Design, synthesis, and pharmacological evaluation of novel 1,2,4-triazol-3-amine derivatives as potential agonists of GABAA subtype receptors with anticonvulsant and hypnotic effects

Article abstract of DOI:10.1016/j.bioorg.2020.104212

In the current study, a series of novel 1,2,4-triazol-3-amine derivatives were designed, synthesized, and biologically evaluated in vivo for their anticonvulsant and hypnotic effects in the pentylenetetrazole (PTZ)-induced seizures, maximal electroshock (MES)-induced seizures, and pentobarbital-induced sleeping tests. Furthermore, the possible side effects of the most potent compounds on the memory, motor coordination, and muscle strength were evaluated in passive avoidance, rotarod, and grip strength tests, respectively. The designed compounds with the main benzodiazepine pharmacophores including aromatic ring and proton accepting group completely mimiced the structure of zolpidem as an α1-selective agonist of GABA_A receptor. Compounds 5c (ED₅₀ ≈ 52.5 mg/kg) and 5 g (ED₅₀ ≈ 16.5 mg/kg) in the PTZ test were the most potent compounds among the designed compounds. In the MES test, the observed ED_50s for compounds 5c and 5 g were reduced to around 11.8 mg/kg and 10.5 mg/kg, respectively. The considerable hypnotic effect in a dose-dependent manner was observed following the administration of newly synthesized compounds. In all experiments administration of flumazenil as an antagonist of benzodiazepines receptor fully antagonized observed effects which indicated the involvement of GABA_A receptors. Since there was no negative effect on memory, motor coordination, and muscle strength following the administration of compounds 5c and 5g as the most potent compounds, it could be concluded that the novel compounds most likely act through α1-containing GABA_A receptors and possess no affinity for α5-containing receptors. The newly designed compounds could be considered as leading compounds in synthesizing novel GABA_A receptor agonists with minimum side effects.

Full text of DOI:10.1016/j.bioorg.2020.104212

BioorganicChemistry104(2020)104212
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, and pharmacological evaluation of novel 1,2,4-triazol-3-
amine derivatives as potential agonists of GABAA subtype receptors with
anticonvulsant and hypnotic effects
Reza Jahania, Seyed Reza Abtahia, Manijeh Nematpourb, Hossein Fasihi Dastjerdic,d
,
⁎
Mohsen Chamanaraa, Zahra Hamia, Babak Paknejada,
a Department of Pharmacology and Toxicology, Faculty of Medicine, AJA University of Medical Sciences, Tehran, Iran
b Department of Chemistry, Tarbiat Modares University, Tehran, Iran
c Department of Pharmaceutical Chemistry, School of Pharmacy, Shahid Beheshti University of Medical Sciences, Tehran, Iran
d Biomaterial and Medicinal Chemistry Research Center, Aja University of Medical Science, Tehran, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords:
In the current study, a series of novel 1,2,4-triazol-3-amine derivatives were designed, synthesized, and biolo-
gically evaluated in vivo for their anticonvulsant and hypnotic effects in the pentylenetetrazole (PTZ)-induced
seizures, maximal electroshock (MES)-induced seizures, and pentobarbital-induced sleeping tests. Furthermore,
the possible side effects of the most potent compounds on the memory, motor coordination, and muscle strength
were evaluated in passive avoidance, rotarod, and grip strength tests, respectively. The designed compounds
with the main benzodiazepine pharmacophores including aromatic ring and proton accepting group completely
1,2,4-triazol-3-amine
Anticonvulsant
Benzodiazepine
Sedative-hypnotic
Synthesis
Toxicity
mimiced the structure of zolpidem as an α1-selective agonist of GABAA receptor. Compounds 5c (ED50
≈
52.5 mg/kg) and 5 g (ED50 ≈ 16.5 mg/kg) in the PTZ test were the most potent compounds among the designed
compounds. In the MES test, the observed ED50s for compounds 5c and 5 g were reduced to around 11.8 mg/kg
and 10.5 mg/kg, respectively. The considerable hypnotic effect in a dose-dependent manner was observed
following the administration of newly synthesized compounds. In all experiments administration of flumazenil
as an antagonist of benzodiazepines receptor fully antagonized observed effects which indicated the involvement
of GABAA receptors. Since there was no negative effect on memory, motor coordination, and muscle strength
following the administration of compounds 5c and 5g as the most potent compounds, it could be concluded that
the novel compounds most likely act through α1-containing GABAA receptors and possess no affinity for α5-
containing receptors. The newly designed compounds could be considered as leading compounds in synthesizing
novel GABAA receptor agonists with minimum side effects.
