Design, synthesis, and evaluation of anti-inflammatory and ulcerogenicity of novel pyridazinone derivatives

Article abstract of DOI:10.1007/s00044-011-9895-7

A series of pyridazinone-containing compounds were designed and synthesized as congeners for diclofenac, the most potent and widely used NSAID. The target compounds were evaluated for their anti-inflammatory activity on rat paw edema inflammation model against diclofenac as a reference compound. Seven of the tested compounds demonstrated more than 50% inhibition of carrageenan-induced rat paw edema at a dose 10 mg/kg. The compounds, 6-(2-βromophenylamino) pyridazin-3(2H)-one 2a and 6-(2,6-dimethylphenylamino)pyridazin-3(2H)-one 2e, displayed 74 and 73.5% inflammationinhibitory activity, respectively, which is comparable to diclofenac (78.3%) at the same dose level after 4 h. The most active compounds as anti-inflammatory agents, 2a, 2e, and 6a, displayed fewer number of ulcers and milder ulcer score than indomethacin in ulcerogenicity screening. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9895-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3581–3590
DOI 10.1007/s00044-011-9895-7
ORIGINAL RESEARCH
Design, synthesis, and evaluation of anti-inflammatory
and ulcerogenicity of novel pyridazinone derivatives
•
K. A. M. Abouzid N. A. Khalil E. M. Ahmed
•
•
•
A. Esmat A. M. Al-Abd
Received: 9 August 2011 / Accepted: 8 November 2011 / Published online: 25 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A series of pyridazinone-containing com-
pounds were designed and synthesized as congeners for
diclofenac, the most potent and widely used NSAID. The
target compounds were evaluated for their anti-inflamma-
tory activity on rat paw edema inflammation model against
diclofenac as a reference compound. Seven of the tested
compounds demonstrated more than 50% inhibition of
carrageenan-induced rat paw edema at a dose 10 mg/kg.
Introduction
Non-steroidal anti-inflammatory drugs (NSAIDs) are the
most commonly prescribed medications for the treatment of
pain, fever, and inflammation, however, their clinical useful-
ness is still restricted due to their gastrointestinal side effects
as gastric irritation, ulceration, bleeding, and in some cases
may lead to life-threatening conditions, (Lanas et al., 2006)
(Fig. 1a). Based on the side effects observed by NSAIDs, it
has been suggested that selective COX-2 inhibitors (coxibs)
may act as safer NSAIDs devoid of ulcerogenic side effects;
however, long-term use of these drugs has shown kidney and
hepatotoxicity as well as an increased risk of cardiovascular
events (Dogne et al., 2000). These observations raised serious
concerns about safety of selective COX-2 inhibitors, since
some of these agents have been withdrawn from the market
(Graham et al., 2005; Hsiao et al., 2009). Therefore, devel-
opment of novel classical anti-inflammatory agents with an
improved safety profileis still a necessity. In thisdirection, the
efforts of our research team were continued to develop safer
and effective anti-inflammatory drug candidates that could be
introducedasasubstitutefor coxibs. Our strategywasdirected
towards structural modification of the well-known anti-
inflammatory/antipyretic/analgesic drug, diclofenac, which is
the most potent and widely prescribed medication for the
treatment of different inflammatory conditions such as
osteoarthritis,rheumatoidarthritis,andankylosingspondylitis
(Allison et al., 1992).
The
compounds,
6-(2-bromophenylamino)pyridazin-
3(2H)-one 2a and 6-(2,6-dimethylphenylamino)pyridazin-
3(2H)-one 2e, displayed 74 and 73.5% inflammation-
inhibitory activity, respectively, which is comparable to
diclofenac (78.3%) at the same dose level after 4 h. The
most active compounds as anti-inflammatory agents, 2a,
2e, and 6a, displayed fewer number of ulcers and milder
ulcer score than indomethacin in ulcerogenicity screening.
Keywords Pyridazinone ꢀ Anti-inflammatory ꢀ
Ulcerogenicity ꢀ Diclofenac
K. A. M. Abouzid (&)
Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Ain Shams University, Cairo 11566, Egypt
e-mail: abouzid@yahoo.com
N. A. Khalil ꢀ E. M. Ahmed
Organic Chemistry Department, Faculty of Pharmacy,
Cairo University, Cairo, Egypt
In previous research work in the anti-inflammatory
area, pyridazinone-based compounds were discovered as
promising candidates in this field, (Gleave et al., 2010;
Chintakunta et al., 2002; Su¨ku¨roglu et al., 2005; Abouzid
A. Esmat
Pharmacology Department, Faculty of Pharmacy,
Ain Shams University, Cairo, Egypt
¨
and Bekhit, 2008; Gokce et al., 2009) (Fig. 1b).
A. M. Al-Abd
Department of Pharmacology, Medical Division,
National Research Center, Dokki, Cairo, Egypt
In this study, and based on the above-mentioned
findings, we attempt to design pyridazinone-containing
123

3582
Med Chem Res (2012) 21:3581–3590
Fig. 1 a Chemical structures of
representative NSAIDs.
b Pyridazinone-containing
compounds with anti-
H
C
3
O S O
HN
(a)
H
OOC
H
OOC
H
OOC
inflammatory activities
N
O2
O
HN
HN
HN
N
C
l
C
l
C
H
C
3
H
3
H
C
F
C
3
3
Mefenamic acid
Diclofenac
Flunixin
Nimesulide
(b)
O
O
O
l
C
NH
H
C
3
N
N
NH
N
H
3C
O
RS-57067
Syntex compound
R1
R2
F
F
O
O
N
H
N
N
O
N
N
O
R3
H
Benzylpyridazinone derivative
Pyridazinone scaffold
compounds as potential candidates for the treatment of
inflammatory conditions (Fig. 2). To achieve this goal, we
introduced certain modification to the skeleton of the lead
compound, diclofenac, via the replacement of aryl acetic
acid moiety with 3(2H)-pyridazinone or pyridazinone-3-
acetic acid moieties. Another approach was to replace the
NH between the two aryl groups of diclofenac with isos-
teric hetero atom like oxygen and addition of a carboxylic
or carboxamide moieties on position-2 of the aryl group.
Also, replacing the SP3 carbon between pyridazinone and
the aryl or heteroaryl ring in the pyridazinone anti-
inflammatory drug candidate with oxygen and nitrogen
hetroatoms.
In Scheme 1, the physical and spectral properties of
6-chloro-3-(4-chlorophenylamino)pyridazine 1b and 6-(4-
chlorophenylamino)pyridazine-3(2H)-one 2b were in
accordance with the literature (Boissier et al., 1963).
