The antinociceptive effect of 4-substituted derivatives of 5-(4-chlorophenyl)-2-(morpholin-4-ylmethyl)-2,4-dihydro-3H-1,2, 4-triazole-3-thione in mice

Article abstract of DOI:10.1007/s00210-013-0938-0

The aim of the present experiments was to examine the antinociceptive activity of 4-substituted derivatives of 5-(4-chlorophenyl)-2-(morpholin-4- ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione in mice. The compounds were synthesized using the so-called Mannich reaction and their structures were confirmed using IR and 1H-NMR spectra. The antinociceptive activity was investigated in two behavioral tests: the hot plate test and the writhing test. For preliminary estimation of other behavioral effects, the locomotor activity of mice, the motor coordination in the rota-rod test, and the myorelaxation in the chimney test were also studied. The changes in body temperature of animals were also recorded. We demonstrated that all examined compounds produced antinociceptive effect, both in the hot plate test and in the writhing test, without impact on the motor coordination and myorelaxation of animals. The pharmacological effect of all drugs has been developed within 60 min after administration of drugs; and in two cases (T-103 and T-104), it has been a short-lasting effect (up to 90 min). Two compounds (T-100 and T-102) also inhibited the locomotor activity of animals. T-104 induced the changes in body temperature of mice. Generally, we demonstrated that combination of two different heterocyclic systems (morpholine and 1,2,4-triazole) might be beneficial for reduction of nociception.

Full text of DOI:10.1007/s00210-013-0938-0

Naunyn-Schmiedeberg's Arch Pharmacol
DOI 10.1007/s00210-013-0938-0
ORIGINAL ARTICLE
The antinociceptive effect of 4-substituted derivatives
of 5-(4-chlorophenyl)-2-(morpholin-4-ylmethyl)
-2,4-dihydro-3H-1,2,4-triazole-3-thione in mice
Listos Joanna & Sylwia Talarek & Jolanta Orzelska &
Sylwia Fidecka & Monika Wujec & Tomasz Plech
Received: 20 June 2013 /Accepted: 6 November 2013
#
The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract The aim of the present experiments was to examine
the antinociceptive activity of 4-substituted derivatives of
5-(4-chlorophenyl)-2-(morpholin-4-ylmethyl)-2,4-dihydro-
3H-1,2,4-triazole-3-thione in mice. The compounds were
synthesized using the so-called Mannich reaction and their
structures were confirmed using IR and 1H-NMR spectra. The
antinociceptive activity was investigated in two behavioral
tests: the hot plate test and the writhing test. For preliminary
estimation of other behavioral effects, the locomotor activity
of mice, the motor coordination in the rota-rod test, and the
myorelaxation in the chimney test were also studied. The
changes in body temperature of animals were also recorded.
We demonstrated that all examined compounds produced
antinociceptive effect, both in the hot plate test and in the
writhing test, without impact on the motor coordination and
myorelaxation of animals. The pharmacological effect of all
drugs has been developed within 60 min after administration
of drugs; and in two cases (T-103 and T-104), it has been a
short-lasting effect (up to 90 min). Two compounds (T-100
and T-102) also inhibited the locomotor activity of animals. T-
104 induced the changes in body temperature of mice. Gen-
erally, we demonstrated that combination of two different
heterocyclic systems (morpholine and 1,2,4-triazole) might
be beneficial for reduction of nociception.
Introduction
Pain is an unpleasant sensory and emotional experience that
serves to alert an individual to actual or potential tissue dam-
age. This damage can be caused by exposure to noxious
chemical, mechanical, or thermal stimuli or by the presence
of a pathologic process (tumor, muscle spasm, inflammation,
nerve damage etc.; Mao 2009; Rabow and Pantilat 2014). In
order to select and apply appropriate therapy, pain is distin-
guished by different types, such as somatic, visceral, neuro-
pathic pain, etc. Each kind of pain is associated with different
mechanisms, like inflammation, noxious stimulus, or nerve
damage; thus, each situation requires different treatment
schemes (Mao 2009; Rabow and Pantilat 2014).
