Synthesis and antinociceptive activities of some pyrazoline derivatives

Article abstract of DOI:10.1016/j.ejmech.2008.09.002

In the present study, some pyrazoline derivatives were synthesized to investigate their potential antinociceptive activities. 1-[(Benzoxazole/benzimidazole-2-yl)thioacetyl]pyrazoline derivatives were obtained by reacting 3,5-diaryl-1-(2-chloroacetyl)pyrazolines with 2-marcaptobenzoxazole/benzimidazole. The chemical structures of the compounds were elucidated by IR, ¹H NMR and FAB⁺-MS spectral data and Elemental Analyses. All of the compounds (100 mg/kg) exhibited significant antinociceptive activities in both hot plate and acetic acid-induced writhing tests. Naloxone (5 mg/kg) pre-treatment reversed the antinociceptive activities suggesting the involvement of opioid system in the analgesic actions. None of the compounds impaired motor coordination of animals when assessed in the Rota-Rod model. These results support the previous papers reporting the opioid sensitive antinociceptive activities of various benzoxazole/benzimidazole-pyrazoline derivative compounds.

Full text of DOI:10.1016/j.ejmech.2008.09.002

European Journal of Medicinal Chemistry 44 (2009) 2606–2610
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Short communication
Synthesis and antinociceptive activities of some pyrazoline derivatives
,
a
a
b
Zafer Asim Kaplancikli a , Gu¨lhan Turan-Zitouni , Ahmet Ozdemir , Ozgu¨r Devrim Can ,
*
¨
¨
Pierre Chevallet c
a Anadolu University, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, 26470 Eskis¸ehir, Turkey
b Anadolu University, Faculty of Pharmacy, Department of Pharmacology, 26470 Eskis¸ehir, Turkey
c Institut des Biomole´cules Max Mousseron, UMR 5247, CNRS-Universite´ Montpellier 1 et 2, Facult´e de Pharmacie, Montpellier Cedex 5, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 14 April 2008
Received in revised form 22 July 2008
Accepted 1 September 2008
Available online 12 September 2008
In the present study, some pyrazoline derivatives were synthesized to investigate their potential anti-
nociceptive activities. 1-[(Benzoxazole/benzimidazole-2-yl)thioacetyl]pyrazoline derivatives were
obtained by reacting 3,5-diaryl-1-(2-chloroacetyl)pyrazolines with 2-marcaptobenzoxazole/benzimid-
azole. The chemical structures of the compounds were elucidated by IR, 1H NMR and FABþ-MS spectral
data and Elemental Analyses.
All of the compounds (100 mg/kg) exhibited significant antinociceptive activities in both hot plate and
acetic acid-induced writhing tests. Naloxone (5 mg/kg) pre-treatment reversed the antinociceptive
activities suggesting the involvement of opioid system in the analgesic actions. None of the compounds
impaired motor coordination of animals when assessed in the Rota-Rod model.
These results support the previous papers reporting the opioid sensitive antinociceptive activities of
various benzoxazole/benzimidazole–pyrazoline derivative compounds.
Keywords:
Pyrazoline
Benzoxazole
Benzimidazole
Hot plate
Writhing test
Rota-Rod
Ó 2008 Elsevier Masson SAS. All rights reserved.
1. Introduction
possess analgesic activities mediated by peripheral mechanisms
such as inhibitions of cyclooxygenase enzyme activity, arachidonic
Pain is a disagreeable and subjective sensation resulting from
a harmful sensorial stimulation that alerts the body about a current
or potential damage to its tissues or organs. Despite the painful
sensation, which can be efficiently solved by the removal of the
main reason, the pain-causing stimulus cannot always be either
easily defined or quickly removed. Contemporary analgesics, like
opiates and nonsteroidal anti-inflammatory drugs have some
limitations in clinical use, especially for opiates, such as addiction,
tolerance and side effects [1]. Therefore, experimental researches
for the development of safer and more effective analgesic agents
are a great deal of interest for many researchers.