1. Introduction
that are used to achieve or maintain seizure control are frequently as-
sociated with unwanted side effects such as memory impairment, loss of
Epilepsy is characterized by a lasting predisposition to generate
spontaneous epileptic seizures which affects over 65 million individuals
worldwide [1] and has numerous neurobiological, cognitive, and psy-
chosocial consequences [2]. The presence of comorbidities such as
depression, anxiety disorder, psychosis, autism, and insomnia is the
norm in more than 50% of patients who suffer from seizure [3]. Anti-
seizure medicines with the aim of stopping seizures at the earliest op-
portunity and reducing morbidity are the main treatment modality in
epilepsy [4,5]. Despite a large number of available drugs in different
categories for the treatment of epilepsy, only 66% of individuals are
treated with the current drugs [6]. Unfortunately, antiepileptic drugs
coordination, muscle relaxation, fatigue, dizziness, loss of libido, and
drug dependency [7]. Therefore, compliance with medication is an
important issue in anticonvulsant therapy [8].
Benzodiazepines (BZDs) as a class of agents that work in the central
nervous system are widely used in various conditions such as epilepsy,
anxiety, and sleep disorders [9]. They act by binding to a specific
binding site known as the BZD receptor on the gamma-aminobutyric
acid-A (GABAA) receptors, opening of a selective chloride ion channel,
and hyperpolarization of the postsynaptic membrane [10]. Since like
other antiepileptic agents, BZDs have their own unwanted side effects,
the design and synthesis of novel compounds with BZDs structure are a
⁎ Corresponding author.
E-mail address: Dr.babakpaknejad@gmail.com (B. Paknejad).
https://doi.org/10.1016/j.bioorg.2020.104212
Received 16 May 2020; Accepted 24 August 2020
Availableonline01September2020
0045-2068/©2020ElsevierInc.Allrightsreserved.

R. Jahani, et al.
BioorganicChemistry104(2020)104212
Fig. 1. The structure of zolpidem and novel synthesized 1,2,4-triazol-3-amine derivatives. The aromatic ring (A) and coplanar proton accepting group (B) are
highlighted.
great deal of attention [11,12]. Based on the structure–activity re-
lationship of BZDs, for any high-affinity benzodiazepine ligand, some
pharmacophore groups such as an aromatic ring that is located in a
suitable distance (5 Angstrom) from a coplanar proton accepting
functional group in the same plane is required. The presence of a second
out-of-plane aromatic ring potentiates binding to the GABAA receptors
[13].
elemental analyzer (Costech, Italy) were used, respectively. 1H and 13C
NMR spectra were recorded in CDCl3 by a Bruker FT-500 MHz instru-
ment (Bruker Biosciences, USA). MS was recorded using an Agilent
Technology (HP) mass instrument operating at 70 eV.
2.3. General procedure for the synthesis of novel 1,2,4-triazol-3-amine
derivatives
In this research, based on the previous studies conducted on the
triazole nucleus [14] we designed novel 1,2,4-triazol-3-amine deriva-
tives according to the structure–activity relationship (SAR) of benzo-
diazepines with all suggested requirements for binding to GABAA re-
ceptors [15,16] (Fig. 1). To confirm whether the designed compounds
could mimic the structure of a BZD agonist, conformational analysis
and superimposition of energy minima conformers of them on zolpidem
as a known BZD agonist were performed.
A
mixture of aniline derivatives (2.0 mmol) and tri-
chloroacetonitrile (3.0 mmol) was stirred in dimethylformamide sol-
vent (3.0 mL) for 20 min at room temperature (RT) to afford tri-
chloroacetamidine analogs. Then the obtained derivatives were reacted
with different hydrazonoyl chlorides analogs (2.0 mmol) in di-
methylformamide solvent (2.0 mL) in the presence of trimethylamine
(1.0 mmol) and CuI (0.10 mmol) as a catalyst for 2 h at room tem-
perature to obtain target compounds. After the end of the reaction, the
mixture washed with water (4 mL), ammonium chloride solution
(4 mL), and dichloromethane (4 mL). Finally, the non-aqueous layer
was separated and evaporated under reduced pressure and the pre-
cipitate washed with n-hexane solvent. Further purification was ob-
tained by recrystallization from absolute ethanol.
The potential anticonvulsant activity of the synthesized compounds
was evaluated in the pentylenetetrazole (PTZ)-induced lethal convul-
sion and maximal electroshock (MES) tests. Pentobarbital-induced sleep
test was employed to evaluate the hypnotic effect of novel compounds.
Step-through passive avoidance, grip strength, and rotarod tests were
carried out in order to assess whether the novel compounds induce
anterograde memory impairment, muscle relaxation, and impairment
in motor coordination, respectively. Finally, the effect of flumazenil as a
BZD receptor antagonist was determined to confirm the possible me-
chanism of action of the synthesized compounds.
