Synthesis and analgesic and anti-inflammatory activities 6-substituted-3(2H)-pyridazinone-2-acetyl-2-(p-substituted/nonsubstituted benzal)hydrazone derivatives

Article abstract of DOI:10.1016/j.ejmech.2009.04.048

In this study new 6-substituted-3(2H)-pyridazinone-2-acetyl-2-(p-substituted benzal)hydrazone V derivatives were synthesized as analgesic and anti-inflammatory agents. The structures of compounds were elucidated by spectral and elemental analysis. Compoun

Full text of DOI:10.1016/j.ejmech.2009.04.048

European Journal of Medicinal Chemistry 44 (2009) 3760–3764
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and analgesic and anti-inflammatory activities 6-substituted-3(2H)-
pyridazinone-2-acetyl-2-(p-substituted/nonsubstituted benzal)
hydrazone derivatives
,
b
c
Mehtap Go¨kçe a , Semra Utku , Esra Ku¨peli
*
a Gazi University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 06330 Ankara, Turkey
b Mersin University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 33169 Mersin, Turkey
c Gazi University, Faculty of Pharmacy, Department of Pharmacognosy, 06330 Ankara, Turkey
a r t i c l e i n f o
a b s t r a c t
Article history:
In this study new 6-substituted-3(2H)-pyridazinone-2-acetyl-2-(p-substituted benzal)hydrazone
V
Received 17 December 2008
Received in revised form
20 February 2009
Accepted 30 April 2009
Available online 9 May 2009
derivatives were synthesized as analgesic and anti-inflammatory agents. The structures of compounds
were elucidated by spectral and elemental analysis. Compounds Va, Vb and Vc were exhibited more
potent analgesic activity than ASA. Also these derivatives demonstrated anti-inflammatory activity as
well as standard compound indomethacin. Side effects of the compounds were examined on gastric
mucosa. None of the compounds showed gastric ulcerogenic effect compared with reference nonste-
roidal anti-inflammatory drugs (NSAIDs). On the basis of available data, the structure–activity rela-
tionship of V derivatives was also discussed.
Keywords:
Pyridazinone
Benzal hydrazone
Analgesic
Ó 2009 Elsevier Masson SAS. All rights reserved.
Anti-inflammatory activity
1. Introduction
inhibiting cyclooxygenases (COXs) [4]. The chronic use of NSAIDs
may elicit appreciable GI toxicity. Therefore synthetic approaches
Pain is a problem not solved by medicine and pain is the most
common reason that patients seek advice from pharmacist and
other professionals and represent important medical and economic
cost for the community. According to the World Health Organiza-
tion, 90% of diseases are associated with pain. Despite growing
knowledge of endogenous nociceptive and antinociceptive systems,
many pain syndromes like rheumatoid arthritis and certain
advanced cancers are still no adequate treatment [1].
Currently available nonsteroidal anti-inflammatory drugs
(NSAIDs) like ibuprofen, indomethacin and naproxen exhibit
gastric toxicity. Long-term use of these drugs has been associated
with gastrointestinal (GI) ulceration, bleeding and nephrotoxicity
[2]. The GI damage from NSAIDs is generally attributed to two
factors, i.e. local irritation by the carboxylic acid moiety common to
most NSAIDs (topical effect); and decreased tissue prostaglandin
production, which undermines the physiological role of cytopro-
tective prostaglandins in maintaining GI health and homeostasis
[3]. The pharmacological activity of NSAIDs is related to the
suppression of prostaglandin biosynthesis from arachidonic acid by
based upon chemical modification of NSAIDs have been taken with
the aim of improving their safety profile.
It is known that some pyrazolone derivatives like dipyrone and
phenylbutazone possess analgesic and anti-inflammatory activities,
but several side effects have limited the clinical use of these drugs.
Pyridazinone derivatives which are related structurally to pyr-
azolone derivatives in the point of ring enlargement of pyrazolone
to pyridazinone. A lot of 3(2H)-pyridazinone derivatives have been
reported as analgesic and anti-inflammatory agents without
gastrointestinal side effect. This is agreement with in our experi-
ence in the pyridazinone field [5–9]. Among the pyridazinone
derivatives endowed with antinociceptive effects, Emorfazone has
emerged the latter, which was launched in Japan, displays an
interesting profile because its activity is not mediated by interac-
tion with the prostaglandins system or by affinity for opioids
receptors [10,11]. Many authors reported that pyridazinone deriv-
atives bearing an arylpiperazine moiety at side chain on the lactam
nitrogen of the ring had significant analgesic activity [12–20].
Moreover, they claimed that these derivatives exhibited better
analgesic activity if they bear a carbon chain between the nitrogen
atom of the lactam and amine component of the side chain [21].
These results led us to design new structurally related derivatives,
keeping the 6-(substitute arylpiperazinyl)-3(2H)-piridazinone
* Corresponding author. Tel.: þ90 0312 2023226; fax: þ90 0312 2235018.
