Design, synthesis, and evaluation of anticonvulsant activities of benzoxazole derivatives containing the 1,2,4-triazolone moiety

Article abstract of DOI:10.1002/ardp.201800313

A novel series of benzoxazole derivatives containing 1,2,4-triazolone (5a-m) was designed. These compounds were synthesized in order to screen their anticonvulsant activities by the maximal electroshock seizure (MES) model and the subcutaneous pentylenete

Full text of DOI:10.1002/ardp.201800313

Received: 25 October 2018
Revised: 15 April 2019
Accepted: 1 May 2019
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DOI: 10.1002/ardp.201800313
F U L L P A P E R
Design, synthesis, and evaluation of anticonvulsant activities
of benzoxazole derivatives containing the 1,2,4‐triazolone
moiety
Ming‐Xia Song1
Xian‐Qing Deng1
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Yuping Huang2
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Shiben Wang3
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Zeng‐Tao Wang4
1Medical College, Jinggangshan University,
Abstract
Jiʼan, China
2Department of Biochemistry and Molecular
Biology, Gannan Medical College, Ganzhou,
China
A novel series of benzoxazole derivatives containing 1,2,4‐triazolone (5a‒m) was
designed. These compounds were synthesized in order to screen their anticonvulsant
activities by the maximal electroshock seizure (MES) model and the subcutaneous
pentylenetetrazole (sc‐PTZ) seizure model in mice. The rotarod test was used to
evaluate their neurotoxicities. Most of the compounds showed anti‐MES activities at
100 and 300 mg/kg. Compound 5f, which showed potential anticonvulsant activity in
the MES model with ED50 values of 22.0 mg/kg, was considered as the most
promising one in this study. It exhibited greater safety than that of carbamazepine
and valproate regarding neurotoxicity. The efficacy of compound 5f in inhibiting the
tonic seizures and death induced by the convulsants 3‐mercaptopropionic acid and
BIC was also verified. In an enzyme‐linked immunosorbent assay, compound 5f and
the positive drug phenytoin significantly increased the γ‐aminobutyric acid (GABA)
level in the mouse brain. Further, pretreatment with an inhibitor of the GABA
synthesizing enzyme dramatically raised the ED50 value of 5f in the MES model.
These results confirmed that the compound 5f plays its anticonvulsive action via
regulating the GABA function in the brain. Also, a docking study of the compound 5f
in the benzodiazepine (BZD) binding site of the GABAA receptor confirmed possible
binding of the compound 5f with BZD receptors.
3School of Pharmacy, Liaocheng University,
Liaocheng, China
4College of Pharmacy, Jiangxi University of
Traditional Chinese Medicine, Nanchang,
China
Correspondence
Xian‐Qing Deng, Medical College,
Jinggangshan University, No. 28 Xueyuan
Road, Ji’an, 343009 Jiangxi, China.
Email: dengxianqing1121@126.com
Funding information
National Natural Science Foundation of China,
Grant/Award Number: 21562028; Doctoral
Foundation of Jinggangshan University,
Grant/Award Number: JZB1316
K E Y W O R D S
Anticonvulsant, benzodiazepine receptors, docking, maximal electroshock seizure, sc‐PTZ,
triazolone
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1
INTRODUCTION
Moreover, their use is often associated with undesirable side effects,
ranging from indisposition (naupathia) to life‐threatening (hepato-
Epilepsy, one of the most common neurological disorders, affects
approximately 1% of the worldʼs population.[1,2] At present,
anticonvulsant drugs have been the mainstay of therapy. However,
the currently available anticonvulsants are effective in reducing the
severity and number of seizures in less than 70% of patients.
toxicity or megaloblastic anemia).[3–5] Therefore, the continued
search for more effective and safer anticonvulsant drugs is both an
urgent and necessary task for medicinal chemistry.
γ‐Aminobutyric acid (GABA) is the major inhibitory neurotrans-
mitter in the adult mammalian central nervous system. The
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Arch Pharm Chem Life Sci. 2019;e1800313.
