Synthesis and radioligand-binding assay of 2,5-disubstituted thiadiazoles and evaluation of their anticonvulsant activities

Article abstract of DOI:10.1002/ardp.202000066

In this study, a number of 2,5-disubstituted 1,3,4-thiadiazoles were synthesized using an appropriate synthetic route, and their anticonvulsant activity was determined by the maximal electroshock seizure (MES) test and their neurotoxicity was evaluated by the rotarod test. Additionally, their hypnotic activity was tested using the pentobarbital-induced sleep test. Compounds 7 (ED₅₀ = 1.14 and 2.72 μmol/kg in the MES and sleep tests, respectively) and 11 (ED₅₀ = 0.65 and 2.70 μmol/kg in the MES and sleep tests, respectively) were the most potent ones in the sleep test and anticonvulsant test, showing a comparable activity with diazepam as the reference drug. The results of in vivo studies, especially the antagonistic effects of flumazenil, and also the radioligand-binding assay confirmed the involvement of benzodiazepine (BZD) receptors in the anticonvulsant and hypnotic activity of compounds 7 and 11. Finally, the docking study of compound 11 in the BZD-binding site of the GABA_A (gamma-aminobutyric acid) receptor confirmed the possible binding of the compound to the BZD receptors. We concluded that the novel 1,3,4-thiadiazole derivatives with appropriate substitution at positions 2 and 5 of the heterocyclic ring had a good affinity to BZD receptors and showed significant efficacy in the pharmacological tests.

Full text of DOI:10.1002/ardp.202000066

Received: 14 March 2020
Revised: 3 August 2020
Accepted: 10 August 2020
|
|
DOI: 10.1002/ardp.202000066
F U L L P A P E R
Synthesis and radioligand‐binding assay of 2,5‐disubstituted
thiadiazoles and evaluation of their anticonvulsant activities
Mahsa Toolabi1 | Mona Khoramjouy2 | Ayoub Aghcheli3 | Adileh Ayati3
Setareh Moghimi3 | Loghman Firoozpour3 | Soraya Shahhosseini4
|
|
Rouhallah Shojaei5 | Ali Asadipour6 | Kouros Divsalar6 | Mehrdad Faizi2
|
Alireza Foroumadi6,7
1Department of Medicinal Chemistry, School
of Pharmacy, Ahvaz Jundishapur University of
Abstract
Medical Sciences, Ahvaz, Iran
In this study, a number of 2,5‐disubstituted 1,3,4‐thiadiazoles were synthesized using an
appropriate synthetic route, and their anticonvulsant activity was determined by the
maximal electroshock seizure (MES) test and their neurotoxicity was evaluated by the
rotarod test. Additionally, their hypnotic activity was tested using the pentobarbital‐
induced sleep test. Compounds 7 (ED50 = 1.14 and 2.72 μmol/kg in the MES and sleep
tests, respectively) and 11 (ED50 = 0.65 and 2.70 μmol/kg in the MES and sleep tests,
respectively) were the most potent ones in the sleep test and anticonvulsant test,
showing a comparable activity with diazepam as the reference drug. The results of in
vivo studies, especially the antagonistic effects of flumazenil, and also the radioligand‐
binding assay confirmed the involvement of benzodiazepine (BZD) receptors in the
anticonvulsant and hypnotic activity of compounds 7 and 11. Finally, the docking study
of compound 11 in the BZD‐binding site of the GABAA (gamma‐aminobutyric acid)
receptor confirmed the possible binding of the compound to the BZD receptors. We
concluded that the novel 1,3,4‐thiadiazole derivatives with appropriate substitution at
positions 2 and 5 of the heterocyclic ring had a good affinity to BZD receptors and
showed significant efficacy in the pharmacological tests.
2Department of Pharmacology and Toxicology,
School of Pharmacy, Shahid Beheshti
University of Medical Sciences, Tehran, Iran
3Drug Design and Development Research
Center, The Institute of Pharmaceutical
Sciences (TIPS), Tehran University of Medical
Sciences, Tehran, Iran
4Department of Pharmaceutical Chemistry and
Radiopharmacy, School of Pharmacy, Shahid
Beheshti University of Medical Sciences,
Tehran, Iran
5Parsian Pharmaceutical Co., Alborz, Iran
6Neuroscience Research Center, Institute of
Neuropharmacology, Kerman University of
Medical Sciences, Kerman, Iran
7Department of Medicinal Chemistry, Faculty
of Pharmacy, Tehran University of Medical
Sciences, Tehran, Iran
Correspondence
Mehrdad Faizi, Department of Pharmacology
and Toxicology, School of Pharmacy, Shahid
Beheshti University of Medical Sciences,
1991953381 Tehran, Iran.
K E Y W O R D S
Email: m.faizi@sbmu.ac.ir
1,3,4‐thiadiazole, anticonvulsant agents, benzodiazepine receptors, flumazenil,
radioligand‐binding assay
Alireza Foroumadi, Department of Medicinal
Chemistry, Faculty of Pharmacy, Tehran
University of Medical Sciences, 14174 Tehran,
Iran.
Email: aforoumadi@yahoo.com
|
1
INTRODUCTION
currently available anticonvulsant drugs can only suppress the symptoms
of epilepsy and called “symptomatic” agents. Also, most of these existing
Epilepsy is a neurological disorder that affects approximately 60 million
people around the world.[1,2] Epilepsy is characterized by epileptic sei-
zures due to abnormal nerve cell activity and neuronal discharge, which
are accepted as pathogenesis of epileptic seizures.[3] Brain injury, tumors,
infections, stroke, and birth defects are known as causes of epilepsy.
However, for most patients, the main causes remain unknown.[4] The
drugs are effective in less than 70% of patients and suffer from in-
adequate potency, toxicity, and a variety of side effects.[5] Although
medication is known as the only treatment for epilepsy, many patients do
not respond to monotherapy and need polytherapy with multiple antic-
onvulsants agents.[6] These limitations precisely explain the need for
further investigations to find new and effective anticonvulsant agents.
|
Arch Pharm. 2020;e2000066.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
1 of 9
https://doi.org/10.1002/ardp.202000066

2 of 9
TOOLABI ET AL.
|
Numerous therapeutic agents have been approved for clinical
usage as anticonvulsants that belong to different chemical structures
including hydantoins, barbiturates, benzodiazepines (BZDs), succini-
mides, valproate, imides, oxazolidinediones, sulfonamides, and gamma‐
aminobutyric acid (GABA) analogs.[7] BZDs are an important class of
anticonvulsant agents with
a great activity against generalized
tonic–clonic and partial seizures, which act through binding to the
GABAA receptors. This binding increases the frequency of chloride ion
channel opening, which facilitates the inhibitory effects of GABA.
