Synthesis and evaluation of the anticonvulsant activities of 4-(2-(alkylthio)benzo[d]oxazol-5-yl)-2,4-dihydro-3H-1,2,4-triazol-3-ones

Article abstract of DOI:10.3390/molecules23040756

In this study, a novel series of 4-(2-(alkylthio)benzo[d]oxazol-5-yl)-2,4-dihydro-3H-1,2,4-triazol-3-ones (4a–m) was designed and synthesized. The anticonvulsant activities of these compounds were evaluated by using the maximal electroshock seizure (MES) and subcutaneous pentylenetetrazole (scPTZ) seizure models in mice. The neurotoxicity of these compounds was evaluated using the rotarod neurotoxicity test. The majority of compounds showed anti-MES activities at 100 or 300 mg/kg. Compound 4g was considered to be the most promising, based on its potency against MES- and PTZ-induced seizures with ED₅₀ values of 23.7 and 18.9 mg/kg, respectively. The TD₅₀ value of 4g was 284.0 mg/kg, which resulted in a higher protective index (PI = TD₅₀/ED₅₀) value than that of carbamazepine and valproate. In an ELISA test, compound 4g significantly increased the γ-aminobutyric acid (GABA) content in mouse brain. In addition, pretreatment with thiosemicarbazide (an inhibitor of the GABA synthesizing enzyme) significantly decreased the activity of 4g in the MES model, which suggests that the mechanism through which compound 4g elicits its anticonvulsive action is at least in part through increasing the GABA level in the brain.

Full text of DOI:10.3390/molecules23040756

molecules
Article
Synthesis and Evaluation of the Anticonvulsant
Activities of 4-(2-(Alkylthio)benzo[d]oxazol-5-yl)-
2,4-dihydro-3H-1,2,4-triazol-3-ones
Ming-Xia Song 1, Zhen-Yuan Wang 2, Shi-Hui He 1, Sheng-Wang Yu 1, Shi-Long Chen 1,
Dong-Fu Guo 1, Wen-Hao Zhao 1 and Xian-Qing Deng 1,
*
1
Medical College, Jinggangshan University, No. 28, Xueyuan Road, Ji’an 343009, Jiangxi, China;
freexiaoxiao83@aliyun.com (M.-X.S.); 18770606620@163.com (S.-H.H.); ysw1779326748@163.com (S.-W.Y.);
chensl@126.com (S.-L.C.); guoran147369@163.com (D.-F.G.); Zhaowh@126.com (W.-H.Z.)
Siping Institute of Food and Drug Control, No. 357, Nanhu Road, Sipin 136000, Jilin, China;
sy6180343@sina.com
2
*
Correspondence: xqdeng@jgsu.edu.cn; Tel.: +86-796-8117893
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 1 March 2018; Accepted: 23 March 2018; Published: 25 March 2018
Abstract: In this study, a novel series of 4-(2-(alkylthio)benzo[d]oxazol-5-yl)-2,4-dihydro-3H-1,2,4-
triazol-3-ones (4a–m) was designed and synthesized. The anticonvulsant activities of these
compounds were evaluated by using the maximal electroshock seizure (MES) and subcutaneous
pentylenetetrazole (scPTZ) seizure models in mice. The neurotoxicity of these compounds was
evaluated using the rotarod neurotoxicity test. The majority of compounds showed anti-MES
activities at 100 or 300 mg/kg. Compound 4g was considered to be the most promising, based
on its potency against MES- and PTZ-induced seizures with ED50 values of 23.7 and 18.9 mg/kg,
respectively. The TD50 value of 4g was 284.0 mg/kg, which resulted in a higher protective index
(PI = TD50/ED50) value than that of carbamazepine and valproate. In an ELISA test, compound
4g significantly increased the γ-aminobutyric acid (GABA) content in mouse brain. In addition,
pretreatment with thiosemicarbazide (an inhibitor of the GABA synthesizing enzyme) significantly
decreased the activity of 4g in the MES model, which suggests that the mechanism through which
compound 4g elicits its anticonvulsive action is at least in part through increasing the GABA level in
the brain.
Keywords: anticonvulsant; maximal electroshock; pentylenetetrazole; benzoxazole; triazolone
1. Introduction
Epilepsy, a group of neurological disorders characterized by epileptic seizures, afflicts over
50 million people worldwide [1,2]. The cause of most cases of epilepsy is unknown. The accepted
pathogenesis of epileptic seizures is the imbalance of excitatory and inhibitory neurotransmitters
in the central nervous system, which leads to abnormal nerve cell activity and neuronal discharge,
resulting in seizures [3]. Until now, anticonvulsants (also commonly known as antiepileptic drugs or
antiseizure drugs), the “symptomatic” agents that suppress the symptoms of epilepsy (i.e., seizures),
have been the main strategy for the treatment of epilepsy. However, the available anticonvulsants
are only effective in reducing the severity and number of seizures in <70% of patients. Furthermore,
the use of anticonvulsants is often associated with numerous undesirable side effects [
life-threatening conditions [ ]. Research to identify more effective and safer antiepileptic drugs is
therefore an imperative yet challenging task for medicinal chemistry.
In our previous study, we reported the synthesis and anticonvulsant activity of 2-thioalkyl-5-(4H-
4,5] and even
6
1,2,4-triazol-4-yl)benzo[d]oxazoles (Figure 1,
I) [7]. This series of compounds exhibited potent
Molecules 2018, 23, 756; doi:10.3390/molecules23040756
www.mdpi.com/journal/molecules