In the chemical literature, derivatives of nitrogenated hetero-
cyclic aromatics of five members have been described with the
inhibition of prostaglandin biosynthesis. Some of these azole
derivatives are pyrrols [2–4], imidazoles [5], pyrazoles [6,7] and
pyrazolines, which are pyrazole derivatives [8,9]. It has been
suggested that biological evaluation of new bioactive molecules
containing pyrazol nucleus is important for the creation of future-
promising new analgesic agents [10–12]. Some pyrazoline-derived
compounds including dipyrone (metamizol) have been shown to
acid cascade and prostaglandin biosynthesis [2–9]. However, some
other pyrazoline-derived compounds have been reported as cen-
trally acting analgesic agents [10–12]. Decrease of on/off cell firing
in the periaqueductal gray [11], activation of endogen opioid
mechanisms [10] and spinal noradrenergic and serotonergic
systems [12] are some of the suggested mechanisms about this
centrally mediated analgesia. On the other hand, different pyrazo-
line derivatives without anti-inflammatory activities or with
analgesic actions unrelated to opioids have also been reported [11].
In addition, benzoxazoles/benzimidazoles have been investi-
gated extensively for their analgesic and anti-inflammatory actions
and have become a promising group to induce analgesia [13–16].
Some benzoxazole derivatives were reported to inhibit nitric oxide
synthase (NOS) activity that contribute to acute and chronic
inflammations [17] and they were evaluated as a novel class of non-
amino acid based NOS inhibitors [18]. Besides their peripherally
mediated antinociceptive actions, some benzimidazole-derived
compounds have also been reported to possess centrally mediated
analgesic effects like pyrazoline-derived ones [19–21].
In the interest of above, the synthesis of the compact-structured
pyrazole system by combining these two bioactive components,
benzoxazoles/benzimidazoles and pyrazolines, and investigation of
their possible analgesic activities were aimed, in this study.
* Corresponding author. Tel.: þ90 222 335 05 80/3779; fax: þ90 222 335 07 50.
E-mail address: zakaplan@anadolu.edu.tr (Z.A. Kaplancikli).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.09.002

Z.A. Kaplancikli et al. / European Journal of Medicinal Chemistry 44 (2009) 2606–2610
2607
O
O
O
R2
R3
R2
R3
NaOH
H
+
1
R1
R1
R2
R3
1
H2N-NH2
+
R1
N
N
H
2
R2
R3
O
N(CH3)3
2
+
Cl
Cl
N
R1
N
O
Cl
3
R2
R3
N
R4
K2CO3
HS
3
+
N
O
R1
N
X
N
R4
S
4 a-f
X
Scheme 1.
2. Chemistry
study. Centrally and peripherally mediated nociceptive activities
were measured by hot plate [24–26] and acetic acid-induced
writhing tests [27,28], respectively. Morphine sulphate (Sigma
Chemical Company, USA) was used as a reference drug and naloxone
used as an opioid antagonist (Sigma Chemical Company, USA) for
nociceptive tests. Rota-Rod test was performed for the examination
of probable neurological deficits due to the test compounds, which
may interfere with the test results to give false positives such as
muscle relaxant or impairment of motor coordination [11,29].
In the present work, 1,3-diaryl-2-propen-1-ones (1) were
prepared by reacting acetophenones and benzaldehydes in accor-
dance with the method described in the literature [22]. 3,5-Diaryl-
2-pyrazolines (2) and 1-(chloroacetyl)-3,5-diaryl-2-pyrazolines (3)
used in the synthesis were prepared according to the methods
reported in the literature [22,23]. The reaction of 1-(chloroacetyl)-
3,5-diaryl-2-pyrazoline (3), benzazol-2-thiole and anhydrous
potassium carbonate in acetone gave the 1-[(benzazole-2-yl)th-
ioacetylamino]-3,5-diaryl-2-pyrazoline derivatives (4a–f), as
shown in Scheme 1. Some characteristics of the synthesized
compounds are shown in Table 1.