However, these types of medicines have several adverse effects such
as sedation, memory impairment, and development of tolerance.[8]
Considering the fact that most of the clinically used BZDs have ne-
gative effects on memory, finding novel anticonvulsant agents as new
ligands for the BZD‐binding site of the GABA receptor, with less effect
on memory, has been an interesting field of drug design in the recent
decade.[9,10]
Many five‐membered heterocyclic compounds such as triazoles,[11,12]
oxadiazoles,[13] pyrrolidines, and pyrazoles[14] have been reported for
their anticonvulsant and antidepressant activities. Particularly, thiadiazole
derivatives have been widely used as anticonvulsant and antidepressant
agents. Thiadiazole is a versatile five‐membered heterocyclic compound
with four isomeric structures (1,2,3‐thiadiazole, 1,2,4‐thiadiazole, 1,2,5‐
thiadiazole, and 1,3,4‐thiadiazole) that are present in several biologically
active natural products, medicinal agents, and commercial drugs.[15–17]
Among these isomers, 1,3,4‐thiadiazoles have a very well‐established role
as pharmacologically significant scaffolds that display a broad spectrum of
activities such as antimicrobial, anti‐inflammatory, antitumor, antioxidant,
antitubercular, antiviral as well as enzymes and human platelet ag-
gregation inhibitors.[18–22] In addition, a large number of 1,3,4‐thiadiazole
containing compounds have been synthesized and evaluated as antic-
onvulsant agents.[23–25]
FIGURE 1 Structures of acetazolamide, diazepam, phenytoin, and
the target compounds
which led to the synthesis of 5‐amino‐1,3,4‐thiadiazole‐2‐thiol (b). In
the next step, 5‐amino‐1,3,4‐thiadiazole‐2‐thiol (b) was reacted with
benzyl bromide derivatives, to afford the appropriate 2‐amino‐5‐
benzylthio‐1,3,4‐thiadiazoles (c1–5). Finally, in the condensation re-
action of phenyl isocyanate derivatives with the intermediate com-
pounds (c1–5) in dry dichloromethane, the final derivatives (1–14)
were obtained in good yields (75–85%).
|
2.2
Pharmacology
|
As a part of our ongoing research program to design new an-
ticonvulsant agents,[26–29] herein we reported the design and
synthesis of several 2,5‐disubstituted 1,3,4‐thiadiazoles (1–14). The
design of the newly synthesized compounds was inspired by the
structures of well‐known anticonvulsant drugs such as acet-
azolamide, diazepam, and phenytoin (Figure 1) by the attachment of
the benzylthio‐1,3,4‐thiadiazole core to the aryl urea structure.
The anticonvulsant activity of the novel derivatives was evaluated
by the maximal electroshock seizure (MES) test. We also investigated
the hypnotic effects of the compounds. Furthermore, to determine
the mode of anticonvulsant action of the synthesized compounds, the
effect of flumazenil, a BZD antagonist, on the anticonvulsant activity
of the compounds was evaluated.
2.2.1
Anticonvulsant activity
The most adopted animal model, MES, is employed to screen the new
anticonvulsants agents. MES is the preclinical test that is developed
to identify the effective compounds against generalized seizures of
the tonic–clonic (grand mal) type.[30] To determine the antic-
onvulsant activity of the synthesized compounds, the ability of the
compounds to protect mice against convulsion induced by maximal
electroshock was evaluated. The results were compared with diaze-
pam, a BZD agonist, as the standard anticonvulsant drug that was
effective in the MES test. The synthesized compounds, diazepam or
vehicle, were administered 30 min before the induction of electro-
shock (60 Hz, 50 mA, and 0.2 s). After that time, the occurrences
of hindlimb tonic extension (HLTE) were observed in the MES model.
The anticonvulsant activities of the synthesized compounds are
depicted in Table 1. The alteration of substituents on the phenylurea
and benzylthio rings on positions 2 and 5 of the 1,3,4‐thiadiazole
moiety (R2 and R1, respectively) had a significant effect on the potency
of the compounds against MES‐induced seizures. On the basis of the
substituents on the attached 5‐benzylthio warhead (R1), the designed
compounds could be categorized into four groups: The unsubstituted
(1–6), 3‐methoxy‐ (7–9), chloro‐ (10 and 11), and 3‐nitro‐substituted
|
2
RESULTS AND DISCUSSION
|
2.1
Chemistry
The target compounds (1–14) were synthesized through the syn-
thetic route outlined in Scheme 1. The pathway started from the
reaction of thiosemicarbazide (a) with carbon disulfide in ethanol,

3 of 9
TOOLABI ET AL.
|
SCHEME 1 The synthesis of
compounds 1–14. Reagents and
conditions: (1) (i) CS2, Na2CO3, EtOH, ref,
8 hr; (ii) H2O, HCl; (2) (i) H2O, KOH, r.t.,
15 min; (ii) EtOH, benzyl bromide
derivatives, rt, 30 min; (3) phenyl
isocyanate derivatives, dry CH2Cl2, r.t.,
overnight
2
1
3
derivatives (12–14). In each series, substituents on the 2‐phenylurea
ring were altered to optimize the anticonvulsant activity. In the MES
test, the unsubstituted derivative (1) showed significant anticonvulsant
activity, and alteration of substitutions on the 2‐phenylurea (R2 = CH3,
Cl, F, and 2,4‐diCl) decreased the anticonvulsant activity of compounds
2–4 and 6. However, compound 5 with R2 = 2‐Br was the most potent
compound in this series (ED50 = 1.05 μmol/kg). In the 3‐methoxy‐
containing derivatives (7–9), all the compounds had moderate‐to‐good
activity. However, attachment of 4‐methyl on the 2‐phenylurea
warhead (8) decreased the anticonvulsant activity in the MES test.
Among 3‐nitro‐containing compounds, the unsubstituted phenylurea
derivative (12) exhibited an interesting anticonvulsant activity in the
MES test. However, attachment of 4‐methyl and 2‐bromo on the
2‐phenylurea warhead decreased the anticonvulsant activity of com-
pounds 13 and 14. In all four groups, the best results were seen by
compound 11, with 2,4‐dichloro substitution on the benzylthio ring
and 4‐fluoro group on the phenylurea warhead in the MES test
(ED50 = 0.65 μmol/kg), which showed a comparable activity with dia-
zepam (ED50 = 0.43 μmol/kg). Furthermore, compounds 5 with R1 = H
and R2 = 2‐Br and 7 with R1 = 3‐OCH3 and R2 = H also showed a re-
latively strong anticonvulsant activity in the MES test (ED50 = 1.05
μmol/kg). On the basis of the structure–activity analysis, it could be
TABLE 1 The in vivo anticonvulsant effects of target compounds 1–14
Sleep test
MES test
Compound
R1
R2
ED50 (95% CI; μmol/kg)
ED50 (95% CI; μmol/kg)
1
H
H
6.48 (4.52–9.30)
10.11 (8.14–12.55)
9.91 (6.09–16.13)
13.14 (9.47–18.24)
2.85 (2.10–3.88)
12.66 (8.83–18.15)
2.72 (1.96–3.77)
10.71 (0.76–7.50)
3.42 (2.20–5.32)
4.10 (3.25–5.17)
2.70 (2.16–3.38)
2.61 (1.40–4.85)
6.53 (4.00–10.65)
9.86 (5.62–17.28)
1.86 (1.03–2.15)
2.36 (1.22–4.54)
3.36 (0.89–12.58)
3.14 (2.19–4.49)
3.53 (1.13–11.01)
1.05 (0.37–2.94)
3.38 (2.19–5.24)
1.14 (0.25–5.12)
3.59 (1.40–9.22)
1.27 (0.16–9.90)
2.36 (0.75–7.42)
0.65 (0.23–1.82)
1.15 (0.41–3.20)
2.44 (1.20–4.94)
2.98 (1.93–4.61)
0.43 (0.24–0.77)
2
H
4‐CH3
4‐F
3
H
4
H
4‐Cl
2‐Br
3,4‐diCl
H
5
H
6
H
7
3‐OCH3
3‐OCH3
3‐OCH3
3‐Cl
2,4‐diCl
3‐NO2
3‐NO2
3‐NO2
–
8
4‐CH3
2‐Br
4‐Cl
4‐F
9
10
11
12
H
13
4‐CH3
2‐Br
–
14
Diazepam
Abbreviation: 95% CI, 95% confidence interval; MES, maximal electroshock seizure.

