Synthesis and evaluation of new 1,3,4-thiadiazole derivatives as antinociceptive agents

Article abstract of DOI:10.3390/molecules21081004

In the current work, new 1,3,4-thiadiazole derivatives were synthesized and investigated for their antinociceptive effects on nociceptive pathways of nervous system. The effects of these compounds against mechanical, thermal and chemical stimuli were eval

Full text of DOI:10.3390/molecules21081004

molecules
Article
Synthesis and Evaluation of New 1,3,4-Thiadiazole
Derivatives as Antinociceptive Agents
1
,
2
2
Mehlika Dilek Altıntop *, Özgür Devrim Can , Ümide Demir Özkay and
Zafer Asım Kaplancıklı 1
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University, Eski s¸ ehir 26470,
Turkey; zakaplan@anadolu.edu.tr
Department of Pharmacology, Faculty of Pharmacy, Anadolu University, Eski s¸ ehir 26470, Turkey;
2
ozgurdt@anadolu.edu.tr (Ö.D.C.); udemir@anadolu.edu.tr (Ü.D.Ö.)
*
Correspondence: mdaltintop@anadolu.edu.tr; Tel.: +90-222-3350580 (ext. 3772)
Academic Editor: Panayiotis A. Koutentis
Received: 24 June 2016; Accepted: 28 July 2016; Published: 1 August 2016
Abstract: In the current work, new 1,3,4-thiadiazole derivatives were synthesized and investigated
for their antinociceptive effects on nociceptive pathways of nervous system. The effects of these
compounds against mechanical, thermal and chemical stimuli were evaluated by tail-clip, hot-plate
and acetic acid-induced writhing tests, respectively. In addition, activity cage was performed to
assess the locomotor activity of animals. The obtained data indicated that compounds 3b
and 3h increased the reaction times of mice both in the hot-plate and tail-clip tests, indicating the
centrally mediated antinociceptive activity of these compounds. Additionally, the number of writhing
behavior was significantly decreased by the administration of compounds 3a 3c 3e and 3f, which
pointed out the peripherally mediated antinociceptive activity induced by these four compounds.
According to the activity cage tests, compounds 3a 3c and 3f significantly decreased both horizontal
, 3c, 3d, 3e,
3
g
,
,
,
and vertical locomotor activity of mice. Antinociceptive behavior of these three compounds may be
non-specific and caused by possible sedative effect or motor impairments.
Keywords: activity cage; antinociceptive; hot-plate; tail-clip; thiadiazole; writhing
1
. Introduction
Pain, an unpleasant sensation ranging in intensity from slight through severe to indescribable,
is the most common symptomatic reason to seek medical consultation. Despite the large number of
currently available analgesic agents, the design of analgesic molecules for the management of pain is
still the subject of ongoing research [1–3].
Analgesics are mainly divided into two groups: opioid analgesics and nonsteroidal
anti-inflammatory drugs (NSAIDs). The fear of addiction, and tolerance related to morphine and
related drugs, has resulted in the restriction and withdrawal of these drugs [1–6]. NSAIDs, which
act primarily by inhibiting cyclooxygenase (COX) enzymes, are frequently prescribed drug groups to
manage inflammatory pain. The long-term use of NSAIDs may lead to severe gastrointestinal side
effects limiting their use. The adverse effects accompanying the use of nonselective NSAIDs arise
from the reduction of the levels of protective prostaglandins in the gastrointestinal (GI) tract due
to the inhibition of COX-1. Although selective COX-2 inhibitors cause less GI adverse effects than
nonselective NSAIDs, their use in the treatment is also limited due to their serious cardiovascular side
effects. Therefore, current research efforts are mainly directed towards the discovery of new potent
analgesic drugs with fewer side effects [1–6].
Thiadiazole has attracted a great deal of interest as a privileged scaffold due to its significant
therapeutic potential for central nervous system (CNS) disorders. 1,3,4-Thiadiazole derivatives have
Molecules 2016, 21, 1004; doi:10.3390/molecules21081004
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been reported to exhibit a wide range of pharmacological effects including analgesic, antidepressant,
anxiolytic, and anticonvulsant activities [ 21]. The sulfur atom of thiadiazole ring imparts improved
7
–
liposolubility, important for the drugs active at CNS level. The mesoionic nature of 1,3,4-thiadiazoles
allows these compounds to cross cellular membranes and interact with biological targets with distinct
affinities [7–10].
Prompted by the aforementioned findings and in continuation of our ongoing research on
1,3,4-thiadiazoles related to their antinociceptive activity [21], herein we reported the synthesis of a
new series of 1,3,4-thiadiazole derivatives and focused on their antinociceptive effects.
2
. Results
2
.1. Chemistry
The synthesis of compounds 3a
–
h
followed the general pathway outlined in Figure 1. Initially,
) was obtained by the reaction of 4-nitrophenyl isothiocyanate
) was synthesized
) with carbon disulphide in
the presence of potassium hydroxide. Finally, the reaction of compound with N-(alkyl/aryl)-2-
chloroacetamide/4-(chloroacetyl)morpholine afforded new thiadiazole derivatives (3a ). The yields
and melting points (M.p.) of the compounds were given in Table 1. The structures of compounds 3a
4-(4-nitrophenyl)thiosemicarbazide (
1
with hydrazine hydrate. 5-(4-Nitrophenyl)amino-1,3,4-thiadiazole-2(3H)-thione (
via the ring closure reaction of 4-(4-nitrophenyl)thiosemicarbazide (
2
1
2
–h
–
h
1
13
were confirmed by IR, H-NMR, C-NMR, mass spectral data, and elemental analysis.
