Anticonvulsant activity of 1,2,4-triazine derivatives with pyridyl side chain: Synthesis, biological, and computational study

Article abstract of DOI:10.1007/s00044-014-1315-3

A series of 5,6-bisaryl-1,2,4-triazine-3-thiol-substituted derivatives were synthesized by condensation of 1,2-diketones and thiosemicarbazide under microwave irradiations and subsequent alkylation of thiol group by chloromethylpyridinium chloride. Evalua

Full text of DOI:10.1007/s00044-014-1315-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-1315-3
ORIGINAL RESEARCH
Anticonvulsant activity of 1,2,4-triazine derivatives with pyridyl
side chain: synthesis, biological, and computational study
•
•
•
Hamid Irannejad Hamid Nadri Nima Naderi Seyedeh Nesa Rezaeian
•
•
Neda Zafari Alireza Foroumadi Mohsen Amini Mehdi Khoobi
•
•
Received: 11 August 2014 / Accepted: 9 December 2014
Ó Springer Science+Business Media New York 2014
Abstract A series of 5,6-bisaryl-1,2,4-triazine-3-thiol-
substituted derivatives were synthesized by condensation
of 1,2-diketones and thiosemicarbazide under microwave
irradiations and subsequent alkylation of thiol group by
chloromethylpyridinium chloride. Evaluation of anticon-
vulsant activity of compounds was performed by maximal
electroshock and pentylenetetrazole-induced seizures tests.
In order to evaluate their neuroprotective potential, the
ability of compounds to inhibit soybean 15-lipoxygenase
was also assessed. Further molecular modeling and dock-
ing study on Na? channel and GABAA receptor was per-
formed to elucidate their mechanisms of action and
necessary interactions in the active site. Compounds 2c and
2d with bis(4-bromophenyl) and pyridyl substituents
showed highest protection up to 70 and 80 % in PTZ and
MES-induced seizures, respectively, compared to the
control group. Molecular docking study revealed their
possible antiseizure mechanism of action through GABAA
receptor, and in silico assessment of their BBB perme-
ability indicated them as CNS active agents.
Keywords Synthesis Á 1,2,4-Triazine Á Anticonvulsant Á
15-LOX Á Molecular docking
Introduction
Epilepsy affects approximately 1 % (50 million people) of
the world’s population, and one in 10 people will suffer a
seizure in their lifetime. Epilepsy is as common as breast
cancer in America and takes many lives. Although
administration of available antiepileptic drugs (AEDs) in
patients successfully controls seizures in 50 % of patients
developing partial seizures and 60–70 % of those devel-
oping generalized seizures, remaining are not responsive to
N. Zafari
Faculty of Medicine, Shahid Beheshti University of Medical
Sciences, Tehran, Iran
H. Irannejad (&)
Department of Medicinal Chemistry, Faculty of Pharmacy and
Pharmaceutical Sciences Research Center, Mazandaran
University of Medical Sciences, Sari, Iran
e-mail: irannejadhamid@gmail.com;
A. Foroumadi Á M. Khoobi
Department of Medicinal Chemistry, Faculty of Pharmacy and
Pharmaceutical Sciences Research Center, Tehran University of
Medical Sciences, 14176 Tehran, Iran
irannejad.hamid@yahoo.com
H. Nadri
Department of Medicinal Chemistry, Faculty of Pharmacy and
Pharmaceutical Sciences Research Center, Shahid Sadoughi
Yazd University of Medical Sciences, Yazd, Iran
M. Amini
Department of Medicinal Chemistry, Faculty of Pharmacy and
Drug Design & Development Research Center, Tehran
University of Medical Sciences, 14176 Tehran, Iran
N. Naderi
Department of Pharmacology and Toxicology, School of
Pharmacy, Shahid Beheshti University of Medical Sciences,
Tehran, Iran
S. N. Rezaeian
Student Research Committee, Faculty of Pharmacy, Mazandaran
University of Medical Sciences, Sari, Iran
123

Med Chem Res
the conventionally available AEDs. Moreover, the efficacy
of many AEDs is limited by the dose-dependent toxicities
and adverse drug reactions such as minimal brain impair-
ment, megaloblastic anemia to death from aplastic anemia,
or hepatic failure. Thus, there is a substantial need for
further development of safer agents and novel mechanistic
approaches in the treatment of epilepsy (Landmark, 2007);
(Siddiqui and Ahsan, 2010).
NH2
N
O
Cl
NH
O
Cl
N
HN
N
NH2
Lamotrigine
Phenytoin
R
R
Y
Z
However, oxidative injury may play a role in the initi-
ation and progression of epilepsy, and therapies targeted at
reducing oxidative stress may ameliorate tissue damage
and favorably alter the clinical course of the disease (Devi
et al., 2008).
N
N
S
N
R = MeO, Me, Cl Y, Z= N, CH
Several in vivo evidences of oxidative stress were found
in animal models of epilepsy. Accordingly, antioxidant
therapies with the aim of reducing oxidative injury have
received remarkable attention in the treatment of epilepsy
(Acharya et al., 2008).