4 of 9
TOOLABI ET AL.
|
concluded that chloro substitution on the benzylthio ring and bromo
substitution on the phenylurea warhead had a favorable impact on the
anticonvulsant activity of the compounds. However, further structural
modifications on the scaffold might lead to the discovery of more
potent anticonvulsant agents.
receptors are evaluated biochemically and physiologically, and
their pharmacological properties are determined. The radioligand
receptor binding assay using [3H]flumazenil is a common method
to evaluate the affinity of newly synthesized BZD ligands to the
receptor in two basic types of experiments: saturation and
competition studies. Saturation studies are used to determine the
affinity of [3H]flumazenil radioligand to a receptor, whereas, in
competition studies, the affinity of nonradioactive ligands to re-
ceptors is measured.[32,33] Encouraged by the acceptable activity
of compounds 7 and 11, the competitive radioligand‐binding as-
say is conducted to understand the mechanisms behind the an-
ticonvulsant activity of these types of derivatives at the receptor
level. In this assay, [3H]flumazenil was used as the specific ligand
for estimating the affinity of the compounds to the BZD receptor.
The results showed that compounds 7 and 11 have an affinity to
the BZD receptor in nanomolar concentrations (IC50 = 0.31 and
0.44 nM and Ki values = 0.18 and 0.26 nM, respectively), which
were comparable with the affinity of diazepam to the BZD re-
ceptor (IC50 = 0.30 nM and Ki value = 0.17 nM). These results
confirmed that the anticonvulsant activity of 7 and 11 was
mediated by BZD receptors (Table 2).
|
2.2.2
Rotarod test
The neurotoxicity of the compounds was assessed by the rotarod test.
The most potent compounds (7 and 11) as anticonvulsants were se-
lected for the rotarod test. TD50 values of compounds 7 and 11 were
29.68 (13.64–64.58) μmol/kg and 20.85 (11.39–38.18) μmol/kg, re-
spectively. Furthermore, the calculated protective index (PI; the ratio
between TD50 and ED50) for anticonvulsant effects of these two com-
pounds (7 and 11) in the MES test was 26.03 and 32.07, respectively.
|
2.2.3
Pentobarbital‐induced sleep test
To assess the hypnotic effects of the compounds, a pentobarbital‐
induced sleep test in mice was performed. Different doses of com-
pounds were injected intraperitoneally (ip), and then animals re-
ceived sodium pentobarbital at a dose of 40 mg/kg. Each mouse was
observed for 120 min after the injection and the time of losing the
righting reflex was recorded. The results were depicted as ED50 va-
lues and compared with diazepam (Table 1). On the basis of the
results, it is observed that all the synthesized compounds had higher
ED50 values than diazepam, which means that all of the newly syn-
thesized derivatives had lower potency as hypnotic agents than the
reference drug. Among the synthesized derivatives, compounds 7, 11,
and 12 showed higher potency as hypnotic agents.
|
2.3
Molecular docking study
To evaluate the binding affinities and interactions of the most
active compound (11) to the BZD‐binding pocket of GABAA re-
ceptor, the docking study was examined by AutoDock 4.2.1
software[34] and the interactions were visualized by Discovery
Studio software. The structure of GABAA receptor with diazepam
(PDB ID: 6HUP) was used and the docking procedure was con-
ducted as previously described.[35] As seen in Figure 2, hydrogen
bonds with Met 261 and Leu 285 residues through urea moiety
were formed. The 4‐F phenyl ring formed π–sulfur interaction
with Met 236 and Met 286 residues. The 2,4‐diCl phenyl ring
made π–π interaction with the aromatic moiety of Phe 289, which
was similar to the binding mode of diazepam's phenyl ring. Fur-
thermore, this compound made other hydrophobic interactions
(van der Waals interactions) with Thr 237, Thr 265, and Leu 269
residues. To summarize, according to the molecular docking
simulation of compound 11 in the BZD‐binding site of the GABAA
receptor, the results were in accordance with the experimental
results.
|
2.2.4
Study on the possible mechanism of action
Effect of flumazenil on anticonvulsant activity of the compounds
To understand the possible mechanism of anticonvulsant activity of
the most potent compounds 7 and 11, we checked if flumazenil
would be able to antagonize the effect of the compounds in the MES
test.[25,31] Flumazenil is a well‐known BZD antagonist, administrated
at a dose of 10 mg/kg, 15 min before the induction of convulsion.
According to the results, in the MES test, the anticonvulsant effects
of 7 and 11 were completely inhibited (100%) by flumazenil at the
dose of 40 mg/kg. These results confirmed that the anticonvulsant
activity of compounds 7 and 11 was antagonized with flumazenil,
which established the involvement of BZD receptors in reported
effects. Furthermore, in the sleep test, flumazenil at 10 mg/kg dose
significantly reduced the hypnotic effects of compounds 7 and 11.
TABLE 2 The binding affinity of compounds 7 and 11 to the
benzodiazepine receptor
Compound
IC50 (95% CI; nM)
0.31 (0.13–0.75)
0.44 (0.14–1.38)
0.30 (0.18–0.51)
Ki (nM)
0.18
7
Radioligand‐binding assay
11
0.26
The binding study by radioligands is a very powerful tool in the
study of receptors and their ligands. In this type of study, the
Diazepam
0.174

5 of 9
TOOLABI ET AL.
|
FIGURE 2 Binding mode of compound 11 with GABAA receptor
|
|
EXPERIMENTAL
3
CONCLUSIONS
4
|
Chemistry
A series of 2,5‐disubstituted 1,3,4‐thiadiazoles was synthesized to
explore prospective anticonvulsant agents. The synthesized com-
pounds were evaluated in the animal model of epilepsy, the MES test.
The results depicted that the anticonvulsant activity was strongly
associated with the type and the position of the substituents at-
tached to the 2‐phenylurea and 5‐benzylthio rings of 1,3,4‐
thiadiazole moiety. The results of this investigation indicated that
1,3,4‐thiadiazole having 2,4‐dichloro substitutions on 5‐benzylthio
ring and 4‐fluoro group on 2‐phenylurea moiety (compound 11) ex-
hibited a promising anticonvulsant activity in the MES test, com-
parable to diazepam. Also, this compound showed a lower hypnotic
effect in the sleep test as compared with diazepam. Additionally, the
ability of flumazenil to antagonize the effects of the compounds and
radioligand‐binding assay confirmed that the anticonvulsant activity
of the two selected compounds 7 and 11 resulted from the direct
modulation of the BZD receptor. The neurotoxicity and PI of these
compounds were also measured. In addition, in silico studies by using
molecular docking simulations showed that the prototype compound
could act as BZD receptor agonist.