Figure 1. The synthetic route for the preparation of compounds 3a–h. Reagents and conditions: (i)
NH NH2 H O, ethanol, rt, 5 h; (ii) (1) CS /KOH, ethanol, reflux, 10 h; (2) HCl, pH 4–5; (iii) ClCH COR,
¨
2
2
2
2
K CO , acetone, rt, 8 h.
2
3
Table 1. The yields and melting points (M.p.) of compounds 3a–h.
˝
Compound
R
Yield (%)
M.p. ( C)
3
a
Diethylamino
92
85
91
92
91
89
89
85
240.1
227.9
233.4
267.0
238.8
235.3
282.5
298.7
3b
(3-Chlorophenyl)amino
(4-Chlorophenyl)amino
(4-Nitrophenyl)amino
(1,3-Benzodioxol-5-ylmethyl)amino
Morpholin-4-yl
3
c
3
3
d
e
3
f
3
g
(Benzothiazol-2-yl)amino
(6-Nitrobenzothiazol-2-yl)amino
3h
2
.2. Pharmacology
As displayed in Figure 2, in tail-clip tests, reference drug morphine and compounds 3b
g and 3h caused prolongation in the response latencies of mice and induced a significant increase in
,
3c, 3d, 3e,
3

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the maximum possible effect % (MPE %) values, with respect to the control values (F (9, 60) = 9.28;
p < 0.001).
Figure 2. Effects of test compounds (100 mg/kg) and morphine (10 mg/kg) on maximum possible
effect (MPE %) values of mice in the tail-clip test. Significance against control group * p < 0.05, ** p < 0.01,
*
** p < 0.001. Values are given as mean ˘ SEM. One-way ANOVA post-hoc Tukey’s test, n = 7.
As shown in Figure 3, in hot-plate tests, reference drug morphine and compounds 3b, 3c, 3d, 3e,
g and 3h caused elongation in the response latencies of mice and induced a significant increase in the
3
MPE % values, when compared to the control values (F (9, 60) = 6.58; p < 0.001).
Figure 3. Effects of test compounds (100 mg/kg) and morphine (10 mg/kg) on maximum possible
effect (MPE %) values of mice in the hot-plate test. Significance against control group * p < 0.05,
*
* p < 0.01, *** p < 0.001. Values are given as mean
˘
SEM. One-way ANOVA post-hoc Tukey’s test, n = 7.
In the acetic acid-induced writhing test, reference drug morphine and compounds 3a
, 3c, 3e and
3
f significantly reduced acetic acid-induced writhing responses of mice, with respect to the control
values (F (9, 59) = 8.98; p < 0.001) (Figure 4).
Figure 4. Effects of test compounds (100 mg/kg) and morphine (10 mg/kg) on number of writhing
behavior of mice in the acetic acid-induced writhing test. Significance against control group * p < 0.05,
*
* p < 0.01, *** p < 0.001. Values are given as mean ˘ SEM. One-way ANOVA post-hoc Tukey’s test, n = 7.

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Following the administrations, compounds 3a
,
3c and 3f significantly decreased both horizontal
(F (8, 54) = 6.48; p < 0.001) and vertical (F (8, 54) = 9.07; p < 0.001) locomotor activities of mice, in the
activity cage measurements (Figure 5).
Figure 5. Effects of test compounds (100 mg/kg) on number of (a) horizontal and (b) vertical activity
of mice in the activity cage test. Significance against control group * p < 0.05, ** p < 0.01. Values are
given as mean ˘ SEM. One-way ANOVA post-hoc Tukey’s test, n = 7.
3
. Discussion
In the present study, compounds 3a
–h were investigated for their probable antinociceptive effects
on nociceptive pathways of nervous system. The effects of these compounds on mechanical, thermal
and chemical stimuli were evaluated by tail-clip, hot-plate and acetic acid-induced writhing tests,
respectively. In addition, an activity cage test was performed to assess the locomotor activity of animals.
A tail-clip test is a frequently preferred method for basic pain studies. The basis of this method is
measuring the reaction time of rodents that feel constant mechanical pressure on their tails. In this
study, the findings obtained from the tail-clip tests exhibited significant increases in the reaction time
of mice following the administrations of compounds 3b, 3c, 3d, 3e, 3g and 3h (Figure 2). The results
indicated the antinociceptive effects of these compounds on nociceptive pathways carrying noxious
mechanical stimuli. These findings also suggest the possible localization of the antinociceptive activity
in the medulla spinalis, since tail-clip tests mainly measure spinal reflexes [22,23]. Alterations in
synaptic transmission of nociceptive input to the dorsal horn, modification in nociceptive processing in
the spinal cord, or changes in spinal sensitization may be some of the possible mechanisms underlying
the spinally mediated antinociceptive effects of compounds 3b, 3c, 3d, 3e, 3g and 3h.
A hot-plate test was performed in order to evaluate antinociceptive potential of the compounds
against thermal noxious stimuli. In this test, thermal pain reflexes due to footpad contact with a
hot surface were assessed. Licking is a rapid reaction to noxious thermal stimuli that is an indicator
of nociceptive threshold, whereas jumping describes a more complex reaction, with a latency, and
includes an emotional part of avoidance [24]. In the present study, the results of the hot-plate tests
showed that the administration of compounds 3b, 3c, 3d, 3e, 3g and 3h significantly increased the
reaction latencies of animals in response to constant exposure to heat (Figure 3), which indicated that
the tested compounds acted on thermal nociceptive pathways. Antinociceptive effects observed in the
hot-plate test seem to be related to supraspinal mechanisms, since this test predominantly measures
supraspinally organized responses [22,23]. Modified transmission in the projections of nociceptive
spinal cord neurons ascending to supraspinal sites, changes in supra-spinal sensitization, or alterations
in the descending inhibitory mechanisms may be some of the possible mechanisms underlying the
supraspinally mediated antinociceptive activities observed in this study.