2.3.1. N-benzyl-2-phenyl-5-p-totyl-2H-1,2,4-triazol-3-amine (5a)
White powder; Yield: 0.91 g (65%); Mp: 189 °C; FT-IR spectrum, ν,
cm−1: 3496, 2996, 1755, 1483; 500 MHz 1H NMR (CDCl3): δ ppm:
2.41(s, 3H, CH3), 4.65(s, 2H, CH2), 6.69(s, 1H, NH), 6.92(t, J = 7.3 Hz,
1H, Ar), 7.15(d, J = 7.6 Hz, 2H, Ar), 7.21(d, J = 8.0 Hz, 2H, Ar),
7.32(t, J = 7.3 Hz, 2H, Ar), 7.38–7.41(m, 3H, Ar), 7.56–7.60(m, 4H,
Ar). 125 MHz 13C NMR (CDCl3) δC:18.88, 45.58, 124.64, 126.68,
128.84, 129.31, 129.84, 132.64, 133.17, 138.12, 138.88, 142.20,
143.86, 145.40, 160.53, 164.21. MS: m/z (%) = 340(M+, 20), 263(90),
234(40), 143(30), 106(100), 91(65). Anal calcd for C22H20N4: C,
77.62; H, 5.92; N, 16.46; Found: C, 77.66; H, 5.96; N, 16.39.
2. Materials and methods
2.1. Conformational analysis
MMX force field method followed by AM1 calculation implemented
in HyperChem 8 software (Hypercube, Inc.) was used for performing
conformational analysis of novel compounds along with zolpidem as a
well-known GABAA receptor agonist to find out whether the designed
molecules are able to mimic the proper conformation for binding to the
benzodiazepine receptor or not. For this purpose, the superimposition
of energy minima conformers of the novel compounds on the corre-
sponding conformer of zolpidem was carried out.
2.3.2. 3-(4-bromophenyl)-N-(4-methylbenzyl)-1-phenyl-1H-1,2,4-triazol-
5-amine (5b)
White powder; Yield: 0.97 g (63%); Mp: 191 °C; FT-IR spectrum, ν,
cm−1: 3406, 3078, 1666, 1591, 1457; 500 MHz 1H NMR (CDCl3): δ
ppm: 2.45(s, 3H, CH3), 4.32(s, 2H, CH2), 5.53(s, 1H, NH), 6.88(t,
J = 7.3 Hz, 1H, Ar), 7.11(d, J = 7.7 Hz, 2H, Ar), 7.18(d, J = 8.1 Hz,
2H, Ar), 7.27–7.30(m, 6H, Ar), 7.55(d, J = 8.1 Hz, 2H, Ar). 125 MHz
13C NMR (CDCl3) δC: 20.81, 45.88, 122.11, 124.27, 126.65, 129.73,
129.79, 130.36, 132.98, 133.04, 138.27, 138.97, 145.23, 155.53,
164.22, 167.12. MS: m/z (%) = 419(M+, 20), 341(55), 327(50),
263(60), 154(50), 120(100). Anal calcd for C22H19BrN4: C, 63.02; H,
4.57; N, 13.36; Found: C, 63.09; H, 4.62; N, 13.31.
2.2. Materials and instrumentation
All chemicals and reagents used in this study were purchased from
Sigma/Aldrich (St. Louis, MO, USA) or Merck (Darmstadt, Germany)
Company and were used without any further purification. An electro-
thermal 9300 apparatus (Ontario, Canada) was used for melting points
measuring. To obtain infrared spectra and elemental analysis for the
synthesized compounds, Shimadzu FT-IR 8400S spectrographs
(Shimadzu, Japan) using the potassium bromide (KBr) disc method and
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R. Jahani, et al.
BioorganicChemistry104(2020)104212
2.3.3. N-(4-chlorobenzyl)-2-phenyl-5-p-tolyl-2H-1,2,4-triazol-3-amine
(5c)
7.48(d, J = 7.3 Hz, 2H, Ar), 7.91(d, J = 7.3 Hz, 2H, Ar). 125 MHz 13
C
NMR (CDCl3) δC: 126.57, 127.46, 128.76, 129.24, 130.70, 131.62,
132.55, 133.42, 136.89, 142.11, 144.51, 154.16, 163.21, 164.39. MS:
m/z (%) = 424(M+, 30), 345(25), 313(25), 154(38), 111(38),
77(100). Anal calcd for C20H14BrClN4: C, 56.43; H, 3.31; N, 13.16;
Found: C, 56.49; H, 3.27; N, 13.11.