Pain, perceived as suffering syndrome, should be alleviated
as uncontrolled pain may induce further pathological changes,
including shock. Generally, there are two groups of analgesics
that are commonly used in the therapy of somatic and visceral
pain (Turk et al. 2011). Non-steroidal anti-inflammatory drugs
and paracetamol are recommended for the therapy of mild to
moderate pains, while opioids, the oldest analgesics, are main-
ly used to alleviate intense acute and severe chronic pains,
often post-operative and cancer-related. Neuropathic pain is
controlled by other drugs, such as tricyclic antidepressants or
anti-seizure drugs (Dworkin et al. 2010). Unfortunately, typ-
ical analgesic drugs produce adverse effects. For example,
non-steroidal anti-inflammatory drugs (NSAIDs) may induce
peptic ulcers, heart failure and hypertension, renal dysfunc-
tions, and allergy, while opioids may produce the state of
tolerance and dependence or, at higher doses, respiratory
depression (Grosser et al. 2011; Yaksh and Wallace 2011).
Thus, in the twenty-first century, pain therapy leaves much
to be desired and is rather far from ideal. Therefore, searching
for new drugs which could improve the quality of pain therapy
seems to be most purposeful and firmly justified. Several
.
.
Keywords Antinociceptive activity The hot plate test The
.
writhing test Behavioral tests
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:
:
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L. Joanna (*) S. Talarek J. Orzelska S. Fidecka M. Wujec
T. Plech
Medical University, Lublin, Poland
e-mail: a.listos@umlub.pl

Naunyn-Schmiedeberg's Arch Pharmacol
reports have demonstrated that the search for new analgesics
has already been undertaken and the antinociceptive activity
of different derivatives of 1,2,4-triazole has repeatedly been
demonstrated (Gowda et al. 2011; Hussein et al. 2011;
Uzgören-Baran et al. 2012; Vijesh et al. 2013). A very similar
effect has also been induced by pyrazoline (Joshi et al. 2010)
and morpholine derivatives (Ahmadi et al. 2011; Xu et al.
2008).
Poland) and the obtained results were within 0.4 % of their
theoretical value.
General procedures for the synthesis of educts
Respective thiosemicarbazide and 1,2,4-triazole-3-thione de-
rivatives were obtained according to the method described
earlier (Plech et al. 2011). A solution of 10 mmol (1.70 g) of
4-chlorobenzoic acid hydrazide and equimolar amount of
appropriate aryl isothiocyanate in 25 ml of anhydrous EtOH
was heated under reflux for 15 min. Next, the ethanolic
solution was cooled and the solid formed was filtered off,
washed with diethyl ether, dried, and recrystallized from
EtOH.
Therefore, the aim of the present experiments was to ex-
amine new compounds from the point of view of their
antinociceptive activity. All the studied compounds were 4-
substituted derivatives of 5-(4-chlorophenyl)-2-(morpholin-4-
ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione. Thus, all
drugs are the combination of 1,2,4-triazole and morpholine
groups which support their significance in pain control. The
antinociceptive activity of the examined drugs was investigat-
ed in mice in the hot plate test and the writhing test, i.e., two
generally accepted behavioral tests that are widely used in
experimental neuropsychopharmacology to evaluate the
antinociceptive effects of drugs. The hot-plate test used for
evaluation of the centrally acting analgesics (Bastia et al.
2002; Grecksch et al. 2006; Liu et al. 2003, Thomsen et al.
2007) is a neurogenic-modulated model that produces, at
constant temperature, two types of behavioral response (paw
licking and jumping), both of which are considered to be
supraspinally integrated. In turn, the writhing test is a chem-
ical method used to induce pain of the peripheral origin by
injection of irritant agents, such as acetic acid, in mice (Bastia
et al. 2002; Liu et al. 2003; Reichert et al. 2001). Therefore,
both tests were applied in the described experiments to extend
knowledge on antinociceptive effects of the examined drugs
and to recognize their influence on the centrally and periph-
erally acting stimuli. Also, the locomotor activity of mice, the
motor coordination in rota-rod test, the miorelaxant effect in
chimney test, and the changes in body temperature were
studied in mice for preliminary estimation of other behavioral
effects of new drugs.