wileyonlinelibrary.com/journal/ardp
© 2019 Deutsche Pharmazeutische Gesellschaft
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https://doi.org/10.1002/ardp.201800313

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FIGURE 1 The structures of
compounds I, diazepam, and target
compounds (5a‒m)
_
Compounds I
Diazepam
Target compounds (5a m)
development of GABAergic drugs is an active area of research in the
fields of medicinal chemistry and neurodegenerative disease. GABA
type A receptors are valuable for their rich pharmacology as they are
targets for a range of therapeutically important drugs, including the
benzodiazepines (BZDs), barbiturates, neuroactive steroids, and
anesthetics.[6,7] Triazole is a functionalized fragment in a variety of
different compounds in medicinal chemistry and has been reported to
exhibit a broad range of biological properties.[8] Since the approval and
sales of triazolam and alprazolam (two of BZDs) as anticonvulsants in
the 1980s, more and more medicinal chemists paid their attention to
design and synthesis of the triazole‐derivatives for their antic-
onvulsant activity.[9]
[d]oxazole‐2‐thiol (3). Then, the thiol group of compound 3 was
alkylated by various alkylating agents to obtain compound 4. Finally,
compound 4 was treated with methyl hydrazine carboxylate and
triethyl orthoformate in the presence of sodium methoxide to
provide the target compounds 5a‒m.[13] Their chemical structures
were characterized using 1H‐NMR, 13C NMR, and mass spectrometry
analysis techniques. The detailed physical and analytical data of these
compounds has been provided in Section 4.
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2.2
Anticonvulsant activity screens
The MES, subcutaneous pentylenetetrazole (sc‐PTZ) model, and
rotarod test were used to evaluate the anticonvulsant activities
and neurotoxicities of all the target compounds. As shown in
Table 1, most compounds showed anticonvulsant activity in the
MES model at different doses. Among them, compounds 5a and
5f showed the best activity with the protection at the dose of
30 mg/kg, and kept active after 4 hr at the higher dose.
Compounds 5b, 5e, 5g, and 5m displayed medium anticonvulsant
activity at a dosage of 100 mg/kg in the MES test at 0.5 hr, and also
In previous work, the anticonvulsant activity of a series of
benzoxazole derivatives containing triazole were reported
(Figure 1; I).[10] Among these compounds, 2‐((4‐fluobenzyl)thio)‐6‐
(4H‐1,2,4‐triazol‐4‐yl)benzo[d]oxazole was the most active one with
an ED50 of 12.7 mg/kg and a protective index (PI) value (TD50/ED50
)
of 38.7 in the maximal electroshock seizure (MES) test. Mechanism
research suggested the GABAergic system may contribute to the
anticonvulsive action of this compound. As a continuation of this
work, a series of benzoxazoles containing 1,2,4‐triazolone moiety
(5a‒m) were designed and synthesized in the current study, in which
the triazole moiety in compounds of I were replaced with triazolone
moiety.
remained active at
a dose of 300 mg/kg after 4 hr. Those
remaining, except 5c and 5k, all displayed weak activity at the
dosage of 300 mg/kg in the MES test. In the sc‐PTZ screens,
compounds 5a–c, 5f, 5h, 5j, and 5l–m protected the mice against
sc‐PTZ induced seizure at 300 mg/kg at 0.5‐hr interval. Com-
pounds 5a and 5m kept active after 4 hr at the same dose. In the
rotarod test, none of the target compounds exhibited neurotoxi-
city at the maximum dose of 300 mg/kg.
Presumably, the designed compounds will act as anticonvulsant
agents through BZD receptors, because they have the essential
features like diazepam for binding to those receptors: an aromatic
ring (A), a coplanar proton‐accepting group in a suitable distance
(B), and second out‐of‐plane aromatic ring (C). The pharmacophore
moieties A and B are essential for interaction with the GABAA
receptor, while the part C could potentiate binding to the receptor
(Figure 1).[11,12]
It was observed that several compounds kept active at the 4‐hr
interval, which indicates the protective effects with long duration. In
order to assess the anticonvulsant activity more accurately, the time
80
60
Thus we report here, the synthesis, pharmacological evaluation,
and docking study of 4‐(2‐(alkylthio)benzo[d]oxazol‐6‐yl)‐2H‐1,2,4‐
triazol‐3(4H)‐one derivatives (5a–m) as potential anticonvulsant
agents.
5a
5f
40
20
0
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2
RESULTS AND DISCUSSION
Chemistry
0.5
1
2
2.5
time (hr)
3
4
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2.1
As seen in Scheme 1, 2‐amino‐4‐nitrophenol (1) was used as the
starting material, which reacted with CS2 in the presence of
EtOH/H2O/KOH to obtain 6‐nitrobenzo[d]oxazole‐2‐thiol (2). The
compound 2 reduced by Na2S.9H2O in ethanol to give 6‐aminobenzo‐
FIGURE 2 Time‐course of compounds 5a and 5f (30 mg/kg) in the
maximal electroshock seizure test (i.p.). The data illustrates % animal
protected relative to animal tested (8 for each point) at various times
after injection

SONG ET AL.