Molecules 2018, 23, 756
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anticonvulsant activities. Among them, 2-(3-fluobenzyl)thio-5-(4H-1,2,4-triazol-4-yl)benzo[d]oxazole
was the most potent with an ED50 value of 11.4 and 31.7 mg/kg in maximal electroshock seizure (MES)
and subcutaneous pentylenetetrazole induced seizure (sc-PTZ) models, respectively. Preliminary
research investigating the mechanism of action suggested that the GABAergic system may contribute
to the anticonvulsive action of this compound. As a continuation of the above study, in the current
study, a series of benzoxazole derivatives containing triazolone (4a–m) were designed and synthesized,
which replaced the triazole in compounds of with triazolone. This design is based on a hypothesis
I
that the carbonyl group in the triazolone may increase the affinity to the receptor, and thus increase
the anticonvulsant activity, which has already been confirmed in previous studies [8,9].
Figure 1. The leading compounds and target compounds (4a–m).
MES and scPTZ models, along with a toxicity screen (rota-rod in mice), were employed to screen
the new anticonvulsants. These are the most widely used animal models in the discovery of new
anticonvulsant drugs at the initial stage [10,11]. To further confirm the anticonvulsant activity of 4g,
it was also tested in 3-mercaptopropionic acid (3-MP), and bicuculline (BIC)-induced seizure models.
In addition, studies investigating the involvement of the GABAergic system in the anticonvulsant
mechanism of 4g were also performed.
2. Results and Discussion
2.1. Chemistry
The target compounds were synthesized according to Scheme 1. The starting material, 2-amino-
5-nitrophenol, was reacted with CS2 to obtain compound
in ethanol to obtain compound 12]. Alkylation of compound
alkylating agents produced compounds , which were then treated with triethyl orthoformate
and methyl hydrazine carboxylate in the presence of sodium methoxide to give rise to the target
1
, which was reduced further by Na2S
·
9H2O
2
[
2
with a variety of different
3
compounds 4a–m [
13]. The chemical structures of these compounds were characterized using 1H-NMR,
13C-NMR, and mass spectrum (MS) analysis techniques. A detailed overview of the physical and
analytical data from these compounds has been provided in the Materials and Methods section.
Scheme 1. The synthesis route of target compounds (4a–m).

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2.2. Anticonvulsant Activity in MES and scPTZ Models and Neurotoxicity in the Rota-Rod Test
MES and scPTZ models, along with toxicity screen (rota-rod in mice), were employed to screen
the new compounds [10 11]. Compounds 4a were initially administered at doses of 30, 100 and
300 mg/kg. As shown in Table 1, compounds 4a and 4e displayed noticeable anticonvulsant activity
,
–m
–
i
at a dosage of 100 mg/kg in the MES model, with some compounds remaining active after 4 h.
Compounds 4b, 4j and 4k displayed weak activity at a dosage of 300 mg/kg in the MES model. In the
scPTZ model, compounds 4a, 4h and 4l protected the mice from sc-PTZ induced seizure at a dosage
of 300 mg/kg. Compounds 4e and 4f prevented seizures at a dose of 100 mg/kg in the scPTZ model
at 0.5 h, and remained active after 4 h at the same dose. Compound 4g was the most promising
compound. Compound 4g prevented seizures at a dose of 30 mg/kg in both models, though it was
also the only compound that showed neurotoxicity at a dose of 300 mg/kg. In the toxicity screen,
all compounds except 4g showed no neurotoxicity at a dose of 300 mg/kg. Additionally, common side
effects of anticonvulsant drugs, such as sedation, hypnosis, and anxiety (with features of bradykinesia
or running), were not observed for any of the compounds at a dose of 100 mg/kg. However, at a
higher dose of 300 mg/kg, compounds 4e–4f caused sedation in the test animals to a certain extent.
Table 1. Anticonvulsant activity and neurotoxicity of compounds 4a–m administered intraperitoneally
to mice.
Intraperitioneal Injection in Mice a
scPTZ Screening c
NT Screening d
Compds.
MES Screening b
R
0.5 h
4 h
0.5 h
4 h
0.5 h
4 h
4a
4b
4c
4d
4e
4f
4g
4h
4i
C3H7
C5H9
C7H15
CH2C6H5
100
300
-
300
-
-
-
-
300
-
-
-
100
100
30
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CH2C6H4(2-F)
CH2C6H4(3-F)
CH2C6H4(4-F)
CH2C6H4(2-Cl)
CH2C6H4(3-Cl)
CH2C6H4(4-Cl)
CH2C6H3(2,4-Cl2)
CH2C6H4(4-CH3)
CH2C6H4(4-OCH3)
100
100
100
100
100
300
300
300
-
100
100
30
300
-
-
30
100
100
-
300
300
-
-
-
-
-
-
-
-
-
-
-
-
4j
4k
4l
-
-
-
300
-
300
-
4m
a
Number of animal used = 3–5, Doses of 15, 30, 100 and 300 mg/kg were administered. The figure in the table
indicates the minimum dose whereby bioactivity was demonstrated in half or more of the mice. The animals were
examined at 0.5 and 4 h after injection was administered. A dash indicates the absence of anticonvulsant activity
and neurotoxicity at the maximum dose administered (300 mg/kg); b Maximal electroshock test; c Subcutaneous
pentylenetetrazole test; d Neurotoxicity screening (rotarod test).
From the data in Table 1, it can be seen that some compounds, including 4g, remained active
until the 4 h time point. This indicates that these compounds possessed anticonvulsant activity with
protective effects for a long duration. To evaluate the anticonvulsant activity of 4g more accurately,
the time to peak effect (TPE) was tested. Compound 4g reached the TPE at 2.5 h after i.p. administration
(Figure 2). Therefore, the 2.5 h time interval was chosen as the assessment time for compound 4g in
further quantification tests.