1 -(4 -Chlorobenzoyl )-4 -(2 ,4 -dichlorophenyl )
thiosemicarbazide
Yield: 90 % (3.36 g). Elemental analysis data for
C14H10Cl3N3OS (374.67); calculated/found (%): C 44.88/
44.70, H 2.69/2.85, N 11.22/11.08. All information on the
compound may be retrieved in the CAS database (CAS Reg-
istry Number: 891643-33-7).
4 - ( 4 - B ro m o p h e n y l ) - 1 - ( 4 - c h l o ro b e n z o y l )
thiosemicarbazide
Yield: 92 % (3.54 g). Elemental analysis data for
C14H11BrClN3OS (384.68); calculated/found (%): C 43.71/
43.79, H 2.88/2.67, N 10.92/10.86. All information on the
compound may be retrieved in the CAS database (CAS Reg-
istry Number: 356576-31-3).
1-(4-Chlorobenzoyl)-4-[4-chloro-3-(trifluoromethyl)
phenyl]thiosemicarbazide
Yield: 79 % (3.22 g). 1H NMR (DMSO-d6): 7.03–7.74 (m,
7H, Ar-H), 10.04, 10.57, 11.56 (3 s, 3H, 3NH). Elemental
analysis data for C15H10Cl2F3N3OS (408.22); calculated/
found (%): C 44.13/44.02, H 2.47/2.67, N 10.29/10.18.
1 -(4 -Chlorobenzoyl )-4 -(3 ,4 -dichlorophenyl )
thiosemicarbazide
Yield: 82 % (3.07 g). Elemental analysis data for
C14H10Cl3N3OS (374.67); calculated/found (%): C 44.88/
44.75, H 2.69/2.78, N 11.22/11.34. All information on the
compound may be retrieved in the CAS database (CAS Reg-
istry Number: 891643-96-2).
Materials and methods
Chemistry
1 - ( 4 - C h l o ro b e n z o y l ) - 4 - ( 3 - c h l o ro p h e n y l )
thiosemicarbazide
All the used reagents and solvents were purchased from Alfa
Aesar (Ward Hill, USA) and Merck Co. (Darmstadt, Germa-
ny). Melting points were determined by a Fisher-Johns appa-
ratus (Fisher Scientific, Schwerte, Germany) and were uncor-
Yield: 85 % (2.89 g). Elemental analysis data for
C14H11Cl2N3OS (340.23); calculated/found (%): C 49.42/
49.58, H 3.26/3.45, N 12.35/12.19. All information on the
compound may be retrieved in the CAS database (CAS Reg-
istry Number: 443298-33-7).
In order to obtain 1,2,4-triazole-3-thiones, a solution of
respective thiosemicarbazide derivative (10 mmol) in 2 %
NaOH was heated under reflux for 2 h. After cooling, the
mixture was neutralized with 3 M HCl. The precipitate formed
1
rected. The H NMR spectra were recorded on a 250-MHz
Bruker Avance spectrometer (Bruker BioSpin GmbH, Germa-
ny), using TMS as an internal standard and DMSO-d6 or
CDCl3 as solvent. IR spectra were obtained on a Perkin-
Elmer 1725X FTIR spectrophotometer. Elemental analyses
were performed on an AMZ 851 CHX analyser (PG, Gdansk,

Naunyn-Schmiedeberg's Arch Pharmacol
was filtered off and washed with distilled water. The com-
pounds were recrystallized from EtOH.
4-(2,4-Dichlorophenyl)-5-(4-chlorophenyl)-2,4-dihydro
-3H-1,2,4-triazole-3-thione
Yield: 83 % (2.96 g), m.p. 238-240 °C, 1H NMR (DMSO-
d6): 7.39 (d, 2H, Ar-H, J =8.6 Hz), 7.55 (d, 2H, Ar-H, J =
8.6 Hz), 7.70–7.95 (m, 3H, Ar-H), 14.39 (s, 1H, NH). Ele-
mental analysis data for C14H8Cl3N3S (356.66); calculated/
found (%): C 47.15/47.13, H 2.26/2.38, N 11.78/11.60.
4-(4-Bromophenyl)-5-(4-chlorophenyl)-2,4-dihydro-
3H-1,2,4-triazole-3-thione
Yield: 81 % (2.97 g). Elemental analysis data for
C14H9BrClN3S (366.66); calculated/found (%): C 45.86/
45.70, H 2.47/2.60, N 11.46/11.37. All information on the
compound may be retrieved in the CAS database (CAS Reg-
istry Number: 537017-82-6).