4.1
|
4.1.1
General
All nuclear magnetic resonance (NMR) experiments (1H and 13C NMR
spectra) were carried out on Bruker 500 MHz at 20°C in dimethyl
sulfoxide (DMSO)‐d6 or CDCl3 as the solvents, using tetramethylsilane
as an internal standard, and chemical shifts were reported in δ parts
per million (ppm). Melting points were determined with a Kofler
hot‐plate microscope apparatus and were uncorrected. Infrared (IR)
spectroscopy was performed using a Nicolet FT‐IR Magna 550 spec-
trograph (KBr disks). Elemental analysis was recorded by a Perkin
Elmer 2400 (automatic elemental analyzer). Commercially available
reagents and solvents used for the synthesis were purchased from
Merck and Sigma‐Aldrich companies without further purification. The
progress of reactions and the purity of synthesized compounds were
checked by thin‐layer chromatography on silica gel 250 mm, F254
plastic sheets. The spots were monitored under ultraviolet irradiation
(λ = 254 nm). Mass spectra were obtained at ionization potential of

6 of 9
TOOLABI ET AL.
|
70 eV using HP Agilent Technologies 5937. The purity of the final
compounds was determined by the high‐performance liquid chroma-
tographic (HPLC) analysis; the HPLC column (Pronaos EP‐C18;
Changhai, China, 250 × 4.6 mm, inner diameter 5 µm) was employed
for all analyses. The data acquisition was performed using ChemSta-
tion software (Agilent Technologies). The mobile phase was composed
of KH2PO4 buffer (20 mM, pH 3.5) and methanol (20:80, v/v). The
isocratic elution mode was used for detection at a flow rate of
1.0 ml/min and with the detector wavelength set at 272 nm (Agilent
1200; Agilent Technologies).
137.7, 150.9, 156.1, and 160.1. Anal. calcd. for C16H14N4OS2: C,
56.12; H, 4.12; N, 16.36. Found: C, 56.31; H, 4.04; N, 16.14; mass
spectroscopy (MS) electrospray ionization (ESI) m/z 343.5 [M+H].
1‐[5‐(Benzylthio)‐1,3,4‐thiadiazol‐2‐yl]‐3‐(p‐tolyl)urea (2)
Yield: 81%; HPLC (purity: 96%); mp 238–240°C; IR (KBr, cm−1): 3,381
(N–H), 3,217 (N–H), 1,726 (C=O), 1,593, 1,548, and 1,317. 1H NMR
(DMSO‐d6, 500 MHz) δ: 2.24 (s, 3H), 4.43 (s, 2H), 7.12 (d, 2H,
J = 8.0 Hz), 7.29–7.24 (m, 1H), 7.30–7.34 (m, 4H), 7.39 (d, 2H,
J = 8.2 Hz), 8.91 (s, 1H), and 10.93 (s, 1H). 13C NMR (DMSO‐d6,
125 MHz): 19.8, 37.2, 118.4 (2C), 126.9, 127.9 (2C), 128.4 (2C), 128.7
(2C), 131.5, 135.1, 136.2, 150.7, 156.0, and 160.0. Anal. calcd. for
C17H16N4OS2: C, 57.28; H, 4.52; N, 15.72. Found: C, 57.41; H, 4.37;
N, 15.81; MS (ESI) m/z 357.2 [M+H].
The original spectra of the investigated compounds are provided
as Supporting Information, together with their InChI codes and some
biological activity data.
|
4.1.2
General procedure for the synthesis of
1‐[5‐(Benzylthio)‐1,3,4‐thiadiazol‐2‐yl]‐3‐(4‐fluorophenyl)urea (3)
Yield: 79%; HPLC (purity: 100%); mp 249–251°C; IR (KBr, cm−1):
3,385 (N–H), 3,221 (N–H), 1,712 (C═O), 1,589, 1,556, and 1,321. 1H
NMR (DMSO‐d6, 500 MHz) δ: 4.43 (s, 2H), 7.14 (t, 2H, J = 8.7 Hz),
7.27–7.24 (m, 1H), 7.31 (t, 2H, J = 8.1 Hz), 7.39 (d, 2H, J = 8.1 Hz),
7.46–7.48 (m, 2H), 9.06 (s, 1H), and 11.03 (s, 1H). 13C NMR (DMSO‐
d6, 125 MHz): 36.3, 114.1 (d, 2C, J = 20.0 Hz), 122.8 (d, 2C,
J = 8.0 Hz), 125.3, 126.3 (2C), 127.1 (2C), 134.2, 135.6 (d, J = 3.1 Hz),
150.0, 154.1, 158.4 (d, J = 251.8 Hz), and 160.8. Anal. calcd. for
C16H13FN4OS2: C, 53.32; H, 3.64; N, 15.54. Found: C, 53.47; H,
3.31; N, 15.74; MS (ESI) m/z 361.7 [M+H].
5‐amino‐1,3,4‐thiadiazole‐2‐thiol (b)
To an ethanol solution of sodium carbonate (4.5 mmol) and thiose-
micarbazide (5 mmol) (a), carbon disulfide (1 ml) was added dropwise.
The reaction mixture was heated under reflux for 8 hr. The solid
product obtained after the evaporation of the reaction mixture was
dissolved in HCl and filtered and washed with water.
|
4.1.3
General procedure for the synthesis of
2‐amino‐5‐(benzylthio)‐1,3,4‐thiadiazol (c1–5)
1‐[5‐(Benzylthio)‐1,3,4‐thiadiazol‐2‐yl]‐3‐(4‐chlorophenyl)urea (4)
Yield: 75%; HPLC (purity: 97%); mp 264–266°C; IR (KBr, cm−1): 3,374
(N–H), 3,227 (N–H), 1,727 (C═O), 1,594, 1,548, and 1,307. 1H NMR
(DMSO‐d6, 500 MHz) δ: 4.44 (s, 2H), 7.26 (t, 1H, J = 7.3 Hz), 7.32 (t,
2H, J = 7.6 Hz), 7.37–7.34 (m, 2H), 7.39 (d, 2H, J = 7.0 Hz), 7.52–7.45
(m, 2H), 9.20 (s, 1H), and 11.07 (s, 1H). 13C NMR (DMSO‐d6,
125 MHz): 37.1, 119.3, 119.9 (2C), 126.1, 126.9, 127.9 (2C), 128.0,
128.1 (2C), 128.3 (2C), 136.2, 136.8, and 157.5. Anal. calcd. for
C16H13ClN4OS2: C, 50.99; H, 3.48; N, 14.87. Found: C, 51.72; H, 3.59;
N, 14.54; MS (ESI) m/z 377.5 [M+H].
Benzyl bromide derivatives (5.2 mmol) were slowly added to the
solution of 5‐amino‐1,3,4‐thiadiazole‐2‐thiol (5 mmol) (b) and KOH
(5.3 mmol) in ethanol (3.5 ml) at 0°C. The reaction mixture was stir-
red at room temperature for 30 min and then diluted with cold water.
The obtained solid was filtered, washed with water, and then crys-
tallized from ethanol.
|
4.1.4
General procedure for the synthesis of
1‐(5‐(benzylthio)‐1,3,4‐thiadiazol‐2‐yl)‐3‐phenylurea
derivatives (1–14)
1‐[5‐(Benzylthio)‐1,3,4‐thiadiazol‐2‐yl]‐3‐(2‐bromophenyl)urea (5)
Yield: 78%; HPLC (purity: 95%); mp 197–199°C; IR (KBr, cm−1): 3,343
(N–H), 3,215 (N–H), 1,708 (C═O), 1,587, 1,531, and 1,320. 1H NMR
(DMSO‐d6, 500 MHz) δ: 4.45 (s, 2H), 7.06 (t, 1H, J = 7.7 Hz), 7.26 (t,
1H, J = 6.6 Hz), 7.32 (t, 2H, J = 7.9 Hz), 7.41–7.34 (m, 3H), 7.65 (d, 1H,
J = 8.0 Hz), 7.99 (d, 1H, J = 8.2 Hz), 8.56 (s, 1H), and 11.70 (s, 1H). 13C
NMR (DMSO‐d6, 125 MHz): 37.1, 113.3, 122.1, 124.9, 126.9, 127.7,
127.9 (2C), 128.4 (2C), 132.1, 135.2, 136.2, 150.5, 156.6, and 159.7.