White powder; Yield: 0.85 g (60%); Mp: 174 °C; FT-IR spectrum, ν,
cm−1: 3320, 3066, 1818, 1449; 500 MHz 1H NMR (CDCl3): δ ppm:
2.45(s, 3H, CH3), 4.52(s, 2H, CH2), 6.16(s, 1H, NH), 7.31–7.34(m, 5H,
Ar), 7.40(d, J = 7.9 Hz, 2H, Ar), 7.49(d, J = 8.1 Hz, 2H, Ar), 7.78(d,
J = 8.1 Hz, 2H, Ar), 7.92(d, J = 8.5 Hz, 2H, Ar). 125 MHz 13C NMR
(CDCl3) δC: 19.99, 45.20, 127.44, 128.74, 129.21, 130.15, 130.27,
130.68, 132.53, 133.14, 134.65, 142.11, 145.97, 147.28, 161.02,
163.66. MS: m/z (%) = 297(80), 283(100), 263(50), 234(55),
140(90), 134(80). Anal calcd for C22H19ClN4: C, 70.49; H, 5.11; N,
14.95; Found: C, 70.43; H, 5.07; N, 14.90.
2.4. Docking study
Molecular docking using the Lamarckian genetic algorithm search
method implemented in Auto Dock 4.2.3. Software (http://autodock.
scripps.edu) was performed, using the previously described method
[11]. Docking study was performed in order to find out the mode of
corresponding interactions of designed compounds with amino acids of
the BZD-binding pocket of the GABAA receptor (α1β2γ2).
2.3.4. N-benzyl-5-(4-chlorophenyl)-2-phenyl-2H-1,2,4-triazol-3-amine
(5d)
Cream powder; Yield: 0.88 g (70%); Mp: 169 °C; FT-IR spectrum,
ν, cm−1: 3496, 1621, 1476, 1397; 500 MHz 1H NMR (CDCl3): δ ppm:
4.45(s, 2H, CH2), 6.01(s, 1H, NH), 7.38(d, J = 8.4 Hz, 2H, Ar),
7.47–7.50(m, 7H, Ar), 7.59(t, J = 7.4 Hz, 1H, Ar), 7.91(d, J = 8.4 Hz,
2H, Ar), 7.97(d, J = 8.5 Hz, 2H, Ar). 125 MHz 13C NMR (CDCl3) δC:
46.64, 127.48, 128.75, 129.03, 129.30, 130.67, 131.62, 131.90,
132.30, 134.42, 140.34, 142.14, 147.23, 162.13, 164.64. MS: m/z
(%) = 360(M+, 20), 283(30), 250(50), 235(30), 138(100), 107(30),
77(70). Anal calcd for C21H17ClN4: C, 69.90; H, 4.75; N, 15.53; Found:
C, 69.95; H, 4.68; N, 15.59.
2.5. Pharmacology
2.5.1. Animals and drugs
All experiments were performed with male NMRI mice (weighing
18–25 g). Animals were housed in plexiglass cages under the same
ambient temperature of 22
2 °C, 50% 10% humidity, and a 12 h:
12 h light–dark cycle. They had free access to food and water.
Experimental animals were randomly allocated to groups of ten and
were transferred to the animal laboratory at least 1 h prior to the be-
ginning of each experiment to get acclimatized to the laboratory con-
dition. Novel synthesized compounds, diazepam, and flumazenil were
dissolved in dimethyl sulfoxide and administrated in the volume of
5 mL/kg by intraperitoneal route (i.p.). Pentobarbital and pentylene-
tetrazole (PTZ) were prepared in normal saline. The injection volume
for pentobarbital and pentylenetetrazole was 10 mL/kg. All experi-
ments were carried out based on the National Institutes of Health (NIH)
guidelines for the Care and Use of Laboratory Animals. Each mouse was
used once and possible efforts were made to decrease animal numbers
and distress.
2.3.5. 5-(4-chlorophenyl)-2-phenyl-N-p-tolyl-2H-1,2,4-triazol-3-amine
(5e)
White powder; Yield: 0.81 g (65%); Mp: 211 °C; FT-IR spectrum, ν,
cm−1: 3444, 2963, 1606, 1472; 500 MHz 1H NMR (CDCl3): δ ppm:
2.60(s, 3H, CH3), 6.12(s, 1H, NH), 7.29–7.34(m, 5H, Ar), 7.47–7.50(m,
5H, Ar), 7.60(t, J = 7.4 Hz, 1H, Ar), 7.96(d, J = 8.5 Hz, 2H, Ar).
125 MHz 13C NMR (CDCl3) δC: 19.49, 128.77, 129.25, 129.32, 129.35,
133.22, 133.35, 134.11, 134.41, 136.11, 137.61, 141.56, 145.21,
162.15, 165.72. MS: m/z (%) = 360(M+, 50), 282(60), 267(30),
248(65), 119(65), 106(75), 91(100). Anal calcd for C21H17ClN4: C,
69.90; H, 4.75; N, 15.53; Found: C, 69.94; H, 4.80; N, 15.48.