4 -[4 -Chloro -3 -(trifluoromethyl )phenyl ]-5 -(4 -
chlorophenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione
Yield: 76 % (2.96 g), m.p. 242-245 °C, 1H NMR (DMSO-
d6): 6.93–7.56 (m, 7H, Ar-H), 14.11 (s, 1H, NH). Elemental
analysis data for C15H8Cl2F3N3S (390.21); calculated/found
(%): C 46.17/46.28, H 2.07/2.00, N 10.77/10.85.
4 -(3 ,4 -Dichlorophenyl )-5 -(4 -chlorophenyl )-2 ,4 -
dihydro-3H-1,2,4-triazole-3-thione
Yield: 86 % (3.07 g), m.p. 280–282 °C, 1H NMR (DMSO-
d6): 7.06–7.68 (m, 7H, Ar-H), 14.08 (s, 1H, NH). Elemental
analysis data for C14H8Cl3N3S (356.66); calculated/found
(%): C 47.15/47.26, H 2.26/2.09, N 11.78/11.64.
4-(3-Chlorophenyl)-5-(4-chlorophenyl)-2,4-dihydro-
3H-1,2,4-triazole-3-thione
Yield: 80 % (2.58 g), m.p. 272–273 °C, 1H NMR (DMSO-
d6): 7.10–7.61 (m, 8H, Ar-H), 14.13 (s, 1H, NH). Elemental
analysis data for C14H9Cl2N3S (322.21); calculated/found
(%): C 52.19/52.30, H 2.82/2.76, N 13.04/12.90.
General procedure for the synthesis of compounds named as
T-100–T-104
To a solution of corresponding 5-(4-chlorophenyl)-4-substitut-
ed-2,4-dihydro-3H-1,2,4-triazole-3-thione (10 mmol) in anhy-
drous ethanol (35 ml) equimolar amounts of morpholine and
formaldehyde (37 %) were added. The obtained mixture was
stirred at room temperature for 2 h. Next, distilled water (5 ml)
was added and the precipitate formed was filtered off and
recrystallized from ethanol. Spectral and physicochemical data
of compounds T-100–T-104 are listed in Table 1.
Pharmacology
Animals
The experiments were carried out on male albino Swiss mice
(20–30 g). The animals were kept 8–10 per cage at room

Naunyn-Schmiedeberg's Arch Pharmacol
Table 2 ED50 values and doses of the drugs used in behavioral
experiments
The control animals received same volumes of saline at the
respective time before the test.
The acute toxicity of the studied compounds was assessed
in mice, according to the Litchfield and Wilcoxon method
(1949), as ED50 calculated for mortality within 48 h. In the
pharmacological experiments, each compound was injected in
doses equivalent to 0.1 and 0.05 ED50 (Table 2).
Drug
ED50 value (mg/kg)
The doses of drug used in
behavioral experiments (mg/kg)
T-100
T-101
T-102
T-103
T-104
100
5 and 10
250
12.5 and 25
100 and 200
100 and 200
100 and 200
2,000
2,000
2,000
Behavioral experiments
The lack of the significant changes in time spending on the rotating rod
and in the chimney confirms that all of the studied compounds did not
influence on the motor coordination and myorelaxation of mice
The antinociceptive activity of the examined compounds was
studied in two tests:
The hot plate test was performed according to the meth-
od described by Woolfe and MacDonald, (1944). In that
test, the animals were placed on hot (55 °C), metal plate.
The time to the first reaction (licking or jumping) of mice to
a nociceptive stimulus was measured. The test was per-
formed 30, 60 and 90 min after injection of a studied
compound. The cut-off time was set to 60 s to avoid severe
pain or tissue damage.
The writhing test (Siegmund et al. 1957) is a chemical
method, generally used to study peripheral pain, induced by
ip injection of irritant acetic acid (0.6 %), as a nociceptive
stimulus, inducing characteristic writhing episodes. In that
test, all the examined compounds were administered (sc)
50 min before injection of acetic acid (ip), and 5 min after
acetic acid administration, the number of writhing episodes
was recorded for a period of 10 min.
temperature of 22 1 °C, on natural day–night cycle (spring).