Fig. 1 Schematic representation of anticonvulsant drugs phenytoin
and lamotrigine, and the previously synthesized compounds as
anticonvulsant agents
According to this fact, anticonvulsant agents with neu-
roprotective properties have received considerable atten-
tion in the treatment of epilepsy. Several studies reported
that neurodegeneration is the major neurobiological
abnormality in epileptic brain (Naegele, 2007). Since epi-
leptic seizure and neurodegeneration are very similar in
some aspects of their basic pathophysiology therefore,
some anticonvulsive agents are equally effective neuro-
protectives (Stanislaw et al., 2007).
Recently, we synthesized and evaluated anticonvulsant
activity of some 1,2,4-triazine-substituted derivatives
(Fig. 1). Among the compounds tested, we found out the
prominent anticonvulsant activity of 3-((pyridin-4-ylme-
thyl)thio)-5,6-di-p-tolyl-1,2,4-triazine as a lead compound
in both maximal electroshock (MES) and pentylenetetraz-
ole (PTZ)-induced seizures tests (Irannejad et al., 2014).
Motivated by these observations and in continuation of our
previous ongoing research studies on novel neuroprotec-
tives and anticonvulsants, we synthesized some new 5,6-
bisaryl-1,2,4-triazine-3-thiol derivatives (Fig. 2) and eval-
uated their anticonvulsant and 15-LOX inhibition activi-
ties. Evaluation of anticonvulsant activity was performed
by two conventional and well-established methods, MES
and PTZ-induced seizures tests. Neurotoxicity of synthe-
sized compounds was assessed by rota-rod test, and the
ability of compounds to inhibit soybean 15-LOX was also
evaluated. To find out their possible anticonvulsant
mechanism of action, molecular docking was performed on
the two main targets of conventional antiseizure drugs,
Na ? channel and GABAA receptor.
Previous studies on oxidative stress model in neuronal
cells have revealed a role for the lipoxygenases (LOX),
lipid oxidizing enzymes, to produce neuronal damage.
15-LOX is one of the key inflammatory mediators in
neurodegenerative disease because it is stimulated by
reactive oxygen species (ROS). Activation of 15-LOX
tends to generate lipid hydroperoxides (15-HPETE) that
help to amplify oxidative stress (Loscalzo, 2008).
Increased level of 15-LOX has been found in early
stages of Alzheimer disease in human and in experimental
model of stroke in mice (Van Leyen et al., 2006; Pratico
et al., 2004). Therefore, inhibition of 15-LOX may protect
a variety of neuronal cell types, and 15-LOX inhibitors
could be potential neuroprotective agents.
Several heterocycle-containing compounds have been
reported to have considerable antiepileptic activities. 1,2,4-
triazine is among the usually occurred heterocyclic nuclei
possessing the wide range of biological applications such
as anticonvulsant (Irannejad et al., 2014), antiparkinsonism
(Congreve et al., 2012), and potent neuroprotective (Ir-
annejad et al., 2010) activities. Among these biological
activities, 1,2,4-triazine derivatives have been a promising
scaffold which is mainly used in the novel anticonvulsant
drugs such as lamotrigine (Fig. 1). Lamotrigine shares
similar mode of action on neuronal sodium channel as
phenytoin.
X
S
S
Y
Z
Y
Z
N
N
S
N
N
S
N
N
X
4a,b
Y, Z = N, CH
2a-d
X= F, Br
Y, Z = N, CH
Fig. 2 New compounds (2a–d and 4a–b) as novel anticonvulsant and
neuroprotective agents
123

Med Chem Res
Materials and methods
Chemistry
5 ml of water) and appropriate chloromethylpyridinium
chloride (2 mmol) were added, and the resulting mixture
was stirred at room temperature for 3 h. At the end of this
time, it was concentrated under vacuum and neutralized
with HCl 1 N, and the resulting aqueous phase was
extracted with chloroform, dried with sodium sulfate. Upon
concentrating the organic phase under vacuum, the result-
ing material was purified by column chromatography (sil-
ica gel, mesh 230–400) eluting with dichloromethane–
methanol (9:1) (Irannejad et al., 2014).
Chemical reagents and solvents were purchased from
Sigma-Aldrich or Merck. NMR spectra were recorded in
DMSO-d6 on a Brucker DRX400 MHz instrument. TMS
was used as an internal standard in reporting chemical
shifts (d). Mass spectra were obtained with Agilent 7890A
spectrometer. Progress of reaction and purity of com-
pounds were monitored on TLC silica gel F254 plates. The
microwave irradiation was carried out using MicroSYNTH
Laboratory System (MILESTONE S.r.l) in Teflon closed
vessel at 300 W power.
5,6-Bis(4-fluorophenyl)-3-(pyridin-3-ylmethylthio)-1,2,4-
triazine (2a)
M.p.: 188–190 °C; IR (KBr): 3125, 2870, 1667 (C=N),
1560 (N=N). H NMR (400 MHz, DMSO-d6) d: 3.73 (s,
1
General procedure for preparation of 3 and 1a,b
2H, SCH2), 7.18 (d, J = 7.24 Hz, 2H, phenyl), 7.22 (d,
J = 7.28 Hz, 2H, phenyl), 7.32 (dd, J = 6.76 Hz, 4.36 Hz,
2H, phenyl), 7.43 (dd, J = 6.8 Hz, 4.36 Hz, 2H, phenyl),
7.70 (d, J = 6.12 Hz, 1H, pyridine), 8.44 (d, J = 3.6 Hz,
1H, pyridine), 8.47 (s, 1H, pyridine). 13C NMR (100 MHz,
DMSO-d6) d: 32.31, 115.10 (d, J = 6.24 Hz), 115.27 (d,
J = 6.24 Hz), 123.49, 131.12 (d, J = 6.79 Hz), 131.96 (d,
J = 6.79 Hz), 132.30, 136.30, 141.50, 148.05, 149.71,
154.32, 161.20, 162.38, 163.15, 164.36, 165.33. Anal.