Anal. calcd. for C16H13BrN4OS2: C, 45.61; H, 3.11; N, 13.30. Found:
C, 45.32; H, 3.28; N, 13.57; MS (ESI) m/z 422.3 [M+H].
To
a mixture of 2‐amino‐5‐(benzylthio)‐1,3,4‐thiadiazol (c1–5)
(0.45 mmol) in 1 ml of dry CH2Cl2, phenyl isocyanate derivatives
(0.3 mmol) dissolved in 1 ml dry CH2Cl2 were added drop‐by‐drop.
The reaction mixture was stirred overnight. After the consumption of
reagents, the obtained solid was filtered and washed with CH2Cl2.
1‐[5‐(Benzylthio)‐1,3,4‐thiadiazol‐2‐yl]‐3‐phenylurea (1)
Yield: 85%; HPLC (purity: 99%); mp 209–211°C; IR (KBr, cm−1): 3,365
(N–H), 3,227 (N–H), 1,721 (C═O), 1,599, 1,547, and 1,314. 1H NMR
(DMSO‐d6, 500 MHz) δ: 4.44 (s, 2H), 7.04 (t, 1H, J = 7.5 Hz),
7.25–7.34 (m, 5H), 7.39 (d, 2H, J = 7.5 Hz), 7.45 (d, 2H, J = 8.0 Hz),
9.02 (s, 1H), and 10.97 (s, 1H). 13C NMR (DMSO‐d6, 125 MHz): 37.2,
118.3 (2C), 122.5, 126.9, 127.9 (2C), 128.3 (2C), 128.4 (2C), 136.2,
1‐[5‐(Benzylthio)‐1,3,4‐thiadiazol‐2‐yl]‐3‐(3,4‐dichlorophenyl)
urea (6)
Yield: 75%; HPLC (purity: 99%); mp 255–257°C; IR (KBr, cm−1): 3,376
(N–H), 3,205 (N–H), 1,732 (C═O), 1,591, 1,543, and 1,303. 1H NMR

7 of 9
TOOLABI ET AL.
|
(DMSO‐d6, 500 MHz) δ: 4.44 (s, 2H), 7.26 (t, 1H, J = 7.3 Hz), 7.32 (t,
2H, J = 8.0 Hz), 7.42–7.38 (m, 3H), 7.54 (d, 1H, J = 8.7 Hz), 7.86 (s, 1H),
9.40 (s, 1H), and 11.29 (s, 1H). 13C NMR (DMSO‐d6, 125 MHz): 37.0,
118.3, 119.4, 123.8, 126.9, 127.9 (2C), 128.3 (2C), 130.0, 130.5,
136.1, 138.1, 152.0, 156.0, and 160.6. Anal. calcd. for
C16H12Cl2N4OS2: C, 46.72; H, 2.94; N, 13.62. Found: C, 46.58; H,
2.69; N, 13.95; MS (ESI) m/z 412.2 [M+H].
127.2, 127.3 (2C), 128.4, 132.9, 137.0, 137.5, 153.0, 154.5, and
166.2. Anal. calcd. for C16H12Cl2N4OS2: C, 46.72; H, 2.94; N, 13.62.
Found: C, 46.94; H, 2.62; N, 13.81; MS (ESI) m/z 412.3 [M+H].
1‐{5‐[(2,4‐Dichlorobenzyl)thio]‐1,3,4‐thiadiazol‐2‐yl}‐3‐(4‐
fluorophenyl)urea (11)
Yield: 79%; HPLC (purity: 100%); mp 228–230°C; IR (KBr, cm−1):
3,378 (N–H), 3,219 (N–H), 1,702 (C═O), 1,595, 1,553, and 1,323. 1H
NMR (DMSO‐d6, 500 MHz) δ: 4.48 (s, 2H), 7.15 (t, 2H, J = 8.7 Hz),
7.40 (dd, 1H, J = 8.2, 2.2 Hz), 7.50–7.42 (m, 3H), 7.66 (d, 1H,
J = 2.3 Hz), 9.06 (s, 1H), and 11.08 (s, 1H). 13C NMR (DMSO‐d6,
125 MHz): 34.7, 114.8 (d, 2C, J = 22.2 Hz), 120.3 (d, 2C, J = 7.8 Hz),
126.9 (2C), 128.4 (2C), 132.0, 132.6, 133.0, 133.7, 134.0 (d,
J = 3.3 Hz), 155.0, and 157.5 (d, J = 236.7 Hz). Anal. calcd. for
C16H11Cl2FN4OS2: C, 44.76; H, 2.58; N, 13.05. Found: C, 44.58; H,
2.81; N, 13.21; MS (ESI) m/z 430.4 [M+H].
1‐{5‐[(3‐Methoxybenzyl)thio]‐1,3,4‐thiadiazol‐2‐yl}‐3‐phenylurea (7)
Yield: 80%; HPLC (purity: 99%); mp 195–197°C; IR (KBr, cm−1):
3,365 (N–H), 3,212 (N–H), 1,720 (C═O), 1,599, 1,544, and 1,315. 1H
NMR (DMSO‐d6, 500 MHz) δ: 3.72 (s, 3H), 4.41 (s, 2H), 6.87–6.80
(m, 1H), 6.99–6.93 (m, 2H), 7.04 (t, 1H, J = 7.4 Hz), 7.24 (t, 1H,
J = 8.1 Hz), 7.31 (t, 2H, J = 7.4 Hz), 7.46 (d, 2H, J = 8.0 Hz), 9.14 (s,
1H), and 11.03 (s, 1H). 13C NMR (DMSO‐d6, 125 MHz): 37.2, 54.4,
112.4, 114.0, 118.3 (2C), 120.5, 122.5, 128.3 (2C), 128.9, 129.0,
137.7, 151.2, 156.1, 158.7, and 160.1. Anal. calcd. for
C17H16N4O2S2: C, 54.82; H, 4.33; N, 15.04. Found: C, 54.59; H, 4.52;
N, 15.27; MS (ESI) m/z 373.2 [M+H].
1‐{5‐[(3‐Nitrobenzyl)thio]‐1,3,4‐thiadiazol‐2‐yl}‐3‐phenylurea (12)
Yield: 80%; HPLC (purity: 95%); mp 201–203°C; IR (KBr, cm−1): 3,347
(N–H), 3,221 (N–H), 1,719 (C═O), 1,598, 1,544, and 1,315. 1H NMR
(DMSO‐d6, 500 MHz) δ: 4.47 (s, 2H), 6.99 (t, 1H, J = 7.0 Hz), 7.27 (t,
2H, J = 6.9 Hz), 7.44 (d, 2H, J = 6.8 Hz), 7.48 (t, 1H, J = 7.7 Hz), 7.63 (d,
1H, J = 7.7 Hz), 8.13 (d, 1H, J = 7.7 Hz), 8.30 (s, 1H), 8.89 (s, 1H), and
10.92 (s, 1H). 13C NMR (DMSO‐d6, 125 MHz): 37.4, 117.0 (2C),
120.3, 120.7, 121.4, 126.6 (2C), 127.2, 132.2, 136.1, 136.7, 146.0,
152.0, 153.5, and 165.2. Anal. calcd. for C16H13N5O3S2: C, 49.60; H,
3.38; N, 18.08. Found: C, 49.87; H, 3.12; N, 18.27; MS (ESI) m/z 388.1
[M+H].