Hydrolysis of 1a–h was carried out upon heating in
glacial acetic to afford 2a–h. The formation of these
compounds was confirmed by IR spectra which showed
the appearance of C=O band in the range of
1,670–1,685/cm. 1H-NMR spectra revealed two D2O
exchangeable signals corresponding to the two NH pro-
tons. Alkylation of pyridazinones 2a–h was performed
using ethyl bromoacetate in the presence of anhydrous
K2CO3 in acetone at reflux temperature to provide the
target acetic acid esters 3a–g. The esters in their IR
spectra displayed an additional C=O band of ester in the
1
Results and discussion
range of 1,728–1,743/cm. In H-NMR spectra, additional
signals derived from ester group were observed at
1.21 ppm (OCH2CH3) and 4.18 ppm (OCH2CH3) inte-
grating for three protons and two protons, respectively.
In Scheme 2, compounds 5a, b were prepared via
reaction of 3,6-dichloropyridazine with either salicylic
acid or salicylamide in the presence of anhydrous
Chemistry
The synthetic pathways leading to the 6-substituted-pyr-
idazine and 3(2H)-pyridazinone derivatives are outlined in
Schemes 1 and 2.
123

Med Chem Res (2012) 21:3581–3590
3583
Fig. 2 Structures of
representative designed target
compounds
EtOOC
XOC
O
H
N
N
O
O
N
N
HN
HN
N
CH3
Cl
H3C
Cl
NH
O
X= OH, NH2
Scheme 1 Reagents and
conditions: a isopropanol/
K2CO3/reflux 4 h, b acetic acid/
reflux 5 h, c BrCH2COOC2H5/
K2CO3, reflux 24 h
Cl
O
Cl
Cl
O
OC2H5
N
N
a
NH
N
b
N
N
c
N
ArNH2
N
O
NHAr
NHAr
NHAr
1a-h
2a-h
3a-g
,
Ar = 2-BrC6H4, 4-ClC6H4 , 2,4-Cl2C6H3, 2,6-Cl2C6H3, 2,6-(CH3)2C6H3
N
N
,
,
N
S
N
H
Cl
Scheme 2 Reagents and
conditions: a isopropanol/
K2CO3/reflux 4 h,
XOC
XOC
Cl
Cl
O
b CH3COOH/reflux 5 h
O
XOC
N
N
b
N
N
a
N
HO
NH
Cl
4a,b
O
5a,b
6a,b
4a : X=OH
5a : X= OH
5b :X= NH2
6a : X=OH
6b : X= NH2
4b : X= NH2
H2NOC
O
Cl
N
a
N
N
4b
+
N
Ph
ph
7
K2CO3. Hydrolysis of 5a, b with acetic acid gave 6a,
b. Finally, compound 7 was synthesized by reacting
3-chloro-6-phenylpyridazine with salicylamide. The IR
spectra of 5a, b, 6a, b, and 7 were very informative.
The carboxylic acid derivatives, 5a and 6a, displayed the
O–H stretching band of acid in the range of
2,515–3,200/cm. The amide derivatives 5b, 6b, and 7
showed the additional C=O of amide at 1,645–1,678/cm.
Further spectroscopic details of these compounds are
presented in ‘‘Experimental’’ section.
123

3584
Med Chem Res (2012) 21:3581–3590
In vivo anti-inflammatory activity
compared to that of the corresponding amide, 6b (29.3%).
The low anti-inflammatory activity observed by the amide
6b was substantiated by the comparable anti-inflammatory
effect of the amide derivative 7 (31.3%).
Some of the synthesized compounds (2a, 2c–h, 3a–g,
6a, b, and 7) were screened for their anti-inflammatory
activities against carrageenan-induced rat paw edema at a
dose 10 mg/kg. The anti-inflammatory properties were
compared to that of diclofenac (in a dose 10 mg/kg) as a
reference standard. The test compounds 2a, 2c–h have
elicited significant anti-inflammatory activity of variant
degree (20.7–74%). Esterification of the latter compounds
to their corresponding esters 3a–g showed low to moderate
anti-inflammatory activities (17.2–61.2%). In general, they
exhibited lower degree of inhibition of edema compared to
their parent compounds except for compound 3c and 3d
(Table 1).
Ulcerogenicity studies
Three of the test compounds that exhibited the most potent
anti-inflammatory activity were tested for gastric ulcerative
effect on rat stomach.
The ulcerative effect of test compounds (2a, 2e, and 6a)
has been inspected visually and histopathologically relative
to the known ulcerogenic drug, indomethacin. After gross
visual inspection, all test compounds showed fewer number
of ulcers than indomethacin and milder ulcer score as well.
Compound 2a showed the strongest gross visual ulcero-
genic score (4.2 1.34); yet, milder than indomethacin
(7.2 0.65). All test compounds showed less than two
gross ulcers per stomach, while indomethacin showed
average of 2.6 0.27 ulcers per stomach (Table 2).
However, microscopically examined sections of all test
compounds and indomethacin showed considerable signs
of gastric ulcerogenic activity such as thinning of gastric
mucosa layer and microscopic cone shape mucosal ulcers.
Thinning of the gastric mucosa is considered early gastric
The presence of bromine atom at position 2 (2a) or 2,6-
dimethyl group (2e) in the aromatic ring gave rise to an
increased anti-inflammatory activity (74 and 73.5%),
respectively. It is also obvious that adopting a 2-chloro-
pyridyl function at the 6-aminopyridazinone 2f seems
preferable for obtaining an effective anti-inflammatory
agent.
On the other hand, the pyridazinone derivative 6a
carrying acid function in the 2-position of the aromatic
ring exhibited higher anti-inflammatory activity (65.6%)
Table 1 Anti-inflammatory activity of the test compounds assessed in comparison to diclofenac as reference
Group
1 h
2 h
4 h
4 h
Paw vol. (mL)
% Edema
inhibition
Paw vol. (mL)
% Edema
inhibition
Paw vol. (mL)
% Edema
inhibition
Potency
Control
1.530a 0.032
1.340a,b 0.034
1.473a,b 0.005
0.00
65.3
19.6
15.8
8.9
1.605a 0.015
1.302a,b 0.013
1.461a,b 0.024
1.558a 0.018
1.584a,b 0.009
1.372a,b 0.006
1.480a,b 0.007
1.561a 0.017
1.523a,b 0.022
1.495a,b 0.004
1.514a,b 0.013
1.481a,b 0.007
1.533a,b 0.02
1.552a,b 0.019
1.591a 0.01
0.00
73.5
34.9
11.4
5
1.817a 0.088
1.279a,b 0.019
1.308a,b 0.02
1.6a,b 0.021
0.00
78.3
74
–
Diclofenac
100
2a
2c
2d
2e
2f
94.4
1.484a,b
0.024
31.6
20.7
73.5
62.4
31.1
49.1
52.8
51.9
61.2
49.6
32.1
17.2
29.1
65.6
29.3
31.3
40.35
25.54
93.86
79.69
39.71
62.7
1.504a 0.023
1.405a,b 0.009
1.473a,b 0.005
1.488a 0.012
1.465a,b 0.021
1.476a,b 0.013
1.483a,b 0.018
1.474a,b 0.007
1.457a,b 0.024
1.502a 0.018
1.504a 0.015
1.498a 0.018
1.471a,b 0.005
1.495a 0.02
1.5a 0.016
1.675a,b 0.065
1.312a,b 0.038
1.388a,b 0.022
1.603a,b 0.026
1.479a,b 0.021
1.454a,b 0.065
1.460a,b 0.025
1.396a,b 0.027
1.476a,b 0.073
1.596a,b 0.025
1.699a,b 0.016
1.617a,b 0.027
1.387a,b 0.019
1.616a,b 0.029
1.602a,b 0.027
42.9
19.6
14.4
22.3
18.6
16.2
19.2
25.1
9.6
56.6
30.3
10.7
19.9
26.9
22.1
30.1
17.5
12.8
3.4
2g
2h
3a
3b
3c
3d
3e
3f
67.43
66.16
78.16
63.34
40.99
21.96
37.16
83.78
37.42
39.97
8.9
3g
6a
6b
7
11
1.561a 0.026
1.483a,b 0.006
1.564a 0.019
1.561a 0.028
10.7
29.6
9.9
20.3
12
10.3
10.8
a
Statistically significant difference from the control group at p \ 0.05
b
Statistically significant difference from the carrageenan-induced group at p \ 0.05
123

Med Chem Res (2012) 21:3581–3590
3585
routine H&E staining for paraffin embedded sections.