Calcd for C21H14F2N4S: C, 64.27; H, 3.60; N, 14.28.
Found: C, 64.41; H, 3.53; N, 14.39.
Preparation of 5,6-bisaryl-1,2,4-triazine-3-thiol derivatives
was performed by a rapid, simple, and green procedure
reported previously by us (Scheme 1). In general, a few
drops of concentrated hydrochloric acid were added to a
mixture of the appropriate 1,2-diketone (1 eq) and thio-
semicarbazide (1.2 eq) in an appropriate ratio of water and
ethanol and then the mixture was irradiated under micro-
wave conditions for 10 min at 120 °C (300 W). At the end
of this time, the precipitated solid was filtered and washed
with water several times to give the desired product
(Irannejad et al., 2014).
5,6-Bis(4-fluorophenyl)-3-(pyridin-4-ylmethylthio)-1,2,4-
General procedure for preparation of compound 2a–
d and 4a,b
triazine (2b)
M.p.: 172–174 °C; IR (KBr): 3,122, 2,884, 1,665 (C=N),
1,569 (N=N). H NMR (400 MHz, DMSO-d6) d: 3.78 (s,
2H, SCH2), 7.11 (t, J = 7.5 Hz, 2H, Ar), 7.14 (t,
1
To a stirred solution of compound 3 or 1a,b (1.7 mmol) in
20 ml of ethanol, sodium hydroxide solution (170 mg in
Scheme 1 Synthesis of 5,6-
bisaryl-1,2,4-triazine-3-thiol
derivatives. Reagents and
conditions: a thiosemicarbazide,
HCl, MW, 120 °C, 10 min; b 4-
chloromethylpyridinium
chloride or
X
X
X
X
Y
Z
N
N
S
N
N
SH
O
b
a
N
N
O
X
X
3-chloromethylpyridinium
chloride, NaOH, EtOH, rt
2a: X=F, Y=CH, Z=N
2b: X=F, Y=N, Z=CH
2c: X=Br, Y=CH, Z=N
2d: X=Br, Y= N, Z=CH
1a: X=F
1b: X=Br
X=F, Br
S
S
S
Y
N
N
SH
O
b
a
N
N
S
Z
S
N
N
O
S
S
4a: Y=CH, Z=N
4b: Y=N, Z=CH
3
123

Med Chem Res
J = 7.5 Hz, 2H, Ar), 7.56 (dd, J = 6.8 Hz, 4.5 Hz, 2H,
Ar), 7.59 (dd, J = 6.8 Hz, 4.5 Hz, 2H, Ar), 7.82 (d,
J = 4.7 Hz, 2H, pyridine), 8.44 (d, J = 4.8 Hz, 2H, pyri-
dine). 13C NMR (100 MHz, DMSO-d6) d: 34.33, 117.45
(d, J = 6.29 Hz), 117.56 (d, J = 6.29 Hz), 122.42, 133.57
(d, J = 6.75 Hz), 134.12 (d, J = 6.75 Hz), 134.77, 136.16,
137.29, 142.46, 147.28, 149.63, 157.51, 160.53, 162.07.
Anal. Calcd for C21H14F2N4S: C, 64.27; H, 3.60; N, 14.28.
Found: C, 64.32; H, 3.48; N, 14.12.
147.07, 148.03, 149.08, 150.51, 168.54. Anal. Calcd for
C17H12N4S3: C, 55.41; H, 3.28; N, 15.20. Found: C, 55.69;
H, 3.16; N, 15.49.
3-(Pyridin-4-ylmethylthio)-5,6-di(thiophen-2-yl)-1,2,4-
triazine (4b)
M.p.: 178–180 °C; IR (KBr): 3,091, 2,899, 1,681 (C=N),
1
1,556 (N=N). H NMR (400 MHz, DMSO-d6) d (ppm):
3.68 (s, 2H, SCH2), 6.96 (d, J = 3.0 Hz, 1H, Ar), 7.08 (t,
J = 3.96 Hz, 1H, Ar), 7.18 (t, J = 4.08 Hz, 1H, Ar), 7.30
(d, J = 2.48 Hz, 1H, Ar), 7.80 (d, J = 4.16 Hz, 1H, Ar),
7.95 (d, J = 4.16 Hz, 1H, Ar), 7.37 (d, J = 4.6 Hz, 2H,
pyridine), 8.49 (d, J = 4.6 Hz, 2H, pyridine). 13C NMR
(100 MHz, DMSO-d6) d: 38.48, 126.82, 127.43, 128.06,
129.22, 129.31, 131.58, 132.49, 134.82, 135.25, 145.67,
147.71, 148.73, 148.86, 166.72. Anal. Calcd for
C17H12N4S3: C, 55.41; H, 3.28; N, 15.20. Found: C, 55.53;
H, 3.41; N, 15.38.