4. Result
The structures of compounds 4a–f were confirmed by elemental
analyses, MS-FABþ, IR and 1H NMR spectral data. All compounds
gave satisfactory elemental analysis. Mass spectra (MS (FAB)) of
compounds showed M þ 1 peaks, in agreement with their molec-
ular formula. IR spectra of compounds showed NH, C]O and C]N,
C]C bands at 3399–3095 cmꢀ1, 1685–1669 cmꢀ1 and 1581–
3. Pharmacology
As stated in Section 1, antinociceptive potential of the some
newly synthesized pyrazoline derivatives were investigated, in this
Table 1
Some characteristics of the compounds
Compound
R1
R2
R3
R4
X
Yield (%)
M.p. (ꢁC)
Molecular formula
24H18ClN3O2S
C26H22ClN3O2S
C25H20ClN3O3S
C24H20N4OS
Molecular weight
4a
4b
4c
4d
4e
4f
H
H
OCH3
H
H
H
CH3
H
H
CH3
H
H
CH3
H
H
CH3
H
Cl
Cl
Cl
H
H
H
O
O
O
NH
NH
NH
80
82
78
83
85
75
155–157
143–144
97–99
127–129
203–205
152–154
C
447
475
477
412
440
442
C26H24N4OS
C25H22N4O2S
OCH3

2608
Z.A. Kaplancikli et al. / European Journal of Medicinal Chemistry 44 (2009) 2606–2610
Table 2
Furthermore, compounds 4e (P < 0.001) and 4b (P < 0.01) also had
highest antinociceptive activities in hot plate tests.
In addition, test compounds did not significantly change the fall-
latency in Rota-Rod tests (related data not showed here) and none
of the animals died when 100 mg/kg doses of the test compounds
were applied.
Effects of compounds on hot plate response in mice
Treatment
% Analgesia (mean ꢂ SEM)
Control
4a (10 mg/kg)
4b (10 mg/kg)
4c (10 mg/kg)
4d (10 mg/kg)
4e (10 mg/kg)
4f (10 mg/kg)
Naloxone (5 mg/kg) þ 4a (10 mg/kg)
Naloxone (5 mg/kg) þ 4b (10 mg/kg)
Naloxone (5 mg/kg) þ 4c (10 mg/kg)
Naloxone (5 mg/kg) þ 4d (10 mg/kg)
Naloxone (5 mg/kg) þ 4e (10 mg/kg)
Naloxone (5 mg/kg) þ 4f (10 mg/kg)
Morphine (10 mg/kg)
ꢀ1.74 ꢂ 1.55
59.7 ꢂ 7.4*
72.6 ꢂ 19.9**
47.7 ꢂ 7.9**
61.9 ꢂ 14.1*
82.5 ꢂ 16.3***
60.6 ꢂ 18.3*
ꢀ1.32 ꢂ 7.94
ꢀ0.97 ꢂ 6.78
ꢀ2.95 ꢂ 5.5
ꢀ1.5 ꢂ 7.7
5. Discussion
According to the results of the analgesia experiments, it can be
evaluated that 100 mg/kg doses of the all test compounds possess
significant antinociceptive activities against chemical and thermal
noxious stimuli (Tables 2 and 3). The compounds did not impair the
motor performance in Rota-Rod test, indicating that the observed
antinociception unlikely occurred due to motor abnormalities.
All of the test compounds caused significant increases in the
reaction times of mice against thermal noxious stimulus in hot
plate tests (Table 2). Compounds 4b and e showed higher, 4f,d and
a moderate, 4c relatively lower antinociceptive actions at the
applied dose. It is a common knowledge that hot plate test
measures centrally mediated transient pain and supraspinally
organized responses [30]. Therefore, it can be concluded that the
tested compounds in this study may act centrally.
On the other hand, in chemical noxious stimulus-induced
writhing test, compounds 4e, b, f almost totally and 4d powerfully
protected animals from writhing. 4a and c were the least active
compounds in the serial. Compounds 4e, b, f and d (at 100 mg/kg
doses) were as effective as morphine (10 mg/kg) in terms of anal-
gesic action, since no statistically significant differences were seen
between morphine and these groups in ANOVA test.
The mouse writhing model involves different nociceptive
mechanisms, such as sympathetic system (biogenic amines
release), cyclooxygenases (COX) and their metabolites and opioid
mechanisms. Acetic acid acts indirectly by inducing the release of
endogenous mediators, which stimulate the nociceptive neurons
sensitive to NSAIDs and/or opioids [31]. When the results of
writhing and hot plate tests were considered together, it can be
concluded that the antinociceptive activities of the tested
compounds may occur by both central and peripheral mechanisms.