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10 hr
4 hr
24 hr
4 hr
10 hr
SCHEME 1 The synthesis route of target compounds (5a‒m)
to peak effect (TPE) of compounds 5a and 5f was tested. As shown in
Figure 2, compounds 5a and 5f both reached the TPE at 2‐hr interval
after i.p. administration. Hence, compounds 5a and 5f were
quantitatively measured for their anticonvulsant activity at 2‐hr
time interval in the next assay.
aspect of safety, compounds 5a and 5f showed superior performance
to carbamazepine and valproate.
3‐Mercaptopropionic acid (3‐MP), as a competitive inhibitor of
glutamate decarboxylase, can inhibit the synthesis of GABA,
subsequently decreasing the levels of this neurotransmitter in the
brain.[14] Bicuculline (BIC), a competitive GABAA receptor antagonist,
is known to produce seizures in mice after subcutaneous injection at
low dose.[15] To further evaluate the anticonvulsive effects of 5f,
3‐MP‐ and BIC‐induced seizure tests were performed. As shown in
Table 3, 3‐MP (60 mg/kg) alone induced clonic and tonic seizures in
100% of the tested mice, while the combined treatment with 5f led to
a marketable decrease in tonic convulsions and death (from 100 to
Based on the notable anticonvulsant activity of 5a and 5f shown in
the initial trials, the quantitative evaluation trials were performed to
obtain the median effective dose (ED50) and median toxic dose (TD50).
As shown in Table 2, the ED50 values of 5a and 5f were measured as
26.4 and 22.0 mg/kg in the MES model, respectively. The TD50 values
of 5a and 5f were measured as 340.8 and 366.6 mg/kg, respectively,
which resulted in the PI values of 12.9 and 16.7, respectively. In the
TABLE 1 Anticonvulsant activity and neurotoxicity data of compounds 5a–m administered intraperitoneally to mice
Intraperitioneal injection in micea
MES screeningb
sc‐PTZ screeningc
NT screeningd
Compounds
R
0.5 hr
30
4 hr
100
300
–
0.5 hr
300
300
300
–
4 hr
300
–
0.5 hr
4 hr
–
5a
5b
5c
5d
5e
5f
C3H7
–
–
–
–
–
–
–
–
–
–
–
–
–
C5H9
100
–
–
C7H15
–
–
CH2C6H5
300
100
30
300
300
100
300
–
–
–
CH2C6H4(o‐F)
CH2C6H4(m‐F)
CH2C6H4(p‐F)
CH2C6H4(o‐Cl)
CH2C6H4(m‐Cl)
CH2C6H4(p‐Cl)
CH2C6H4(2,4‐Cl2)
CH2C6H4(p‐CH3)
CH2C6H4(p‐OCH3)
–
–
–
300
–
–
–
5g
5h
5i
100
300
300
300
–
–
–
300
–
–
–
–
–
–
5j
–
300
–
–
–
5k
5l
–
–
–
300
100
–
300
300
–
–
5m
300
300
–
aThree mice were used for each group, doses of 30, 100, and 300 mg/kg were administered. The figure in the table indicates the minimum dose whereby
bioactivity was demonstrated in 2/3 or 3/3 of the mice. The animals were examined at 0.5 and 4 hr after injection was administered. A dash indicates the
absence of anticonvulsant activity and neurotoxicity at the maximum dose administered (300 mg/kg).
bMaximal electroshock seizure test.
cSubcutaneous pentylenetetrazole test.
dNeurotoxicity screening (rotarod test).

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TABLE 2 Quantitative anti‐MES activity of 5a and 5f in mice
administered intraperitoneally
a
b
Compounds
5a
ED50
TD50
PIc
26.4 (24.0–29.1)
22.0 (20.1–24.1)
9.8 (8.9–10.8)
340.8 (309.9–374.9)
366.6 (333.3–403.3)
44.0 (40.2–48.1)
418 (369–450)
12.9
16.7
4.5
5f
Carbamazepine
Valproate
264 (247–338)
1.6
aED50: median effective dose.
bTD50: median toxic dose.
cPI: protective index (TD50/ED50).
10%, p < 0.001; and from 80 to 30%, p < 0.05, for seizures and death,
respectively). As shown in Table 4, when administered with 5.4 mg/kg
of BIC, the mice produced clonic seizures and tonic seizures in 100%,
and death in 90%. By comparison, after combined treatment with 5f,
the tonic seizures and death of the mice were inhibited significantly
(from 100 to 0%, p < 0.001; and from 90 to 10%, p < 0.01, for tonic
seizures and death, respectively). The inhibitory action of compound
5f against the tonic seizures and death induced by 3‐MP and BIC
further confirmed the anticonvulsant activity of 5f. Furthermore, the
suppression of 5f on the BIC‐induced seizure suggested that it may
play its anticonvulsive action via acting on the GABAA active site or
increasing the concentration of GABA in the brain.