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Figure 2. Time-course of compound 4g (50 mg/kg) in the maximal electroshock seizure test (i.p.).
The data illustrates % animal protected relative to animal tested (10 for each point) at various times
after injection.
Based on the considerable anticonvulsant activity of 4g that was demonstrated in the preliminary
screens, further quantitative evaluation trials for the quantification of anticonvulsant activity (indicated
by ED50) and neurotoxicity (indicated by TD50) in mice were performed. The ED50 of 4g was measured
as 23.7 mg/kg in the MES model (Table 2). Compound 4g showed a higher activity than the positive
drugs carbamazepine and valproate in the scPTZ model, with an ED50 of 18.9 mg/kg. The TD50 of 4g
was measured as 284.0 mg/kg, which resulted in PI values of 12.0 and 15.0 for the MES and scPTZ
models, respectively. Compound 4g showed superior performance to phenobarbital and valproate in
terms of safety.
Table 2. Quantitative anticonvulsant date of 4g (anti-MES and anti-PTZ) in mice administered
intraperitoneally.
ED50
PI c
a
b
Compounds
TD50
MES
scPTZ
MES
scPTZ
4g
23.7 (21.5–26.0)
9.8 (8.9–10.8)
264 (247–338)
18.9 (17.2–20.8)
>100
149 (123–177)
284.0 (258.2–312.4)
44.0 (40.2–48.1)
418 (369–450)
12.0
4.5
1.6
15.0
<0.44
2.8
Carbamazepine
Valproate
a
Ten animals were used for each group; polyethylene glycol 400 (0.1 mL, i.p.) was used as solvent. ED50—median
effective dose required to assure anticonvulsant protection in 50% animals; b TD50—median toxic dose eliciting
minimal neurological toxicity in 50% animals; c PI—protective index (TD50/ED50).
2.3. Anticonvulsant Activity Evaluation of Compound 4g in Other Seizure Models
To further evaluate the anticonvulsive ability of 4g, 3-mercaptopropionic acid (3-MP)- and
bicuculline (BIC)-induced seizure tests were carried out. The 3-MP is a competitive inhibitor of
glutamate decarboxylase (GAD) that can inhibit the synthesis of GABA, decreasing the level of this
neurotransmitter in the brain [14]. 3-MP at a dose of 60 mg/kg induced convulsions in 100% of the
mice (Table 3). Combined treatment with 3-MP and 4g led to a significant decrease in tonic convulsions
and death when compared to the results following administration of 3-MP alone (from 100% to 20%,
p < 0.01; and from 60% to 30%, p < 0.05, for convulsions and death, respectively). BIC is a competitive
GABAA receptor antagonist that is known to cause seizures in mice [15]. BIC induced convulsions in
100% of the mice at a dose of 5.4 mg/kg. As shown in Table 4, carbamazepine inhibited tonic seizure
and death significantly. Comparably, compound 4g also inhibited tonic seizure and death significantly
(from 100% to 0, p < 0.001; and from 100% to 30%, p < 0.01, for convulsions and death, respectively).
The efficacy of compound 4g in inhibiting tonic seizures and death induced by 3-MP and BIC, at a
level comparable with carbamazepine, further confirmed the antiepileptic action of 4g.

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Table 3. Effect of compound 4g on 3-mercaptopropionic-acid-induced seizures in mice.
Doses
(mg/kg) Time (h)
Test
Clonic
Seizures (%)
Tonic
Seizures (%)
Lethality
(%)
Compound
DMSO
Carbamazepine
4g
-
30
30
0.5
0.5
0.5
100
100
100
100
0 ***
20 **
60
0 **
30 *
Results are expressed as a percentage of animals that showed clonic and tonic convulsions and death among all
animals tested. Ten mice were included in each group. Significance determined with Fisher’s exact test. * p < 0.05,
** p < 0.01, *** p < 0.001 when vs. control group (3-MP, 60 mg/kg).
Table 4. Effect of compound 4g on Bicuculline-induced seizures in mice.
Doses
(mg/kg) Time (h)
Test
Clonic
Seizures (%)
Tonic
Seizures (%)
Lethality
(%)
Compound
DMSO
Carbamazepine
4g
-
30
30
0.5
0.5
0.5
100
100
100
100
0 ***
0 ***
100
20 **
30 **
Results are expressed as a percentage of animals that showing clonic and tonic convulsions and death among all
animals tested. The number of animals tested in each group was ten. Significance determined with Fisher’s exact
test. ** p < 0.01, *** p < 0.001 when vs. control group (BIC, 5.4 mg/kg).
2.4. The Role of the GABAergic System in the Anticonvulsant Mechanism of 4g
In our previous study, the GABAergic system was involved in the anticonvulsive action of the
compounds I [7]. Because of the structural similarity between the target compounds and the previous
compounds, there is reason to believe that the mechanism through which these compounds produce
an anticonvulsive action also involves the GABAergic system. An ELISA assay was performed
to investigate the effects of 4g on the GABA content in the mouse brain. As shown in Figure 3,
compound 4g and phenytoin significantly increased the GABA content in the mouse brain when
compared to the results from the control group (p < 0.01 and p < 0.01, following treatment with 4g and
phenytoin respectively).
Figure 3. Effect of 4g and phenytoin on whole brain GABA concentration in mice. Data are presented
as mean ± SEM. of six animals. * p < 0.05, ** p < 0.01 vs. control (animals treated with saline).
To further confirm the anticonvulsant mechanism of 4g, the effects of thiosemicarbazide (TSC)
on the anticonvulsive action of 4g were determined. As shown in Figure 4, pretreatment with TSC
(25 mg/kg/day for 3 days) significantly decreased the seizure-preventing action of 4g in the MES

Molecules 2018, 23, 756
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model. TSC is a competitive inhibitor of the GABA synthesis enzyme, glutamate decarboxylase (GAD),
and it inhibits the synthesis of GABA, resulting in a decrease in the GABA level in the brain [16].
The above results further confirmed that the mechanism through which compound 4g produces its
anticonvulsive action is at least in part through increasing the GABA level in the brain.
Figure 4. Effect of the thiosemicarbazide (TSC) on the anticonvulsive action of 4g against maximal
electroshock-induced seizures in mice. All data are presented as median effective dose (ED50
All drugs were administered i.p. in a single injection, 30 min before maximal electroshock-induced
)
±
SEM.
seizures; *** p < 0.001 vs. control group (pretreatment with saline).
3. Materials and Methods
3.1. Instruments and Reagents
All of the reagents and solvents were purchased from Aladdin (Shanghai, China) or Sinopharm
Chemical Reagent Co. Ltd. (Shanghai, China), and were used as received. Melting points were
determined in open capillary tubes and are uncorrected. Reaction courses were monitored by thin-layer
chromatography on silica gel-precoated F254 plates (Merck, Darmstadt, Germany). Developed plates
were examined with UV lamps (254 nm). Nuclear magnetic resonance spectroscopy was performed on
an AV-300 spectrometer (Bruker, Zurich, Switzerland) operating at 300 MHz for 1H and 75 MHz for 13
C
and using DMSO-d6 as solvent and tetramethylsilane as the internal standard. An MALDI-TOF/TOF
mass spectrometer (Bruker Daltonik, Bremen, Germany) was used to measure the high resolution
mass spectroscopy.
3.2. Synthesis Method and Spectral Data
3.2.1. General Procedure for the Preparation of Intermediates 1, 2 and 3
Intermediates 1, 2 and 3 were synthesized according to the literature [7].
3.2.2. General Procedure for the Preparation of Target Compounds 4a–m
Compounds
3 (10 mmol), methyl hydrazinocarboxylate (50 mmol), and triethyl orthoformate
(50 mmol) were placed into a round-bottomed flask containing 30 mL anhydrous alcohol and refluxed
for 24 h. After cooling, sodium methylate (50 mmol) was added and refluxed. Following completion
of the reaction (the mixtu◦re turning pink is a sign of completion), the mixture was cooled down
to room temperature (20 C) and diluted in 120 mL water and neutralized with HCl solution to
pH 5–6. The precipitate that formed was filtered and washed with water, which was then purified by
recrystallization with ethanol to obtain compounds 4a–m.

