepared as suspension (1% w/v
in 0.9% saline, injected as 0.05 mL/mouse).

N. A. Khalil et al. / Bioorg. Med. Chem. 22 (2014) 2080–2089
2089
between the mean score of each treated group and the mean score
References and notes
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Stomach sections were dehydrated with ethanol, passed through
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4.3.6. In vitro COX-1 and COX-2 inhibition assay
In vitro inhibition of COX-1 and COX-2 enzymes was deter-
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the biosynthesis of arachidonic acid to PGH2. The PGF2a produced
from PGH2 by reduction with stannous chloride is measured by
EIA. The tested compounds were dissolved in DMSO. The enzyme
COX-1 and COX-2 (10
added to the supplied reaction buffer solution (950
l
L), heme (10
l
L) and samples (20
lL) were
lL, 0.1 M Tris–
HCl, pH 8 containing 5 mM ethylenediamine tetraacetate (EDTA)
and 2 mM phenol). The mixture of these solutions were incubated
for a period of 10 min at 37 °C, after that COX reactions were initi-
ated by adding arachidonic acid (10
100 M) solution. The COX reactions were stopped by addition of
HCl (1 M, 50 L) after 2 min and then saturated stannous chloride
(100 L) was added and again incubated for 5 min at room temper-
ature. The PGF2 formed in the samples by COX reactions was
lL, making final concentration
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Data were collected, tabulated and expressed as mean SEM.
One-way analysis of variance (ANOVA) followed by Bonferroni’s
post hoc test were used to test statistical differences between the
groups for parameters with Gaussian distribution. However,
parameters with non-Gaussian distribution were analyzed using
Kruskal–Wallis test (non-parametric ANOVA) followed by Dun-
nett’s test for multiple comparisons. Linear regression lines were
plotted using Microsoft Excel 2007 whereas other statistical anal-
yses were performed employing the Statistical Package for Social
Sciences, version 17 (SPSS Inc., Chicago, IL, USA). All P values re-
ported are two-tailed and P < 0.05 was considered significant.
Acknowledgment
40. Laine, L.; Weinstein, W. M. Gastroenterology 1988, 94, 1254.
41. COX (ovine) inhibitor screening assay kit (Catalog No. 560101), Cayman
Chemical Co., USA.
Authors wish to acknowledge EIPICo Pharmaceutical Company
for the generous gift of diclofenac sodium.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2014.02.042.