Opioids are known to show analgesic activities in both hot plate
and writhing tests by acting on central and peripheral nociceptive
pathways, respectively [31,32]. So, naloxone was used as an opioid
receptor antagonist, for the investigation of the possible involve-
ment of opioid system in the antinociceptive activities of the tested
compounds. It was observed that, analgesic activities of all tested
compounds were reversed completely by naloxone pre-treatment
ꢀ0.58 ꢂ 9.52
ꢀ1.34 ꢂ 6.16
92.9 ꢂ 9.03***
Values are mean ꢂ SEM. *P < 0.05, **P < 0.01, ***P < 0.001, compared with control.
One-way ANOVA, post-hoc Newman-Keul’s test, n ¼ 7.
1301 cmꢀ1 regions, respectively. In the 250 MHz 1H NMR spectrum
of compounds, the C4 protons of the pyrazoline ring resonated as
multiplet at 3.20–3.40 ppm (Ha), 3.80–4.05 ppm (Hb). The CH2
protons of acetyl which are on 1 position of pyrazolines are
observed at 4.55–4.95 ppm as double doublets (J ¼ 15.54–16.16 Hz,
J ¼ 15.54–16.17) this geminal coupling is the result from steric
structure of compound. These geminal protons are observed as
double doublet because of possible two different conformations
since rigid protons occurred. The C5 (Hx) proton of pyrazoline
appeared as multiplet at
d 5.55–5.70 ppm due to vicinal coupling
with the two magnetically non-equivalent protons of the methy-
lene group at position 4 of the pyrazoline ring. The benzimidazole
derivatives showed specific NH proton at 12.55–12.60 ppm as
broad or singlet. All the other aromatic and aliphatic protons were
observed at expected regions.
All of the compounds in the serial had statistically significant
antinociceptive effects in both hot plate (Table 2) and acetic acid-
induced writhing tests (Table 3). Additionally, naloxone pre-treat-
ment reversed the antinociceptive activities of all compounds in
both of the tests (Tables 1 and 2). Morphine (10 mg/kg) had both
peripheral and central antinociceptive actions supporting its use as
a standard agent for both chemical and thermal painful models,
causing significant increase in the reaction times in hot plate tests
(P < 0.001) and decreased the numbers of abdominal contractions
in writhing tests (P < 0.001).
Among the tested compounds, 4e (P < 0.001), 4b (P < 0.001) and
4f (P < 0.001) inhibited writhing behaviour almost totally.
Table 3
Effects of compounds on writhing test in mice
Treatment
Number of writhing (10 min) (Mean ꢂ SEM)
% Protection
Control
4a (10 mg/kg)
4b (10 mg/kg)
4c (10 mg/kg)
4d (10 mg/kg)
4e (10 mg/kg)
4f (10 mg/kg)
Naloxone (5 mg/kg) þ 4a (10 mg/kg)
Naloxone (5 mg/kg) þ 4b (10 mg/kg)
Naloxone (5 mg/kg) þ 4c (10 mg/kg)
Naloxone (5 mg/kg) þ 4d (10 mg/kg)
Naloxone (5 mg/kg) þ 4e (10 mg/kg)
Naloxone (5 mg/kg) þ 4f (10 mg/kg)
Morphine (10 mg/kg)
30.7 ꢂ 3.6
_
13.0 ꢂ 3.57***,a
0.57 ꢂ 0.42***
17.43 ꢂ 2.32**,b
4.57 ꢂ 1.44***
0.71 ꢂ 0.56***
0.85 ꢂ 0.34***
28.43 ꢂ 2.11
24.14 ꢂ 1.91
57.72
98.14
43.32
85.13
97.67
97.21
7.44
21.39
5.58
12.56
19.07
22.32
87.45
29.0 ꢂ 1.25
26.86 ꢂ 2.13
24.86 ꢂ 2.45
23.86 ꢂ 1.87
3.85 ꢂ 0.5***
Values are mean ꢂ SEM. **P < 0.01, ***P < 0.001 compared with control.
a
P < 0.01 compared with morphine. One-way ANOVA, post-hoc Newman-Keul’s test, n ¼ 7.