Molecules 2018, 23, 756
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3.2.3. Spectral Data
4-(2-(Propylthio)benzo[d]oxazol-5-yl)-2H-1,2,4-triazol-3(4H)-one (4a). White solid; m.p. 202 ◦C, yield 31%.
1H-NMR (DMSO-d6, 300 MHz):
1.01 (t, 3H, J = 7.3 Hz, CH2CH3), 1.78–1.86 (m, 2H, CH2CH3), 3.32 (t,
2H, J = 7.1 Hz, SCH2), 7.63 (d, 1H, J = 7.4 Hz, Ph-H), 7.75 (d, 1H, J = 7.4 Hz, Ph-H), 7.94 (s, 1H, Ph-H),
δ
8.38 (s, 1H, CH=N), 11.99 (s, 1H, CO-NH). 13C-NMR (DMSO-d6, 75 MHz):
δ 166.8, 153.5, 150.1, 142.2,
137.1, 131.3, 118.6, 112.3, 110.9, 34.1, 22.8, 13.4. IR (KBr) cm−1: 3208 (N-H), 1703 (C=O). ESI-HRMS
calcd. for C12H13N4O2S+ ([M + H]+): 277.0754; found: 277.0761.
4-(2-(Pentylthio)benzo[d]oxazol-5-yl)-2H-1,2,4-triazol-3(4H)-one (4b). White solid; m.p. 206–207 ◦C, yield
1
63%. H-NMR (DMSO-d6, 300 MHz):
δ 0.87 (t, 3H, J = 6.8 Hz, CH2CH3), 1.31–1.37 (m, 4H, (CH2)2CH3),
1.75–1.80 (m, 2H, SCH2CH2), 3.32 (t, 2H, J = 7.0 Hz, SCH2), 7.62 (d, 1H, J = 8.5 Hz, Ph-H), 7.75
(d, 1H, J = 8.5 Hz, Ph-H), 7.94 (s, 1H, Ph-H), 8.38 (s, 1H, CH=N), 12.00 (s, 1H, CO-NH). 13C-NMR
(DMSO-d6, 75 MHz):
δ 166.8, 153.5, 150.1, 142.2, 137.1, 131.3, 118.6, 112.3, 110.9, 32.2, 30.6, 29.0, 22.1,
14.3. IR (KBr) cm−1: 3207 (N-H), 1702 (C=O). ESI-HRMS calcd for C14H17N4O2S+ ([M + H]+): 305.1067;
found: 305.1076.
4-(2-(Heptylthio)benzo[d]oxazol-5-yl)-2H-1,2,4-triazol-3(4H)-one (4c). White solid; m.p. 201–202 ◦C, yield
1
53%. H-NMR (DMSO-d6, 300 MHz):
δ 0.85 (t, 3H, J = 6.4 Hz, CH2CH3), 1.26–1.41 (m, 8H, (CH2)4CH3),
1.73–1.89 (m, 2H, SCH2CH2), 3.33 (t, 2H, J = 7.2 Hz, SCH2), 7.63 (dd, 1H, J1 = 8.7 Hz, J2 = 1.8 Hz, Ph-H),
7.74 (d, 1H, J = 8.7 Hz, Ph-H), 7.94 (d, 1H, J = 1.8 Hz, Ph-H), 8.38 (s, 1H, CH=N), 11.96 (s, 1H, CO-NH).
13C-NMR (DMSO-d6, 75 MHz):
δ 166.8, 153.5, 150.0, 142.2, 137.1, 131.3, 118.4, 112.2, 110.9, 32.3, 31.6,
29.3, 28.6, 28.4, 22.5, 14.4. IR (KBr) cm−1: 3206 (N-H), 1701 (C=O). ESI-HRMS calcd for C16H21N4O2S+
([M + H]+): 333.1380; found: 333.1373.
4-(2-(Benzylthio)benzo[d]oxazol-5-yl)-2H-1,2,4-triazol-3(4H)-one (4d). White solid; m.p. 185–186 ◦C, yield
1
28%. H-NMR (DMSO-d6, 300 MHz):
δ 4.63 (s, 2H, SCH2), 7.28–7.52 (m, 5H, benzyl-H), 7.66 (dd, 1H,
J1 = 8.5 Hz, J2 = 1.6 Hz, Ph-H), 7.76 (d, 1H, J = 8.5 Hz, Ph-H), 8.05 (d, 1H, J = 1.6 Hz, Ph-H), 8.42 (s, 1H,
CH=N), 12.07 (s, 1H, CO-NH). 13C-NMR (DMSO-d6, 75 MHz):
δ 165.5, 153.4, 151.6, 140.2, 137.0, 136.9,
130.9, 129.5, 129.1, 128.2, 119.0, 118.9, 104.8, 36.1. IR (KBr) cm−1: 3192 (N-H), 1703 (C=O). ESI-HRMS
calcd for C16H13N4O2S+ ([M + H]+): 325.0754; found: 325.0746.
◦
4-(2-(2-Fluorobenzylthio)benzo[d]oxazol-5-yl)-2H-1,2,4-triazol-3(4H)-one (4e). White solid; m.p. 198–199 C,
yield 41%. H-NMR (DMSO-d6, 300 MHz):
1
δ 4.67 (s, 2H, SCH2), 7.16–7.38 (m, 3H, benzyl-H), 7.46–7.61
(m, 1H, benzyl-H), 7.66 (dd, 1H, J1 = 8.6 Hz, J2 = 1.6 Hz, Ph-H), 7.78 (d, 1H, J = 8.6 Hz, Ph-H), 7.98
(d, 1H, J = 1.6 Hz, Ph-H), 8.40 (s, 1H, CH=N), 12.00 (s, 1H, CO-NH). 13C-NMR (DMSO-d6, 75 MHz):
δ
165.6, 160.9 (d, 1Jc-f = 243.8 Hz), 153.6, 150.2, 142.1, 137.1, 131.8, 131.6 (d, 2Jc-f = 29.3 Hz), 130.6 (d,
3Jc-f = 8.0 Hz), 125.1 (d, 4Jc-f = 3.4 Hz), 123.9 (d, 3Jc-f = 14.1 Hz), 119.0, 116.0 (d, 2Jc-f = 20.1 Hz), 112.5,
111.1, 30.0. IR (KBr) cm−1: 3190 (N-H), 1705 (C=O). ESI-HRMS calcd for C16H12FN4O2S+ ([M + H]+):
343.0660; found: 343.0652.
◦
4-(2-(3-Fluorobenzylthio)benzo[d]oxazol-5-yl)-2H-1,2,4-triazol-3(4H)-one (4f). White solid; m.p. 220–222 C,
yield 46%. H-NMR (DMSO-d6, 300 MHz):
1
δ 4.66 (s, 2H, SCH2), 7.16–7.64 (m, 4H, benzyl-H), 7.67 (dd,
1H, J1 = 8.6 Hz, J2 = 1.7 Hz, Ph-H), 7.78 (d, 1H, J = 8.6 Hz, Ph-H), 8.07 (d, 1H, J = 1.7 Hz, Ph-H), 8.43 (s,
1H, CH=N), 11.98 (s, 1H, CO-NH). 13C-NMR (DMSO-d6, 75 MHz):
δ
164.9, 160.9 (d, 1Jc-f = 244.5 Hz),
153.5, 151.6, 140.1, 137.0, 131.8 (d, 3Jc-f = 3.5 Hz), 130.9 (d, 2Jc-f = 20.5 Hz), 130.6, 125.1 (d, 4Jc-f = 3.4 Hz),
123.8 (d, 3Jc-f = 14.5 Hz), 119.1, 119.0, 116.0 (d, 2Jc-f = 20.8 Hz), 104.8, 29.9. IR (KBr) cm−1: 3189 (N-H),
1709 (C=O). ESI-HRMS calcd for C16H12FN4O2S+ ([M + H]+): 343.0660; found: 343.0656.
◦
4-(2-(4-Fluorobenzylthio)benzo[d]oxazol-5-yl)-2H-1,2,4-triazol-3(4H)-one (4g). White solid; m.p. 218–219 C,
yield 43%. H-NMR (DMSO-d6, 300 MHz):
1
δ 4.63 (s, 2H, SCH2), 7.15–7.21 (m, 2H, benzyl-H), 7.54–7.59
(m, 2H, benzyl-H), 7.67 (dd, 1H, J1 = 8.6 Hz, J2 = 2.0 Hz, Ph-H), 7.77 (d, 1H, J = 8.6 Hz, Ph-H), 8.05 (d,
1H, J = 2.0 Hz, Ph-H), 8.42 (s, 1H, CH=N), 11.99 (s, 1H, CO-NH). 13C-NMR (DMSO-d6, 75 MHz):
165.4,
162.1 (d, 1Jc-f = 242.7 Hz), 153.6, 151.6, 140.1, 136.9, 133.3 (d, 4Jc-f = 3.0 Hz), 131.6 (d, 3Jc-f = 8.3 Hz), 131.0,
δ