E-mail address: mgokce@gazi.edu.tr (M. Go¨kçe).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.04.048

M. Go¨kçe et al. / European Journal of Medicinal Chemistry 44 (2009) 3760–3764
3761
framework and modifying the substitutent on the 2 position of the
pyridazinone ring. As part of a program aimed at developing simple
and efficient syntheses of pharmacologically useful pyridazinones,
we synthesized new 6-substituted-3(2H)-pyridazinone-2-acetyl-2-
(p-substituted benzal)hydrazone V derivatives. The analgesic and
anti-inflammatory activities were investigated for the title
compounds utilizing the phenylbenzoquinone-induced writhing
test (PBQ test) and the carrageenan-induced foot paw edema test
(CPE test) respectively. All the compounds were also tested for the
irritative and ulcerogenic action on gastric mucosa.
was used [26]. ASA and indomethacin were used as reference drugs
in the tests respectively. All the pyridazinone derivatives have also
been evaluated for acute toxicity and gastric ulcerogenic effect tests.
The observed data on analgesic and anti-inflammatory activity of
the compounds and reference drugs are given in Tables 2 and 3.
4. Results, discussions and conclusions
In this study, novel derivatives of 3(2H)-pyridazinone have been
synthesized in attempt to find new derivatives having analgesic
and anti-inflammatory activities. One of the most interesting
characteristic of these novel compounds is their basic nature, which
differentiates them from the classical acidic nonsteroidal anti-
inflammatory agents (NSAIDs). It is interest, therefore to study
analgesic-anti-inflammatory properties of these novel compounds.
The synthesis pathway leading to the title compounds is given in
Scheme 1. 6-Substituted-3-chloropyridazines I were synthesized in
our laboratory according to reports in the literature [22,23]. As
a starting material, 6-substituted-3(2H)-pyridazinone-2-yl aceto-
hydrazide IV derivatives were used to produce new 6-substituted-
3(2H)-pyridazinone-2-acetyl-2-(p-substituted benzal)hydrazone V
derivatives. The structure of the compounds was elucidated by IR,
1H NMR spectral data and elemental analysis. In the IR spectra of all
new compounds, hydrazone C]O bands were observed at about
1682–1685 cmꢀ1 region and lactam C]O bands were observed at
about 1652–1660 cmꢀ1 region. Furthermore, all of the V derivatives
have N–H band at about 3172–3196 cmꢀ1 region in their IR spec-
trum. The absorption bands associated with other functional
groups appeared in the expected regions.
2. Chemistry
New 6-substituted-3(2H)-pyridazinone-2-acetyl-2-(p-substitu
ted/nonsubstituted benzal)hydrazone V derivatives were synthe-
sized according to Scheme 1. Initially, nucleophilic displacement
reaction of commercial 3,6-dichloropyridazine with arylpiperazines
in ethanol afforded 3-chloro-6-substitutedpyridazines I. The physical
and spectral properties of 3-chloro-6-substitutedpyridazine I were in
accordance with the literature [22,23]. Therefore we carried out the
next steps of the reaction without any further analysis. Synthesis
procedure of all II, III, IV derivatives reported by us in our previous
study [5,9,24]. Hydrolysis of 3-chloro-6-substituted pyridazines I was
carried out upon heating in glacial acetic acid to afford 6-substituted-
3(2H)-pyridazinone II derivatives. The formation of these
compounds was confirmed by IR spectra of a C]O signal at about
1660 cmꢀ1. Esterification of pyridazinones II was performed using
ethyl bromoacetate in the presence of potassium carbonate in
acetone at reflux temperature [9]. 6-Substituted-3(2H)-pyr-
idazinone-2-ylacetohydrazide IV derivatives were prepared by the
reaction of ethyl 6-substituted-3(2H)-pyridazinone-2-ylacetate III
derivatives with hydrazine hydrate in ethanol at room temperature
[24]. Finally, the target 6-substituted-3(2H)-pyridazinone-2-acetyl-
According to the literature [27,28], the hydrazones may exist as
Z/E geometrical isomers about C]N double bonds and cis/trans
amide conformers. It has been reported that when hydrazones are
dissolved in dimethyl-d6 sulfoxide solution, the E geometrical of
these compounds undergo a rapid cis/trans amide equilibrium, in
which the cis conformer predominates. 1H NMR spectra of the 6-
2-(p-substituted/nonsubstituted benzal)hydrazone
V derivatives
were synthesized via condensation of acetohydrazides IV with p-
substituted benzaldehydes. (Scheme 1). The physical data, yield, and
molecular formula of all compounds are reported Table 1.