2.5.2. The anticonvulsant activity
The anticonvulsant activity of the novel compounds was evaluated
in PTZ-induced seizures and MES-induced seizures tests as described
before [17]. To summarize, mice were treated with different doses of
novel compounds, diazepam, or vehicle 30 min before the induction of
seizure in each model. In the PTZ and MES models, the seizure was
induced by the administration of pentylenetetrazole (100 mg/kg) and
application of electroshock (10 Hz, 37.2 mA, and 0.3 s) through the ear
electrodes, respectively. Protection of mice against the lethal dose of
pentylenetetrazole was considered as the potential anti-seizure activity
in the PTZ model while in the MES model the potential activity of
compounds was determined by the reduction in the number of hind
limb tonic extension (HLTE). In both models of seizure, flumazenil as a
GABAA receptor antagonism was used for the evaluation of the possible
mechanism of action.
2.3.6. 5-(4-bromophenyl)-N,2-diphenyl-2H-1,2,4-triazol-3-amine (5f)
Cream powder; Yield: 0.79 g (61%); Mp: 183 °C; FT-IR spectrum,
ν, cm−1: 3507, 1621, 1382, 1319; 500 MHz 1H NMR (CDCl3): δ ppm:
6.40(s, 1H, NH), 7.30–7.35(m, 7H, Ar), 7.48(d, J = 8.5 Hz, 2H, Ar),
7.60(t, J = 7.7 Hz, 1H, Ar), 7.45(t, J = 7.4 Hz, 2H, Ar), 8.04(d,
J = 8.5 Hz, 2H, Ar). 125 MHz 13C NMR (CDCl3) δC: 126.17, 127.42,
127.76, 128.75, 129.23, 130.00, 130.14, 132.56, 133.35, 135.68,
142.24, 144.86, 155.10, 162.31, 164.55. MS: m/z (%) = 391(M+, 10),
313(20), 298(35), 235(25), 153(25), 91(100), 77(50). Anal calcd for
C20H15BrN4: C, 61.39; H, 3.86; N, 14.32; Found: C, 61.45; H, 3.81; N,
14.37.
2.3.7. N-(4-chlorophenyl)-2,5-diphenyl-2H-1,2,4-triazol-3-amine (5 g)
Cream powder; Yield: 0.86 g (68%); Mp: 180 °C; FT-IR spectrum, ν,
cm−1: 3216, 1941, 1442, 1364; 500 MHz 1H NMR (CDCl3): δ ppm:
6.35(s, 1H, NH), 7.38(t, J = 8.4 Hz, 1H, Ar), 7.47–7.50(m, 8H, Ar),
7.60(t, J = 7.4 Hz, 1H, Ar), 7.91(d, J = 8.4 Hz, 2H, Ar), 7.97(d,
J = 7.4 Hz, 2H, Ar). 125 MHz 13C NMR (CDCl3) δC: 127.48, 128.75,
129.03, 129.23, 129.30, 130.67, 131.62, 131.90, 132.30, 134.39,
134.42, 156.34, 163.12, 165.67. MS: m/z (%) = 235(50), 220(85),
111(100), 77(75). Anal calcd for C20H15ClN4: C, 69.26; H, 4.36; N,
16.15; Found: C, 69.20; H, 4.42; N, 16.09.
2.5.3. Potentiation of pentobarbital sleeping time
The experiment was performed with a slight modification of the
previously described method [18]. Mice were administered a single
dose of novel compounds with different concentrations 30 min prior to
the administration of pentobarbital (50 mg/kg). Pentobarbital was used
to induce the loss of the righting reflex (LRR). Each mouse was observed
and the LRR duration (the time between the loss and the recovery of the
righting reflex) was recorded for each mouse. To examine the possible
mechanism of action of novel compounds in the pentobarbital-induced
sleeping model, flumazenil was administrated at 10 mg/kg and 15 min
prior to injection of pentobarbital.