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119.0, 118.9, 116.1 (d, 2Jc-f = 21.4 Hz), 104.7, 35.2. IR (KBr) cm−1: 3186 (N-H), 1710 (C=O). ESI-HRMS
calcd for C16H12FN4O2S+ ([M + H]+): 343.0660; found: 343.0650.
4-(2-(2-Chlorobenzylthio)benzo[d]oxazol-5-yl)-2H-1,2,4-triazol-3(4H)-one (4h). White solid; m.p. 200–201 ◦C,
1
yield 40%. H-NMR (DMSO-d6, 300 MHz):
δ 4.63 (s, 2H, SCH2), 7.36–7.60 (m, 4H, benzyl-H), 7.66 (dd,
1H, J1 = 8.5 Hz, J2 = 1.7 Hz, Ph-H), 7.76 (d, 1H, J = 8.5 Hz, Ph-H), 8.05 (d, 1H, J = 1.7 Hz, Ph-H), 8.41 (s,
1H, CH=N), 12.05 (s, 1H, CO-NH). 13C-NMR (DMSO-d6, 75 MHz):
δ 165.3, 153.4, 151.6, 140.1, 139.7,
136.9, 133.5, 133.4, 131.0, 130.9, 129.3, 128.2, 119.1, 118.9, 104.8, 35.3. IR (KBr) cm−1: 3194 (N-H), 1711
(C=O). ESI-HRMS calcd for C16H12ClN4O2S+ ([M + H]+): 359.0364; found: 359.0372.
4-(2-(3-Chlorobenzylthio)benzo[d]oxazol-5-yl)-2H-1,2,4-triazol-3(4H)-one (4i). White solid; m.p. 188–190 ◦C,
1
yield 34%. H-NMR (DMSO-d6, 300 MHz):
δ 4.64 (s, 2H, SCH2), 7.35–7.50 (m, 3H, benzyl-H), 7.60
(s, 1H, benzyl-H), 7.64 (dd, 1H, J1 = 8.8 Hz, J2 = 1.7 Hz, Ph-H), 7.77 (d, 1H, J = 8.8 Hz, Ph-H), 7.98 (d,
1H, J = 1.7 Hz, Ph-H), 8.39 (s, 1H, CH=N), 11.99 (s, 1H, CO-NH). 13C-NMR (DMSO-d6, 75 MHz):
δ
165.9, 153.5, 150.2, 142.1, 139.8, 137.1, 133.5, 131.4, 130.9, 129.3, 128.2, 128.0, 118.9, 112.4, 111.1, 35.3.
IR (KBr) cm−1: 3197 (N-H), 1712 (C=O). ESI-HRMS calcd for C16H12ClN4O2S+ ([M + H]+): 359.0364;
found: 359.0356.
4-(2-(4-Chlorobenzylthio)benzo[d]oxazol-5-yl)-2H-1,2,4-triazol-3(4H)-one (4j). White solid; m.p. 225–227
1
◦C, yield 54%. H-NMR (DMSO-d6, 300 MHz):
δ 4.63 (s, 2H, SCH2), 7.41 (d, 2H, J = 8.4 Hz, benzyl-H),
7.55 (d, 2H, J = 8.4 Hz, benzyl-H), 7.67 (dd, 1H, J1 = 8.6 Hz, J2 = 1.9 Hz, Ph-H), 7.76 (d, 1H, J = 8.6 Hz,
Ph-H), 8.05 (d, 1H, J = 1.9 Hz, Ph-H), 8.41 (s, 1H, CH=N), 12.02 (s, 1H, CO-NH). 13C-NMR (DMSO-d6,
75 MHz):
δ 165.4, 153.5, 151.6, 140.2, 137.0, 136.3, 132.9, 131.4, 130.9, 129.1, 119.1, 119.0, 104.8, 35.3.
IR (KBr) cm−1: 3195 (N-H), 1711 (C=O). ESI-HRMS calcd for C16H12ClN4O2S+ ([M + H]+): 359.0364;
found: 359.0361.
4-(2-(2,4-Dichlorobenzylthio)benzo[d]oxazol-5-yl)-2H-1,2,4-triazol-3(4H)-one (4k). White solid; m.p.
1
171–172 ◦C, yield 45%. H-NMR (DMSO-d6, 300 MHz):
δ 4.69 (s, 2H, SCH2), 7.41 (dd, 1H, J1 = 8.6 Hz,
J2 = 1.8 Hz, Ph-H), 7.65–7.72 (m, 3H, benzyl-H), 7.76 (d, 1H, J = 8.6 Hz, Ph-H), 8.05 (d, 1H, J = 1.8 Hz,
Ph-H), 8.42 (s, 1H, CH=N), 12.06 (s, 1H, CO-NH). 13C-NMR (DMSO-d6, 75 MHz):
δ 164.8, 153.4, 151.6,
140.0, 136.9, 134.8, 134.0, 133.5, 133.1, 131.1, 129.6, 128.1, 120.5, 119.0, 104.7, 33.8. IR (KBr) cm−1: 3208
(N-H), 1707 (C=O). ESI-HRMS calcd for C16H11Cl2N4O2S+ ([M + H]+): 392.9974; found: 392.9986.
4-(2-(4-Methylbenzylthio)benzo[d]oxazol-5-yl)-2H-1,2,4-triazol-3(4H)-one (4l). White solid; m.p. 230–231 ◦C,
1
yield 55%. H-NMR (DMSO-d6, 300 MHz):
δ 2.27 (s, 3H, Ar-CH3), 4.59 (s, 2H, SCH2), 7.15 (d, 2H,
J = 7.4 Hz, benzyl-H), 7.39 (d, 2H, J = 7.4 Hz, benzyl-H), 7.64 (d, 1H, J = 8.6 Hz, Ph-H), 7.76 (d, 1H,
J = 8.6 Hz, Ph-H), 7.97 (s, 1H, Ph-H), 8.39 (s, 1H, CH=N), 11.99 (s, 1H, CO-NH). 13C-NMR (DMSO-d6,
75 MHz):
δ 166.2, 153.6, 150.1, 142.2, 137.5, 137.1, 133.8, 131.4, 129.6, 129.4, 118.8, 112.4, 111.0, 35.9,
21.2. IR (KBr) cm−1: 3204 (N-H), 1713 (C=O). ESI-HRMS calcd for C17H15N4O2S+ ([M + H]+): 339.0910;
found: 339.0909.
4-(2-(4-Methoxybenzylthio)benzo[d]oxazol-5-yl)-2H-1,2,4-triazol-3(4H)-one (4m). White solid; m.p. 227–228 ◦C,
1
yield 57%. H-NMR (DMSO-d6, 300 MHz):
δ 3.72 (s, 3H, Ar-OCH3), 4.58 (s, 2H, SCH2), 6.90 (d, 2H,
J = 8.4 Hz, benzyl-H), 7.43 (d, 2H, J = 8.4 Hz, benzyl-H), 7.63 (d, 1H, J = 8.7 Hz, Ph-H), 7.65 (d, 1H,
J = 8.7 Hz, Ph-H), 7.97 (s, 1H, Ph-H), 8.40 (s, 1H, CH=N), 12.02 (s, 1H, CO-NH). 13C-NMR (DMSO-d6,
75 MHz):
δ 166.2, 159.3, 153.6, 150.1, 142.2, 137.1, 131.3, 130.8, 128.6, 118.8, 114.5, 112.4, 111.0, 55.5,
35.7. IR (KBr) cm−1: 3202 (N-H), 1714 (C=O). ESI-HRMS calcd for C17H15N4O3S+ ([M + H]+): 355.0859;
found: 355.0867.
3.3. Pharmacology
3.3.1. Animals and Experimental Conditions
Experiments were carried out on KunMing mice that weighed 18–24 g. The mice were housed
collectively in polycarbonate cages, in groups of ten, where they were maintained under a 12 h