substituted-3(2H)-pyridazinone-2-acetyl-2-(p-substituted
ben-
zal)hydrazone V derivatives that are taken in DMSO d6. In the 1H
NMR of the compounds Va–i, the signals belonging to benzylidene
group were observed at aromatic region while signals belonging to
the –NHNH2 disappeared. Two sets of signals each belonging to the
CH2 and ]CH group of cis and trans conformers were observed at
4.60–4.70/8.10–8.18 and 4.99–5.03/7.92–7.99 ppm. The upfield
lines of ]CH protons were assigned to cis conformer of the amide
structure and downfield lines of the protons of the same group
3. Pharmacology
The analgesic activity of the compounds was studied by using
phenylbenzoquinone-induced writhing test in mice [25]. In order to
screen the anti-inflammatory activity of the synthesized
compounds, carragenin-induced hind paw edema model in mice
N
N
N
N
Cl
Cl
+
H
N
N R
Cl
N
N R
I
CH3COOH
CH2COOCH2CH3
H
N
N
N
N
BrCH2COOCH2CH3
O
N
N
R
O
N
N R
II
III
NH2NH2.H2O
CH2CONH2NH2
O
C
N
N
CH2 CO NH
N
CH
R1
O
N
N
R
R1
O
N
N
H
N
N R
IV
V
Scheme 1. Synthesis of new 6-substituted-3(2H)-pyridazinone-2-acetyl-2-(p-substituted/nonsubstituted benzal) hydrazone V derivatives.

3762
M. Go¨kçe et al. / European Journal of Medicinal Chemistry 44 (2009) 3760–3764
Table 1
bearing methoxy substituent on benzal hydrazone ring exhibi-
ted no inhibition against p-benzoquinone-induced writhings in
mice. However, the mechanism underlying their antinociceptive
activity remains to some degree unknown. Thus further studies
are essential to ascertain the mechanism involved in the
analgesic properties of the ring system.
The anti-inflammatory activity evaluation was carried out using
carrageenin-induced paw oedema assay, and compounds Va, Vb, Vc
exhibited good anti-inflammatory activity. Anti-inflammatory
activity of 6-[4-(3-chlorophenyl)piperazine]-3(2H)-pyridazinone-2-
acetyl-2-benzal hydrazone Va has been found slight superior to that
of indomethacin although it was tested at a dosage much higher than
indomethacin. In light of these results, one that can say the p-
substitution of benzal hydrazone aromatic ring with methoxy or
methyl groups decreases either analgesic activity or anti-inflam-
matory activity.
We also screened all compounds for ulcerogenic adverse effect
at 200 mg/kg dose level. After microscopic elimination, no ulcera-
tion risk was seen in compounds Va–i. It is well known that most of
anti-inflammatory drugs provide an ulcerogenic activity. In the
present experiment ASA and indomethacin used as reference
showed marked ulcerogenic effect. Compounds Va, Vb, Vc
possessed anti-inflammatory activity as well as indomethacin and
more potent analgesic activity than ASA and did not induce any
gastric lesions or death in the observation period. Since our findings
are preliminary results; further studied need to be carried out to
investigate the other specifications such as in vitro assays, toxico-
logical studies or side effect-activity profiles of these compounds.
Physical constant of 6-substituted-3(2H)-pyridazinone-2-acetyl-2-(p-substituted/
nonsubstituted benzal)hydrazone V derivatives.
CH2 CO NH N CH
N N
R1
O
N
N R
V
Com.
R
R1
Yield (%)
Mp (ꢂC)
Molecular formula
(M. W)
Va
Vb
Vc
Vd
Ve
Vf
Vg
Vh
Vi
3-Chlorophenyl
4-Chlorophenyl
2-pyridyl
3-Chlorophenyl
4-Chlorophenyl
2-pyridyl
3-Chlorophenyl
4-Chlorophenyl
2-pyridyl
H
H
H
CH3
CH3
67
70
73
62
69
72
61
56
64
194
191
196
168
159
241
211
171
165
C23H23ClN6O2
C23H23ClN6O2
C22H23N7O2
C24H25ClN6O2
C24H25ClN6O2
C23H25N7O2
C24H25ClN6O3
C24H25ClN6O3
C23H25N7O3
CH3
OCH3
OCH3
OCH3
Melting points were determined on electrothermal melting point apparatus and
uncorrected. The C, H, N were performed at Scientific and Technical Council of
Turkey, Instrumental Analysis Center and within ꢁ0.4% of the theoretical values.
were assigned to trans conformer of the amide structure [27]. NH
proton of the hydrazone moiety was seen as singlet at about 11.28–
11.34 ppm. All the other aromatic and aliphatic protons were
observed at the expected region. Further spectroscopic details of
these compounds are presented in the Experimental part.
All the newly synthesized compounds were tested for anti-
inflammatory and analgesic activities. These results of anti-inflam-
matory and analgesic activities of all compounds are statistically
significant. Analgesic activity evaluation was carried out using
phenylquinone-induced writhing assay. 6-[4-(3-Chlorophenyl)piper-
azine]-3(2H)-pyridazinone-2-acetyl-2-benzal hydrazone Va, 6-[4-
(4-chlorophenyl)piperazine]-3(2H)-pyridazinone-2-acetyl-2-benzal
hydrazone Vb and 6-[4-(pyridyl)piperazine]-3(2H)-pyridazinone-
2-acetyl-2-benzal hydrazone Vc derivatives have been shown
better analgesic activity than reference compound ASA.