Standard food (Murigran pellets, Bacutil, Motycz) and tap
water were freely available. All the experiments were made
between 9 a.m. and 2 p.m. After 1 week of adaptation and
handling, the animals were divided into groups (10–14
animals/group) and prepared for the tests.
The study was performed according to the National Insti-
tute of Health Guidelines for the Care and Use of Laboratory
Animals and the European Community Council Directive for
Care and Use of Laboratory Animals and was approved by
local ethics committee (The Medical University of Lublin
Committee on the Use and Care of Animals).
Drugs
Other parameters were also assessed in behavioral tests, in
order to extend the knowledge about new compounds, includ-
ing the locomotor activity, the motor coordination, the
myorelaxation and the changes in body temperature of mice.
The locomotor activity in mice was measured in round
actometer cages (32 cm in diameter, Multiserv, Lublin, Poland),
which were kept in a sound-attenuated experimental room. The
Before behavioral experiments, all the examined compounds
were suspended in a little amount (3–4 drops) of Tween 80
and then diluted in saline. All the drugs were administered
intraperitoneally (ip), except the new compounds in the writh-
ing test that were subcutaneously (sc) injected. All the drugs
were applied in volume of 10.0 ml/kg.
O
Schemes 1 Synthetic route to
target compounds T-100–T-104.
Reagents and conditions: a
NCS
O
a
NH
NH
NH
NH
EtOH, reflux, 15 min; b 2 %
NaOH, reflux, 2 h; and c HCHO,
morpholine, EtOH, 2 h, rt. List of
substituents: R =2,4-diCl (T-100),
4-Br (T-101), 3-CF3-4-Cl (T-102),
3,4-diCl (T-103), 3-Cl (T-104)
+
NH2
Cl
S
Cl
R
R
b
O
N
S
H
N
N
N
N
c
S
N
N
Cl
Cl
-
T-100 T-104
R
R

Naunyn-Schmiedeberg's Arch Pharmacol
cages were equipped with one row of infrared light-sensitive
photocells located 1 cm above the floor. The locomotor activity
was measured 30 min after compound injection for a total
period of 30 min (Rump and Kleinrok 1982). The motor
coordination of mice was assessed in the rota-rod test (Ataner
Zakład Uslugowy Elektroniki Inż K. Fic, Lublin, Poland) and
the miorelaxant effects were measured in the chimney test. The
rota-rod test (Dunham and Miya 1957) assesses the ability of
animals to keep balance on a rotating rod (20 mm diameter)
during 60 s. The rod revolves with constant speed of 18 rpm.
The time period that the mice were spent on the rotating rod,
was measured and recorded. The chimney test (Listos et al.
2010) evaluates the ability of mice to go backwards, vertically
from the tube. Accordingly, the time period that the mice were
spent in a tube (maximum 60 s) was measured and recorded.
The tube was made of Plexiglas with rough surface (30 mm
diameter and 25 cm length). Both tests were performed 60 min
after injection (ip) of the examined drugs.
a sharp singlet, integrated for two protons of the methylene
group (linking the morpholine and the 1,2,4-triazole rings)
was visible in the range of 5.25–5.29 ppm. Chemical shifts of
the morpholine hydrogens occurred between 2.92 and
3.76 ppm, while the aromatic protons resonated as multiplets
at 7.08–7.87 ppm. In IR spectra, absorption bands, character-
istic for C=S, C=N, –CH2– groups, were visible in ranges of 1,
312–1,323, 1,597–1,613, and 2,840–2,945 cm−1, respectively.
Effects of the new derivatives in the hot plate test
Thirty minutes after the injection of examined drugs, the one-
way ANOVA did not show any significant effect, while after
60 min significant changes were demonstrated: T-100: F2,25
=
Before the rota-rod test and the chimney test, the animals
had been trained: each mouse was placed on the rod and in
the tube for 3 min. The number of trials for each mouse was
unlimited. Then, consequently, only those animals were
approved that were able to stay on the rotating rod for
60 s or were able to leave the chimney without much
problem (up to 15 s).