2.3.8. N-(4-bromophenyl)-5-(4-chlorophenyl)-2-phenyl-2H-1,2,4-triazol-
3-amine (5 h)
White powder; Yield: 0.94 g (59%); Mp: 171 °C; FT-IR spectrum, ν,
cm−1: 3328, 3089, 1643, 1494; 500 MHz 1H NMR (CDCl3): δ ppm:
6.63(s, 1H, NH), 7.29–7.34(m, 7H, Ar), 7.38(d, J = 8.1 Hz, 2H, Ar),
2.5.4. Step-through passive avoidance test
The Step-through passive avoidance test was carried out using a
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Fig. 2. Superimposition of compound 5g and zolpidem as a well-known GABAA receptor agonist.
common procedure with a slight modification [19]. On the training
day, mice received the novel compounds at doses corresponding to their
ED50 and twice ED50 values from the PTZ test, diazepam (2 mg/kg), or
vehicle. After 30 min, mice were placed individually in the light
chamber of apparatus and the gate between the two compartments was
opened after 30 s. Animals were allowed to explore the light com-
partment for 8 min and they got punishment by an adequate electric
footshock (0.2 mA, 50 Hz, 2 s) when they entered the dark compart-
ment. On the test day (24 h later), the experiment was repeated on the
pre-trained animals while the gate between light and dark compart-
ments was open. The time that the mice took to enter the dark box was
recorded and considered as an index for retention of the learned ex-
perience.
connected to an isometric force transducer. Animals were lifted by their
tail so that they grasp the tension pad and then pulled back until the
pad was released. This procedure was repeated in triplicate and the
averaged force (gram) was recorded automatically by the apparatus.
2.5.8. Preliminary evaluation of toxicity
The toxicity of the most potent compounds was evaluated in 10
mice. Mice were treated with 105 mg/kg of compound 5c and 70 mg/kg
of compound 5 g which were 10 times more than ED50s of hypnotic
doses and were observed for 24 h for any possible toxicity.
2.6. Data analysis
GraphPad Prism Software (Graph Pad Prism software, San Diego,
CA; version 5.0) was used for data analysis. ED50s with their 95%
confidence limits were estimated using the log-probit method. The re-
sults were statistically compared using one-way analysis of variance
(ANOVA), followed by Tukey post-test, when appropriate. The nu-
2.5.5. Open field test
Thirty minutes and 24 h after administration of novel compounds at
doses corresponding to their ED50 and twice ED50 values, vehicle, and
diazepam (2 mg/kg), the locomotor activity of animals was examined in
a Plexiglas box (42 cm × 42 cm × 42 cm). The apparatus was equipped
merical results of the experiment were expressed as mean
standard
with
a
video-based Ethovision System (Noldus, Wageningen,
error of the mean (SEM). Statistical significance was considered when
p-values were < 0.05.
Netherlands). The camera recorded the animals' movement while they
were exploring the open filed arena for ten minutes. The total loco-
motor activity of animals was expressed as a total distance movement in
centimeter [20]. Ethanol 70% was used to clean the open field arena
between the two subjects.
3. Results and discussion
3.1. Conformational analysis and molecular modeling (docking) studies
2.5.6. Rotarod test
As shown in Fig. 2, following superimposing energy minima con-
formers of novel compounds on zolpidem, aromatic ring and proton
accepting group as two main benzodiazepine pharmacophores of
compound 5 g and zolpidem are completely matched. The protein–li-
gand interaction of zolpidem and the most potent designed compound
(5 g) with amino acids of the BZD-binding site of the GABAA receptor is
illustrated in Fig. 3. As Fig. 3 depicts, zolpidem and compound 5 g
establish the same hydrogen binding with Thr109 and Thr206. Fur-
thermore, compound 5 g interacts with Tyr159 and His101 through π-π
stacking and hydrophobic interactions, respectively.
The possible unwanted effect of novel compounds on motor co-
ordination of animals was assessed using the rotarod test [21]. Mice
were trained for 3 days on an accelerating rotarod (rod diameter: 2 cm)
that rotated at 6 revolutions /min. During each training session, mice
were trained for 3 min with an unlimited number of trials. On the test
day (24 h after the last training), mice were intraperitoneally pretreated
with the test compounds, vehicle, and diazepam (2 mg/kg). Following
30 min, the animals were tested on the rotarod apparatus. Mice were
observed with a cut-off time of 3 min and the latency to the first fall
from the rod was recorded. Motor impairment was defined as the in-
ability to remain on the rotating rod compared to the control group.
3.2. Chemistry
2.5.7. Grip strength test
The newly designed compounds (5a-5 h) were synthesized ac-
cording to the following synthetic pathway (Scheme 1). Briefly, the
synthetic pathway was started with the reaction of different aniline
derivatives 1 and trichloroacetonitrile 2 in dimethylformamide solvent
for 20 min at room temperature to afford required compound 3.
The effect novel compounds at doses corresponding to their ED50
and twice ED50 values from the PTZ test, vehicle, and diazepam (2 mg/
kg) on skeletal muscular strength in mice were quantified in the grip-
strength test [22]. Thirty minutes after each treatment, mice were
placed on the tension pad (8 cm × 8 cm) of the apparatus which was
Compound
3 then participated in the reaction with different
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R. Jahani, et al.