These compounds have no substituent on benzal hydrazone
aromatic ring. While the compounds Va, Vb, Vc exhibited
remarkable analgesic activity, 6-[4-(3-chlorophenyl)piperazine]-
3(2H)-pyridazinone-2-acetyl-2-(p-methyl benzal)hydrazone Vd,
6-[4-(4-chlorophenyl)piperazine]-3(2H)-pyridazinone-2-acetyl-2-
(p-methyl benzal)hydrazone Ve, 6-[4-(pyridyl)piperazine]-3
(2H)-pyridazinone-2-acetyl-2-(p-methyl benzal)hydrazone Vf
derivatives showed moderate analgesic activity. Vh and Vi
5. Experimental
All chemical melting points of the compounds were determined
on Electrothermal 9200 melting points apparatus (Southent, Great
Britain) and the values given are uncorrected.
The IR spectra of the compounds were recorded on a Bruker
Vector 22 IR Spectrophotometer (Bruker Analytische Messtechnik,
Karlrure, Germany). The 1H NMR of the compounds spectra were
recorded on a Bruker 400 MHz-NMR Spectrometer (Rheinstetten,
Karlrure, Germany) using tetramethylsilane as an internal standard.
All the chemical shifts were recorded as
d (ppm). Elemental anal-
yses were performed with Leco-932 (C, H, N, S, O-Elemental
analyzer, St. Joseph, USA) at Scientific and Technical Research
Council of Turkey, Instrumental Analysis Center (Ankara, Turkey)
and within ꢁ0.4% of the theoretical values.
5.1. General procedure for the synthesis of compounds
5.1.1. Synthesis of 6-substituted-3(2H)-pyridazinone
derivatives IIa–i
Table 2
Analgesic activity of 6-substituted-3(2H)-pyridazinone-2-acetyl-2-(p-substituted/
nonsubstituted benzal)hydrazone V derivatives against p-benzoquinone-induced
writhings in mice.
A solution of 0.05 mol of a 6-substituted-3-chloropyridazinone I
derivative in 30 ml glacial acetic acid was refluxed for 6 h. The
acetic acid was removed under reduced pressure, the residue dis-
solved in water and extracted with CHCl3. The organic phase was
dried over Na2SO4 and evaporated under reduced pressure. The
residue was purified by recrystallization from ethanol.
Test samples
Dose
(mg/kg)
Number
of writhings ꢁ SEM
Inhibitory
ratio (%)
Ratio of
ulceration
Control
Va
Vb
Vc
Vd
Ve
Vf
Vg
Vh
Vi
42.7 ꢁ 4.92
20.3 ꢁ 1.98
21.5 ꢁ 1.85
19.4 ꢁ 2.13
31.0 ꢁ 2.25
33.5 ꢁ 4.02
26.1 ꢁ 3.09
35.7 ꢁ 2.41
47.4 ꢁ 4.16
51.2 ꢁ 3.98
21.9 ꢁ 1.92
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
0/6
5/6
100
100
100
100
100
100
100
100
100
100
52.5***
49.6***
54.6***
27.4
21.5
38.9*
16.4
5.1.2. Synthesis of ethyl 6-substituted-3(2H)-pyridazinone-2-
ylacetate derivatives IIIa–I
A
mixture of required 6-substitited-3(2H)pyridazinones II
(0.01 mol), ethyl bromoacetate (0.02 mol) and potassium carbonate
(0.02 mol) in acetone (40 ml) was refluxed overnight. After the mixture
was cooled, the organic salts were filtered off, the solvent evaporated,
and the residue purified by recrystallization with appropriate alcohol
to give the esters.
–
–
ASA
48.7***
*p < 0.05, **p < 0.01, ***p < 0.001 significant from the control value.

M. Go¨kçe et al. / European Journal of Medicinal Chemistry 44 (2009) 3760–3764
3763
Table 3
Antiiflammatory activity of 6-substituted-3(2H)-pyridazinone-2-acetyl-2-(p-substituted/nonsubstituted benzal)hydrazone V derivatives against carrageenan-induced paw
edema in mice.