5,6-Bis(4-bromophenyl)-3-(pyridin-3-ylmethylthio)-1,2,4-
triazine (2c)
M.p.: 212–214 °C; IR (KBr): 3,093, 2,897, 1,680 (C=N),
1
1,564 (N=N). H NMR (400 MHz, DMSO-d6) d: 3.72 (s,
2H, SCH2), 7.22 (d, J = 6.72 Hz, 2H, phenyl), 7.31 (d,
J = 6.72 Hz, 2H, phenyl), 7.51 (t, J = 6.34 Hz, 1H, pyr-
idine), 7.55 (d, J = 6.68 Hz, 2H, phenyl), 7.59 (d,
J = 6.84 Hz, 2H, phenyl), 7.70 (d, J = 6.08 Hz, 1H, pyr-
idine), 8.44 (d, J = 2.84 Hz 1H, pyridine), 8.47 (s, 1H,
pyridine). 13C NMR (100 MHz, DMSO-d6) d: 32.32,
122.26, 123.50, 124.57, 130.24, 130.92, 131.18, 131.24,
131.37, 133.90, 134.99, 136.30, 141.35, 148.04, 149.71,
154.53, 165.06. Anal. Calcd for C21H14Br2N4S: C, 49.05;
H, 2.74; N, 10.90. Found: C, 49.28; H, 2.68; N, 10.54 s.
Docking study
The homology model of the Diazepam bound GABAA
receptor developed by Richter et al. (2012) and lamotrigine
bound Na? channel by Lipkind and Fozzard (2010) was
retrieved from the supplementary material of their pub-
lished paper.
5,6-Bis(4-bromophenyl)-3-(pyridin-4-ylmethylthio)-1,2,4-
triazine (2d)
Protein structure was prepared with Pymol 1.6.0 (The
¨
PyMOL Molecular Graphics System, Schrodinger, LLC).
M.p.: 209–211 °C; IR (KBr): 3,104, 2,833, 1,684 (C=N),
1,562 (N=N). H NMR (400 MHz, DMSO-d6) d: 4.6 (s,
HyperChem v.8, with AM1 force field was used to generate
the lowest energy conformations. Docking study has been
performed with AutoDock 4.2 software, using AutoDock
Tools (ADT) in MGLTools 1.5.6. For the protein and the
ligand, Kollman and Gasteiger charges were computed,
respectively. The Lamarckian genetic algorithm (LGA)
was used as a search engine. AutoGrid 4.2 was utilized for
defining active site, and the grid size was set to
20 9 20 9 20 points with a grid spacing of 1.0 on the
center of the X-ray ligand in the crystal structure complex.
The other parameters were as follows:
1
2H, SCH2), 7.40 (d, J = 6.72 Hz, 2H, phenyl), 7.42 (d,
J = 6.72 Hz, 2H, phenyl), 7.63 (d, J = 6.72 Hz, 4H,
phenyl), 7.51 (d, J = 4.72 Hz, 2H, pyridine), 8.51 (d,
J = 4.8 Hz, 2H, pyridine). 13C NMR (100 MHz, DMSO-
d6) d: 32.66, 123.17, 123.98, 124.85, 131.38, 131.53,
131.74, 133.92, 134.16, 146.70, 149.61, 153.37, 154.89,
169.24. Anal. Calcd for C21H14Br2N4S: C, 49.05; H, 2.74;
N, 10.90. Found: C, 49.23; H, 2.64; N, 10.69.
3-(Pyridin-3-ylmethylthio)-5,6-di(thiophen-2-yl)-1,2,4-
Maximum number of energy evaluations: 2,500,000,
independent GA runs: 20, maximum number of LGA
operations: 27,000, population size: 150, crossover: 0.80,
mutation: 0.02, elitism: 1.
triazine (4a)
M.p.: 175 °C; IR (KBr): 3,086, 2,852, 1,671 (C=N), 1,523
1
(N=N). H NMR (400 MHz, DMSO-d6) d (ppm): 3.62 (s,
2H, SCH2), 7.02 (d, J = 3.2 Hz, 1H, Ar), 7.14 (t,
J = 3.6 Hz, 1H, Ar), 7.24 (t, J = 3.6 Hz, 1H, Ar), 7.34 (d,
J = 3.3 Hz, 1H, Ar), 7.57 (d, J = 4.0 Hz, 1H, Ar), 7.87 (d,
J = 4.0 Hz, 1H, Ar), 7.51 (t, J = 4.5 Hz, 1H, pyridine),
7.81 (d, J = 4.0 Hz, 1H, pyridine), 8.31 (s, 1H, pyridine),
8.43 (d, J = 4.5 Hz, 1H, pyridine). 13C NMR (100 MHz,
DMSO-d6) d: 36.82, 125.93, 127.69, 128.45, 129.11,
129.27, 132.51, 132.59, 133.20, 134.62, 136.38, 138.63,
Pharmacology
In vivo seizure test was performed on male NMRI mice
weighting 20–30 g (Pasteur Institute, Tehran, Iran). Mice
were kept in animal facility at controlled temperature
(22 2 °C) with 12–12 h light–dark cycle and free access
to food and water. The behavioral experiments were done
between 9 a.m. and 4 p.m. Each mouse was used only once
123

Med Chem Res
in all experiments. Solutions were prepared in a vehicle
consisted of polyethyleneglycol (PEG) 30 % v/v in dis-
tilled water and were administered at the dose of 100 mg/
kg to mice. All compounds (or vehicle in control group)
were administered through intraperitoneal (i.p.) injection
30 min before performing seizure test. Procedures involv-
ing animals were conducted in accordance with the Shahid
Beheshti University Guidelines for Care and Use of Lab-
oratory Animals.
animals were placed on rods (rotating at the speed of 10
rounds per minute). Failure to keep staying on the rotating
rod for a period of 90 s was considered as drug-induced
motor impairment.