Normal group (A) showed normal gastric mucosal
appearance. Group treated with the test compounds 2a (B),
2e (C), 6a (D), and indomethacin (E) showed thinning in
the mucosal layer, cone-shape ulcers (star), and mucosal
dislodgments (arrows). Gastric mucosal thickness and
depth of ulcers were calculated using image-JTM software
and compared to control untreated group (F). Scale bar
equals 100 lm. Data are presented as mean SEM.
Table 2 Gross visual gastric ulcerative assessment
Compound number
Ulcer number
Ulcer score
Control
0.0 0.0
0.0 0.0
Indomethacin
2.6 0.27
1.8 0.55
1.4 0.89
1.6 1.04
7.2 0.65
4.2 1.34
1.8 0.42
2.0 1.27
2a
2e
6a
ulceration sign; herein, gastric mucosal thickness has been
calculated microscopically after treatment with test com-
pounds and compared to normal gastric mucosal tissues.
All test compounds showed significant gastric mucosal
thinning down to 20% of the original mucosal thickness.
Again, compound 2a showed the most profound gastric
mucosal thinning among all test compounds. Besides
mucosal thickness, the microscopic depth of ulcer has been
evaluated after treatment with test compounds and com-
pared to control untreated group. All test compounds
showed narrower and shallower gastric ulcers than indo-
methacin. Surprisingly, indomethacin-induced gastric
mucosal thinning was less than all test compounds. How-
ever, indomethacin-induced ulcers were much deeper than
all other test compounds (Fig. 3).
Conclusion
The anti-inflammatory activity evaluation was carried out
using carrageenan-induced paw edema assay. The screen-
ing data revealed that all investigated compounds exhibit
considerable anti-inflammatory properties with the per-
centage inhibition of edema ranging from 17.2 to 74, while
the reference diclofenac showed 78.3 inhibition. The
test compounds, 6-(substitutedamino)pyridazin-3(2H)-ones,
except 2c and 2d were found to be more potent than their
corresponding esters 3a–g. Compounds 2a and 2e showed
remarkable activities with potency of 94.5 and 93.86%,
respectively. The esters 3a–g exhibited anti-inflammatory
activity of the potency range from 21.96 to 78.16%. The
carboxylic acid derivative 6a displayed higher potency
than the corresponding amide 6b.
Histopathological assessment for gastric ulceration was
carried out in fundus glandular region of gastric tissues by
Fig. 3 Histological assessment of gastric ulceration
123

3586
Med Chem Res (2012) 21:3581–3590
The ulcerogenicity studies revealed that compounds 2a,
2e, and 6a showed fewer number of ulcers and milder ulcer
score than indomethacin.
6-Chloro-3-(2,4-dichlorophenylamino)pyridazine 1cyield
75%; oil; IR(KBr)/cm: 3310 (N–H), 3155, 3033 (C–H
aromatic), 1617 (C=N);1H-NMR (DMSO-d6, 300 MHz) d
(ppm): 5.44 (br s, 1H, NH, D2O exchangeable); 6.76 (d,
1H, pyridazine H-4); 7.01 (d, 1H, Ar–H); 7.05 (d, 1H,
Ar–H); 7.23 (d, 1H, pyridazine H-5); 8.00 (s, 1H, Ar–H).
Anal calcd for C10H6Cl3N3 (274.53): C, 43.75; H, 2.20; N,
15.31; Found: C, 43.90; H, 2.45; N, 15.50.
Since our findings are preliminary results; further stud-
ies need to be carried out to investigate the other specifi-
cations such as in vitro assays, toxicological studies, or side
effect-activity profiles of these compounds.
6-Chloro-3-(2,6-dichlorophenylamino)pyridazine
1d
Experimental
yield 70%; oil; IR (KBr)/cm: 3325 (N–H), 3145, 3068 (C–
H aromatic), 1616 (C=N); 1H-NMR (DMSO-d6, 300 MHz)
d (ppm): 5.34 (s, 1H, NH, D2O exchangeable); 6.53 (d, 1H,
pyridazine H-4); 6.56 (d, 1H, Ar–H); 7.16 (d, 2H, Ar–H);
7.92 (d, 1H, pyridazine H-5). Anal calcd for C10H6Cl3N3
(274.53): C, 43.75; H, 2.20; N, 15.31; Found: C, 43.80; H,
2.05; N, 15.65.
Melting points were determined on Griffin apparatus and
the values given are uncorrected. IR spectra were deter-
mined on Shimadzu IR 435 spectrophotometer (KBr, /cm).
1H-NMR spectra were carried out using a Varian Gemini
200 MHz Spectrophotometer and Varian Mercury-300
(300 MHz) Spectrophotometer using TMS as internal
standard. Chemical shift values are recorded in ppm on d
scale, Microanalytical Center, Cairo University, Egypt.
Mass spectra were recorded on a GCMP-QP1000 EX Mass
spectrometer, Microanalytical Center, Cairo University,
Egypt. Elemental analyses were carried out at the Micro-
analytical Center, Cairo University, Egypt. Progress of the
reactions was monitored using TLC sheets precoated with
UV fluorescent silica gel Merck 60F 254 using acetone/
benzene (1:9) and were visualized using UV lamp.
All chemicals were obtained from Aldrich, Fluka, or
Merck chemicals.