Test samples
Dose (mg/kg)
Swelling thickness (ꢃ10ꢀ2mm) ꢁ SEM (inhibition %)
90 min
180 min
270 min
360 min
Control
Va
Vb
Vc
Vd
Ve
Vf
Vg
49.1 ꢁ 4.16
56.4 ꢁ 4.93
64.9 ꢁ 5.02
73.5 ꢁ 5.27
100
100
100
100
100
100
100
100
100
10
37.6 ꢁ 2.50 (23.4)
40.8 ꢁ 2.94 (16.9)
39.6 ꢁ 2.51 (19.3)
49.6 ꢁ 3.02
39.3 ꢁ 2.82 (30.3)*
42.7 ꢁ 3.03 (24.2)
41.2 ꢁ 2.64 (26.9)
57.3 ꢁ 3.26
40.5 ꢁ 3.14 (37.6)**
45.6 ꢁ 3.22 (32.7)**
44.8 ꢁ 2.16 (30.9)*
61.2 ꢁ 3.91 (5.7)
57.5 ꢁ 3.06 (11.4)
49.3 ꢁ 3.02 (24.0)
54.1 ꢁ 3.19 (16.6)
65.8 ꢁ 4.81
43.3 ꢁ 3.19 (41.0)***
49.2 ꢁ 3.17 (33.1)**
49.6 ꢁ 3.20 (32.5)**
69.8 ꢁ 3.24 (5.0)
59.9 ꢁ 3.73 (18.5)
51.1 ꢁ 3.16 (30.5)*
60.2 ꢁ 4.01 (18.1)
70.9 ꢁ 3.92 (3.5)
74.1 ꢁ 3.27
45.2 ꢁ 2.83 (7.9)
41.7 ꢁ 2.64 (15.1)
45.1 ꢁ 2.64 (8.1)
50.2 ꢁ 4.11
49.6 ꢁ 2.53 (12.1)
45.8 ꢁ 2.91 (18.8)
49.8 ꢁ 3.02 (11.7)
59.4 ꢁ 4.72
Vh
Vi
47.6 ꢁ 3.24 (3.1)
32.2 ꢁ 3.14 (34.4)*
54.2 ꢁ 2.98 (3.9)
37.9 ꢁ 3.02 (32.8)*
67.9 ꢁ 3.15
Indomethacin
40.6 ꢁ 2.94 (37.4)**
43.8 ꢁ 2.71 (40.4)***
*p < 0.05, **p < 0.01, ***p < 0.001 significant from the control value.
5.1.3. Synthesis of 6-substituted-3(2H)-pyridazinone-2-yl acetohy
drazide derivatives IVa–I
protons þ pyridazinone H5), 7.97 and 8.12 (1H, s, s, N]CH), 11.57
(1H, s, NH), Anal. Calc. for C24H25ClN6O2: C: 62.00, H: 5.42, N: 18.08.
Found: C: 62.13, H: 5.51, N: 18.33.
To methanolic solution of ethyl 6-substituted-3(2H)-pyr-
idazinone-2-ylacetate derivatives III (25 ml, 0.01 mol) was added
hydrazine hydrate (99%) (3 ml) and stirred for 3 h in the room
temperature. The precipitate obtained was filtered off, washed with
water, dried and recrystallized from ethanol.
5.1.4.5. Compound Ve. IR (KBr) nmax (cmꢀ1): 3186 N–H, 1680 C]O
hydrazone, 1660 C]O lactam, 1592–1509 C]N, 1H NMR (300 MHz)
(DMSO d6) d (ppm): 2.29 (s, 3H, CH3), 3.23–3.36 (m, 4H, piperazine
b þ b0), 3.41–3.47 (m, 4H, piperazine a þ a0), 4.61 and 5.03 (2H, s, s,
CH2), 6.76–6.89 (d, 1H, pyridazinone H4), 6.96–7.62 (m, 9H, phenyl
protons þ pyridazinone H5), 7.93 and 8.13 (1H, s, s, N]CH), 11.56
(1H, s, NH), Anal. Calc. for C24H25ClN6O2: C: 62.00, H: 5.42, N: 18.08.
Found: C: 61.96, H: 5.30, N: 18.24.
5.1.4. Synthesis of 6-substituted-3(2H)-pyridazinone-2-acetyl-2-
(p-substituted benzal)hydrazone V derivative
‘Mixture of 6-substituted-3(2H)-pyridazinone-2-yl acetohy-
drazide derivatives IV (0.01 mol) and appropriate benzaldehyde
(0.01 mol) was refluxed in ethanol (15 ml) for 6 h. Then the mixture
was poured into ice-water. The precipitate formed was recrystallized
from ethanol.
5.1.4.6. Compound Ve. IR (KBr) nmax (cmꢀ1): 3182 N–H, 1677 C]O
hydrazone, 1658 C]O lactam, 1589–1509 C]N, 1H NMR (300 MHz)
(DMSO d6) d (ppm): 2.30 (s, 3H, CH3), 3.24–3.35 (m, 4H, piperazine
5.1.4.1. Compound Va. IR (KBr) nmax (cmꢀ1): 3185 N–H, 1681 C]O
hydrazone, 1652 C]O lactam, 1595–1515 C]N, 1H NMR (300 MHz)
b þ b0), 3.42–3.48 (m, 4H, piperazine a þ a0), 4.61 and 5.03 (2H, s, s,
CH2), 6.76–6.89 (d, 1H, pyridazinone H4), 6.96–7.62 (m, 9H, phenyl
protons þ pyridazinone H5), 7.93 and 8.13 (1H, s, s, N]CH), 11.58
(1H, s, NH), Anal. Calc. for C24H25ClN6O2: C: 62.00, H: 5.42, N: 18.08.
Found: C: 61.96, H: 5.30, N: 18.24.
(DMSO d6)
d
(ppm): 3.20–3.27 (m, 4H, piperazine b þ b0), 3.28–3.34
(m, 4H, piperazine a þ a0), 4.82 and 5.03 (2H, s, s, CH2), 6.88–6.91 (d,
1H, pyridazinone H4), 6.96–7.70 (m, 10H, phenyl proto-
ns þ pyridazinone H5), 7.98 and 8.18 (1H, s, s, N]CH), 11.64 (1H, s,
NH). Anal. Calc. for C23H23ClN6O2: C: 61.26, H: 5.14, N: 18.64. Found:
C: 61.38, H: 5.21, N: 18.83.