The antinociceptive effects of the compounds on chemical noxious stimuli were examined by
acetic acid-induced writhing test. In this inflammatory/irritative pain model, i.p. administration of
acetic acid causes a reaction characterized by contraction of the abdominal muscles with simultaneous
extension of the hind limbs and elongation of the body. Acetic acid is known to cause the release of
several mediators (neurotransmitters/neuromodulators, kinins, histamine, acetylcholine, substance P,

Molecules 2016, 21, 1004
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and prostaglandins, etc.), reducing the threshold of the nociception and stimulating the nociceptive
neurons [24 25]. In this study, the data obtained from the acetic acid-induced writhing test showed
that compounds 3a 3c 3e and 3f significantly reduced acetic acid-induced writhing responses of
mice (Figure 4). The reduction in the number of writhes after the administration of these compounds
suggests that the mechanism of compounds 3a 3c 3e and 3f may be related with a decrease in the
,
,
,
,
,
release of inflammatory mediators in peripheral tissues or by direct blockage of its receptors, which
results in antinociceptive activity. Furthermore, this activity could be produced by enhancement of the
nociceptive threshold or by interruption of the transmission of the pain stimulus to the nerve fibre [25].
In the activity cage test, notable decreases were observed in the horizontal and vertical locomotor
activities of mice following the administration of compounds 3a, 3c and 3f (Figure 5). These findings
indicated that the antinociceptive behavior of these three compounds might be caused by possible
sedative effect or motor impairments. In other words, antinociceptive effects induced by compounds
3
a, 3c and 3f may not be specific.
Absence of deaths or unwanted adverse effects such as paralysis, ataxia, convulsions, and diarrhea
pointed out the negligible toxicity of the tested compounds [24]. Nevertheless, the probable side effects
of these compounds should be clarified with further detailed studies.
4
. Materials and Methods
4
.1. Chemistry
All reagents were purchased from commercial suppliers and were used without further
purification. Melting points (M.p.) were determined on an Electrothermal 9100 melting point
apparatus (Weiss-Gallenkamp, Loughborough, UK) and are uncorrected. IR spectra were recorded on
1
an IRPrestige-21 Fourier Transform Infrared spectrophotometer (Shimadzu, Tokyo, Japan). H-NMR
and 13C-NMR spectra were recorded on a Varian Mercury-400 FT-NMR spectrometer (Agilent, Palo
Alto, CA, USA). Chemical shifts were expressed in parts per million (ppm) and tetramethylsilane
was used as an internal standard. Mass spectra were recorded on an Agilent LC-MSD-Trap-SL Mass
spectrometer (Agilent Technologies, Palo Alto, CA, USA). Elemental analyses were performed on a
Perkin Elmer EAL 240 elemental analyzer (Perkin-Elmer, Norwalk, CT, USA).
General Procedure for the Synthesis of the Compounds
4
-(4-Nitrophenyl)thiosemicarbazide (1) 4-(4-Nitrophenyl)thiosemicarbazide (1) was synthesized via the
reaction of 4-nitrophenyl isothiocyanate (0.1 mol) with hydrazine hydrate (0.2 mol) as previously
reported [26].
5
-(4-Nitrophenyl)amino-1,3,4-thiadiazole-2(3H)-thione (2) [27] 4-(4-Nitrophenyl)thiosemicarbazide (1) was
dissolved in a solution of potassium hydroxide in ethanol. Carbon disulfide was then added while
stirring and the reaction mixture was heated under reflux for 10 h. The solution was cooled and
acidified to pH 4-5 with hydrochloric acid solution and crystallized from ethanol.
N-(Alkyl/Aryl)-2-[(5-((4-nitrophenyl)amino)-1,3,4-thiadiazol-2-yl)thio]acetamide derivatives (3a–h). A mixture
of N-(alkyl/aryl)-2-chloroacetamide/4-(chloroacetyl)morpholine (2 mmol) and 5-(4-nitrophenyl)amino-
1
,3,4-thiadiazole-2(3H)-thione (2) (2 mmol) in acetone was stirred at room temperature for 8 h in the
presence of potassium carbonate. The reaction mixture was filtered. The residue was washed with
water and crystallized from ethanol.
N,N-Diethyl-2-[(5-((4-nitrophenyl)amino)-1,3,4-thiadiazol-2-yl)thio]acetamide (3a) IR νmax (cm 1): 3271,
´
3
223 (N-H stretching), 2978, 2936, 2916, 2845, 2803 (Aliphatic C-H stretching), 1653 (C=O stretching),
1
1628, 1586, 1505, 1497 (N-H bending, C=N, C=C stretching and NO asymmetric stretching). H-NMR
2
(
400 MHz, DMSO-d6) δ (ppm): 1.04 (t, J = 7.2 Hz, 6.8 Hz, 3H, CH ), 1.17 (t, J = 7.2 Hz, 6.8 Hz, 3H, CH ),
3 3
3
2
.28–3.41 (m, 4H, N-CH ), 4.35 (s, 2H, S-CH ), 7.79 (d, J = 9.2 Hz, 2H, aromatic), 8.25 (d, J = 9.2 Hz,
2 2
13
H, aromatic), 11.09 (s, 1H, N-H). C-NMR (100 MHz, DMSO-d ): 12.89 (CH ), 14.05 (CH ), 37.40
6
3
3

Molecules 2016, 21, 1004
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(
(
CH ), 40.03 (CH ), 41.88 (CH ), 116.88 (2CH), 125.50 (2CH), 140.83 (C), 146.05 (C), 155.69 (C), 163.56
2 2 2
+
C), 165.44 (C). MS (ESI) (m/z): [M + H] 368.4. For C H N O S Calculated: C, 45.76; H, 4.66; N,