1‐{5‐[(3‐Methoxybenzyl)thio]‐1,3,4‐thiadiazol‐2‐yl}‐3‐(p‐tolyl)
urea (8)
Yield: 78%; HPLC (purity: 98%); mp 210–212°C; IR (KBr, cm−1):
3,381 (N–H), 3,224 (N–H), 1,722 (C═O), 1,587, 1,534, and 1,314. 1H
NMR (DMSO‐d6, 500 MHz) δ: 2.24 (s, 3H), 3.72 (s, 3H), 4.40 (s, 2H),
6.84–6.82 (m, 1H), 6.96–6.94 (m, 2H), 7.11 (d, 2H, J = 8.0 Hz), 7.24
(t, 1H, J = 7.8 Hz), 7.33 (d, 2H, J = 8.0 Hz), 9.06 (s, 1H), and 11.00 (s,
1H). 13C NMR (DMSO‐d6, 125 MHz): 19.8, 37.2, 54.4, 112.5, 114.0,
115.4, 118.3, 120.5, 125.4, 128.7 (2C), 129.0, 131.5, 135.1, 137.7,
156.0, 158.7, and 160.2. Anal. calcd. for C18H18N4O2S2: C, 55.94; H,
4.69; N, 14.50. Found: C, 55.75; H, 4.84; N, 14.73; MS (ESI) m/z
387.5 [M+H].
1‐{5‐[(3‐Nitrobenzyl)thio]‐1,3,4‐thiadiazol‐2‐yl}‐3‐(p‐tolyl)urea (13)
Yield: 75%; HPLC (purity: 96%); mp 223–225°C; IR (KBr, cm−1):
3,381 (N–H), 3,225 (N–H), 1,722 (C═O), 1,587, 1,534, and 1,314. 1
H
NMR (DMSO‐d6, 500 MHz) δ: 2.24 (s, 3H), 4.59 (s, 2H), 7.11 (d, 2H,
J = 8.1 Hz), 7.33 (d, 2H, J = 8.1 Hz), 7.62 (t, 1H, J = 8.0 Hz), 7.84 (d,
1H, J = 7.5 Hz), 8.11 (d, 1H, J = 8.3 Hz), 8.28 (s, 1H), 8.96 (s, 1H), and
10.97 (s, 1H). 13C NMR (DMSO‐d6, 125 MHz): 19.7, 36.0, 118.4 (2C),
121.7, 123.0, 128.7 (2C), 129.3, 131.5, 135.0, 135.0, 139.2, 147.1,
151.0, 155.2, and 160.4. Anal. calcd. for C17H15N5O3S2: C, 50.86; H,
3.77; N, 17.45. Found: C, 50.52; H, 3.97; N, 17.61; MS (ESI) m/z
402.4 [M+H].
1‐(2‐Bromophenyl)‐3‐{5‐[(3‐methoxybenzyl)thio]‐1,3,4‐thiadiazol‐2‐
yl}urea (9)
Yield: 75%; HPLC (purity: 96%); mp 186–188°C; IR (KBr, cm−1): 3,352
(N–H), 3,204 (N–H), 1,705 (C═O), 1,581, 1,531, and 1,317. 1H NMR
(DMSO‐d6, 500 MHz) δ: 3.71 (s, 3H), 4.41 (s, 2H), 6.83 (d, 1H,
J = 7.1 Hz), 6.97–6.94 (m, 2H), 7.06 (t, 1H, J = 7.4 Hz), 7.24 (t, 1H,
J = 7.8 Hz), 7.36 (t, 1H, J = 7.4 Hz), 7.64 (d, 1H, J = 8.0 Hz), 7.97 (d, 1H,
J = 8.2 Hz), 8.61 (s, 1H), and 11.75 (s, 1H). 13C NMR (DMSO‐d6,
125 MHz): 37.2, 54.4, 112.5, 113.5, 114.0, 120.5, 122.3, 124.9, 127.6,
129.0, 132.1, 135.2, 137.7, 150.7, 156.5, 158.7, and 159.8. Anal.
calcd. for C17H15BrN4O2S2: C, 45.24; H, 3.35; N, 12.41. Found: C,
45.51; H, 3.12; N, 12.73; MS (ESI) m/z 452.5 [M+H].
1‐(2‐Bromophenyl)‐3‐{5‐[(3‐nitrobenzyl)thio]‐1,3,4‐thiadiazol‐2‐yl}
urea (14)
Yield: 77%; HPLC (purity: 99%); mp 205–207°C; IR (KBr, cm−1):
3,397 (N–H), 3,229 (N–H), 1,755 (C═O), 1,593, 1,546, and 1,325. 1H
NMR (DMSO‐d6, 500 MHz) δ: 4.46 (s, 2H), 7.15 (t, 1H, J = 7.6 Hz),
7.42 (t, 1H, J = 7.6 Hz), 7.49–7.46 (m, 2H), 7.57 (d, 1H, J = 7.8 Hz),
7.62 (d, 1H, J = 7.7 Hz), 8.11 (d, 1H, J = 7.7 Hz), 8.30 (s, 1H), 8.81 (s,
1H), and 11.11 (s, 1H). 13C NMR (DMSO‐d6, 125 MHz): 35.9, 113.6,
121.8, 122.9, 123.9, 124.7, 127.4, 129.2, 131.9, 135.0, 136.2, 138.9,
146.94, 150.0, 155.9, and 159.9. Anal. calcd. for C16H12BrN5O3S2:
C, 41.21; H, 2.59; N, 15.02. Found: C, 41.54; H, 2.31; N, 15.27; MS
(ESI) m/z 466.3 [M+H].
1‐{5‐[(3‐Chlorobenzyl)thio]‐1,3,4‐thiadiazol‐2‐yl}‐3‐(4‐chlorophenyl)
urea (10)
Yield: 77%; HPLC (purity: 99%); mp 267–269°C; IR (KBr, cm−1): 3,375
(N–H), 3,202 (N–H), 1,715 (C═O), 1,582, 1,547, and 1,317. 1H NMR
(DMSO‐d6, 500 MHz) δ: 4.42 (s, 2H), 7.20 (d, 1H, J = 7.5 Hz),
7.35–7.22 (m, 4H), 7.54–7.46 (m, 3H), 8.77 (s, 1H), and 11.36 (s, 1H).