FIGURE 3 Effects of 5f and phenytoin on whole brain
γ‐aminobutyric acid (GABA) concentration in mice. Data are
presented as mean SEM. of seven animals. **p < 0.01 versus
control (animals treated with saline)
for 3 days) significantly increased the ED50 value of 5f in the MES
model (p < 0.001). This result further suggested that compound 5f
plays its anticonvulsive action via regulating the GABA function in
the brain.
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Docking study
2.3
Anticonvulsant mechanism of 5f involved in
2.4
the GABAergic system
To confirm whether the anticonvulsant activity of the synthesized
compounds mediates through BZD receptors, the molecular
docking of representative 5f with the BZD‐binding site of GABAA
receptor (α1β2γ2) was performed by Discovery Studio 4.5 version.
Interactions of the compound 5f with amino acids of BZD‐binding
pocket of GABAA receptor were illustrated and shown in Figure 5.
As can be seen in Figure 5, the most important residues in the
binding mode of compound 5f are Thr206, Tyr 209, Phe77, His101,
Tyr159, Heu140, Val190, and Glu189. The carbonyl group of
compound 5f, as a H‐bond acceptor, was involved in an interaction
with the hydroxy group of Thr206, while the trizolone ring was
responsible for π‐π interaction with Tyr209 and Heu140. In
addition to above, the benzoxazole and terminal phenyl moiety
also showed π‐π interactions with some critical amino acid
residues (Phe77, Tyr159, His101, and Val190). Moreover, the
hydrogen bonding between the F atom of the compound 5f and
To investigate whether the concentration of GABA in the brain was
involved in the anticonvulsive action of compound 5f, an enzyme‐
linked immunosorbent assay (ELISA) was carried out to measure the
effect of 5f on the GABA level in mouse brain. As shown in Figure 3,
when compared to the results of the control group, compounds 5f
and positive control drug phenytoin both significantly increased the
level of GABA in mouse brain (p < 0.01).
Thiosemicarbazide (TSC), a competitive inhibitor of the GABA
synthesis enzyme, can inhibit the synthesis of GABA, resulting in a
decrease of GABA level in the brain.[16] To further confirm the
anticonvulsive mechanism of 5f involved in the GABAergic system,
the influence of TSC on the anti‐MES activity of 5f was investigated.
As shown in Figure 4, the ED50 value of 5f in the normal mice in MES
model was 22.0 mg/kg, while the ED50 value of 5f in the treated mice
was 273.86 mg/kg. Obviously, pretreatment with TSC (25 mg/kg/day
TABLE 3 Effects of compound 5f on 3‐MP‐induced seizures in mice
Compound
DMSO
Doses (mg/kg)
Test time (hr)
Clonic seizures (%)
Tonic seizures (%)
Lethality (%)
–
0.5
0.5
0.5
100
100
100
100
80
Carbamazepine
5f
30
30
0***
0**
30*
10***
Note: Results are expressed as a percentage of animals that showed clonic and tonic convulsions and death among all animals tested. Ten mice were used
in each group. Significance determined with Fisherʼs exact test. *p < 0.05, **p < 0.01, ***p < 0.001 versus control group (3‐MP, 60 mg/kg).
Abbreviation: DMSO, dimethyl sulfoxide

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4
EXPERIMENTAL
Chemistry
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4.1
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4.1.1
General
Melting points were measured by a capillary tube. Infrared spectro-
scopy was determined (in KBr using an FTIR1730 spectrometer
[Perkin Elmer, Waltham, MA]). 1H NMR and 13C NMR spectroscopies
were measured on an AV‐300 spectrometer (Bruker, Switzerland)
using tetramethylsilane as the internal standard. An MALDI‐TOF/
TOF mass spectrometer (Bruker Daltonik, Germany) was used to
measure high resolution mass spectroscopy. The major chemicals
were bought from Aldrich Chemical Corporation (St. Louis).
FIGURE 4 Effects of the thiosemicarbazide on the anticonvulsant
activity of 5f against maximal electroshock‐induced seizures in mice.
All data are presented as median effective dose (ED50
) SEM. All
drugs were administered i.p. in a single injection, 30 minutes before
maximal electroshock‐induced seizures; ***p < 0.001 versus control
group (pretreatment with saline)
The InChI codes of the investigated compounds together with
some biological activity data are provided as Supporting Information.