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◦
2 C) laboratory with free access to food and
light/dark cycle in a temperature-controlled (25
±
water. Each animal was used only once. Procedures involving animals and their care were conducted
in accordance with the Guide for the Care and Use of Laboratory Animals, 8th Edition, National
Academies Press, Washington, DC, USA. Local ethical committee approval was also obtained
(No. 20170701). Additionally, all efforts were made to minimize animal suffering and to use only the
number of animals necessary to produce reliable scientific data. Electro-convulsions were produced
by an electric stimulation generator (JTC-1, Chengdu, China). The following drugs were used in this
study: pentylenetetrazole, 3-mercaptopropionic acid, thiosemicarbazide, and bicuculline (these drugs
were obtained from Aladdin Industrial Inc., Shanghai, China), valproate sodium, carbamazepine,
phenytoin (these drugs were obtained from melongpharma, Dalian, China).
3.3.2. The Maximal Electroshock (MES) Test
Seizures were elicited in mice with ear stimulation using a 0.2 s 60-Hz 50-mA alternating current.
Protection against the spread of MES-induced seizures was defined as the abolition of the hind limb
tonic extension spasm [17,18].
3.3.3. Subcutaneous Pentylenetetrazole Induced Seizures Test (scPTZ)
Pentylenetetrazole was dissolved in 0.9% saline to allow subcutaneous injections to mice at a dose
of 85 mg/kg. The failure to observe even a threshold seizure (a single episode of clonic spasms of at
least 5 s duration) is defined as protection [19].
3.3.4. Neurotoxicity Screening
The neurotoxicity of the compounds was measured in mice using the rotarod test. The mice were
trained to stay on an accelerating rotarod, with a diameter of 3.2 cm, that rotates at 10 rpm. Trained
animals were given an intraperitoneal injection of the test compounds. Neurotoxicity was indicated
by the inability of the animal to maintain equilibrium on the rod for at least 1 min in each of the
trials [17,18].
3.3.5. Quantification Trials
Quantitative anticonvulsant activity was expressed in terms of the median effective dose (ED50),
that is, the dose of drug that is required to produce the biological responses in 50% of animals.
Quantitative neurotoxicity was expressed as the median toxic dose (TD50). For determination of the
ED50 and TD50, groups of ten mice were given a range of doses of the tested compounds until at least
three points were established in the range of 10–90% seizure protection or neurotoxicity. From these
data, the respective ED50 and TD50 values, and the 95% confidence intervals were calculated using the
Graphpad prism 5. The PI values were calculated by dividing the TD50 by the ED50 [20].
3.3.6. 3-Mercaptopropionic Acid-Induced Seizures Test
In the 3-Mercaptopropionic acid induced seizures test, the vehicle, carbamazepine (30 mg/kg)
and 4g (30 mg/kg) were administered, via i.p. injection, to groups of ten mice 30 min before the
injection of 3-MP (60 mg/kg). The mice were placed in individual cages and observed for 60 min.
The numbers of clonic seizures (categorized as behavior ranging from exaggerated twitches of the
limbs to violent shaking or vibrating of the stiffened extremities) and tonic seizures (categorized as the
extremities pulling towards the body or rigidly push away from it, usually with maximal extension of
the hind leg), as well as the number of deaths were noted [14].
3.3.7. Bicuculline-Induced Seizures Test
In the bicuculline induced seizures test, the vehicle, carbamazepine (30 mg/kg) and 4g (30 mg/kg)
were administered, via i.p. injection, to groups of ten mice 30 min before the injection of bicuculline