5.1.4.7. Compound Vf. IR (KBr) nmax (cmꢀ1): 3181 N–H, 1679 C]O
hydrazone, 1661 C]O lactam, 1590–1508 C]N, 1H NMR (300 MHz)
(DMSO d6) d (ppm): 2.30 (s, 3H, CH3), 3.24–3.35 (m, 4H, piperazine
5.1.4.2. Compound Vb. IR (KBr) nmax (cmꢀ1): 3182 N–H, 1683 C]O
hydrazone, 1653 C]O lactam, 1590–1510 C]N, 1H NMR (300 MHz)
b þ b0), 3.41–3.47 (m, 4H, piperazine a þ a0), 4.61 and 5.01 (2H, s, s,
CH2), 6.77–6.87 (d, 1H, pyridazinone H4), 6.93–7.62 (m, 9H, pyridyl
protons þ pyridazinone H5), 7.95 and 8.14 (1H, s, s, N]CH), 11.61
(1H, s, NH), Anal. Calc. for C23H25N7O2: C: 64.02, H: 5.84, N: 22.72.
Found: C: 64.21, H: 5.60, N: 22.31.
(DMSO d6)
d
(ppm): 3.26–3.33 (m, 4H, piperazine b þ b0), 3.34–3.40
(m, 4H, piperazine a þ a0), 4.63 and 5.01(2H, s, s, CH2), 6.78–6.86 (d,
1H, pyridazinone H4), 6.91–7.70 (m, 10H, phenyl proto-
nsþ þ pyridazinone H5), 7.99 and 8.18 (1H, s, s, N]CH), 11.61 (1H, s,
NH). Anal. Calc. for C23H23ClN6O2: C: 61.26, H: 5.14, N: 18.64. Found:
C: 61.46, H: 5.10, N: 18.43.
5.1.4.8. Compound Vg. IR (KBr) nmax (cmꢀ1): 3180 N–H, 1679 C]O
hydrazone, 1659 C]O lactam, 1591–1510 C]N, 1H NMR (300 MHz)
(DMSO d6)
d
(ppm): 3.18–3.35 (m, 4H, piperazine b þ b0), 3.36–3.43
5.1.4.3. Compound Vc. IR (KBr) nmax (cmꢀ1): 3185 N–H, 1682 C]O
hydrazone, 1658 C]O lactam, 1593–1512 C]N, 1H NMR (300 MHz)
(m, 4H, piperazine a þ a0), 3.75 (s, 3H, OCH3), 4.59 and 4.99 (2H, s, s,
CH2), 6.86–6.94 (d, 1H, pyridazinone H4), 6.97–7.62 (m, 9H, phenyl
protons þ pyridazinone H5), 7.91 and 8.10 (1H, s, s, N]CH), 11.50
(1H, s, NH), Anal. Calc. for C24H25ClN6O3: C: 59.94, H: 5.24, N: 17.47.
Found: C: 59.63, H: 5.43, N: 17.13.
(DMSO d6)
d
(ppm): 3.25–3.33 (m, 4H, piperazine b þ b0), 3.34–3.40
(m, 4H, piperazine a þ a0), 4.68 and 5.02 (2H, s, s, CH2), 6.78–6.80 (d,
1H, pyridazinone H4), 6.96–7.70 (m, 10H, pyridyl proto-
ns þ pyridazinone H5), 7.98 and 8.17 (1H, s, s, N]CH), 11.60 (1H, s,
NH), Anal. Calc. for C22H23N7O2: C: 63.30, H: 5.55, N: 23.49. Found:
C: 63.42, H: 5.62, N: 23.83.
5.1.4.9. Compound Vh. IR (KBr) nmax (cmꢀ1): 3181 N–H, 1680 C]O
hydrazone, 1660 C]O lactam, 1590–1510 C]N, 1H NMR (300 MHz)
(DMSO d6)
d
(ppm): 3.24–3.34 (m, 4H, piperazine b þ b0), 3.38–3.48
5.1.4.4. Compound Vd. IR (KBr) nmax (cmꢀ1): 3189 N–H, 1678 C]O
hydrazone, 1661 C]O lactam, 1590–1510 C]N, 1H NMR (300 MHz)
(m, 4H, piperazine a þ a0), 3.76 (s, 3H, OCH3), 4.60 and 4.99 (2H, s, s,
CH2), 6.76–6.90 (d, 1H, pyridazinone H4), 6.95–7.63 (m, 9H, phenyl
protons þ pyridazinone H5), 7.92 and 8.11 (1H, s, s, N]CH), 11.51
(1H, s, NH), Anal. Calc. for C24H25ClN6O3: C: 59.94, H: 5.24, N: 17.47.
Found: C: 59.47, H: 5.18, N: 17.24.