Statistical analysis
Statistical analysis was done using GraphPad Prism 5.0
(GraphPad Software Inc., CA, USA). In PTZ test, the
results were shown as mean SEM. One-way analysis of
variance (ANOVA) followed by Dunnett’s post-test was
used to compare various groups with the control group. In
MES test, data were analyzed using Fisher’s exact test.
p values less than 0.05 were considered as statistically
significant.
Pentylenetetrazole (PTZ)-induced seizure test
Thirty minutes after intraperitoneal injection (at a volume
of 10 ml/kg) of synthesized compounds or vehicle (PEG
30 % v/v in distilled water), animals were subject to PTZ-
induced seizure test. The threshold for clonic seizures was
determined by infusion of a 1 % solution of PTZ in dis-
tilled water through the tail vein of unrestricted freely
moving mouse at a constant rate of 0.25 ml/min with an
infusion pump (model 53140, Stoelting, USA). The
required dose of PTZ (in mg/kg) that caused generalized
clonic seizure with loss of righting reflex was considered as
the threshold for the clonic seizure (Naderi et al., 2011).
15-Lipoxygenase inhibition assay
LOX inhibitory activity of target compounds was studied
by a spectrophotometric assay. The tested compounds were
dissolved in DMSO (1 ml) and phosphate buffer (9 ml,
0.1 M, pH 8). This stock solution was added to test solution
containing enzyme (final concentration: 167 U/ml) and
phosphate buffer (pH 8) to achieve concentrations in range
of 10-3–10-6 M. After incubation of test solution for
4 min, substrate (linoleic acid, final concentration:
134 lM) was added, and change in the absorbance was
measured for 60 s at 234 nm. The enzyme solution was
kept in ice, and controls were measured at intervals
throughout the experimental periods to ensure that the
enzyme activity was constant (Assadieskandar et al.,
2012).
Maximal electroshock (MES)-induced seizure test
Electroshock was induced by generating an alternating
current (intensity 40 mA, pulse duration 0.2 s, frequency
50 Hz, pulse width 0.5 ms) through ear clip electrodes by a
stimulator (Borj Sanat, Iran). In order to improve electrode
conduction, electrodes were moistened by saline before
attaching to the ear of mouse. The end-point for the
occurrence of seizure was the observation of hind-limb
tonic extension (HLTE) in mice (Woodbury and Daven-
port, 1952); (Woodbury and Swinyard, 1952). To select
proper current intensity for electroconvulsions, 5 groups of
mice consisting of five animals per group were challenged
with electroshocks of various intensities (25, 30, 35, 40,
and 45 mA) that yield seizure in minimum of 20 % and
maximum of 100 % of animals in a current-dependent
manner. After plotting a current intensity-seizure curve, the
CS97 (40 mA), which was a current intensity that produced
HLTE in 97 % of animals, was determined according to
log-Probit method by Litchfield and Wilcoxon (1949) and
was applied in all electroshock experiments. Data were
shown as percent protection, which is the percentage of
animals in each group that did not have HLTE after
receiving electroshock.
Results and discussion
Antiseizure effects of compounds in PTZ and MES-
induced seizure test in mice
Protective effect of new compounds against convulsion
was evaluated in MES and PTZ-induced seizures in mice.
The required dose of PTZ to induce seizure, 30 min after
the intraperitoneal administration of drugs, through con-
tinuous i.v. infusion for each compound is shown in
Table 1. One-way ANOVA revealed a significant change
in the dose of PTZ required for seizure induction between
different groups [F(7,41) = 2.567; p = 0.027]. Further
analysis using Dunnett’s test revealed a significant increase
in PTZ dose for seizure induction in mice pretreated with
compound 2c (70 mg/kg, p \ 0.05) compared with the
control group. Compound 2c bearing bromine atom at para
position of phenyl rings and 3-pyridylmethylthio side chain
had the highest protective effect in PTZ-induced seizure
Rota-rod test
To evaluate the effect of compounds on motor coordina-
tion, rota-rod test was conducted before seizure test. The
123

Med Chem Res
seizures. Obtained results clearly indicated that the pyri-
dylmethyl side chain is necessary for optimal anticonvul-
sive effect, and a lipophilic group such as bromine atom at
para positions of phenyl rings could improve the protective
effect of compounds. The results of rota-rod test revealed
no drug-induced motor impairment at the administered
dose of 100 mg/kg (data not shown).