14 17 5 3 2
1
9.06. Found: C, 45.75; H, 4.64; N, 19.06.
N-(3-Chlorophenyl)-2-[(5-((4-nitrophenyl)amino)-1,3,4-thiadiazol-2-yl)thio]acetamide (3b) IR νmax (cm 1):
´
3
258, 3212 (N-H stretching), 3077, 3023 (Aromatic C-H stretching), 1672 (C=O stretching), 1596, 1568,
1
1
δ
7
539, 1505 (N-H bending, C=N, C=C and NO asymmetric stretching). H-NMR (400 MHz, DMSO-d )
2
6
(ppm): 4.21 (s, 2H, S-CH ), 7.14–7.16 (m, 1H, aromatic), 7.37 (t, J = 8.4 Hz, 7.6 Hz, 1H, aromatic),
2
.44–7.47 (m, 1H, aromatic), 7.78–7.84 (m, 3H, aromatic), 8.23–8.26 (m, 2H, aromatic), 10.55 (s, 1H, N-H),
1.12 (s, 1H, N-H). 13C-NMR (100 MHz, DMSO-d ): 38.19 (CH ), 116.93 (2CH), 117.56 (CH), 118.64
1
6 2
(
1
CH), 123.34 (CH), 125.46 (2CH), 130.54 (CH), 133.19 (C), 140.16 (C), 140.86 (C), 145.96 (C), 154.95 (C),
+
63.80 (C), 165.94 (C). MS (ESI) (m/z): [M + H] 422.8. For C H ClN O S Calculated: C, 45.55; H,
16
12
5
3 2
2
.87; N, 16.60. Found: C, 45.54; H, 2.88; N, 16.61.
N-(4-Chlorophenyl)-2-[(5-((4-nitrophenyl)amino)-1,3,4-thiadiazol-2-yl)thio]acetamide (3c) IR νmax (cm 1):
´
3
271, 3221 (N-H stretching), 3165, 3098, 3030 (Aromatic C-H stretching), 2876 (Aliphatic C-H stretching),
1
653 (C=O stretching), 1616, 1597, 1562, 1514, 1501, 1489 (N-H bending, C=N, C=C and NO asymmetric
2
1
stretching). H-NMR (400 MHz, DMSO-d )
δ
(ppm): 4.20 (s, 2H, S-CH ), 7.38–7.41 (m, 2H, aromatic),
6
2
7
1
.61–7.64 (m, 2H, aromatic), 7.77–7.80 (m, 2H, aromatic), 8.23–8.26 (m, 2H, aromatic), 10.48 (s, 1H, N-H),
1.11 (s, 1H, N-H). 13C-NMR (100 MHz, DMSO-d ): 38.18 (CH ), 116.94 (2CH), 120.72 (2CH), 125.48
6
2
(
(
2CH), 127.21 (C), 128.76 (2CH), 137.69 (C), 140.86 (C), 145.98 (C), 155.01 (C), 163.79 (C), 165.67 (C). MS
+
ESI) (m/z): [M + H] 422.8. For C H ClN O S Calculated: C, 45.55; H, 2.87; N, 16.60. Found: C,
16
12
5
3 2
4
5.55; H, 2.85; N, 16.62.
N-(4-Nitrophenyl)-2-[(5-((4-nitrophenyl)amino)-1,3,4-thiadiazol-2-yl)thio]acetamide (3d) IR νmax (cm 1):
343 (N-H stretching), 3078 (Aromatic C-H stretching), 2899, 2835, 2797 (Aliphatic C-H stretching), 1697
C=O stretching), 1614, 1584, 1553, 1489 (N-H bending, C=N, C=C and NO asymmetric stretching).
´
3
(
2
1
H-NMR (400 MHz, DMSO-d6)
m, 2H, aromatic), 8.22–8.26 (m, 4H, aromatic), 10.97 (s, 1H, N-H), 11.14 (s, 1H, N-H). C-NMR (100
MHz, DMSO-d ): 38.24 (CH ), 116.86 (2CH), 118.86 (2CH), 124.90 (2CH), 125.30 (2CH), 140.86 (C),
δ (ppm): 4.28 (s, 2H, S-CH ), 7.77–7.80 (m, 2H, aromatic), 7.84–7.87
2
13
(
6
2
+
1
42.44 (C), 144.73 (C), 145.90 (C), 154.70 (C), 163.77 (C), 166.48 (C). MS (ESI) (m/z): [M + H] 433.4. For
C H N O S Calculated: C, 44.44; H, 2.80; N, 19.43. Found: C, 44.44; H, 2.81; N, 19.42.