(DMSO d6)
d (ppm): 2.28 (s, 3H, CH3), 3.18–3.35 (m, 4H, piperazine
b þ b0), 3.42–3.49 (m, 4H, piperazine a þ a0), 4.60 and 5.00 (2H, s, s,
CH2), 6.85–6.91 (d, 1H, pyridazinone H4), 6.97–7.63 (m, 9H, phenyl

3764
M. Go¨kçe et al. / European Journal of Medicinal Chemistry 44 (2009) 3760–3764
5.1.4.10. Compound Vf. IR (KBr) nmax (cmꢀ1): 3182 N–H, 1682 C]O
hydrazone, 1662 C]O lactam, 1592–1510 C]N, 1H NMR (300 MHz)
groups were compared with mean values of a control group and
analyzed using statistical methods. Indomethacin (10 mg/kg) was
used as reference drug.
(DMSO d6)
d
(ppm): 3.23–3.34 (m, 4H, piperazine b þ b0), 3.40–3.49
(m, 4H, piperazine a þ a0), 3.75 (s, 3H, OCH3), 4.60 and 5.00 (2H, s, s,
CH2), 6.76–6.89 (d, 1H, pyridazinone H4), 6.93–7.62 (m, 9H, pyridyl
protons þ pyridazinone H5), 7.91 and 8.10 (1H, s, s, N]CH), 11.49
(1H, s, NH), Anal. Calc. for C23H25N7O3: C: 61.73, H: 5.63, N: 21.91.
Found: C: 61.90, H: 5.42, N: 21.61.
5.2.4. Acute toxicity
Animals employed in the carrageen-induced paw edema
experiment were observed during 24 h and mortality was recor-
ded, if happens, for each group at the end of observation period.
5.2. Analgesic and anti-inflammatory activity
5.2.5. Gastric ulcerogenic effect
After the analgesic activity experiment mice were killed under
deep ether anesthesia and stomachs were removed. Then the
abdomen of each mouse was opened through the great curvature
and examined under dissecting microscope for lesions or bleedings.
5.2.1. Animals
Male Swiss albino mice (20–25 g) were purchased from the
animal breeding laboratories of Refik Saydam Central Institute of
Health (Ankara, Turkey). The animals left for two days for accli-
matization to animal room conditions were maintained on stan-
dard pellet diet and water ad libitum. The food was withdrawn one
day before the experiment, but allowed free access of water. Six
animals at least were used in each group. Throughout the experi-
ments, animals were processed according to the suggested ethical
guidelines for the care of laboratory animals.
5.2.6. Statistical analysis of data
Data obtained from animal experiments were expressed as
mean standard error (ꢁSEM). Statistical differences between the
treatments and the control were evaluated by ANOVA and
Students–Newman–Keels post-hoc tests. p < 0.05 was considered
to be significant [*p < 0.05; **p < 0.01; ***p < 0.001].
5.2.2. Preparation of test samples for bioassay
References
Test samples were given orally to test animals after suspending
in a mixture of distilled H2O and 0.5% sodium carboxymethylcel-
lulose (CMC). The control group animals received the same exper-
imental handling as those of the test groups except that the drug
treatment was replaced with appropriate volumes of the dosing
vehicle. Either indometacin (CAS 53-86-1) (10 mg/kg) or acetylsa-
licylic acid (CAS 50-78-2; ASA) (100 mg/kg) in 0.5% CMC was used
as reference drug.
[1] C. Vergelli, M.P. Giovannoni, S. Pieretti, A. Di Giannuario, V. Dal Piaz, Bioorg.
Med. Chem. 15 (16) (2007) 5563–5575.
[2] T.K. Motawi, H.M. Abd Elgawad, N.N. Shahin, J. Biochem. Mol Toxicol. 21 (5)
(2007) 280–288.
[3] Y. Nakano, E. Kuroda, T. Kito, S. Uematsu, S. Akira, A. Yokota, S. Nishizawa,
U. Yamashita, J. Neurosurg. 108 (2) (2008) 311–319.
[4] P. Singh, A. Mittal, Mini. Rev. Med. Chem. 8 (1) (2008) 73–90 Review.
[5] M. Go¨kçe, D. Dog˘ruer, M.F. S¸ahin, IL Farmaco 56 (2001) 233–237.
[6] M. Go¨kçe, M.F. S¸ahin, E. Ku¨peli, E. Yes¸ilada, Arzneim. Forsch. Drug Res. 54
(2004) 396–401.
[7] M.F. S¸ahin, B. Badıçog˘lu, M. Go¨kçe, E. Ku¨peli, E. Yes¸ilada, Arch. Pharm. Pharm.
Med. 337 (2004) 445–452.
[8] M. Go¨kçe, G. Bakır, M.F. S¸ahin, E. Ku¨peli, E. Yes¸ilada, Arzneim. Forsch. Drug Res.
55 (2005) 318–325.
[9] Y. Du¨ndar, M. Go¨kçe, E. Ku¨ peli, M.F. S¸ahin, Arzneim. Forsch. Drug Res. 57
(2007) 777–781.