6-Chloro-3-(2,6-dimethylphenylamino)pyridazine
1e
yield 50%; oil; IR (KBr)/cm: 3312 (N–H), 3150, 3050 (C–
H aromatic), 2950, 2844 (C–H aliphatic), 1618 (C=N);
(DMSO-d6, 300 MHz) d (ppm): 2.04 (s, 6H, 2CH3); 4.70
(br s, 1H, NH, D2O exchangeable); 6.42 (d, 1H, pyridazine
H-4); 6.58 (d, 1H, Ar–H); 6.89 (d, 2H, Ar–H); 7.94 (d, 1H,
pyridazine H-5). Anal Calcd for C12H12ClN3 (233.70): C,
61.67; H, 5.18; N, 17.98; Found: C, 61.95; H, 5.43; N,
18.25.
6-Chloro-3-(2-chloropyridin-3-ylamino)pyridazine 1f
yield 50%; mp 45–46 °C; IR (KBr)/cm: 3309 (N–H), 3178,
3030 (C–H aromatic), 1616 (C=N); 1H-NMR (CDCl3,
300 MHz) d (ppm): 4.11 (br s, 1H, NH, D2O exchange-
able); 7.00 (d, 1H, pyridazine H-4); 7.03–7.75 (m, 4H, 3H
pyridine and 1H pyridazine H-5). Anal Calcd for
C9H6Cl2N4, (241.08): C, 44.84; H, 2.51; N, 23.24; Found:
C, 44.93; H, 2.65; N, 23.50.
6-Chloro-3-(4-chlorophenylamino)pyridazine 1b and
6-(4-chlorophenylamino)pyridazine-3(2H)-one 2b were
prepared according to reported procedure (Boissier et al.,
1963).
6-Chloro-3-substituted-aminopyridazines (1a–h)
N-(6-Chloropyridazin-3-yl)-4,5-dihydrothiazol-2-amine
1g yield 60%; mp 43–44 °C; IR (KBr)/cm: 3305 (N–H),
3066, 3039 (C–H aromatic), 2943, 2854 (C–H aliphatic),
1623 (C=N); (DMSO-d6, 300 MHz) d (ppm): 3.43 (t, 2H,
CH2–thiazoline); 3.84 (t, 2H, CH2–thiazoline); 3.96 (br s,
1H, NH, D2O exchangeable); 7.72 (d, 1H, pyridazine
H-4); 8.03 (d, 1H, pyridazine H-5). MS (EI) m/z (% rel.
General procedure
A mixture of 3,6-dichloropyridazine (1.48 g, 0.01 mol),
anhydrous K2CO3 (0.02 mol) and the appropriate substi-
tuted amine (0.01 mol) in isopropanol (30 mL) was heated
under reflux for 4 h. The reaction mixture was concentrated
under reduced pressure to half its volume, poured onto
water (50 mL) and extracted by methylene chloride
(3 9 10 mL). The combined extract was dried (Na2SO4)
and distilled off under diminished pressure to give 1a–h.
3-(2-Bromophenylamino)-6-chloropyridazine 1a yield
60%; oil; IR (KBr)/cm: 3315 (N–H), 3135, 3085 (C–H
aromatic), 1615 (C=N); 1H-NMR (DMSO-d6, 300 MHz) d
(ppm): 5.10 (br s, 1H, NH, D2O exchangeable); 6.43 (d,
1H, Ar–H); 6.77- 6.81 (m, 2H, Ar–H); 7.06 (d, 1H, Ar–H);
7.31 (d, 1H, pyridazine H-4); 7.99 (d, 1H, pyridazine H-5).
Anal calcd for C10H7BrClN3 (284.54): C, 42.21; H, 2.48;
N, 14.77; Found: C, 42.51; H, 2.60; N, 14.85.
?
Int.): 214 (Mꢀ , 0.44); 216 (M?2?, 0.97). Anal Calcd for
C7H7ClN4S (214.68): C, 39.16; H, 3.29; N, 26.10; Found:
C, 39.40; H, 3.45; N, 26.35.
N-(6-Chloropyridazin-3-yl)-1H-benzo[d]imidazol-2-
amine 1h yield 50%; mp 183–184 °C; IR (KBr)/cm:
3317 (N–H), 3143, 3066 (C–H aromatic), 1620 (C=N);
1H-NMR (CDCl3, 300 MHz) d (ppm): 6.05 (s, 1H, NH,
D2O exchangeable); 6.80–7.09 (m, 5H, 4 Ar–H, and 1H
pyridazine H-4); 8.01 (d, 1H, pyridazine H-5); 10.62 (br
s, 1H, NH, D2O exchangeable). MS (EI) m/z (% rel. Int.):
245 (M?, 0.15). Anal Calcd for C11H8ClN5 (245.67): C,
53.78; H, 3.28; N, 28.51; Found: C, 53.80; H, 3.40; N,
28.73.
123

Med Chem Res (2012) 21:3581–3590
6-(Substituted-amino)pyridazin-3(2H)-ones (2a–h)
General procedure
3587
3120, 3055 (C–H aromatic), 1674 (C=O), 1581 (C=N); 1H-
NMR (DMSO-d6, 300 MHz) d (ppm): 5.50 (s, 1H, NH,
D2O exchangeable); 6.95 (d, 1H, J = 9.9 Hz, pyridazine
H-4); 7.07–7.11 (m, 2H, Ar–H); 7.40 (d, 1H, J = 9.9 Hz,
pyridazine H-5); 7.56 (d, 1H, Ar–H); 13.14 (s, 1H, NH,
Compounds 1a–h (0.01 mol) were heated under reflux in
glacial acetic acid (25 mL) for 5 h. The reaction mixture
was concentrated to half its volume then cooled. The pre-
cipitated crystalline solid was filtered, washed with water,
and recrystallized from ethanol to give 2a–h.
?
D2O exchangeable). MS (EI) m/z (% rel. Int.): 222 (Mꢀ ,
33.85), 224 (M?2, 23.05). Anal Calcd for C9H7ClN4O
(222.63): C, 48.55; H, 3.17; N, 25.17; Found: C, 48.77; H,
3.50; N, 25.45.
6-(2-Bromophenylamino)pyridazin-3(2H)-one 2a yield
60%; mp 175–176 °C; IR (KBr)/cm: 3398, 3221(N–H),
3035, 3005 (C–H aromatic), 1685 (C=O), 1589 (C=N); 1H-
NMR (DMSO-d6, 300 MHz) d (ppm): 7.15 (d, 1H,
J = 9.6 Hz, pyridazine H-4); 7.29–7.61 (m, 4H, Ar–H);
7.65 (d, 1H, J = 9.6 Hz, pyridazine H-5); 8.00 (s, 1H, NH,
D2O exchangeable); 8.91 (s, 1H, NH, D2O exchangeable).