BioorganicChemistry104(2020)104212
Fig. 3. The binding modes of zolpidem (yellow color) and the most potent novel agent 5 g (blue color) with amino acids in the BZD-binding pocket of the GABAA
receptor. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
hydrazonoyl chlorides analogs 4 in dimethylformamide solvent in the
presence of trimethylamine and CuI catalyzer for 2 h at room tem-
perature to obtain target compounds in good yields (59–70%). The
structures of novel synthesized compounds were characterized by the
aid of elemental analysis, 1H NMR, 13C NMR, FT-IR, and mass spec-
trometry analysis.
or tonic-clonic epileptic seizures [23]. In both models of seizure, flu-
mazenil as an antagonist of benzodiazepines receptor significantly an-
tagonized anticonvulsant effects of the novel compounds which in-
dicate the involvement of GABAA receptors in the observed effects.
3.3.2. Potentiation of pentobarbital sleeping time
The hypnotic effect of all compounds was evaluated in the pento-
barbital induced loss of righting test. The results were compared to
diazepam as a standard hypnotic agent. ED50 of each tested compound
which refers to the dose that doubles the sleeping time compared to the
control group with the calculated 95% confidence interval is reported
in Table 1. In all compounds, the sleeping time was increased in a dose-
dependent manner. Compounds 5 g (ED50 ≈ 7.1 mg/kg) and compound
5c (ED50 ≈ 10.5 mg/kg) were found more potent than other agents.
The observed pharmacological effect was completely blocked following
the administration of flumazenil which reconfirms the responsibility of
benzodiazepines receptors.
3.3. Pharmacology
3.3.1. The anticonvulsant activity
The novel synthesized compounds were screened for their possible
anticonvulsant activity in the PTZ and MES models of seizure.
Diazepam was used as a standard anticonvulsant agent. The results are
presented as ED50 values with 95% confidence intervals. ED50 refers to
the median effective dose which protects 50% of animals against in-
duced seizure. As illustrated in Table 1, compound 5c (ED50
≈
52.5 mg/kg) and 5 g (ED50 ≈ 16.5 mg/kg) had potent anticonvulsant
activity in the PTZ-induced seizures model while other compounds
were almost inactive. In the MES-induced seizures model, all com-
pounds were found to have anticonvulsant activity but compounds 5c
and 5 g with ED50 values of 11.8 mg/kg and 10.50 mg/kg, respectively
were the most potent compounds among the novel 1,2,4-triazol-3-
amine derivatives. The higher potency of the tested agents in the MES
test compared to the PTZ test could be related to the type of induced
epilepsy. The MES induces epileptic seizure represents the grand mal
type of epilepsy while PTZ induces epileptic seizure produces petit mal
Considering the obtained ED50s in the PTZ, MES and anticonvulsant
and hypnotic activities of the novel compounds, the presence of a ha-
logen substituent at the para position of phenyl ring at R2 (especially Cl)
increases the observed anticonvulsant and hypnotic effects while a
methyl substituent diminishes the activity. Replacement of phenyl ring
by benzyl ring increases activity probably due to better hydrophobic
interaction. Furthermore, the presence of an electron-donating group
like a methyl substituent at R2 position produces more potent com-
pounds compared to electron-withdrawing groups such as halogens.
Scheme 1. Reagents and conditions: (a) Dimethylformamide, RT, 20 min; (b) Triethylamine, CuI, Dimethylformamide, RT, 2 h.
5

R. Jahani, et al.
BioorganicChemistry104(2020)104212
Table 1
The anticonvulsant and hypnotic activities of novel synthesized compounds and diazepam.
ED50 mg/kg(95% confidence intervals)
Pentobarbital test
MES test
> 100
PTZ test
> 100
R2
R1
Compound
5a
80.54 (68.66–109.70)
CH3
95.24 (79.32–139.50)
10.53 (8.92–14.93)
> 100
> 100
Br
5b
5c
11.80 (8.26–15.92)
52.48 (36.14–72.27)
CH3
31.30 (25.06–41.04)
96.98 (88.10–160.30)
50.52 (32.28–73.90)
7.11 (5.93–8.70)
27.92 (16.21–42.53)
> 100
> 100
Cl
Cl
Br
–
5d
5e
5f
> 100
47.42 (31.26–67.76)
10.54 (7.74–14.32)
> 100
16.55 (13.03–19.54)
5 g
32.29 (24.53–43.85)
1.60 (0.98–2.01)
> 100
> 100
Cl
5 h
1.02 (0.73–1.26)
0.96 (0.66–1.20)
diazepam
3.3.3. Passive avoidance test
involvement of α5-containing GABAA receptors which affect cognitive
functions [24,25].