[10] M. Sato, Y. Ishizuka, A. Yamagucci, Arzneim. Forsch. Drug Res. 31 (1981)
1738–1745.
[11] M.C. Viaud, P. Jamoneau, C. Flouzat, J.G. Bizod-Espiard, B. Pfeiffer, P. Renard,
D.H. Caignard, G. Adam, G. Guillaument, J. Med. Chem. 38 (8) (1995) 1278–1286.
[12] F. Rohet, C. Rubat, P. Coudert, E. Albuisson, J. Couquelet, Chem. Pharm. Bull.
(Tokyo) 44 (5) (1996) 980–986.
[13] V. Dal Piaz, C. Vergelli, M.P. Giovannoni, M.A. Scheider, G. Petrone, P. Zaratin,
Eur. J. Med. Chem. 58 (2003) 1063–1071.
[14] C. Rubat, P. Coudert, P. Tronche, J. Bastide, P. Bastide, A.M. Privat, Chem. Pharm.
Bull. 37 (10) (1989) 2832–2835.
[15] C. Rubat, P. Coudert, E. Albussion, J. Bastide, J. Couquelet, P. Tronche, J. Pharm.
Sci. 81 (1992) 1084–1087.
5.2.3. Analgesic and anti-inflammatory activity tests
5.2.3.1. p-Benzoquinone-induced abdominal constriction test in mice
[25]. 60 min after the oral administration of test samples, the mice
were intraperitoneally injected with 0.1 ml/10 g body weight of
2.5% (v/v) p-benzoquinone (PBQ; Merck) solution in distilled H2O.
Control animals received an appropriate volume of dosing vehicle.
The mice were then kept individually for observation and the total
number of abdominal contractions (writhing movements) was
counted for the next 15 min, starting on the 5th min after the PBQ
injection. The data represent average of the total number of
writhings observed. The antinociceptive activity was expressed as
writhing percentage of that of control0s. 100 mg/kg acetylsalicylic
acid (ASA) was used as the reference.
[16] V. Dal Piaz, M.P. Giovannoni, G. Ciciani, D. Barlocco, G. Giardina, G. Petrone,
G.D. Clarke, Eur. J. Med. Chem. 31 (1996) 65–70.
[17] N. Cesari, C. Biancalani, C. Vergelli, V. Dal Piaz, A. Graziano, P. Biagini, C. Ghe-
lardini, N. Galeotti, M.P. Giovannoni, J. Med. Chem. 49 (26) (2006) 7826–7835.
[18] M.P. Giovannoni, C. Vergelli, C. Chelardini, G. Nicoletta, A. Bartolini, V. Dal Piaz,
J. Med. Chem. 46 (2003) 1055–1059.
5.2.3.2. Carrageenan-induced hind paw edema [26]. The method of
Kasahara et al. (1985) was used with modifications in measuring
periods. The difference in footpad thickness between the right and
left foot was measured with a pair of dial thickness gauge calipers
(Ozaki Co., Tokyo, Japan). Mean values of treated groups were
compared with mean values of a control group and analyzed using
statistical methods. 60 min after the oral administration of test
sample or dosing vehicle each mouse was injected with freshly
[19] N. A Santagati, F. Duro, A. Caruso, S. Trombadore, M. Amico-Roxas, IL Farmaco
Ed. Sci. 40 (1985) 921–929.
¨
[20] E. Banog˘lu, Ç. Akog˘lu, S. Unlu¨, E. Ku¨peli, E. Yes¸ilada, M.F. S¸ahin, Arch. Pharm.
Pharm. Med. Chem. 337 (2004) 7–14.
[21] D.S. Dogruer, M.F. Sahin, S. Unlu, S. Ito, Arch. Pharm. Pharm. Med. Chem. 333
(2000) 79–86.
prepared (0.5 mg/25
Louis, Missouri, USA) in physiological saline (154 nmol/l NaCl) into
subplantar tissue of the right hind paw. As the control, 25 l saline
ml) suspension of carrageenan (Sigma, St.
[22] J.R. Boissier, R. Ratouis, C. Dumont, J. Med. Chem. 6 (1963) 541–544.
[23] B. Elvio, F. Paravicini, T. Emilio, Farmaco Ed. Sci. 24 (1969) 919–929.
[24] G. Yelken, Msc. Thesis, Gazi University, Institute of Health Science, Ankara,
Turkey, 2008.
[25] R. Okun, S.C. Liddon, L. Lasagnal, J. Pharmacol. Exp. Ther. 139 (1963) 107–114.
[26] Y. Kasahara, H. Hikino, S. Tsurufujı, M. Watanabe, Planta Med. 51 (1985) 325.
[27] G. Palla, G. Predieri, G. Domiano, Tetrahedron 42 (1986) 3649–3654.
[28] E. Wryzykiewicz, D. Prakula, J. Heterocycl. Chem. 35 (1988) 381–386.
m
solutions were injected into that of the left hind paw. Paw edema
was measured in every 90 min during 6 h after induction of
inflammation. The difference in footpad thickness was measured by
a gauge calipers (Ozaki Co., Tokyo, Japan). Mean values of treated