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Procedures for the synthesis of compounds
2, 3, and 4
residue Glu189 may help to explain the best activity of 5f among
this series of compounds. According to the reports of Richter and
Mohammadi‐Khanaposhtani,[17,18] Thr206, Tyr209, Phe77,
His101, and Tyr159 are the critical residues in the binding mode
of diazepam. Therefore, the binding mode of compound 5f in the
BZD‐binding pocket of GABAA receptor resembled to that of
diazepam, and the synthesized compounds may well play its
anticonvulsant activity via mediating BZD receptors.
4.1.2
Compounds 2, 3, and 4 were previously reported.[10]
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4.1.3
General procedure for the synthesis of 4‐(2‐
(alkylthio)benzo[d]oxazol‐5‐yl)‐2,4‐dihydro‐3H‐1,2,4‐
triazol‐3‐ones (5a‒m)
Compounds 4 (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 hr. After cooling, sodium methylate (50 mmol) was added and
refluxed. After the reaction was completed (the mixture went pink as
the sign), the mixture was cooled down to room temperature and
diluted by 120 ml water followed the neutralizing with HCl solution
to pH 6. The precipitate formed was filtered and washed with water,
which was then purified by recrystallization with ethanol to obtain
compounds 5a‒m. The yield, melting point, analytical data, and the
spectral data of each compound are given below.
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3
CONCLUSION
In summary, we have synthesized a series of 4‐(2‐(alkylthio)benzo[d]‐
oxazol‐6‐yl)‐2H‐1,2,4‐triazol‐3(4H)‐one (5a‒m) and investigated their
anticonvulsant activities using MES and PTZ models. In the current
work, compound 5f was considered as the most promising one, which
displayed outstanding anticonvulsant activity in the MES model with
an ED50 value of 22.0 mg/kg. The protective action of 5f in 3‐MP‐ and
BIC‐induced seizure models further confirmed its anticonvulsant
activity. Furthermore, experiments investigating the anticonvulsant
mechanism of 5f suggested that the compound 5f plays its
anticonvulsive action by increasing the GABA content or regulating
the GABA function in brain. The docking of 5f in the BZD‐binding site
of GABAA receptor demonstrated that this compound has a similar
binding to diazepam, thus it may well play its anticonvulsant activity
via mediating BZD receptors.
4‐(2‐(Propylthio)benzo[d]oxazol‐6‐yl)‐2H‐1,2,4‐triazol‐3(4H)‐one
(5a)
Mp 168–169°C, yield 59%. 1H‐NMR (CDCl3, 300 MHz): δ 1.12 (t, 3H,
J = 7.4 Hz, CH2CH3), 1.87–1.94 (m, 2H, CH2CH3), 3.33 (t, 2H,
J = 7.2 Hz, SCH2), 7.41 (dd, 1H, J1 = 8.5 Hz, J2 = 1.9 Hz, Ph‐H), 7.70
(d, 1H, J = 8.5 Hz, Ph‐H), 7.76 (s, 1H, CH=N), 7.79 (d, 1H, J = 1.9 Hz,
TABLE 4 Effects of compound 5f on BIC‐induced seizures in mice
Compound
DMSO
Doses (mg/kg)
Test time (hr)
Clonic seizures (%)
Tonic seizures (%)
Lethality (%)
–
0.5
0.5
0.5
100
100
100
100
0***
0***
90
Carbamazepine
5f
30
30
20**
10**
Note: 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 10. Significance determined with Fisherʼs exact test. **p < 0.01, ***p < 0.001 when versus control group (BIC, 5.4 mg/kg).
Abbreviation: DMSO, dimethyl sulfoxide

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FIGURE 5 The binding modes of compound 5f within the benzodiazepine‐binding pocket of GABAA (A for 2D model; B for 3D model). GABA,
γ‐aminobutyric acid
Ph‐H), 10.82 (s, 1H, CO‐NH). 13H‐NMR (CDCl3, 75 MHz): δ 167.4,
154.1, 151.8, 141.4, 136.0, 129.3, 118.9, 118.8, 105.2, 34.3, 22.7,
13.2. IR (KBr) cm−1: 3211 (N‐H), 1710 (C=O). ESI‐HRMS calcd for
C12H13N4O2S+ ([M+H]+): 277.0754; found: 277.0758.
(KBr) cm−1
: 3206 (N‐H), 1711 (C=O). ESI‐HRMS calcd for
C16H13N4O2S+ ([M+H]+): 325.0754; found: 325.0753.