Table 1 Effects of i.p. administration of compounds in protection
against PTZ and MES-induced seizures in mice
X
S
N
N
S
N
N
S
R
R
N
N
The results of anticonvulsant evaluation of compounds
revealed that presence of a lipophilic atom such as bromine
at para position of phenyl rings could improve the anti-
seizure ability of compounds 2c and 2d. Presence of thio-
phene ring instead of 4-bromophenyl moiety has also
increased the anticonvulsant property in compound 4a due
to its lipophilic character. Similar results were obtained in
our previous study on 1,2,4-triazine-substituted derivatives
in which lipophilic substituents such as chloro atom and
methyl group at para position of phenyl ring showed higher
anticonvulsant action (Irannejad et al., 2014). Presence of a
suitable substitution at thiol group is necessary for opti-
mum antiseizure property. According to our previous study
on new antiepileptic compounds, 3-pyridylmethyl or
4-pyridylmethyl groups could increase antiepileptic char-
acter of compounds and are preferred over methyl group or
non-substituted compounds at thiol groups (compounds
1a,b and 3). Investigation of structure–activity relationship
of 5,6-diaryl-1,2,4-triazine derivatives as new anticonvul-
sant compounds has shown that incorporation of groups
capable of constituting polar interactions such as hydrogen
bonds (in this case, nitrogen atom of pyridine ring) can
substantially increase the anticonvulsant effect.
S
X
2a-d
4a,b
Compound
X
R
% protection in PTZ dose
MES
(mg/kg)
2a
F
F
3-Pyridylmethyl 20
41
2b
4-Pyridylmethyl 58**
52
2c
Br 3-Pyridylmethyl 60**
Br 4-Pyridylmethyl 80**
70*
59
2d
4a
–
–
F
3-Pyridylmethyl 71**
4-Pyridylmethyl 40
56
4b
66
1a
H
40
43
1b
Br H
34
47
3
–
–
H
–
0
33
Control
0
45
Lamotrigine –
–
100**
82***
p values are related to each group compared with the control group
* p \ 0.05 significant difference compared with the control group;
** p \ 0.01 significant difference compared with the control group;
*** p \ 0.005 significant difference compared with the control group
Molecular docking study
test among all compounds. The required dose of PTZ to
induce seizure in control group was 45 mg/kg and in group
receiving the reference drug lamotrigine was 82 mg/kg.
Compound 4b was in the second order of protective effect
and required 66 mg/kg of PTZ to induce seizure. The other
compounds had less anticonvulsive effect especially com-
pounds 3 and 1a,b without the pyridylmethyl side chain on
thiol group.
In order to find out their possible mechanism of anticon-
vulsion, molecular docking was performed on the two main
targets of conventional anticonvulsant drugs, Na? channel
and GABAA receptor. Compound 2d was chosen for this
purpose due to its high anticonvulsive property.
Na? channel is a multisubunit complex. Channel’s pore
and especially the principal drug-binding site are composed
of a-subunit with approximately 2,000 amino acids. Four
highly homologous domains (I–IV) make the a-subunit,
and each domain is composed of six presumably trans-
membrane a-helical segments (S1–S6), assembled in a
clockwise pattern around the central pore. This pore has a
large outer vestibule, formed by the four extracellular P
loop segments. These P loops fold back into the membrane
to make a shallow funnel so that in deeper part becomes
The results of MES-induced seizure test are also shown
in Table 1. Control group (receiving PEG 30 % in water)
did not show any protective effect against MES-induced
seizures, while group receiving the reference drug lamo-
trigine revealed 100 % protection. Among compounds
tested, compound 2d with bromine atom at phenyl rings
had the highest protective effect up to 80 % with p value
less than 0.01. Compounds 4a, 2c, and 2b could protect
animals up to 71, 60, and 58 %, respectively (p value
\0.01). Similar to PTZ-induced seizure test, compounds 3
and 1a,b without the pyridylmethyl side chain on thiol
group had the least protective effect against MES-induced
˚
narrow and makes the selectivity filter with 4 9 6 A size.
The large outer vestibule of this pore was used as
binding site for docking of anticonvulsant drugs. After
docking calculation, compound 2d was located in the
middle of sodium channel and could occlude the inner pore
123

Med Chem Res
Fig. 4 Binding mode of compound 2d in a1–c2 subunits interface.
The a-subunit is shown in blue and c-subunit in red (Color figure
online)
Fig. 3 Location of compound 2d inside the inner pore of the Na?
Channel (top view). The backbones of S6 a-helices of domains I–IV
are shown by red, blue, green, and yellow ribbons, respectively. The
same colors are used for amino acid residues in each domain (Color
figure online)
binding site on GABAA receptor to exert their antiseizure
action as revealed by the binding free energy calculation.
However, other important anticonvulsant targets such as
glutamate receptors and calcium channels are not evaluated
here, but it remains to be cleared in our next study.
as shown in Fig. 3. Binding mode of compound 2d showed
that 4-bromophenyl moieties of this compound are inter-
acting with Phe91 and Leu88 of IIS6 and IIIS6 and Phe84
and Val88 in IVS6 through hydrophobic and p–p interac-
tions. Pyridyl ring has twisted toward Val87, Val88, and
Tyr91 of IVS6 and Phe84 from IS6 and constitutes van der
waals interactions mainly by its aromatic p electrons.
Finally, triazine ring is hydrogen bonded to Asn88 side
chain in IS6. The estimated free energy of binding to
sodium channel was -6.38 kcal/mol.