The body temperature of animals was monitored using an
electronic thermometer with a rectal probe during the total
period 240 min (60 min before and 180 min after drug admin-
istration). The average of the first two measurements (60 and
30 min before drug injection) was determined as an initial
temperature (Δi). After drug administration the final temper-
ature (Δf) was measured six times every 30 min (up to
180 min) in each mice. The changes in body temperature
(Δ) were calculated according to the formula: Δ=Δf−Δi.
The changes in body temperature were studied after adminis-
tration of high doses of all the studied compounds.
Statistical analysis
All behavioral results, presented in the figures and tables as
mean SEM, were statistically calculated using the one-way
analysis of variance (ANOVA). Post hoc comparisons were
carried out by means of the Tukey test. The probability (p)
value of 0.05 or less was considered as statistically significant.
Each group of animals consisted of 8–10 mice.
Results
Fig. 1 Effects of T-100 (5, 10 mg/kg), T-101 (12.5, 25 mg/kg), T-102
(100, 200 mg/kg), T-103 (100, 200 mg/kg), and T-104 (100, 200 mg/kg)
on response latency in the hot- plate test in mice. Reaction latency values
were recorded at 30 (a), 60 (b), and 90 min (c) following the adminis-
tration of drugs. Data are presented as mean SEM. ***p <0.001, **p <
0.01, *p <0.05 vs vehicle-treated group (Tukey’s test)
The synthesis pathway, leading to compounds T-100–T-104
was depicted in Scheme 1. The structure and purity of the
compounds were characterized, using IR and 1H-NMR spec-
tra, combined with an elemental analysis. In 1H-NMR spectra,

Naunyn-Schmiedeberg's Arch Pharmacol
Fig. 2 Effects of T-100 (5,
10 mg/kg), T-101 (12.5, 25 mg/
kg), T-102 (100, 200 mg/kg), T-
103 (100, 200 mg/kg), and T-104
(100, 200 mg/kg) on acetic acid-
induced abdominal writhing in
mice. Data are presented as mean
SEM of writhing episodes.
***p <0.001, **p <0.01, *p <
0.05 vs. the vehicle-treated group
(Tukey’s test)
4.17, p =0.0285; T-101: F2,24=7.401, p =0.0003; T-102: F2,
24=8.006, p =0.0024; T-103: F2,23=6.921, p =0.0049; T-104:
demonstrated significant antinociceptive effect after each dose
of the administered: T-100 (5 and 10 mg/kg—p <0.05); T-101
(12.5 and 25 mg/kg—p <0.001 and p <0.01, respectively); T-
102 (100 and 200 mg/kg—p <0.01 and p <0.05, respective-
ly); T-103 (100 and 200 mg/kg—p <0.001); T-104 (100 and
200 mg/kg—p <0.05 and p <0.01, respectively; Fig. 2).
F
2,23=13.64, p =0.0002. In turn, after 90 min from the injec-
tions the one-way analysis demonstrated significant changes
in three cases only: T-100: F2,25=5.157, p =0.0141; T-101:
F
2,24=6.992, p =0.0045; T-102: F2,24=7.552, p =0.0032. A
post hoc analysis showed that 60 min after the administration
of examined drugs, significant and dose-dependent changes
were induced by either dose of T-101 (12.5 and 25 mg/kg—p
<0.05, p <0.01, respectively) and T-104 (100 and 200 mg/
kg—p <0.01 and p <0.001, respectively). In the case of the
other drugs, significant effects were demonstrated only after
high doses: T-100 (10 mg/kg—p <0.05), T-102 (200 mg/kg—
p <0.01), T-103 (200 mg/kg—p <0.05). In 90 min after the
administration of T-100, T-101, and T-102, the antinociceptive
effects were similar to the previous effect; however, the effects
of T-103 and T-104 were completely diminished (Fig. 1).
Effects of the new derivatives in other tests (locomotor
activity, rota-rod test, chimney test, the changes in body
temperature)
The one-way ANOVA showed significant changes in the
locomotor activity of studied mice after two drugs: T-100
and T-102: F2,27=19.12, p <0.001 and F2,24=12.6, p <
0.0002, respectively. A post hoc analysis demonstrated that
high dose of T-100 (p <0.001) and both doses of T-102 (p <
0.001 and p <0.05, respectively) significantly inhibited the
locomotor activity in studied animals, while the other three
drugs (T-101, T-103, and T-104) did not produce any signif-
icant effects on the locomotor activity of the mice (Fig. 3). No
significant changes in motor coordination of mice in the rota-
rod test and in myorelaxation in the chimney test (Table 3).