4‐(2‐(2‐Fluorobenzylthio)benzo[d]oxazol‐6‐yl)‐2H‐1,2,4‐triazol‐
3(4H)‐one (5e)
4‐(2‐(Pentylthio)benzo[d]oxazol‐6‐yl)‐2H‐1,2,4‐triazol‐3(4H)‐one
(5b)
Mp 215–216°C, yield 57%. H‐NMR (CDCl3+DMSO‐d6, 300 MHz): δ
4.52 (s, 2H, SCH2), 6.98–7.27 (m, 3H, benzyl‐H), 7.42 (dd, 1H,
J1 = 8.5 Hz, J2 = 2.0 Hz, Ph‐H), 7.45–7.51 (m, 1H, benzyl‐H), 7.59 (d,
1H, J = 8.5 Hz, Ph‐H), 7.80 (d, 1H, J = 2.0 Hz, Ph‐H), 7.86 (s, 1H,
CH=N), 11.79 (s, 1H, CO‐NH). 13H‐NMR (CDCl3+DMSO‐d6, 75 MHz):
Mp 160–162°C, yield 66%. 1H‐NMR (CDCl3, 300 MHz): δ 0.95 (t, 3H,
J = 7.1 Hz, CH2CH3), 1.32–1.59 (m, 4H, (CH2)2CH3), 1.82–1.91 (m,
2H, SCH2CH2), 3.34 (t, 2H, J = 7.3 Hz, SCH2), 7.40 (dd, 1H, J1 = 8.4 Hz,
J2 = 1.7 Hz, Ph‐H), 7.70 (d, 1H, J = 8.4 Hz, Ph‐H), 7.76 (s, 1H, CH=N),
7.79 (d, 1H, J = 1.7 Hz, Ph‐H), 10.63 (s, 1H, CO‐NH). 13H‐NMR
(CDCl3, 75 MHz): δ 167.3, 154.0, 151.8, 141.5, 135.8, 129.2, 118.9,
118.8, 105.2, 32.4, 30.8, 28.9, 22.1, 13.9. IR (KBr) cm−1: 3213 (N‐H),
1711 (C=O). ESI‐HRMS calcd for C14H17N4O2S+ ([M+H]+): 305.1067;
found: 305.1075.
1
δ 165.4, 160.9 (d, Jc‐f = 246.3 Hz), 153.6, 151.8, 140.6, 135.7, 131.3,
131.2, 130.3, 130.1(d, 3Jc‐f = 8.2 Hz), 124.5 (d, 4Jc‐f = 3.6 Hz), 123.2 (d,
3
2
2Jc‐f = 14.5 Hz), 118.6 (d, Jc‐f = 11.0 Hz), 115.6 (d, Jc‐f = 20.0 Hz),
104.7, 29.8. IR (KBr) cm−1: 3200 (N‐H), 1711 (C=O). ESI‐HRMS calcd
for C16H12FN4O2S+ ([M+H]+): 343.0660; found: 343.0665.
4‐(2‐(3‐Fluorobenzylthio)benzo[d]oxazol‐6‐yl)‐2H‐1,2,4‐triazol‐
3(4H)‐one (5f)
4‐(2‐(Heptylthio)benzo[d]oxazol‐6‐yl)‐2H‐1,2,4‐triazol‐3(4H)‐one
(5c)
Mp 209°C, yield 69%. H‐NMR (CDCl3+DMSO‐d6, 300 MHz): δ 4.47
(s, 2H, SCH2), 6.88–7.28 (m, 4H, benzyl‐H), 7.40 (dd, 1H, J1 = 8.5 Hz,
J2 = 1.9 Hz, Ph‐H), 7.58 (d, 1H, J = 8.5 Hz, Ph‐H), 7.78 (d, 1H,
J = 1.9 Hz, Ph‐H), 7.82 (s, 1H, CH=N), 11.76 (s, 1H, CO‐NH). 13H‐
NMR (CDCl3+DMSO‐d6, 75 MHz): δ 165.3, 162.6 (d, 1Jc‐f = 244.6 Hz),
Mp 167–168°C, yield 67%. 1H‐NMR (CDCl3+DMSO‐d6, 300 MHz): δ
0.83 (t, 3H, J = 6.7 Hz, CH2CH3), 1.18–1.46 (m, 8H, (CH2)4CH3),
1.68–1.85 (m, 2H, SCH2CH2), 3.25 (t, 2H, J = 7.3 Hz, SCH2), 7.50 (dd,
1H, J1 = 8.5 Hz, J2 = 1.9 Hz, Ph‐H), 7.57 (d, 1H, J = 8.5 Hz, Ph‐H), 7.87
(d, 1H, J = 1.9 Hz, Ph‐H), 8.09 (s, 1H, CH=N), 11.87 (s, 1H, CO‐NH).
13H‐NMR (CDCl3+DMSO‐d6, 75 MHz): δ 166.3, 153.5, 151.6, 140.6,
136.1, 130.4, 118.4, 118.4, 104.4, 32.3, 31.6, 29.3, 28.6, 28.4, 22.5,
14.2. IR (KBr) cm−1: 3215 (N‐H), 1712 (C=O). ESI‐HRMS calcd for
C16H21N4O2S+ ([M+H]+): 333.1380; found: 333.1373.