16
12
6
5 2
N-(1,3-Benzodioxol-5-ylmethyl)-2-[(5-((4-nitrophenyl)amino)-1,3,4-thiadiazol-2-yl)thio]acetamide (3e) IR νmax
´
1
(
1
cm ): 3264, 3216 (N-H stretching), 3090 (Aromatic C-H stretching), 1654 (C=O stretching), 1641, 1620,
1
599, 1578, 1518, 1501 (N-H bending, C=N, C=C and NO asymmetric stretching). H-NMR (400 MHz,
2
DMSO-d6)
δ (ppm): 3.98 (s, 2H, CH ), 4.19 (d, J = 5.6 Hz, 2H, CH ), 5.92 (s, 2H, O-CH -O), 6.70 (d,
2 2 2
J = 8.0 Hz, 1H, aromatic), 6.79 (d, J = 7.6 Hz, 2H, aromatic), 7.77 (d, J = 9.6 Hz, 2H, aromatic), 8.23 (d,
J = 8.8 Hz, 2H, aromatic), 8.63–8.65 (m, 1H, N-H), 11.09 (s, 1H, N-H). 13C-NMR (100 MHz, DMSO-d6):
3
7.23 (CH ), 42.35 (CH ), 100.80 (CH ), 107.90 (CH), 107.94 (CH), 116.94 (2CH), 120.44 (CH), 125.49
2 2 2
(
2CH), 132.79 (C), 140.86 (C), 146.05 (C), 146.09 (C), 147.26 (C), 155.21 (C), 163.74 (C), 166.60 (C). MS
+
(
ESI) (m/z): [M + H] 446.4. For C H N O S Calculated: C, 48.53; H, 3.39; N, 15.72. Found: C, 48.51;
18
15
5
5 2
H, 3.40; N, 15.71.
1
-(Morpholin-4-yl)-2-[(5-((4-nitrophenyl)amino)-1,3,4-thiadiazol-2-yl)thio]ethan-1-one (3f) IR νmax (cm 1):
´
3
271, 3225 (N-H stretching), 2963, 2916, 2845, 2806 (Aliphatic C-H stretching), 1654 (C=O stretching),
1
1630, 1589, 1512, 1501 (N-H bending, C=N, C=C and NO asymmetric stretching). H-NMR (400 MHz,
2
DMSO-d6)
8
δ (ppm): 3.43–3.61 (m, 8H, morpholine), 4.34 (s, 2H, S-CH ), 7.74–7.78 (m, 2H, aromatic),
2
.21–8.23 (m, 2H, aromatic), 11.08 (s, 1H, N-H). 13C-NMR (100 MHz, DMSO-d ): 37.09 (CH ), 42.05
6
2
(
(
CH ), 45.87 (CH ), 65.93 (2CH ), 116.86 (2CH), 125.44 (2CH), 140.83 (C), 145.99 (C), 155.18 (C), 163.70
2 2 2
+
C), 165.30 (C). MS (ESI) (m/z): [M + H] 382.4. For C H N O S Calculated: C, 44.09; H, 3.96; N,
14 15 5 4 2
1
8.36. Found: C, 44.10; H, 3.97; N, 18.35.

Molecules 2016, 21, 1004
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N-(Benzothiazol-2-yl)-2-[(5-((4-nitrophenyl)amino)-1,3,4-thiadiazol-2-yl)thio]acetamide (3g) IR νmax (cm 1):
´
3
206 (N-H stretching), 3159, 3078, 3069, 3059 (Aromatic C-H stretching), 2957, 2934, 2914, 2843, 2805,
2
725 (Aliphatic C-H stretching), 1684 (C=O stretching), 1613, 1597, 1568, 1506 (N-H bending, C=N,
1
C=C stretching and NO asymmetric stretching). H-NMR (400 MHz, DMSO-d )
δ (ppm): 4.37 (s, 2H,
2
6
S-CH ), 7.31–7.35 (m, 1H, aromatic), 7.44–7.48 (m, 1H, aromatic), 7.76–7.79 (m, 3H, aromatic), 8.00
2
(
d, J = 8.0 Hz, 1H, aromatic), 8.21–8.25 (m, 2H, aromatic), 11.14 (br, 1H, N-H), 12.72 (br, 1H, N-H).
1
3
C-NMR (100 MHz, DMSO-d ): 36.98 (CH ), 116.97 (2CH), 120.67 (CH), 121.74 (CH), 123.72 (CH),
6
2
1
25.46 (2CH), 126.21 (CH), 131.45 (C), 140.89 (C), 145.92 (C), 148.48 (C), 154.53 (C), 157.69 (C), 163.88
+
(C), 166.99 (C). MS (ESI) (m/z): [M + H] 445.5. For C H N O S Calculated: C, 45.94; H, 2.72; N,
17
12
6
3 3
1
8.91. Found: C, 45.95; H, 2.73; N, 18.90.
N-(6-Nitrobenzothiazol-2-yl)-2-[(5-((4-nitrophenyl)amino)-1,3,4-thiadiazol-2-yl)thio]acetamide (3h) IR νmax
´
1
(
cm ): 3298, 3204 (N-H stretching), 3152, 3084 (Aromatic C-H stretching), 2930, 2911 (Aliphatic
C-H stretching), 1713, 1688 (C=O stretching), 1613, 1597, 1559, 1505 (N-H bending, C=N, C=C and
1
NO asymmetric stretching). H-NMR (400 MHz, DMSO-d )
δ (ppm): 4.41 (s, 2H, S-CH ), 7.59 (d,
2
2
6
J = 9.2 Hz, 1H, aromatic), 7.78 (d, J = 9.2 Hz, 1H, aromatic), 7.91–7.94 (m, 1H, aromatic), 8.21–8.30
m, 3H, aromatic), 9.07 (s, 1H, aromatic), 10.96 and 11.21 (2s, 1H, N-H), 13.15 and 13.97 (2s, 1H, N-H).
(
1
3
C-NMR (100 MHz, DMSO-d ): 36.95 (CH ), 116.88 (2CH), 118.91 (d, CH), 120.69 (d, CH), 121.68 (CH),
6
2
1
1
25.29 (2CH), 132.13 (C), 140.86 (C), 143.07 (C), 145.87 (C), 153.25 (C), 154.24 (C), 163.09 (C), 163.87 (C),
+
67.69 (C). MS (ESI) (m/z): [M + H] 490.5. For C H N O S Calculated: C, 41.71; H, 2.27; N, 20.03.