P < 0.001 compared with morphine. One-way ANOVA, post-hoc Newman-Keul’s test, n ¼ 7.
b

Z.A. Kaplancikli et al. / European Journal of Medicinal Chemistry 44 (2009) 2606–2610
2609
(Tables 2 and 3), which indicates the involvement of the opioid
mechanisms in the analgesic action. This effect could be due to the
direct opioid receptor agonistic activities of the constituents in the
extract and/or induction of endogenous opioid peptide release.
These results support the previous studies suggesting opioid
mediated analgesic activities of some benzoxazole/benzimidazole–
pyrazoline-derived compounds [10,11,21,33].
under reduced pressure and the products were recrystallized from
ethanol.
7.1.1.4. 1-[(Benzazole-2-yl)thioacetylamino]-3,5-diaryl-2-pyrazoline
derivatives (4a–f). A mixture of 1-(chloroacetyl)-3,5-diaryl-2-pyr-
azoline (3) (0.01 mol), benzazol-2-thiole (0.01 mol) and K2CO3
(0.01 mol) in acetone (50 mL) was refluxed for 8 h. After cooling,
the solution was evaporated until dryness. The residue was washed
with water and recrystallized from ethanol.
6. Conclusions
7.1.1.4.1. Compound 4a. IR (KBr, cmꢀ1): 3366 and 3115(NH),
1680 (C]O), 1502–1321 (C]C and C]N).
The synthesis and antinociceptive evaluation of six pyrazoline
derivatives were presented in this study. The synthesized
compounds exhibited statistically significant antinociceptive
activities against thermal and chemical noxious stimuli. As
a general consideration, it can be concluded that the compounds 4b
and e showed highest antinociceptive activities in both nociception
tests. Therefore, it can be suggested that, dimethyl substitution on
phenyl at third position of the pyrazole ring increases the anti-
nociceptive activities. Additionally, substitutional changes in ben-
zoxazole/benzimidazole rings on the basic structure did not affect
the activity.
The results of this investigation prove the hypothesis that ben-
zoxazoles/benzimidazoles and pyrazoline-combined pyrazole
derivatives possess centrally and peripherally mediated anti-
nociceptive activities and supports the previous papers reporting
the opioid sensitive antinociceptive activities of various benzox-
azole/benzimidazole–pyrazoline derivative compounds.
1H NMR (250 MHz) (DMSO-d6)
d (ppm): 3.20–3.40 (1H, m,
pyrazoline C4-H), 3.90–4.05 (1H, m, pyrazoline C4-H), 4.70 and 4.90
(2H, 2d [J ¼ 16.15 Hz and J ¼ 16.16 Hz], COCH2 geminal protons),
5.60–5.70 (1H, m, pyrazoline C5-H), 7.20–7.90 (13H, m, aromatic
protons). MS (FAB) [M þ 1]: m/z 448.
Anal. Calcd for C24H18ClN3O2S: C, 64.35; H, 4.05; N, 9.38. Found:
C, 64.37; H, 4.06; N, 9.35.
7.1.1.4.2. Compound 4b. IR (KBr, cmꢀ1): 3286 and 3095 (NH),
1676 (C]O), 1542–1335 (C]C and C]N).
1H NMR (250 MHz) (DMSO-d6)
d (ppm): 2.30 (6H, s, two CH3),
3.10–3.25 (1H, m, pyrazoline C4-H), 3.85–4.00 (1H, m, pyrazoline
C4-H), 4.70 and 4.95 (2H, 2d [J ¼ 16.16 Hz and J ¼ 16.17 Hz], COCH2
geminal protons), 5.55–5.65 (1H, m, pyrazoline C5-H), 7.30–7.80
(11H, m, aromatic protons). MS (FAB) [M þ 1]: m/z 476.
Anal. Calcd for C26H22ClN3O2S: C, 65.61; H, 4.66; N, 8.83. Found:
C, 65.63; H, 4.68; N, 8.80.
7.1.1.4.3. Compound 4c. IR (KBr, cmꢀ1): 3391 and 3122 (NH),
1682 (C]O), 1533–1301 (C]C and C]N).