3
153.6, 151.8, 140.5, 138.8 (d, Jc‐f = 7.6 Hz), 135.8, 130.5, 130.4,
125.0, 124.9, 118.6 (d, 3Jc‐f = 10.5 Hz), 116.0 (d, 2Jc‐f = 21.9 Hz), 114.8
2
(d, Jc‐f = 20.9 Hz), 104.7, 35.8. IR (KBr) cm−1: 3198 (N‐H), 1710
(C=O). ESI‐HRMS calcd for C16H12FN4O2S+ ([M+H]+): 343.0660;
found: 343.0662.
4‐(2‐(Benzylthio)benzo[d]oxazol‐6‐yl)‐2H‐1,2,4‐triazol‐3(4H)‐one
(5d)
4‐(2‐(4‐Fluorobenzylthio)benzo[d]oxazol‐6‐yl)‐2H‐1,2,4‐triazol‐
3(4H)‐one (5g)
Mp 199°C, yield 59%. 1H‐NMR (CDCl3+DMSO‐d6, 300 MHz): δ 4.50
(s, 2H, SCH2), 7.21–7.41 (m, 5H, benzyl‐H), 7.44 (dd, 1H, J1 = 8.5 Hz,
J2 = 1.9 Hz, Ph‐H), 7.58 (d, 1H, J = 8.5 Hz, Ph‐H), 7.82 (d, 1H,
J = 1.9 Hz, Ph‐H), 7.92 (s, 1H, CH=N), 11.82 (s, 1H, CO‐NH). 13H‐
NMR (CDCl3+DMSO‐d6, 75 MHz): δ 165.6, 153.6, 151.8, 140.6,
135.9, 135.8, 130.3, 129.1, 128.8, 128.0, 118.6, 118.5, 104.6, 36.4. IR
Mp 219–221°C, yield 61%. H‐NMR (CDCl3+DMSO‐d6, 300 MHz): δ
4.53 (s, 2H, SCH2), 6.98–7.04 (m, 2H, benzyl‐H), 7.45–7.50 (m, 2H,
benzyl‐H), 7.56 (dd, 1H, J1 = 8.5 Hz, J2 = 1.8 Hz, Ph‐H), 7.62 (d, 1H,
J = 8.5 Hz, Ph‐H), 7.92 (d, 1H, J = 1.8 Hz, Ph‐H), 8.17 (s, 1H, CH=N),
11.91 (s, 1H, CO‐NH). 13H‐NMR (CDCl3+DMSO‐d6, 75 MHz):
1
δ 165.4, 162.1 (d, Jc‐f = 244.0 Hz), 153.5, 151.7, 140.3, 136.2, 132.6

SONG ET AL.
4
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3
(d, Jc‐f = 3.2 Hz), 131.2 (d, Jc‐f = 8.2 Hz), 130.7, 118.6, 118.5, 115.6
4‐(2‐(4‐Methylbenzylthio)benzo[d]oxazol‐6‐yl)‐2H‐1,2,4‐triazol‐
3(4H)‐one (5l)
2
(d, Jc‐f = 21.4 Hz), 104.4, 35.5. IR (KBr) cm−1: 3144 (N‐H), 1711
(C=O). ESI‐HRMS calcd for C16H12FN4O2S+ ([M+H]+): 343.0660;
Mp 224°C, yield 75%. H‐NMR (CDCl3+DMSO‐d6, 300 MHz): δ 2.25
(s, 3H, Ar‐CH3), 4.46 (s, 2H, SCH2), 7.07 (d, 2H, J = 7.9 Hz, benzyl‐H),
7.28 (d, 2H, J = 7.9 Hz, benzyl‐H), 7.44 (dd, 1H, J1 = 8.5 Hz, J2 = 2.0 Hz,
Ph‐H), 7.57 (d, 1H, J = 8.5 Hz, Ph‐H), 7.81 (d, 1H, J = 2.0 Hz, Ph‐H),
7.93 (s, 1H, CH=N), 11.82 (s, 1H, CO‐NH). 13H‐NMR (CDCl3+DMSO‐
d6, 75 MHz): δ 165.7, 153.6, 151.7, 140.5, 137.0, 135.8, 132.8, 130.3,
129.4, 129.0, 118.6, 118.5, 104.6, 36.3, 21.2. IR (KBr) cm−1: 3199
(N‐H), 1713 (C=O). ESI‐HRMS calcd for C17H15N4O2S+ ([M+H]+):
339.0910; found: 339.0906.
found: 343.0660.