Passive avoidance test which is a well-known model in the assess-
ment of anterograde amnesia was carried out to determine whether the
novel compounds cause memory impairment in mice or not. Since
compounds 5c and 5g were more potent than other agents in the pre-
vious experiments, the possible unwanted effect of these agents on
anterograde memory were evaluated. Following the administration of
compounds 5c and 5g, the latency in the entrance to the dark box did
not change compared to the control group while the administration of
diazepam significantly reduced the latency time (Fig. 4). This finding
reveals that the novel agents most probably act through α1-containing
GABAA receptors that are responsible for the sedative effect and an-
ticonvulsant action of benzodiazepine-like agents and rules out the
3.3.4. Open field test
The assessment of locomotor activity in the open field test re-
presented a significant reduction in the spontaneous motor activities of
animals 30 min after administration of compounds 5c, 5g, and dia-
zepam (Fig. 5A). No obvious reduction in the locomotor activities was
observed on the next day of treatment (Fig. 5B). Therefore, it should be
pinpointed that the latency time in the entrance to the dark box in the
passive avoidance test is not due to reduced locomotor activity caused
by tested compounds.
3.3.5. Rotarod test
The effect of compounds 5c and 5g on the motor coordination of
animals in the rotarod test is shown in Fig. 6. The evaluation of mice in
this test revealed that the treatment of animals with the novel com-
pounds was unable to produce alteration in the motor coordination of
animals. Finally, the administration of diazepam (2 mg/kg) as a re-
ference drug significantly reduced the time mice stayed on the rod, with
respect to the control group.
3.3.6. Grip strength test
The results of the grip strength test revealed that compounds 5c and
5g do not cause significant muscle tone reduction compared to the
control group while administration of diazepam as a standard muscle
relaxant meaningfully affects the skeletal muscular strength in mice
(Fig. 7). Since α5-containing GABAA receptors significantly contribute
to the benzodiazepine induced muscle relaxation [26], it could be
concluded that the novel compounds do not affect this type of re-
ceptors.
3.3.7. Preliminary evaluation of toxicity
Fig. 4. The effect of novel compounds and diazepam (2 mg/kg) on avoidance
No significant toxicity reaction except sedation and hypnosis was
observed following injection of 105 mg/kg of compound 5c and 70 mg/
kg of compound 5g, which were 10 times more than the estimated
latency in the passive avoidance test. Values are reported as mean
SEM. ***
indicates p < 0.001 compared to the control group; n = 10 in all groups.
6

R. Jahani, et al.
BioorganicChemistry104(2020)104212
Fig. 5. The effect of novel compounds and diazepam (2 mg/kg) on the locomotor activity of animals in the open field test 30 min (A) and 24 h (B) after treatment.
Values are reported as mean
SEM. *** indicates p < 0.001 compared to the control group; n = 10 in all groups.
hypnotic doses. Probably the synthesized compounds are safe enough
for future studies but more comprehensive toxicity studies need to be
carried out.
4. Conclusion
In summary, a series of novel 1,2,4-triazol-3-amine derivatives were
designed and synthesized as potential agonists of GABAA subtype re-
ceptors. The biological effects of synthesized compounds were in-
vestigated using animal models. Compounds 5c and 5g in the PTZ test
and compounds 5c, 5d, 5f, and 5g in the MES test revealed significant
anticonvulsant activity while all the designed compounds showed a
considerable hypnotic effect. Furthermore, compounds 5g and 5c were
the most potent compounds in all experiments. The observed effects
were completely antagonized by flumazenil which confirms the in-
volvement of GABAA receptors in the observed effects. No negative
effects on the memory, motor coordination, and muscle strength were
observed in the passive avoidance test, rotarod test, and grip strength
test, respectively. Therefore, we can conclude that the novel com-
pounds most likely act similar to α1-selective agonist zolpidem which
possesses no affinity for α5-containing GABAA receptors. The promising
compounds (especially compound 5g) could be used in designing and
synthesizing novel benzodiazepine receptor agonists with minimum
side effects.
Fig. 6. The effect of novel compounds and diazepam (2 mg/kg) on the motor
coordination of animals in the rotarod test. Values are reported as
mean
SEM. *** indicates p < 0.001 compared to the control group; n = 10
in all groups.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influ-
ence the work reported in this paper.
Acknowledgments
The authors are thankful to AJA University of Medical Sciences for
providing the facility for the research work.
Funding
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
Fig. 7. The effect of novel compounds and diazepam (2 mg/kg) on the skeletal
muscular strength of animals in the grip strength test. Values are reported as
Appendix A. Supplementary material
mean
SEM. *** indicates p < 0.001 compared to the control group; n = 10
in all groups.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2020.104212.
7

R. Jahani, et al.
BioorganicChemistry104(2020)104212
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