7. Experimental
1H NMR (250 MHz) (DMSO-d6)
d (ppm): 3.15–3.30 (1H, m, pyr-
azoline C4-H), 3.65 (3H, s, OCH3), 3.80–4.00 (1H, m, pyrazoline C4-
H), 4.60 and 4.85 (2H, 2d [J ¼ 16.11 Hz and J ¼ 16.12 Hz], COCH2
geminal protons), 5.50–5.65 (1H, m, pyrazoline C5-H), 7.10–7.75
(12H, m, aromatic protons). MS (FAB) [M þ 1]: m/z 478.
Anal. Calcd for C25H20ClN3O3S: C, 62.82; H, 4.22; N, 8.79. Found:
C, 62.84; H, 4.25; N, 8.80.
7.1. Chemistry
All reagents were used as purchased from commercial suppliers
without further purification. Melting points were determined by
using an Electrothermal 9100 digital melting point apparatus and
were uncorrected (Electrothermal, Essex, UK). The compounds
were checked for purity by TLC on silica gel 60 F254. Spectroscopic
data were recorded on the following instruments: IR, Shimadzu 435
IR spectrophotometer (Shimadzu, Tokyo, Japan); 1H NMR, Bruker
250 MHz NMR spectrometer (Bruker Bioscience, Billerica, MA, USA)
in DMSO-d6 using TMS as internal standard; MS-FAB, VG Quattro
mass spectrometer (Fisons Instruments Vertriebs GmbH, Mainz,
Germany), Elemental analyses were performed on a Perkin Elmer
EAL 240 elemental analyser (Perkin–Elmer, Norwalk, CT, USA).
7.1.1.4.4. Compound 4d. IR (KBr, cmꢀ1): 3322 and 3111 (NH),
1669 (C]O), 1566–1385 (C]C and C]N).
1H NMR (250 MHz) (DMSO-d6)
d (ppm): 3.15–3.25 (1H, m, pyr-
azoline C4-H), 3.90–4.00 (1H, m, pyrazoline C4-H), 4.60 and 4.80
(2H, 2d [J ¼ 15.56 Hz and J ¼ 15.57 Hz], COCH2 geminal protons),
5.60–5.70 (1H, m, pyrazoline C5-H), 7.10–7.80 (14H, m, aromatic
protons), 12.60 (1H, br, benzimidazole N–H). MS (FAB) [M þ 1]: m/z
413.
Anal. Calcd for C24H20N4OS: C, 69.88; H, 4.89; N, 13.58. Found: C,
69.90; H, 4.86; N, 13.59.
7.1.1. General procedure for the synthesis of compounds
7.1.1.1. 1,3-Diaryl-2-propen-1-ones (1). A mixture of acetophenone
(0.06 mol), aromatic aldehyde (0.06 mol) and 10% aqueous sodium
hydroxide (10 mL) in ethanol (30 mL) was stirred at room
temperature for about 3 h. The resulting solid was washed, dried
and crystallized from ethanol.
7.1.1.4.5. Compound 4e. IR (KBr, cmꢀ1): 3399 and 3215 (NH),
1685 (C]O), 1545–1311 (C]C and C]N).
1H NMR (250 MHz) (DMSO-d6)
d (ppm): 2.35 (6H, s, 2CH3), 3.10–
3.20 (1H, m, pyrazoline C4-H), 3.80–3.90 (1H, m, pyrazoline C4-H),
4.65 and 4.80 (2H, 2d [J ¼ 15.62 Hz and J ¼ 15.62 Hz], COCH2
geminal protons), 5.60–5.65 (1H, m, pyrazoline C5-H), 7.10–7.70
(12H, m, aromatic protons), 12.55 (1H, s, benzimidazole N–H). MS
(FAB) [M þ 1]: m/z 441.
7.1.1.2. 3,5-Diaryl-2-pyrazolines (2). A solution of the appropriate
chalcone (0.03 mol) (1) and hydrazine hydrate (80%) (0.06 mol) in
ethanol (30 mL) was refluxed for 3 h. The reaction mixture was
cooled and kept at 0 ꢁC overnight. The resulting solid was recrys-
tallized from ethanol.