6-(4,5-Dihydrothiazol-2-ylamino)pyridazin-3(2H)-one
2g yield 55%; mp 117–118 °C; IR (KBr)/cm: 3432, 3350
(N–H), 3120, 3055 (C–H aromatic), 2985, 2850 (C–H
aliphatic), 1670 (C=O), 1581 (C=N). 1H-NMR (DMSO-d6,
300 MHz) d (ppm): 3.42 (t, 2H, CH2 thiazoline), 4.05 (t,
2H, CH2 thiazoline), 6.92 (d, 1H, J = 9.9 Hz, pyridazine
H-4); 7.48 (d, 1H, J = 9.9 Hz, pyridazine H-5); 13.09 (br
s, 2H, 2NH, D2O exchangeable). MS (EI) m/z (% rel. Int.):
197 (M?H, 2.76). Anal Calcd for C7H8N4OS (196.23): C,
42.85; H, 4.11; N, 28.55; Found: C, 42.75; H, 4.45; N,
28.65.
?
MS (EI) m/z (% rel. Int.): 265 (Mꢀ , 1.22), 267(M?2,
1.36); Anal Calcd for C10H8BrN3O (266.09): C, 45.14; H,
3.03; N, 15.79; Found: C, 45.30; H, 3.33; N, 15.90.
6-(2,4-Dichlorophenylamino)pyridazin-3(2H)-one
2c
yield 65%; mp 159–160 °C; IR (KBr)/cm: 3398, 3282 (N–
H), 3078, 3032 (C–H aromatic), 1670 (C=O), 1585 (C=N);
1H-NMR (DMSO-d6, 300 MHz) d (ppm): 6.84 (d, 1H,
J = 9.9 Hz, pyridazine H-4); 7.36–7.65 (m, 3H, Ar–H);
7.96 (d, 1H, J = 9.9 Hz, pyridazine H-5); 8.26 (s, 1H, NH,
D2O exchangeable); 9.05 (s, 1H, NH, D2O exchangeable).
6-(1H-Benzo[d]imidazol-2-ylamino)pyridazin-3(2H)-one
2h yield 58%; mp 119–120 °C; IR (KBr)/cm: 3433, 3350
(N–H), 3140, 3055 (C–H aromatic), 1674 (C=O), 1581
1
(C=N); H-NMR (DMSO-d6, 300 MHz) d (ppm): 6.92 (d,
1H, J = 9.6 Hz, pyridazine H-4); 7.16–7.37 (m, 4H, Ar–
H); 7.48 (d, 1H, J = 9.6 Hz, pyridazine H-5); 13.13 (br s,
3H, 3NH, D2O exchangeable). MS (EI) m/z (% rel. Int.):
228 (M?H, 4.83). Anal Calcd for C11H9N5O (227.22): C,
58.14; H, 3.99; N, 30.82; Found: C, 58.45; H, 4.25; N,
30.65.
?
MS (EI) m/z (% rel. Int.): 256 (Mꢀ , 8.81), 258 (M?2,
4.30), 260 (M?4, 1.33). Anal Calcd for C10H7Cl2N3O
(256.09): C, 46.90; H, 2.76; N, 16.41; Found: C, 47.10; H,
2.82; N, 16.51.
6-(2,6-Dichlorophenylamino)pyridazin-3(2H)-one
2d
yield 62%; mp 64–65 °C; IR (KBr)/cm: 3444, 3352 (N–H),
3059, 3032 (C–H aromatic), 1681 (C=O), 1585 (C=N); 1H-
NMR (DMSO-d6, 300 MHz) d (ppm): 5.43 (s, 1H, NH, D2O
exchangeable); 6.55–6.60 (m, 1H, Ar–H); 6.98 (d, 1H,
J = 10.5 Hz, pyridazine H-4); 7.20 (d, 2H, Ar–H); 7.51 (d,
1H, J = 10.5 Hz, pyridazine H-5); 13.15 (s, 1H, NH, D2O
exchangeable). MS (EI) m/z (% rel. Int.): 256 (M?, 0.35), 258
(M?2, 0.08). Anal Calcd for C10H7Cl2N3O (256.09): C,
46.90; H, 2.67; N, 16.41; Found: C, 47.23; H, 2.73; N, 16.55.
Ethyl 2-[3-(substitutedamino)-6-oxo-1,6-
dihydropyridazin-1-yl] acetates (3a–g)
General procedure
A mixture of the appropriate 2a–g (0.01 mol), anhydrous
K2CO3 (0.04 mol), and ethyl bromoacetate (0.02 mol) and
in dry acetone (40 mL) was heated under reflux for 24 h.
Water (10 mL) was added and the mixture was extracted
with methylene chloride (3 9 5 mL). The combined
organic layers were washed with water (2 9 5 mL), dried
(Na2SO4), filtered, and concentrated in vacuo to give 3a–g.
Ethyl 2-[3-(2-bromophenylamino)-6-oxo-1,6-dihydropy-
ridazin-1-yl] acetate 3a yield 60%; mp 109–110 °C; IR
(KBr)/cm: 3221 (N–H); 3120, 3035 (C–H aromatic); 2954,
2854 (C–H aliphatic); 1743, 1670 (C=O); 1589 (C=N). 1H-
NMR (DMSO-d6, 300 MHz) d (ppm): 1.18 (t, 3H, CH3),
4.13 (q, 2H, CH2); 4.33 (s, 2H, N–CH2), 7.07–7.76 (m, 6H,
4H, 3 Ar–H, and 2H pyridazine); 8.90 (s, 1H, NH, D2O
6-(2,6-Dimethylphenylamino)pyridazin-3(2H)-one
2e
yield 55%; mp 159–160 °C; IR (KBr)/cm: 3452, 3390 (N–
H), 3062, 3039 (C–H aromatic), 2924, 2800 (C–H ali-
1
phatic), 1680 (C=O), 1585 (C=N); H-NMR (DMSO-d6,
300 MHz) d (ppm): 2.21 (s, 6H, 2CH3); 6.77 (d, 1H,
J = 9 Hz, pyridazine H-4); 7.00–7.12 (m, 3H, Ar–H); 7.45
(d, 1H, J = 9 Hz, pyridazine H-5); 8.09 (s, 1H, NH, D2O
exchangeable); 8.68 (s, 1H, NH, D2O exchangeable). MS
?
(EI) m/z (% rel. Int.): 215 (Mꢀ , 0.37). Anal Calcd for
C12H13N3O (215.25): C, 66.96; H, 6.09; N, 19.52; Found:
C, 66.75; H. 5.98; N, 19.65.
?
exchangeable). MS (EI) m/z (% rel. Int.): 351 (Mꢀ , 0.11),
6-(2-Chloropyridin-3-ylamino)pyridazin-3(2H)-one 2f
yield 50%; mp 84–85 °C; IR (KBr)/cm: 3421, 3300 (N–H),
353 (M?2, 0.06). Anal Calcd for C14H14BrN3O3 (352.18):
123

3588
Med Chem Res (2012) 21:3581–3590
C, 47.74; H, 4.01; N, 11.93; Found: C, 47.89; H, 4.30; N,
11.85.