In vitro 15-LOX inhibition assay
As discussed earlier in this section, 15-lipoxygenase (15-
LOX) is a key enzyme in initiation of oxidative stress in
neurodegenerative diseases. Thus, inhibition of 15-LOX by
synthesized 1,2,4-triazine-substituted derivatives was
assessed, and the results are shown in Table 2. All com-
pounds showed mild to moderate inhibition of 15-LOX at
about 100 lM, while quercetin as a reference drug inhib-
ited the 15-LOX at 18 lM. Compound 2c with highest
anticonvulsive property in PTZ test could inhibit 15-LOX
at 89 lM which was again the best one among compounds.
There was not a significant difference in potency among
compounds against 15-LOX, and the IC50 values were
GABAA receptor is one of the main targets for anticonvul-
sive drugs such as benzodiazepines. The majority of GABAA
receptors are composed of a, b, and c-subunits. The benzodi-
azepine-binding site of these receptors is located extra-cellu-
larly at a1–c2 interface. Molecular docking was performed on
the benzodiazepine-binding site of GABAA receptor.
Table 2 The IC50 values of the target compounds against soybean
15-lipoxygenase
Binding mode of compound 2d is visualized in Fig. 4.
As shown, one 4-bromophenyl part of ligand is interacted
with Tyr159 and His101 at a1-subunit, while the other
4-bromophenyl group is in contact with a1-Val202 and
Phe77 at d2-subunit. Triazine ring has p–p stacking with
a1-Tyr209, and the protonated nitrogen atom of pyridine
ring at physiological pH is ionic bonded to d2-Arg132. The
free energy of binding was estimated to be -11.74 kcal/
mol, in benzodiazepine-binding site in GABAA receptor.
The higher free energy of binding for compound 2d in
GABAA receptor than Na? channel may propose its pos-
sible mechanism of anticonvulsant action through binding
to benzodiazepine-binding site on GABAA receptor. On the
other hand, compound 2d and its congeners (compounds
2a–d and 4a,b) possibly bind to the benzodiazepine-
Compound
15-LOX (IC50 lM)
2a
119.8 7.33
2b
97.88
7
2c
89.3 6.5
105 6.69
98.12 6.98
112 5.57
NA
2d
4a
4b
1a
1b
NA
3
NA
Quercetin
18 0.9
NA not-active
123

Med Chem Res
ranging from 89 to 119 lM due to their high structural
similarity protective effect of compounds against MES and
PTZ-induced seizures along with their moderate inhibition
of 15-LOX may provide synergistic effect in prevention of
neurodegenerative diseases progression and to introduce
new generation of anticonvulsant agents.
maximum allowed value of 4. As seen in this table, number
of total hydrogen bonds for all compounds is less than 10.
However, some predicted values such as logD of com-
pounds 2c,d and TPSA for 4a,b fall a bit out of the range,
but it does not mean that these compounds have lack of
CNS activity and only may reduce their extent of passage
through BBB. Moreover, this fact was further confirmed by
the predicted values of logBB and CNS activity. Finally,
considering the pred. logBB values of compounds 2a–
d and 4a,b reported in Table 3, all of them were predicted
to be CNS active.
Prediction of blood–brain barrier permeability
Blood–brain barrier (BBB) permeability is one of the most
important challenges in the pharmacology of CNS active
drugs. The extent of CNS penetration has limited usage of
many drugs whose target is located in the central nervous
system and high percentage of CNS-acting drugs fail to pass
the clinical trials annually due to the CNS penetration failure.
This drawback has imposed high expenses at pharmaceutical
and drug development companies nowadays. Therefore,
evaluating the capability of new compounds to pass the BBB
by in silico methods would be inevitable and helpful.
Conclusion
Development of novel anticonvulsive drugs has become a
challenge of the new century. Available anticonvulsive
drugs are not effective and responsive in most cases.
Therefore, introduction of novel and multitargeted anticon-
vulsive agents offers new opportunity to overcome convul-
sions. In this study, some new 1,2,4-triazine derivatives were
evaluated as dual anticonvulsive and 15-lipoxygenase
inhibitors. Molecular docking study revealed their possible
antiseizure mechanism of action through GABAA receptor.
Finally, in silico assessment of their BBB permeability
indicated them as CNS active agents. Future studies of these
novel neuroprotective inhibitors of 15-LOX may provide
new therapeutic opportunities to combat convulsion and
neurodegenerative diseases.
Several ‘‘rules of thumb’’ have been developed for
prediction of passive BBB permeability. Compounds are
more likely to penetrate the BBB if the following criteria
meet. (1) Molecular weight (MW) less than 400–500 Da.
(2) LogD (logarithm of distribution coefficient) between
-1 and 4. (3) Number of total hydrogen bonds (nHB) less
2
˚
than 10. (4) Polar surface area (TPSA) less than 90 A .
Increasing the number of rules which are not obeyed by
one compound decreases the possibility of its BBB per-
meability (Waterbeemd et al., 1998).
The calculated physiochemical parameters for com-
pounds 2a–d and 4a,b are shown in Table 3 along with
their predicted values of logBB (logarithm of brain/plasma
concentration) and CNS activity by ACD/Lab. As shown,
values of MW are almost in the defined range (\500 Da),
but with a minor and negligible increase for compounds 2c
and 2d, but predicted logD values for these two compounds
(5.96 and 5.94, respectively) are located a bit far from the
Acknowledgments We gratefully acknowledge the financial sup-
port from the Research Council of Mazandaran University of Medical
Sciences, Sari, Iran.