Anal. Calcd for C26H24N4OS: C, 70.88; H, 5.49; N, 12.72. Found: C,
70.80; H, 5.50; N, 12.72.
7.1.1.4.6. Compound 4f. IR (KBr, cmꢀ1): 3341 and 3130 (NH),
1675 (C]O), 1581–1381 (C]C and C]N).
1H NMR (250 MHz) (DMSO-d6)
d (ppm): 3.10–3.20 (1H, m, pyr-
7.1.1.3. 1-(Chloroacetyl)-3,5-diaryl-2-pyrazolines (3). 3,5-Diaryl-2-
pyrazoline (2) (0.02 mol) and triethylamine (0.02 mol) were dis-
solved in dry toluene (30 mL) with constant stirring. Later, the
mixture was cooled in an ice bath, and chloroacetylchloride
(0.02 mol) was added drop wise with stirring. The reaction mixture
thus obtained was further agitated for 1 h at room temperature. The
precipitate was filtrated, the solvent was evaporated to dryness
azoline C4-H), 3.70 (3H, s, OCH3), 3.85–4.00 (1H, m, pyrazoline C4-
H), 4.55 and 4.80 (2H, 2d [J ¼ 15.54 Hz and J ¼ 15.54 Hz], COCH2
geminal protons), 5.55–5.65 (1H, m, pyrazoline C5-H), 6.80–7.80
(13H, m, aromatic protons), 12.55 (1H, br, benzimidazole N–H). MS
(FAB) [M þ 1]: m/z 443.
Anal. Calcd for C25H22N4O2S: C, 67.85; H, 5.01; N, 12.66. Found:
C, 67.84; H, 5.00; N, 12.69.

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Z.A. Kaplancikli et al. / European Journal of Medicinal Chemistry 44 (2009) 2606–2610
7.2. Pharmacology
7.3. Statistical analysis
7.2.1. Animals
Experimental data from hot plate, acetic acid-induced writhing
and Rota-Rod tests were analysed by One-way ANOVA followed by
Newman-Keul’s test. The data used in statistical analysis was
obtained from seven animals for each of the groups. Statistical
evaluation of the data was performed using GraphPad Prism 3.0
(GraphPad Software, San Diego, CA, USA). Experimental results
were expressed as mean ꢂ SEM. Differences between given sets of
data were considered to be statistically significant when P value
was less than 0.05.
Swiss albino mice of both sexes, weighing 30–40 g, were housed
at room temperature of 24 ꢂ1 ꢁC with 12/12 h light/dark cycle.
Twelve hours before each experiment animals received only water,
in order to avoid food interference with substance absorption. The
experimental protocols have been approved by the Local Ethical
Committee on Animal Experimentation of the Eskis¸ehir Osmangazi
University, Turkey.
7.2.2. Assessment of analgesic activity
7.2.2.1. Hot plate test. To evaluate the central component of noci-
ception, hot plate test was applied. Mice (n ¼ 7) were placed indi-
vidually on a hot plate set at 55 ꢂ1.0 ꢁC and the time of licking the
forepaws or eventually jumping out of the glass beaker were
recorded as an index of nociception. Maximum cut-off time was
chosen as 30 s to avoid tissue damage [24–26]. Response latencies
were measured 30 min after the application of sunflower oil as
control; morphine sulphate as reference drug and 100 mg/kg doses
of the each test compounds.
In six separate groups of animals (n ¼ 7 for each), naloxone was
administered intraperitoneally (5 mg/kg) 15 min before the injec-
tion of the compounds in order to examine possible involvement of
opioid system in the analgesic activities. The effects of the
compounds on nociception were determined by converting the hot
plate latencies to percentage analgesic activity according to the
following equation:
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7.2.3. Assessment of motor coordination via Rota-Rod test
For the investigation of possible neurological deficits due to
the test compounds, which may interfere with the test results to
give false positives such as muscle relaxant or impairment of
motor coordination, Rota-Rod test was performed. Before the
experimental session, three trials were given for three consecu-
tive days on the Rota-Rod apparatus (Ugo Basile 7560, Milano,
Italy) set at a rate of 16 revolutions/min. Mice that were able to
remain on the rod longer than 180 s were selected for the test.
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[11,29].