13C NMR (DMSO‐d6, 125 MHz): 38.0, 118.7 (2C), 126.5, 126.5, 126.8,

8 of 9
TOOLABI ET AL.
|
|
4.2
Pharmacological assays
1 min was recorded. The toxic dose values (TD50) were measured for
each novel compound.
|
4.2.1
Anticonvulsant activity
|
Radioligand receptor binding assay
Animals and drugs
4.2.2
Adult male NMRI mice (Pasteur Institute, Iran), weighing 18–25 g, were
kept in colony cages. Mice were housed under standardized conditions in
a temperature‐controlled animal facility (22 2°C) and 12‐hr light/dark
cycle. They were allowed to acclimatize to the animal facility for 1 week
before the experiments. All experimental animals had free access to food
and water except during the experiment. After 7 days of adaptation to
laboratory conditions, animals were divided into experimental groups
randomly (each comprised of 8 mice). For the rest of the experiments, we
used each mouse just one time for the experiments. Diazepam (Darou
Pakhsh Co.), the synthesized compounds, and flumazenil (Hoffmann‐La
Roche) were suspended in a solution of carboxymethylcellulose (1%) and
Tween 80 (0.5%) in distilled water. The compounds and diazepam were
injected 30 min before the induction of convulsion and flumazenil was
injected 15 min before the compounds and diazepam. This study was run
on the basis of protocols approved by the institutional animal care
committee, and the experimenters did their best to minimize the number
of animals used in the study. First, the in vitro test was run; ethical
approval had been granted after obtaining positive results in the in vitro
assays.
The compounds were evaluated for their in vitro affinity to BZD re-
ceptor by the radioligand receptor binding assay. All of the binding
assays were performed in triplicates. Male Sprague Dawley rats with
weights of 200–250 g were anesthetized with isoflurane and then were
killed. The cortical membrane tissue of the brain of the rats was col-
lected and homogenized in 20 ml of ice‐cold Tris‐HCl buffer. The
homogenate was centrifuged and the resulting supernatant was ultra‐
centrifuged (35,000g for 25 min), followed by three centrifugations and
resuspension cycles. The final pellet was resuspended in 30ml Tris‐HCl
buffer (50 mM, pH 7.4). The amount of protein was estimated in the
membrane preparation by the Bradford method using bovine serum
albumin as a standard. There are two fundamental types of receptor
binding experiments: saturation and competition. For the saturation
experiment, eight different concentrations of [3H]flumazenil were in-
cubated with the membrane preparation at 30°C for 35 min. The total
binding (TB; receptor + radioligand), nonspecific binding (NSB;
receptor + radioligand + excess diazepam), and specific binding (SB;
TB‐NSB) were measured at various radioligand concentrations. NSB
was determined in parallel assays performed in the presence of 100 µM
diazepam. The amount of radioligand required to saturate the receptors
was used to determine the receptor binding affinity of [3H]flumazenil
(Kd), and the BZD receptor density (Bmax) was measured in the sa-
turation study based on the nonlinear regression analysis (Kd = 1.41
0.4126 nM, Bmax = 0.728 0.109 pmol/mg). In the competition experi-
ment, the affinity (Ki) of the synthesized ligands and diazepam (Ki) was
determined in comparison with [3H]flumazenil as a standard antagonist
of BZD receptors. The rat brain protein (100 μg) in Tris‐HCl buffer
(50 mM, pH 7.4) was incubated with 8.6 × 10–5 nM of [3H]flumazenil and
increasing concentrations of the synthesized ligands (5 mM–50 pM) for
35 min at 30°C. At the end of the incubation, the reaction mixture was
centrifuged and the radioactivity of pellet was counted, adding Ultima
Gold cocktail (Perkin Elmer), using liquid scintillation counting (Triathler
Multilabel Tester; Hidex, Finland). The concentration of ligands that
inhibits the binding of [3H]flumazenil by 50% is the IC50 value. The data
were analyzed using GraphPad Prism software. To determine IC50 va-
lues, the data were fitted to the equation by using nonlinear regression
analysis, competitive binding. NSB was determined in parallel assays
performed in the presence of 100 µM diazepam.[38,39]
Maximal electroshock‐induced seizure test
The ability of the targeted compounds 1–14 to prevent MES‐induced
seizures was evaluated in mice. The compounds were injected (ip) at
doses of 2.5, 5, 10, and 20 mg/kg. The occurrence of HLTE in mice
was assessed by applying maximal electroshock with a 60‐Hz alter-
nating current of 50‐mA intensity delivered for 0.2 s via ear elec-
trodes. The MES was applied 30 min after (ip) administration of the
compounds and diazepam. Mice were observed for 60 s for the in-
cidence of HLTE. The results were recorded as the number of animals
protected/number of animals tested.[36]
Pentobarbital‐induced sleep test
Hypnotic effects of the compounds were assessed by the
pentobarbital‐induced sleep test. All the compounds were injected ip.
After the pretreatment with different doses of compounds, animals
were given sodium pentobarbital at a dose of 40 mg/kg. After pen-
tobarbital injection, each mouse was observed for the onset of sleep
(loss of righting reflex). The time between loss (sleep onset) and re-
covery of the righting reflex was measured. Animals were observed
for 120 min after the injection.[37]
|
Statistical analysis
Rotarod test
4.2.3
The rotarod test was used to investigate the motor coordination of the
subjects. The various doses of compounds 7 and 11 were injected ip; after
30 min, the animals were placed on a rotating straight rod at a speed of
6 rpm for 1 min. In this experiment, if the mouse fell three times during
the 1‐min trial, it would be considered as an animal with neurotoxicity.
The number of animals that were not able to maintain on the rod during
In this study, results were described as significant when p < .05. In MES,
pentobarbital‐induced sleep, and rotarod tests, the ED50 and TD50 values
were measured by the linear regression analysis of the log dose–response
curve and presented as mean with 95% confidence intervals. To identify
the IC50 values of in vitro studies, we used the nonlinear regression

9 of 9
TOOLABI ET AL.
|
analysis of competitive binding results and we reported the results as
mean with 95% confidence intervals. All statistical analyses were carried
out by the GraphPad Prism software (San Diego, CA; version 5.0).
[20] Y. Hu, C.‐Y. Li, X.‐M. Wang, Y.‐H. Yang, H.‐L. Zhu, Chem. Rev. 2014,
114, 5572. https://doi.org/10.1021/cr400131u
[21] P. Kaur, Int. J. Univers. Pharm. Bio. Sci. 2018, 246.
[22] G. Serban, O. Stanasel, E. Serban, S. Bota, Drug Des. Dev. Ther. 2018,
12, 1545. https://doi.org/10.2147/DDDT.S155958
ACKNOWLEDGMENT
[23] A. Almasirad, N. Vousooghi, S. A. Tabatabai, A. Kebriaeezadeh, A.
Shafiee, Acta Chim. Slov. 2007, 54, 317.
This work was supprted by a grant from Neuroscience Research
Center, Institute of Neuropharmacology, Kerman University of
Medical Sciences.
[24] S. Rybka, J. Obniska, A. Rapacz, B. Filipek, P. Żmudzki, J. Enzyme Inhib.
Med. Chem. 2016, 31, 1038. https://doi.org/10.3109/14756366.
2015.1088842
[25] A. Foroumadi, V. Sheibani, A. Sakhteman, M. Rameshk, M. Abbasi, R.
Farazifard, S. A. Tabatabai, A. Shafiee, Daru 2007, 15, 89.
[26] N. Ahangar, A. Ayati, E. Alipour, A. Pashapour, A. Foroumadi, S.
Emami, Chem. Biol. Drug Des. 2011, 78, 844. https://doi.org/10.1111/
j.1747-0285.2011.01211.x
CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interest.
ORCID
[27] A. Zarghi, S. A. Tabatabai, M. Faizi, A. Ahadian, P. Navabi, V.