References
Acharya MM, Hattiangady B, Shetty AK (2008) Progress in neuroprotec-
tive strategies for preventing epilepsy. Prog Neurobiol 84:363–404
Assadieskandar A, Amini M, Salehi M, Sadeghian H, Alimardani M,
Sakhteman A, Nadri H, Shafiee A (2012) Synthesis and SAR study
of 4,5-diaryl-1H-imidazole-2(3H)-thione derivatives as potent
15-lipoxygenase inhibitors. Bioorg Med Chem 20:7160–7166
Congreve M, Andrews SP, Dore AS, Hollenstein K, Hurrel E,
Langmead CJ, Mason JS, Ng IW, Tehan B, Zhukov A, Weir M,
Marshall FH (2012) Discovery of 1,2,4-triazine derivatives as
adenosine A2A antagonists using structure based drug design.
J Med Chem 55:1898–1903
Devi PU, Manocha A, Vohora D (2008) Seizures, antiepileptics,
antioxidants and oxidative stress: an insight for researchers.
Expert Opin Pharmacother 9:3169–3177
Irannejad H, Amini M, Khodaghi F, Ansari N, Khoramian Tusi S,
Sharifzadeh M, Shafiee A (2010) Synthesis and in vitro eval-
uation of novel 1,2,4-triazine derivatives as neuroprotective
agents. Bioorg Med Chem 18:4224–4230
Table 3 Calculated physicochemical parameters for compounds 2a–
d and 4a,b and prediction of their CNS activity
Compound MW logDa
(pH 7.4)
nHBa TPSAa pred.
logBBa activitya
pred. CNS
2a
2b
2c
2d
4a
4b
392.4 4.11
4
4
4
4
4
4
76.8 -0.25
Active
Active
Active
Active
Active
Active
392.4 4.09
514.2 5.96
514.2 5.94
368.5 4.3
368.5 4.28
76.8 -0.26
76.8 -0.25
76.8 -0.26
133.3 -0.41
133.3 -0.41
nHB number of total hydrogen bonds (acceptors ? donors), logD
logarithm of distribution coefficient, logBB logarithm of brain/plasma
concentration
Irannejad H, Naderi N, Emami S, Foroumadi A, Qobadi Ghadikolaei
R, Zafari T, Mazar-Atabaki A, Dadashpour S (2014) Microwave
a
Calculated or predicted by ACD/Lab (www.acdlab.com)
123

Med Chem Res
assisted synthesis and anticonvulsant activity of 5,6-bisaryl-
Richter L (2012) Diazepam-bound GABA receptor models identify new
benzodiazepine binding-site ligands. Nat Chem Biol 8:455–464
Siddiqui N, Ahsan W (2010) Triazole incorporated thiazoles as a new
class of anticonvulsants: design, synthesis and in vivo screening.
Eur J Med Chem 45:1536–1543
Stanislaw C, Renata F, Barbara B, Kinga KB (2007) Neuroprotective
effects of some newer and potential antiepileptic drugs. J Pre-
Clin Clin Res 1:1–5
1,2,4-triazine-3-thiol derivatives. Med Chem Res 23:2503–2514
Landmark CJ (2007) Targets for antiepileptic drugs in the synapse.
Med Sci Monit 13:RA1–RA7
Lipkind GM, Fozzard HA (2010) Molecular model of anticonvulsant
drug binding to the voltage-gated sodium channel inner pore.
Mol Pharmacol 78:631–638
Litchfield J, Wilcoxon F (1949) A simplified method of evaluating
dose-effect experiments. J Pharmacol Exp Ther 96:99–113
Loscalzo J (2008) Membrane redox state and apoptosis: death by
peroxide. Cell Metab 8:182–183
Van Leyen K, Kim HY, Lee SR, Jin G, Arai K, Lo EH (2006)
Baicalein and 12/15-lipoxygenase in the ischemic brain. Stroke
37:3014–3018
Naderi N, Ahmad-Molaei L, Aziz Ahari F, Motamedi F (2011)
Modulation of anticonvulsant effects of cannabinoid compounds
by GABA-A receptor agonist in acute pentylenetetrazole model
of seizure in rat. Neurochem Res 36:1520–1525
Waterbeemd HV, Camenisch G, Folkers G, Chretien JR, Raevsky OA
(1998) Estimation of blood-brain barrier crossing of drugs using
molecular size and shape, and H-bonding descriptors. J Drug
Target 6:151–165
Naegele JR (2007) Neuroprotective strategies to avert seizure-induced
neurodegeneration in epilepsy. Epilepsia 48:107–117
Pratico D, Zhukareva V, Yao Y, Uryu K, Funk CD, Lawson JA,
Trojanowski JQ, Lee VM (2004) 12/15Lipoxygenase is
increased in Alzheimer’s disease: possible involvement in brain
oxidative stress. Am J Pathol 164:1655–1662
Woodbury LA, Davenport VD (1952) Design and use of a new
electroshock seizure apparatus, and analysis of factors altering
seizure threshold and pattern. Arch Int Pharmacodyn Ther 92:97–107
Woodbury LA, Swinyard CA (1952) Stimulus parameters for
electroshock seizures in rats. Am J Physiol 170:661–667
123