The significant changes in body temperature in mice were
observed only after administration of T-104: F11,90=12, p <
0.0001. Post hoc analysis confirmed that T-104 induced the
Effects of the new derivatives in the writhing test
The one-way analysis revealed significant changes after the
administration of all the examined compounds: T-100: F2,24
5.568, p =0.011; T-101: F2,27=12.29, p =0.000; T-102:
2,29=6.614, p =0.0046; T-103: F2,25=13.34, p =0.0001;
=
F
T-104: F 2,27 =5.719, p =0.009. A post hoc analysis
Fig. 3 Effects of T-100 (5,
10 mg/kg), T-101 (12.5, 25 mg/
kg), T-102 (100, 200 mg/kg), T-
103 (100, 200 mg/kg), and T-104
(100, 200 mg/kg) on locomotor
activity in mice. Data are pre-
sented as mean SEM of beam
breaks recorded for 30 min. ***p
<0.001, *p <0.05 vs. the vehicle-
treated group (Tukey’s test)

Naunyn-Schmiedeberg's Arch Pharmacol
Table 3 The results obtained in the rota-rod test and in the chimney test
the locomotor activity of the studied animals and the other—
T-104—induced the significant reduction of body temperature
in mice.
Drug
Dose of drug The time on the rotating The time in the chim-
(mg/kg)
rod (in seconds)
ney (in seconds)
The obtained results clearly demonstrate that both T-
100, containing 2,4-dichlorophenyl as substituent, and
T-102, containing 4-chloro-(3-trifluoromethyl)phenyl, re-
quire further studies to define their significance with
regards to the central nervous system functions. Both
drugs produced significant effects in the anti-nociceptive
tests. The reduction in the locomotor activity of the
studied mice, could, however, been attributed to drug-
induced sedation or other toxic effects, which may
preclude the use of these compounds as potential drugs.
On the other hand, it is possible that dose reduction of
the drugs may also diminish their toxicity. Taking into
account the clear, antinociceptive effects in both tests,
and the significant reduction in the locomotor activity, it
can be supposed that both compounds produce their
pharmacological effect via some receptors in the central
nervous system, e.g., the opioid receptors. All such
speculations, however, need further studies to unveil
their mechanisms.
T-103, which contains 3,4-dichlorophenyl as substituent,
produced stronger peripheral antinociceptive effect in the
writhing test vs. the central effects in the hot plate test. It
may then be assumed that the drug produced mainly periph-
eral antinociceptive effects, although poor penetration of the
compound across the blood-brain barrier cannot be excluded,
either. Similar properties were observed in the case of T-104
that contains 3-chlorophenyl as substituent. Both drugs (T-103
and T-104) produced short-lasting effects, as after 90 min from
their injections and no effect was observed in the hot plate test.
T-104 significantly reduced the body temperature. The lack of
changes in the locomotor activity, the motor coordination, and
myorelaxation of the studied mice suggests that especially T-
103 may be non-toxic compound, thus prompting further
studies to confirm its safety and efficacy.
Vehicle –
47.000 4.575
58.010 1.990
38.140 5.595
51.820 5.658
52.700 4.686
46.140 5.179
38.570 6.311
31.660 7.488
29.180 7.710
35.250 7.063
50.200 5.430
9.733 0.887
9.178 0.906
12.690 1.954
9.731 0.857
12.17 2.199
10.030 0.969
9.613 1.048
6.640 0.885
7.880 0.930
7.960 0.934
7.676 1.063
T-100
5
10
T-101 12.5
25
T-102 100
200
T-103 100
200
T-104 100
200
significant reduction of body temperature in comparison with
control animals. The changes were observed 30 min (p <
0.05), 60 and 90 min (p <0.001), 120 min (p <0.05), and
150 and 180 min (p <0.01) after drug administration. One-
way ANOVA did not show the significant changes in body
temperature of animals after administration of: T-100, T-101,
T-102, and T-103 (Table 4).