Molecules 2018, 23, 756
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(5.4 mg/kg). The mice were placed in individual cages and observed for 60 min. The numbers of clonic
seizures (categorized as behavior ranging from exaggerated twitches of the limbs to violent shaking or
vibrating of the stiffened extremities) and tonic seizures (categorized as the extremities pulling towards
the body or rigidly push away from it, usually with maximal extension of the hind leg), as well as the
number of deaths were noted [15].
3.3.8. Determination of Brain GABA Concentrations by ELISA
Mice were randomly divided into three groups. The vehicle, phenytoin (25 mg/kg), and 4g
(50 mg/kg) were each administered via i.p. injection, to groups of ten mice. After 30 min, the mice
were decapitated and the brains were rapidly removed, washed with cooled saline, and homogenized
◦
in 6 volumes (g/mL) of saline. The homogenate was centrifuged at 5000
×
g for 10 min at 4 C.
The GABA content of the supernatant was determined using enzyme-linked immunosorbent assay
(ELISA) kits (Biolegend, San Diego, CA, USA) according to the manufacturer’s instructions. The GABA
content of the brain was expressed as µmol/L [21].
3.3.9. Test for the Effects of Thiosemicarbazide on the Anti-MES Action of 4g
Pretreatment with TSC will significantly decrease the GABA levels in the brain of mice. In this
study, thirty mice were pretreated with TSC at a dose of 25 mg/kg/day for three days. On the last day,
the treated mice were used for the determination of the ED50 value of 4g using the MES test. The ED50
value of 4g in mice that were pretreated with TSC were obtained and compared to the previous ED50
value of 4g achieved using the MES test [22].
3.3.10. Statistical Analysis
In order to compare the ED50 values, the standard error of the mean (SEM) values were
transformed from 95% confidence limits, and the ED50 values with the SEM were compared using
one-way analysis of variance (ANOVA) followed by Dunnet’s test. The number of seizures and lethality
from the chemical-induced seizure models were compared using the Fisher’s exact test. Differences
among values were considered to be statistically significant if P
performed using the commercially available GraphPad Prism version 5.0 for Windows (GraphPad
Software Inc., La Jolla, CA, USA).
≤
0.05. All statistical tests were
4. Conclusions
In summary, a series of benzoxazole derivatives containing triazolone (4a-m) were designed and
synthesized. The anticonvulsant effects of these compounds were investigated using several kinds
of seizure models. Compound 4g was considered to be the most promising compound based on
its potency against MES- and PTZ-induced seizures. The ED50 values of 4g in these models were
23.7 and 18.9 mg/kg, respectively. The seizure-preventing action of 4g in the 3-MP- and BIC-induced
seizure models further confirmed its anticonvulsant activity. The protective activity of 4g against
BIC-induced seizures also suggested that the anticonvulsant activity of 4g involves the GABAergic
system. Furthermore, experiments investigating the anticonvulsant mechanism of 4g suggested
that compound 4g can increase GABA levels in the mouse brain and that this compound elicits its
anticonvulsive action at least in part by increasing the GABA levels in the brain. A comparison of
the previously synthesized compounds (I) with these triazolone compounds, with regard to their
anticonvulsant activity, showed that there is no significant difference between them. Although the
expectation of obtaining improved anticonvulsant activity using the triazolone moiety was not reached,
compound 4g exhibited a better anti-PTZ activity than the representative compound obtained in the
previous study (18.9 mg/kg vs. 31.7 mg/kg).