17
11
7
5 3
Found: C, 41.70; H, 2.26; N, 20.02.
4
.2. Pharmacology
4
.2.1. Animals
Adult BALB/c mice (30–35 g) were used for all tests. Animals were maintained under a 12 h
˝
light/dark cycle in a temperature (24
˘
1 C) and humidity controlled room. To warrant adaptation
to the new environment, mice were housed in the laboratory at least 48 h before the experimental
session. Food was withdrawn 12 h before experiments in order to avoid food interference with
substance absorption, though water was allowed ad libitum. The Local Ethical Committee on Animal
Experimentation of Anadolu University, Eski s¸ ehir, Turkey has approved the experimental protocols.
4
.2.2. Administration of the Compounds
Mice assigned randomly to following experimental groups: control group (sunflower oil),
reference drug (morphine sulfate, Sigma-Aldrich, St. Louis, MO, USA, 10 mg/kg, i.p) group and
test compounds (100 mg/kg, i.p) groups. Each group consists of seven animals. Injections were
administrated (i.p) 0.5 h before the tests.
4
.2.3. Assessment of Pain Behavior
Mechanical Nociceptive Test
The mechanical antinociceptive activities of the test compounds were evaluated by the tail-clip
test in mice. A metal artery clamp was applied to the tail of mouse and the time spent before biting
the clamp (response latency) was recorded by a stopwatch [28]. Elongation of response latency is
considered as an antinociception parameter. A sensitivity test was carried out before the experimental
session and animals that did not respond to the clamp within 10 s were discarded from the experiments.
Maximum latency time (cut-off time) for the tail-clip tests was chosen as 10 s to avoid possible tissue
damage [25].

Molecules 2016, 21, 1004
8 of 10
Thermal Nociceptive Test
The thermal antinociceptive activities of the test compounds were evaluated by the hot-plate test
in mice. Mice were placed on a hot-plate analgesiameter (Ugo Basile, Italy, No. 7280), which was set at
˝
5
5
˘
1.0 C and response latencies (time of licking the forepaws or eventually jumping) were recorded.
Elongation of “response latency” is considered as an antinociception parameter. A sensitivity test was
carried out before the experimental sessions and only the animals reacting within 15 s were selected
for the tests. Maximum cut-off time was chosen as 30 s to prevent tissue damage [25].
Antinociceptive effect was calculated by converting the tail-clip and hot-plate latencies to
percentage of the maximum possible effect (MPE %), according to the following equation [24]:
MPE % “ rppost ´ drug latency ´ pre ´ drug latencyq{pcut ´ off time ´ pre ´ drug latencyqs ˆ 10
(1)
Chemical Nociceptive Test
The chemical antinociceptive activities of the test compounds were evaluated by the acetic-acid
induced writhing test in mice [29]. Thirty minutes after the administrations, mice were treated with an
aqueous solution of acetic acid (Merck, Rio de Janeiro, Brazil) (0.6% v/v, i.p) at a dose of 10 mL/kg
to induce contractions. After 5 min, the number of abdominal constrictions and stretches during the
following 10 min was recorded. Significant reduction in the number of writhes was considered as an
antinociception parameter [30].
4
.2.4. Assessment of Spontaneous Locomotor Activity
Activity Cage Test
The spontaneous locomotor activities of mice were monitored by the activity cage apparatus (Ugo
Basile, No.7420, Varese, Italy) containing two pairs of 16 photocells, 3 cm and 12 cm above the floor
under a transparent cover. Interruptions of light beams to the photocells during vertical and horizontal
movements of the animals were automatically recorded for 4 min [31].
4
.2.5. Statistical Analyses
Statistical evaluation was performed using GraphPad Prism (version 6.01 software; GraphPad,
Inc., San Diego, CA, USA). The data used in statistical analyses were acquired from 7 animals for each
group. Data obtained from the pharmacological tests were analyzed by a one-way ANOVA followed
by a post hoc Tukey’s test. The results were expressed as mean
Differences between data sets were considered as significant when p-value was less than 0.05.
˘
standard error of mean (S.E.M).
5
. Conclusions
In the present paper, we described the synthesis of new 1,3,4-thiadiazole derivatives, which were
evaluated for their antinociceptive activities.
Among the tested compounds, (1,3-benzodioxol-5-ylmethyl)amino substituted compound 3e was
the only compound that showed its specific antinociceptive activity by manipulating both central
and peripheral nociceptive mechanisms. In addition, compounds 3b, 3d, 3g and 3h were shown to
inhibit the transmission of nociceptive stimuli by affecting central nociceptive mechanisms without
any interaction with peripheral pathways. These findings supported the previous papers reporting the
centrally and/or peripherally mediated antinociceptive effects of 1,3,4-thiadiazole derivatives [11–14,21].
Since pain is a process concerning numerous neuronal signaling and modulatory pathways [32
various mechanisms (such as opioidergic, tachykininergic, cholecystokinergic, cannabinoidergic,
cholinergic, monoaminergic, nitrergic systems or ion channels such as Ca, Na etc.) [34 37] might
,33],
–
contribute to the antinociceptive effects of these compounds. Therefore, future studies are needed to
clarify underlying mechanisms of the compounds.

Molecules 2016, 21, 1004
9 of 10
Acknowledgments: This study was supported by Anadolu University Scientific Research Projects Commission
under grant No: 1605S299.
Author Contributions: M.D.A. and Z.A.K. designed the research; M.D.A. performed the synthetic work and
mainly wrote the manuscript, Ö.D.C. and Ü.D.Ö. were responsible for the whole pharmacological part of the
manuscript (animal experiments, statistical evaluation, writing and discussion of the pharmacological results).