Discussion
In the described experiment, the antinociceptive activity of
five 4-substituted derivatives of 5-(4-chlorophenyl)-
2-(morpholin-4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-
thione was studied. We demonstrated antinociceptive effects
in all the examined compounds, both in the hot plate test and
the writhing test, without impact on the motor coordination (in
rota-rod test) or myorelaxation (in the chimney test) of ani-
mals. Pharmacological effects of all the applied developed
within 60 min after their administration and, in two cases (T-
103 and T-104), their short-lasting effects (up to 90 min) were
observed. The two compounds (T-100 and T-102) suppressed
The most promising effect was observed in the case of T-
101 that contains 4-bromophenyl as substituent. Strong, dose-
Table 4 The changes in body temperature (°C) after administration of high dose of the studied compounds
Drug
Changes in body temperature [Δ°C]
30’
60’
90’
120’
150’
180’
Vehicle
−0.5 0.187
−0.112 0.279
−0.550 0.931
−0.225 0.392
−0.757 0.212
−0.8375 0.368
−1.586 0.199***
0.537 0.316
0.157 0.193
−0.537 0.2605
0.742 0.249
−0.400 0.151
0.214 0.251
−1.214 0.218*
0.200 0.197
0.125 0.295
0.312 0.430
−0.500 0.264
−0.050 0.172
−1.225 0.076**
0.287 0.222
0.100 0.271
0.225 0.452
−0.350 0.396
−0.150 0.189
−1.257 0.097**
T-100 (10 mg/kg)
T-101 (25 mg/kg)
T-102 (200 mg/kg)
T-103 (200 mg/kg)
T-104 (200 mg/kg)
−0.087 0.386
−0.987 0.405
−1.000 0.126
−1.325 0.464
−1.829 0.458*
−0.237 0.525
0.014 0.392
−0.050 0.056
−0.317 0.290
−1.371 0.300***
***p <0.001, **p <0.01, and *p <0.05 vs. the vehicle-treated group (Tukey’s test)

Naunyn-Schmiedeberg's Arch Pharmacol
dependent and long-lasting antinociceptive effects were ob-
served in the hot plate test and clearly antinociceptive effects
were also demonstrated in the writhing test. Moreover, the
compound did not induce any changes in the locomotor
activity, the motor coordination or myorelaxation of the stud-
ied mice. Thus, T-101 seems to be a relatively safe agent,
although it requires further studies to confirm its safety and
efficacy.
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The search for new analgesic drugs is well grounded.
Admittedly, two groups of analgesics are used in clinical
practice: weaker (NSIADs) and stronger analgesics
(opioids); however, all of them produce some adverse effects
that limit their free usage. Opioids also induce the state of
tolerance and the state of dependence (Grosser et al., 2011,
Yaksh and Wallace, 2011). Therefore, any experiments, which
may lead to formulate new analgesic drugs, give chance to
obtain a new medicinal agent or a group of medicinal agents
with different functionalities and improved pharmacological
properties, substantially helping to extend the range of pain
therapies, their effectiveness, and safety. The presented results
show only a fragment of investigations that would be neces-
sary to identify and understand the complete pharmacological
profile of the examined compounds. Accordingly, a series of
biochemical and pharmacokinetic experiments are planned to
extend the range of our knowledge on the new medicinal
agents. Although further studies are necessary to identify all
the properties of derivatives that contain the combination of
morpholine and 1,2,4-triazole groups, T-101, as long-lasting
drug, and T-103 and T-104, as short-lasting compounds, may
be considered as the most promising for further experiments.
Conclusions
In summary, a synthesis and an identification of five, 4-
substituted derivatives of 5-(4-chlorophenyl)-2-(morpholin-
4-ylmethyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione have
been presented and discussed. Potential antinociceptive activ-
ities of all the studied drugs were demonstrated in two behav-
ioral tests (the hot-plate test and the writhing test) and their
effect on locomotor activity, motor coordination,
myorelaxation and changes in body temperature in mice was
shown. All the examined compounds showed some biological
activity that confirms the beneficial effect of combining two
different heterocyclic systems (morpholine and 1,2,4-triazole)
in nociception. Further experiments are necessary to specify
other properties of the present compounds.
Open Access This article is distributed under the terms of the Creative
Commons Attribution License which permits any use, distribution, and
reproduction in any medium, provided the original author(s) and the
source are credited.

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