N–CH2); 6.89 (d, 1H, J = 9.6 Hz, pyridazine-H4);
6.93–7.27 (m, 3H, Ar–H); 7.73 (d, 1H, J = 9.6 Hz, pyr-
idazine-H5). Anal Calcd for C13H13ClN4O3 (308.72): C,
50.58; H, 4.24; N, 18.15; Found: C, 50.80; H,4.62; N,
18.33.
Ethyl 2-[3-(4-chlorophenylamino)-6-oxo-1,6-dihydro-
pyridazin-1-yl] acetate 3b yield 62%; mp 129–130 °C; IR
(KBr)/cm: 3278 (N–H), 3089, 3051 (C–H aromatic); 2980,
2835 (C–H aliphatic); 1728, 1666 (C=O), 1581 (C=N). 1H-
NMR (DMSO-d6, 300 MHz) d (ppm): 1.20 (t, 3H, CH3);
4.14 (q, 2H, CH2); 4.73 (s, 2H, N–CH2); 7.18–7.76 (m, 6H,
4H Ar–H, and 2H pyridazine); 9.59 (s, 1H, NH, D2O
Ethyl 2-[3-(4,5-dihydrothiazol-2-yl)amino-6-oxo-1,6-di-
hydropyridazin-1-yl] acetate 3g yield 60%; oil; IR (KBr)/
cm: 3432 (N–H); 3120, 3055 (C–H aromatic); 2985, 2854
(C–H aliphatic); 1735, 1670 (C=O); 1581 (C=N). 1H-NMR
(CDCl3, 300 MHz) d (ppm): 1.27 (t, 3H, CH3); 3.81 (t, 2H,
CH2–thiazoline); 3.90 (s, 1H, NH, D2O exchangeable);
3.93 (t, 2H, CH2–thiazoline); 4.23 (q, 2H, CH2); 4.80 (s,
2H, N–CH2); 6.91 (d, 1H, J = 9.6 Hz, pyridazine-H4);
7.23 (d, 1H, J = 9.6 Hz, pyridazine-H5). Anal Calcd for
C11H14N4O3S (282.32): C, 46.80; H, 5.00; N, 19.85;
Found: C, 46.95; H, 5.22; N, 19.98.
?
exchangeable). MS (EI) m/z (% rel. Int.): 307 (Mꢀ , 34.94),
309 (M?2, 16.36). Anal Calcd for C14H14ClN3O3
(307.73): C, 54.64; H, 4.59; N, 13.65; Found: C, 54.75; H,
4.80; N, 13.85.
Ethyl 2-[3-(2,4-dichlorophenylamino)-6-oxo-1,6-dihyd-
ropyridazin-1-yl] acetate 3c yield 65%; mp 105–106 °C;
IR (KBr)/cm: 3390 (N–H), 3109, 3062 (C–H aromatic),
2927, 2862 (C–H aliphatic); 1739, 1670 (C=O); 1589
1
(C=N). H-NMR (CDCl3, 300 MHz) d (ppm): 1.28 (t, 3H,
2-(6-Chloropyridazine-3-yloxy)benzoic acid
CH3); 4.24 (q, 2H, CH2); 4.80 (s, 2H, N–CH2); 6.94 (d, 1H,
J = 9.6 Hz, pyridazine-H4); 7.04–7.45 (m, 2H, Ar–H);
7.53 (d, 1H, J = 9.6 Hz, pyridazine-H5); 8.10 (s, 1H, Ar–
H); 9.30 (s, 1H, NH, D2O exchangeable). Anal Calcd for
C14H13Cl2N3O3 (342.18): C, 49.14; H, 3.83; N, 12.28;
Found: C, 49.30; H, 3.75; N, 12.55.
and benzamide (5a, b)
A mixture of 3,6-dichloropyridazine (1.48 g, 0.01 mol),
anhydrous K2CO3 (0.02 mol) and the desired acid or amide
(0.01 mol) in isopropanol (30 mL) was heated under reflux
for 4 h. The reaction mixture was concentrated under
reduced pressure to half its volume, cooled, and poured
into ice-cold water. The separated solid was filtered, dried,
and crystallized from isopropanol.
Ethyl 2-[3-(2,6-dichlorophenylamino)-6-oxo-1,6-dihyd-
ropyridazin-1-yl] acetate 3d yield 52%; oil; IR (KBr)/cm:
3340 (N–H); 3110, 3054 (C–H aromatic); 2980, 2820 (C–H
aliphatic); 1740, 1675 (C=O), 1585 (C=N). 1H-NMR
(DMSO-d6, 300 MHz) d (ppm): 1.20 (t, 3H, CH3); 4.16 (q,
2H, CH2); 4.95 (s, 2H, N–CH2); 6.58 (d, 1H, Ar–H); 7.10
(d, 1H, J = 9.6 Hz, pyridazine-H4); 7.23 (d, 2H, Ar–H);
7.64 (d, 1H, J = 9.6 Hz, pyridazine-H5); 9.80 (s, 1H, NH,
2-(6-Chloropyridazine-3-yloxy)benzoic acid 5a
Yield 80%; mp 94–95 °C; IR (KBr)/cm: 3200-2534(O–H);
3047 (C–H aromatic); 1681 (C=O); 1581 (C=N). 1H-NMR
(DMSO-d6, 300 MHz) d (ppm): 6.89–6.90 (m, 2H, 1H
aromatic, and 1H pyridazine); 7.44–7.49 (m, 2H, 1H aro-
matic, and 1H pyridazine); 7.75–7.77 (m, 1H, Ar–H); 7.97-
8.00 (m, 1H, Ar–H); 11.26 (br. s, 1H, OH, D2O
exchangeable). Anal Calcd for C11H7ClN2O3 (250.64): C,
52.71; H, 2.82; N, 11.18; Found: C, 52.80; H, 2.53; N,
11.30.
?
D2O exchangeable). MS (EI) m/z (% rel. Int.): 342 (Mꢀ ,
1.95), 344 (M?2, 1.04), 346 (M?4, 1.09); Anal Calcd for
C14H13Cl2N3O3 (342.18): C, 49.14; H, 3.83; N, 12.28;
Found: C, 49.45; H, 3.95; N, 12.45.
Ethyl 2-[3-(2,6-dimethylphenylamino)-6-oxo-1,6-dihyd-
ropyridazin-1-yl] acetate 3e yield 50%; mp 115–116 °C;
IR (KBr)/cm: 3468 (N–H); 3093, 3055 (C–H aromatic);
2962, 2800 (C–H aliphatic); 1743, 1678 (C=O); 1585
1
(C=N). H-NMR (DMSO-d6, 300 MHz) d (ppm): 1.20 (t,
3H, CH3); 2.02 and 2.21 (2s, 6H, 2CH3); 4.14 (q, 2H, CH2);
4.82 (s, 2H, N–CH2); 7.10–7.64 (m, 5H, 3Ar–H, and 2H
pyridazine); 9.20 (s, 1H, NH, D2O exchangeable). MS (EI)
2-(6-Chloropyridazine-3-yloxy)benzamide 5b
?