M.D.A. was also responsible for the correspondence of the manuscript. All authors discussed, edited and approved
the final version.
Conflicts of Interest: The authors declare no conflict of interest.
References
1
.
Guindon, J.; Walczak, J.S.; Beaulieu, P. Recent advances in the pharmacological management of pain. Drugs
007, 67, 2121–2133. [CrossRef] [PubMed]
2
2
3
.
.
Moore, N.D. In search of an ideal analgesic for common acute pain. Acute Pain 2009, 11, 129–137. [CrossRef]
Buschmann, H.; Christoph, T.; Friderichs, E.; Maul, C.; Sundermann, B. Analgesics: From Chemistry and
Pharmacology to Clinical Application; Wiley-VCH: Weinheim, Germany, 2002; pp. 1–264.
4.
5.
6.
7.
Turunen, J.H.O.; Mäntyselkä, P.T.; Kumpusalo, E.A.; Ahonen, R.S. Frequent analgesic use at population level:
Prevalence and patterns of use. Pain 2005, 115, 374–381. [CrossRef] [PubMed]
Simmons, D.L.; Botting, R.M.; Hla, T. Cyclooxygenase Isozymes: The Biology of Prostaglandin Synthesis
and Inhibition. Pharmacol. Rev. 2004, 56, 387–437. [CrossRef] [PubMed]
Dannhardt, G.; Laufer, S. Structural Approaches to Explain the Selectivity of COX-2 Inhibitors: Is There a
Common Pharmacophore? Curr. Med. Chem. 2000, 7, 1101–1112. [CrossRef] [PubMed]
Jain, A.K.; Sharma, S.; Vaidya, A.; Ravichandran, V.; Agrawal, R.K. 1,3,4-Thiadiazole and its Derivatives:
A Review on Recent Progress in Biological Activities. Chem. Biol. Drug Des. 2013, 81, 557–576. [CrossRef]
[PubMed]
8
9
1
1
.
Li, Y.; Geng, J.; Liu, Y.; Yu, S.; Zhao, G. Thiadiazole-a Promising Structure in Medicinal Chemistry.
Chem. Med. Chem. 2013, 8, 27–41. [CrossRef] [PubMed]
Matysiak, J. Biological and Pharmacological Activities of 1,3,4-Thiadiazole Based Compounds. Mini-Rev.
Med. Chem. 2015, 15, 762–775. [CrossRef] [PubMed]
.
0. Haider, S.; Alam, M.S.; Hamid, H. 1,3,4-Thiadiazoles: A potent multi targeted pharmacological scaffold.
Eur. J. Med. Chem. 2015, 92, 156–177. [CrossRef] [PubMed]
1. Shkair, A.M.; Shakya, A.K.; Raghavendra, N.M.; Naik, R.R. Molecular Modeling, Synthesis and
Pharmacological Evaluation of 1,3,4-Thiadiazoles as Anti-inflammatory and Analgesic Agents. Med. Chem.
2
016, 12, 90–100. [CrossRef] [PubMed]
1
1
1
2. Schenone, S.; Brullo, C.; Bruno, O.; Bondavalli, F.; Ranise, A.; Filippelli, W.; Rinaldi, B.; Capuano, A.;
Falcone, G. New 1,3,4-thiadiazole derivatives endowed with analgesic and anti-inflammatory activities.
Bioorg. Med. Chem. 2006, 14, 1698–1705. [CrossRef] [PubMed]
3. Amir, M.; Kumar, H.; Javed, S.A. Non-carboxylic Analogues of Naproxen: Design, Synthesis,
and Pharmacological Evaluation of some 1,3,4-Oxadiazole/Thiadiazole and 1,2,4-Triazole Derivatives.
Arch. Pharm. Chem. Life Sci. 2007, 340, 577–585. [CrossRef] [PubMed]
4. Salgin-Gök s¸ en, U.; Gökhan-Kelekçi, N.; Gökta s¸ , O.; Köysal, Y.; Kiliç, E.; I s¸ ik, S.; Aktay, G.;
Ozalp, M. 1-Acylthiosemicarbazides, 1,2,4-triazole-5(4H)-thiones, 1,3,4-thiadiazoles and hydrazones
containing 5-methyl-2-benzoxazolinones: Synthesis, analgesic-anti-inflammatory and antimicrobial activities.
Bioorg. Med. Chem. 2007, 15, 5738–5751. [CrossRef] [PubMed]
1
5. Yavuz, S.; Ünal, Y.; Pamir, Ö.; Yılmazer, D.; Kurtipek, Ö.; Kavutçu, M.; Arslan, M.; Ark, M.; Yıldırır, Y.
Synthesis and pharmacological evaluation of some novel thebaine derivatives: N-(tetrazol-1H-5-yl)-6,14-
endoethenotetrahydrothebaine incorporating the 1,3,4-oxadiazole or the 1,3,4-thiadiazole moiety.
Arch. Pharm. Chem. Life Sci. 2013, 346, 455–462. [CrossRef] [PubMed]
16. Clerici, F.; Pocar, D. Synthesis of 2-Amino-5-sulfanyl-1,3,4-thiadiazole derivatives and evaluation of their
antidepressant and anxiolytic activity. J. Med. Chem. 2001, 44, 931–936. [CrossRef] [PubMed]
17. Yusuf, M.; Khan, R.A.; Ahmed, B. Syntheses and anti-depressant activity of 5-amino-1,3,4-thiadiazole-2-thiol
imines and thiobenzyl derivatives. Bioorg. Med. Chem. 2008, 16, 8029–8034. [CrossRef] [PubMed]
18. Pattanayak, P.; Sharma, R.; Sahoo, P.K. Synthesis and evaluation of 2-amino-5-sulfanyl-1,3,4-thiadiazoles as
antidepressant, anxiolytic, and anticonvulsant agents. Med. Chem. Res. 2009, 18, 351–361. [CrossRef]

Molecules 2016, 21, 1004
10 of 10
1
2
2
9. Sharma, R.; Misra, G.P.; Sainy, J.; Chaturvedi, S.C. Synthesis and biological evaluation of
2
-amino-5-sulfanyl-1,3,4-thiadiazole derivatives as antidepressant, anxiolytics and anticonvulsant agents.