Zanganeh, A. Shafiee, Bioorg. Med. Chem. Lett. 2005, 15, 1863. https://
doi.org/10.1016/j.bmcl.2005.02.014
Mehrdad Faizi
http://orcid.org/0000-0002-6896-838X
http://orcid.org/0000-0003-2416-5611
Alireza Foroumadi
[28] M. Mohammadi‐khanaposhtani, M. Shabani, M. Faizi, I. Aghaei, R.
Jahani, Z. Sharafi, N. Shamsaei Zafarghandi, M. Mahdavi, T. Akbar-
zadeh, S. Emami, A. Shafiee, A. Foroumadi, Eur. J. Med. Chem. 2016,
13, 91. https://doi.org/10.1016/j.ejmech.2016.01.054
REFERENCES
[1] A. K. Ngugi, C. Bottomley, I. Kleinschmidt, J. W. Sander, C. R. Newton,
Epilepsia 2010, 51, 883. https://doi.org/10.1111/j.1528-1167.2009.
02481.x
[29] E. Rezaee Zavareh, M. Hedayati, L. Hoghooghi Rad, S. Shahhosseini,
M. Faizi, S. A. Tabatabai, Iran. J. Pharm. Res. 2014, 13, 51.
[30] S. N. Mandhane, K. Aavula, T. Rajamannar, Seizure 2007, 16, 636.
https://doi.org/10.1016/j.seizure.2007.05.005
[2] E. Beghi, G. Giussani, Neuroepidemiology 2018, 51, 216. https://doi.
org/10.1159/000493484
[31] T. Akbarzadeh, S. A. Tabatabai, M. J. Khoshnoud, B. Shafaghi, A.
Shafiee, Bioorg. Med. Chem. 2003, 11, 769.
[3] T. W. Strine, R. Kobau, D. P. Chapman, D. J. Thurman, P. Price, L. S.
Balluz, Epilepsia 2005, 46, 1133. https://doi.org/10.1111/j.1528-
1167.2005.01605.x
[32] F. Ahmadi, M. Faizi, S. A. Tabatabai, D. Beiki, S. Shahhosseini, Nucl. Med.
Biol. 2013, 40, 896. https://doi.org/10.1016/j.nucmedbio.2013.06.010
[33] F. Ahmadi, S. Dabirian, M. Faizi, S. A. Tabatabai, D. Beiki, S.
Shahhosseini, Iran. J. Pharm. Res. 2014, 13, 79.
[4] E. M. Goldberg, D. Coulter, Nat. Rev. Neurosci. 2014, 14, 337. https://
doi.org/10.1038/nrn3482
[5] M.‐X. Song, X.‐Q. Deng, J. Enzyme Inhib. Med. Chem. 2018, 33, 453.
https://doi.org/10.1080/14756366.2017.1423068
[6] L. M. Rambo, L. R. Ribeiro, M. S. Oliveira, A. F. Furian, F. D. Lima, M. A.
Souza, L. F. Silva, L. T. Retamoso, C. L. Corte, G. O. Puntel, D. S. de
Avila, F. A. Soares, M. R. Fighera, C. F. Mello, L. F. Royes, Neurochem.
Int. 2009, 55, 333. https://doi.org/10.1016/j.neuint.2009.04.007
[7] M. N. Aboul‐Enein, A. A. El‐Azzouny, O. A. Saleh, Y. A. Maklad, Mini‐
Rev. Med. Chem. 2012, 12, 671. https://doi.org/10.2174/
138955712800626665
[34] G. M. Morris, R. Huey, W. Lindstrom, M. Sanner, R. K. Belew, D. S.
Goodsell, A. J. Olson, J. Comput. Chem. 2009, 16, 2785e2791. https://
doi.org/10.1002/jcc.21256
[35] M. Toolabi, S. Moghimi, T. O. Bakhshaiesh, S. Salarinejad, A. Aghcheli,
Z. Hasanvand, E. Nazeri, A. Khalaj, R. Esmaeili, A. Foroumadi, Eur.
J. Med. Chem. 2020, 185, 111786. https://doi.org/10.1016/j.ejmech.
2019.111786
[36] M. Faizi, M. Sheikhha, N. Ahangar, H. Tabatabaei Ghomi, B. Shafaghi,
A. Shafiee, S. A. Tabatabai, Iran. J. Pharm. Res. 2012, 11, 83.
[37] R. Shi, Y. Han, Y. Yan, H. Qiao, J. He, W. Lian, Front. Pharmacol. 2019,
10, 1. https://doi.org/10.3389/fphar.2019.00409
[8] A. Schmitz, Ment. Health Clin. 2016, 6, 120. https://doi.org/10.9740/
mhc.2016.05.120
[9] J. Guina, B. Merrill, J. Clin. Med. 2018, 7, 17. https://doi.org/10.3390/
jcm7020017
[38] P. Kahnberg, E. Lager, C. Rosenberg, J. Schougaard, L. Camet, O.
Sterner, E. Nielsen, M. Nielsen, T. Liljefors, J. Med. Chem. 2002, 45,
4188. https://doi.org/10.3791/52434
[10] U. Rudolph, F. Knoflach, Nat. Rev. Drug Discov. 2012, 10, 685. https://
doi.org/10.1038/nrd3502
[39] M. McKinney, R. Raddatz, Curr. Protoc. Pharmacol. 2006, 33, 1.3.1.
https://doi.org/10.1002/0471141755.ph0103s33
[11] A. Ayati, S. Emami, A. Foroumadi, Eur. J. Med. Chem. 2016, 109, 380.
https://doi.org/10.1016/j.ejmech.2016.01.009
[12] R. Kharb, P. C. Sharma, M. S. Yar, J. Enzyme Inhib. Med. Chem. 2011,
26, 1. https://doi.org/10.3109/14756360903524304
[13] S. A. Mazumder, M. S. Yar, R. Mazumder, G. S. Chakraborthy, M. J.
Ahsan, M. U. Rahman, Synth. Commun. 2017, 47, 1805. https://doi.
org/10.1080/00397911.2017.1360911
SUPPORTING INFORMATION
Additional supporting information may be found online in the
Supporting Information section.
[14] N. K. Downer‐Riley, Y. A. Jackson, Curr. Top. Med. Chem. 2016, 16,
3617. https://doi.org/10.2174/1568026616666160414122349
[15] K. M. Dawood, T. A. Farghaly, Expert Opin. Ther. Pat. 2017, 27, 477.
https://doi.org/10.1080/13543776.2017.1272575
[16] N. Kushwaha, S. K. S. Kushwaha, A. K. Rai, Int. J. Chemtech. Res. 2012,
4, 517.
How to cite this article: Toolabi M, Khoramjouy M,
Aghcheli A, et al. Synthesis and radioligand‐binding assay
of 2,5‐disubstituted thiadiazoles and evaluation of their
anticonvulsant activities. Arch Pharm. 2020;e2000066.
https://doi.org/10.1002/ardp.202000066
[17] N. Siddiquia, P. Ahujaa, W. Ahsana, S. N. Pandeyab, M. S. Alam,
J. Chem. Pharm. Res. 2009, 1, 19.
[18] A. S. Shawali, J. Adv. Res. 2014, 5, 1. https://doi.org/10.1016/j.jare.
2013.01.004
[19] S. Haider, M. S. Alam, H. Hamid, Eur. J. Med. Chem. 2015, 6, 156.
https://doi.org/10.1016/j.ejmech.2014.12.035