Yield 70%; mp 199–200 °C; IR (KBr)/cm: 3487, 3336
(NH2); 3066 (C–H aromatic); 1670 (C=O); 1582 (C=N).
1H-NMR (DMSO-d6, 200 MHz) d (ppm): 6.40 (d, 1H,
J = 9.6 Hz, pyridazine-H4); 6.18–6.22 (m, 1H, Ar–H);
6.96–7.01 (m, 1H, Ar–H); 7.65–7.72 (m, 2H, Ar–H); 8.04
(s, 2H, NH2, D2O exchangeable); 8.63 (d, 1H, J = 9.4 Hz,
pyridazine-H5). Anal Calcd for C11H8ClN3O2 (249.65): C,
52.92; H, 3.23; N, 16.83; Found: C, 53.10; H, 3.45; N,
16.94.
m/z (% rel. Int.): 301 (Mꢀ , 0.37). Anal Calcd for
C16H19N3O3 (301.34): C, 63.77; H, 6.36; N, 13.94; Found:
C, 63.85; H, 6.60; N, 13.82.
Ethyl 2-[3-(2-chloropyridin-3-yl)amino-6-oxo-1,6-di-
hydropyridazin-1-yl] acetate 3f yield 54%; oil; IR (KBr)/
cm: 3442 (N–H); 3098, 3052 (C–H aromatic); 2980, 2844
(C–H aliphatic); 1738, 1670 (C=O), 1584 (C=N). 1H-NMR
(CDCl3, 300 MHz) d (ppm): 1.24 (t, 3H, CH3); 4.02 (s, 1H,
NH, D2O exchangeable); 4.21 (q, 2H, CH2); 4.90 (s, 2H,
123

Med Chem Res (2012) 21:3581–3590
3589
2-(6-Oxo-1,6-dihydropyridazin-3-yloxy)benzoic acids
Pharmacology
and benzamides (6a, b)
Anti-inflammatory activity
Materials and methods
Either of 5a or 5b (0.01 mol) in glacial acetic acid (25 mL)
was heated under reflux for 5 h. The reaction mixture was
concentrated to half its volume under reduced pressure then
cooled. The precipitated crystalline solid was filtered,
washed with water, and recrystallized from ethanol to give
6a, b.
Inflammatory-grade carrageenan was purchased from FMC
(Rockland, ME, USA). All other chemicals were of the
highest available commercial grade. All test compounds
were dissolved in carboxymethylcellulose (CMC) before
being injected into the animals. Throughout the experi-
ments, adult male Sprague–Dawley rats weighing
180–200 g were used (Pharmacological Department, Fac-
ulty of Pharmacy, Ain Shams University). They were
housed at a temperature of 23 2 °C with free access to
water and standard food pellets. Rats were acclimatized in
our animal facility for at least 1 week prior to the
experiment.
2-(6-Oxo-1,6-dihydropyridazin-3-yloxy)benzoic acid 6a
Yield 85%; mp 148–149 °C; IR (KBr)/cm: 3228-2515 (O–
H); 1678, 1639 (C=O); 1581 (C=N). H-NMR (DMSO-d6,
1
300 MHz) d (ppm): 6.89–6.95 (m, 3H, 2H aromatic, and
1H pyridazine); 7.20 (s, 1H, NH, D2O exchangeable);
7.47–7.53 (m, 2H, Ar–H); 7.80 (d, 1H, pyridazine); 10.58
(br s, 1H, OH, D2O exchangeable). MS (EI) m/z (% rel.
?
Int.): 232 (Mꢀ , 41.07). Anal Calcd for C11H8N2O4
(232.19): C, 56.90; H, 3.47; N, 12.06; Found: C, 56.98; H,
3.55; N, 12.40.
Measurement of paw volume in carrageenan-induced rat
edema model
2-(6-Oxo-1,6-dihydropyridazin-3-yloxy)benzamide 6b
The experimental tests on animals have been performed in
accordance with the Institutional Ethical Committee
approval. The anti-inflammatory effect of the test com-
pounds was evaluated in correspondence to the carra-
geenan-induced paw edema method (Winter et al., 1962).
The rats were equally divided into 14 groups (six animals/
group). The first group was injected with 0.05 mL of 1%
carrageenan in the subplantar tissue of the right-hind paw
and served as untreated control. The positive control group
was given 10 mg/kg diclofenac 1 h before carrageenan
injection.
Yield 80%; mp 229–230 °C; IR (KBr)/cm: 3421, 3271
(NH, NH2); 3082 (C–H aromatic); 1678, 1645 (C=O); 1593
1
(C=N). H-NMR (DMSO-d6, 300 MHz) d (ppm): 6.94 (d,
1H, J = 9.3 Hz, pyridazine-H4); 6.99–7.08 (m, 2H, Ar–
H); 7.92-8.05 (m, 2H, Ar–H); 8.54 (d, 1H, J = 9.3 Hz,
pyridazine-H5); 10.73 (s, 1H, NH, D2O exchangeable);
11.69 (s, 2H, NH2, D2O exchangeable). MS (EI) m/z (%
?
rel. Int.): 231 (Mꢀ , 11.28), 232 (M?H, 2.45). Anal Calcd
for C11H9N3O3 (231.21): C, 57.14; H, 3.92; N, 18.17;
Found: C, 57.25; H, 4.10; N,18.35.
The test compounds were suspended in 0.5% carboxy-
methylcellulose (CMC) and given to the rats at a dose of
10 mg/kg 1 h prior to carrageenan injection. The paw
volume of each rat was measured before 1 h and after 1, 2,
and 4 h of carrageenan treatment using UGO-BASILE
7140 plethysmometer (Comerio, Italy).
2-(6-Phenylpyridazin-3-yloxy)benzamide 7
An equimolar mixture of 3-chloro-6-phenylpyridazine and
salicylamide (0.01 mol each) and anhydrous K2CO3
(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, cooled, and poured
into ice-cold water (20 mL). The solid separated was fil-
tered, dried, and crystallized from isopropanol.
Statistical analysis of data
Quantitative variables from normal distribution were
expressed as means SE (standard error). The significant
difference between groups was tested using one-way
ANOVA and the chosen level of significance was
p \ 0.05. Further comparisons among groups were made
according to post hoc Tukey’s test. All statistical analyses
were performed using Graph Pad Instat software version 3
(ISIÒ software, CA, USA).