Med. Chem. Res. 2011, 20, 245–253. [CrossRef]
0. Rajak, H.; Deshmukh, R.; Aggarwal, N.; Kashaw, S.; Kharya, M.D.; Mishra, P. Synthesis of novel
2
,5-disubstituted 1,3,4-thiadiazoles for their potential anticonvulsant activity: Pharmacophoric model studies.
Arch. Pharm. Chem. Life Sci. 2009, 342, 453–461. [CrossRef] [PubMed]
˙
1. Can, Ö.D.; Altıntop, M.D.; Demir Özkay, Ü.; Üçel, U.I.; Do g˘ ruer, B.; Kaplancıklı, Z.A. Synthesis of Thiadiazole
Derivatives Bearing Hydrazone Moieties and Evaluation of Their Pharmacological Effects on Anxiety,
Depression, and Nociception Parameters in Mice. Arch. Pharm. Res. 2012, 35, 659–669. [CrossRef] [PubMed]
2. Wong, C.H.; Dey, P.; Yarmush, J.; Wu, W.H.; Zbuzek, V.K. Nifedipine-induced analgesia after epidural
injection in rats. Anesth. Analg. 1994, 79, 303–306. [CrossRef] [PubMed]
3. Gabra, B.H.; Sirois, P. Beneficial effect of chronic treatment with the selective bradykinin B1 receptor
antagonists, R-715 and R-954, in attenuating streptozotocin-diabetic thermal hyperalgesia in mice. Peptides
2
2
2
003, 24, 1131–1139. [CrossRef] [PubMed]
2
4. Can, O.D.; Alyu, F.; Turan, N.; Ozdemir, A. Antinociceptive activities of some 4,5-dihydro-1H-pyrazole
derivatives: Involvement of central and peripheral pathways. Lett. Drug Des. Discov. 2016, 13, 411–417.
[CrossRef]
2
2
2
2
5. Demir Özkay, U.; Can, O.D. Anti-nociceptive effect of vitexin mediated by the opioid system in mice.
Pharmacol. Biochem. Behav. 2013, 109, 23–30. [CrossRef] [PubMed]
˙
6. Ermut, G.; Karalı, N.; Çetin, I.; Topçul, M.; Birteksöz, S. Synthesis and chemotherapeutic activities of
-chloro-1H-indole-2,3-dione 3-thiosemicarbazones. Marmara Pharm. J. 2013, 17, 147–154. [CrossRef]
5
7. Tomkinson, A.E.; Chen, X.; Dziegielewska, B.; Mackerell, A.D.; Zhong, S.; Wilson, G.M. Compounds that
Inhibit Human DNA Ligases and Methods of Treating Cancer. US20100099683A1, 22 April 2010.
8. D’Amour, F.E.; Smith, D.L. A method for determining loss of pain sensation. J. Pharmacol. Exp. Ther. 1941, 72,
7
4–79.
2
3
9. Koster, R.; Anderson, M.; De Beer, E.J. Acetic acid for analgesic screening. Fed. Proc. 1959, 18, 412–415.
0. Kaplancikli, Z.A.; Altintop, M.D.; Turan-Zitouni, G.; Ozdemir, A.; Can, O.D. Synthesis and analgesic activity
of some acetamide derivatives. J. Enzyme Inhib. Med. Chem. 2012, 27, 275–280. [CrossRef] [PubMed]
1. Votava, M.; Hess, L.; Slíva, J.; Krsiak, M.; Agová, V. Dexmedetomidine selectively suppresses dominant
behavior in aggressive and sociable mice. Eur. J. Pharmacol. 2005, 523, 79–85. [CrossRef] [PubMed]
3
3
2. Argoff, C. Mechanisms of pain transmission and pharmacologic management. Curr. Med. Res. Opin. 2011
7, 2019–2031. [CrossRef] [PubMed]
,
2
3
3
3. Millan, M.J. Descending control of pain. Prog. Neurobiol. 2002, 66, 355–474. [CrossRef]
4. Mollica, A.; Costante, R.; Novellino, E.; Stefanucci, A.; Pieretti, S.; Zador, F.; Samavati, R.; Borsodi, A.;
Benyhe, S.; Vetter, I.; Lewis, R.J. Design, synthesis and biological evaluation of two opioid agonist and Cav
2
.2 blocker multitarget ligands. Chem. Biol. Drug Des. 2015, 86, 156–162. [CrossRef] [PubMed]
5. Suzuki, H. Tachykininergic neurotransmission in the central nervous system. J. Nippon. Med. Sch. 2002, 69,
22–327. [CrossRef] [PubMed]
6. Noble, F. Pharmacology of CCKRs and SAR studies of peptidic analog ligands. Curr. Top. Med. Chem. 2007
, 1173–1179. [CrossRef] [PubMed]
7. Luo, Z.D.; Cizkova, D. The role of nitric oxide in nociception. Curr. Rev. Pain. 2000, 4, 459–466. [CrossRef]
PubMed]
3
3
3
3
,
7
[
Sample Availability: Samples of the compounds 3a–h are available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).