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Acknowledgments: This study was supported by the National Science Foundation of China (No. 21562028),
the National Training Program of Innovation and Entrepreneurship for Undergraduates (No. 201710419020), and
the Doctoral Foundation of Jinggangshan University (No. JZB1316).
Author Contributions: Xian-Qing Deng designed the experiments and conducted the spectrum analysis of the
target compounds; Ming-Xia Song, Zhen-Yuan Wang, and Shi-Hui He synthesized all the presented compounds;
Sheng-Wang Yu, Shi-Long Chen, Dong-Fu Guo, and Wen-Hao Zhao conducted the anticonvulsant activity
determination; Xian-Qing Deng wrote the article.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
4.
5.
6.
Beghi, E. Addressing the burden of epilepsy: Many unmet needs. Pharmacol. Res. 2016, 107, 79–84. [CrossRef]
[PubMed]
Singh, S.P.; Sankaraneni, R.; Antony, A.R. Evidence-based guidelines for the management of epilepsy.
Neurol. India 2017, 65, S6–S11. [PubMed]
Mcphee, S.J.; Hammer, G.D. Pathophysiology of Disease: An Introduction to Clinical Medicine, 6th ed.;
Open University Press: Maidenhead, UK, 2010.
Gaitatzis, A.; Sander, J.W. The long-term safety of antiepileptic drugs. CNS Drugs 2013, 27, 435–455.
[CrossRef] [PubMed]
Błaszczyk, B.; Lason´, W.; Czuczwar, S. Antiepileptic drugs and adverse skin reactions: An update. Pharmacol. Rep.
2015, 67, 426–434. [CrossRef] [PubMed]
Ferrer, P.; Ballarín, E.; Sabaté, M.; Vidal, X.; Rottenkolber, M.; Amelio, J.; Hasford, J.; Schmiedl, S.; Ibáñez, L.
Antiepileptic drugs and suicide: A systematic review of adverse effects. Neuroepidemiology 2014, 42, 107–120.
[CrossRef] [PubMed]
7.
8.
9.
Tan, Y.D.; He, X.Y.; Rao, B.Q.; Cheng, B.B.; Song, M.X.; Deng, X.Q. Synthesis and Evaluation of the
Anticonvulsant Activities of Triazole-Containing Benzo[d]oxazoles. Chin. J. Org. Chem. 2016, 36, 2449–2455.
[CrossRef]
Cui, L.J.; Xie, Z.F.; Piao, H.R.; Li, G.; Chai, K.Y.; Quan, Z.S. Synthesis and anticonvulsant activity of
1-substituted-7-benzyloxy-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoline. Biol. Pharm. Bull. 2005, 28, 1216–1220.
[CrossRef] [PubMed]
Jin, H.G.; Sun, X.Y.; Chai, K.Y.; Piao, H.R.; Quan, Z.S. Anticonvulsant and toxicity evaluation of some
7-alkoxy-4,5-dihydro-[1,2,4]triazolo[4,3-a]quinoline-1(2H)-ones. Bioorg. Med. Chem. 2006, 14, 6868–6873.
[CrossRef] [PubMed]
10. Dawidowski, M.; Lewandowski, W.; Turło, J. Synthesis of new perhydropyrrolo[1,2-a]pyrazine derivatives
and their evaluation in animal models of epilepsy. Molecules 2014, 19, 15955–15981. [CrossRef] [PubMed]
11. Loscher, W. Critical review of current animal models of seizures and epilepsy used in the discovery and
development of new antiepileptic drugs. Seizure 2011, 20, 359–368. [CrossRef] [PubMed]
12. Katz, L.; Cohen, M.S. Benzoxazole derivatives. I. 2-mercaptobenzoxazoles. J. Org. Chem. 1954, 19, 758–766.
[CrossRef]
13. Shu, B.; Zheng, Y.; Wang, S.B.; Deng, X.Q.; Quan, Z.S. Design, synthesis, and anticonvulsant activity
evaluation of 4-(3-alkoxy-phenyl)-2,4-dihydro-[1,2,4]triazol-3-ones. Arch. Pharm. 2013, 346, 127–133.
[CrossRef] [PubMed]
14. Crick, E.W.; Osorio, I.; Frei, M.; Mayer, A.P.; Lunte, C.E. Correlation of 3-mercaptopropionic acid induced
seizures and changes in striatal neurotransmitters monitored by microdialysis. Eur. J. Pharm. Sci. 2014, 57,
25–33. [CrossRef] [PubMed]
15. Rubio, C.; Rubio-Osornio, M.; Retana-Márquez, S.; Verónica Custodio, M.L.; Paz, C. In vivo experimental
models of epilepsy. Cent. Nerv. Syst. Agents Med. Chem. 2010, 10, 298–309. [CrossRef] [PubMed]
16. Collins, G.S. Effect of aminooxyacetic acid, thiosemicarbazide and haloperidol on the metabolism and
half-lives of glutamate and GABA in rat brain. Biochem. Pharmacol. 1973, 22, 101–111. [CrossRef]
17. Krall, R.L.; Penry, J.K.; White, B.G.; Kupferberg, H.J.; Swinyard, E.A. Antiepileptic drug development: II.
Anticonvulsant drug screening. Epilepsia 1978, 19, 409–428. [CrossRef] [PubMed]
18. Porter, R.J. Antiepileptic drug development program. Prog. Clin. Biol. Res. 1983, 127, 53–66. [CrossRef]
[PubMed]

Molecules 2018, 23, 756
12 of 12
19. Zhang, H.J.; Shen, Q.K.; Jin, C.M.; Quan, Z.S. Synthesis and Pharmacological Evaluation of New
3,4-Dihydroisoquinolin Derivatives Containing Heterocycle as Potential Anticonvulsant Agents. Molecules
2016, 21, 1635. [CrossRef] [PubMed]
20. White, H.S.; Woodhead, J.H.; Wilcox, K.S.; Stables, J.P.; Kupferberg, H.J.; Wolf, H.H. Antiepileptic Drugs;
Levy, R.H., Mattson, R.H., Meldrum, B.S., Perucca, E., Eds.; Lippincott Williams & Wilkins Publishers:
New York, NY, USA, 2002; pp. 36–48.
21. Huisman, H.; Wynveen, P.; Nichkova, M.; Kellermann, G. Novel ELISAs for screening of the biogenic amines
GABA, glycine, beta-phenylethylamine, agmatine, and taurine using one derivatization procedure of whole
urine samples. Anal. Chem. 2010, 82, 6526–6533. [CrossRef] [PubMed]
22. Song, M.X.; Xiao, F.; Yu, H.H.; Liu, B.; Deng, X.Q. The Anticonvulsive Activities of Venlafaxine and its
Interactions with Some Antiepileptic Drugs. Lat. Am. J. Pharm. 2016, 35, 1959–1965.
Sample Availability: Samples of the compounds (4a–m) are available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).