Yield 78%; mp 139–140 °C; IR (KBr)/cm: 3398, 3363
(NH, NH2); 3049 (C–H aromatic); 1674, 1650 (C=O); 1593
1
(C=N). H-NMR (DMSO-d6, 300 MHz) d (ppm): 6.85 (d,
1H, J = 9 Hz, pyridazine-H4); 7.37-8.01 (m, 9H, Ar–H);
8.30 (d, 1H, J = 9 Hz, pyridazine-H5); 13.00 (s, 2H, NH2,
?
D2O exchangeable). MS (EI) m/z (% rel. Int.): 291 (Mꢀ ,
68), 292(M?H, 20.62). Anal Calcd for C17H13N3O2
(291.30): C, 70.09; H, 4.50; N, 14.42; Found: C, 70.44; H,
4.65; N, 14.60.
The anti-inflammatory activity was expressed as per-
centage inhibition of edema volume in treated animals in
comparison with the control group (Table 1).
123

3590
Med Chem Res (2012) 21:3581–3590
% Inhibition of edema = Vc - Vt/Vc 9 100 where Vc
and Vt are the volumes of edema for the control and drug-
treated animal groups, respectively.
the significance of data using SPSSÒ for windows, version
17.0. p \ 0.05 was taken as a cut off value for significance.
Potency of the tested compounds was calculated 4 h
after treatment with a single dose (10 mg/kg) of the tested
compounds relative to diclofenac-treated group.
According to the following equation:
References
Abouzid K, Bekhit SA (2008) Novel anti-inflammatory agents based
on pyridazinone scaffold; design, synthesis and in vivo activity.
Bioorg Med Chem 16:5547–5556
Allison MC, Howatson AG, Torranc CJ, Lee FD, Russell RIG (1992)
Gastrointestinal damage associated with the use of nonsteroidal
antiinflammatory drugs. N Engl J Med 327:749–754
Boissier JR, Ratouis R, Dumont C (1963) Synthesis and pharmaco-
logical study of new piperazine derivatives I benzylpiperazines.
J Med Chem 6:541–544
Potency ¼ % edema inhibition of test
compounds-treated group/
%edema inhibition of diclofenac-treated group:
ð1Þ
Chintakunta VK, Akella V, Vedula MS, Mamnoor PK, Mishra P,
Casturi SR, Vangoori A, Rajagopalan R (2002) 3-O-Substituted
benzyl pyridazinone derivatives as COX inhibitors. Eur J Med
Chem 37:339–347
Gastric ulcerative effect
Materials and methods
Clementi G, Caruso A, Cutuli VMC, deBernardis E, Prato A,
Mangano NG, Amico-Roxas M (1998) Effects of centrally or
peripherally injected adrenomedullin on reserpine-induced gas-
tric lesions. Eur J Pharmacol 360:51–54
Dogne JM, Supuran CT, Pratico D (2000) Adverse cardiovascular
effects of the coxibs. J Med Chem 48:2251–2257
Gleave RJ, Beswick PJ, Brown AJ, Giblin GMP, Goldsmith P,
Haslam CP et al (2010) Synthesis and evaluation of 3-amino-6-
aryl-pyridazines as selective CB(2) agonists for the treatment of
inflammatory pain. Bioorg Med Chem 20:465–468
Male Sprague–Dawley rats (120–130 g wt) were pur-
chased from the animal house facility the National
Research Center (Dokki, Giza, Egypt). Animals were
acclimatized in the animal house unite facility, Faculty of
pharmacy, Ain Shams University, Cairo, Egypt for at least
1 week prior to experimentation. Animals were kept at
22 3 °C and 55 5% relative humidity during the
whole experiment. Standard food pellets and water were
supplied ad labium. All test compounds were dispensed in
0.5% CMC solution in distilled water. Animals’ treatment
protocol was approved by Ain Shams University Animal
Rights committee.
¨
¨
Gokce M, Utku S, Kupeli E (2009) Synthesis and analgesic and anti-
inflammatory activities 6-substituted-3(2H)-pyridazinone-2-
acetyl-2-(p-substituted/non substituted benzal)hydrazone deriva-
tives. Eur J Med Chem 44:3760–3764
Graham DJ, Campen D, Hui R, Spence M (2005) Risk of acute
myocardial infarction and sudden cardiac death in patients
treated with cyclo-oxygenase 2 selective and non-selective non-
steroidal anti-inflammatory drugs: nested case-control study. The
Lancet 365(9458):475–481
Hsiao F-Y, Tsai Y-W, Huang W-F (2009) Changes in physicians’
practice of prescribing cyclooxygenase-2 inhibitor after market
withdrawal of rofecoxib: a retrospective study of physician-
patient pairs in Taiwan. Clin Ther 31(11):2618–2627
Ulcerative effect (Gastric hemorrhagic gross lesions) of
test compounds was assessed after intragastric adminis-
tration of 50 mg/kg in 0.5% CMC solution as previously
described; briefly, 6 h after drug administration, rats were
killed by cervical dislocation, and the stomachs were
removed, inflated, and opened along the greater curvature.
The hemorrhagic lesions were stretched out, and scored
from 0 to 5 according to the method of Clementi et al.
(1998). Gastric tissue samples were fixed in neutral buf-
fered formalin for 24 h. Stomach sections were dehydrated
with graded ethanol, passed through xylene, and embedded
in paraffin. The paraffin sections (5-lm thick) were stained
with hematoxylin and eosin (H&E) (Oyagi et al., 2010).
Mucosal membrane thinning and mucosal microscopic
ulcer depth were further quantified using Image-JTM soft-
ware indomethacin was used as positive control drug.
Lanas A, Rodriguez LAG, Arroyo MT, Gomollon F, Feu F, Gonzalez-
´
Perez A et al (2006) Risk of upper gastrointestinal ulcer bleeding
associated with selective cyclooxygenase-2 inhibitors, traditional
non-aspirin non-steroidal anti-inflammatory drugs, aspirin and
combinations. Gut 55(12):1731–1738
Oyagi A, Ogawa K, Kakino M, Hara H (2010) Protective effects of a
gastrointestinal agent containing Korean red ginseng on gastric
ulcer models in mice. BMC Complement Altern Med 10:45–53
¨
Su¨ku¨roglu M, C¸ aliskanergun B, Unlu¨ S, Sahin MF, Ku¨pelli E,
Yesilada E, Banoglu E (2005) Synthesis, analgesic, and anti-
inflammatory activities of [6-(3, 5-dimethyl-4-chloropyrazole-1-
yl)-3(2H)-pyridazinon-2-yl] acetamides. Arch Pharm Res 28:
509–517
Winter CA, Risley EA, Nuss GW (1962) Carrageenin-induced edema
in hind paw of the rat as an assay for antiinflammatory drugs.
Proc Soc Exp Biol Med 111:544–547
Statistical analysis
Data are presented as mean SEM. Analysis of variance
(ANOVA) with Dunnet’s post hoc test was used for testing
123