4‐(2‐(2‐Chlorobenzylthio)benzo[d]oxazol‐6‐yl)‐2H‐1,2, 4‐triazol‐
3(4H)‐one (5h)
Mp 201–203°C, yield 63%. H‐NMR (CDCl3+DMSO‐d6, 300 MHz): δ
4.59 (s, 2H, SCH2), 7.18–7.23 (m, 2H, benzyl‐H), 7.33 (d, 1H,
J = 7.5 Hz, benzyl‐H), 7.44 (d, 1H, J = 7.6 Hz, benzyl‐H), 7.57 (dd, 1H,
J1 = 8.6 Hz, J2 = 2.2 Hz, Ph‐H), 7.60 (d, 1H, J = 8.6 Hz, Ph‐H), 7.81 (d,
1H, J = 2.2 Hz, Ph‐H), 7.93 (s, 1H, CH=N), 11.89 (s, 1H, CO‐NH). 13H‐
NMR (CDCl3+DMSO‐d6, 75 MHz): δ 165.5, 153.6, 151.8, 140.6,
135.9, 134.1, 133.8, 133.2, 131.4, 130.3, 129.7, 127.3, 118.7, 118.7,
104.7, 34.3. IR (KBr) cm−1: 3205 (N‐H), 1711 (C=O). ESI‐HRMS calcd
for C16H12ClN4O2S+ ([M+H]+): 359.0364; found: 359.0369.
4‐(2‐(4‐Methoxybenzylthio)benzo[d]oxazol‐6‐yl)‐2H‐1,2,4‐triazol‐
3(4H)‐one (5m)
Mp 202–206°C, yield 73%. H‐NMR (CDCl3+DMSO‐d6, 300 MHz): δ
1.17 (s, 3H, Ar‐OCH3), 4.47 (s, 2H, SCH2), 7.23 (d, 2H, J = 8.2 Hz,
benzyl‐H), 7.38 (d, 2H, J = 8.2 Hz, benzyl‐H), 7.45 (d, 1H, J = 8.5 Hz,
Ph‐H), 7.57 (d, 1H, J = 8.5 Hz, Ph‐H), 7.82 (s, 1H, Ph‐H), 7.94 (s, 1H,
CH=N), 11.83 (s, 1H, CO‐NH). 13H‐NMR (CDCl3+DMSO‐d6, 75 MHz):
δ 165.3, 153.5, 151.8, 140.4, 135.9, 135.2, 133.4, 130.7, 130.5, 128.8,
118.7, 118.5, 104.5, 35.5, 29.6. IR (KBr) cm−1: 3200 (N‐H), 1713
(C=O). ESI‐HRMS calcd for C17H15N4O3S+ ([M+H]+): 355.0859;
found: 355.0859.
4‐(2‐(3‐Chlorobenzylthio)benzo[d]oxazol‐6‐yl)‐2H‐1,2,4‐triazol‐
3(4H)‐one (5i)
Mp 196–197°C, yield 72%. H‐NMR (CDCl3+DMSO‐d6, 300 MHz): δ
4.45 (s, 2H, SCH2), 7.18–7.31 (m, 3H, benzyl‐H), 7.41 (s, 1H, benzyl‐
H), 7.40 (dd, 1H, J1 = 8.5 Hz, J2 = 1.9 Hz, Ph‐H), 7.57 (d, 1H, J = 8.5 Hz,
Ph‐H), 7.78 (d, 1H, J = 1.9 Hz, Ph‐H), 7.82 (s, 1H, CH=N), 11.76 (s, 1H,
CO‐NH). 13H‐NMR (CDCl3+DMSO‐d6, 75 MHz): δ 165.3, 153.6,
151.8, 140.6, 138.2, 135.6, 134.2, 130.3, 130.1, 129.0, 128.0, 127.4,
118.7, 118.6, 104.7, 35.7. IR (KBr) cm−1: 3206 (N‐H), 1710 (C=O).
ESI‐HRMS calcd for C16H12ClN4O2S+ ([M+H]+): 359.0364; found:
359.0361.
|
4.2
Pharmacology
|
4.2.1
Animals and experimental conditions
Experiments were carried out on Kunming mice (half male and
female) weighing 18–24 g. The mice were housed collectively in
polycarbonate cages in groups of 10, where they were maintained on
a 12‐hr light/dark cycle in a temperature‐controlled (25 2°C)
laboratory with free access to food and 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, Washing-
ton, DC. Local ethical committee approval was also obtained (No.
20170707). In addition, all efforts were made to minimize animal
suffering and to use only the number of animals necessary to produce
reliable scientific data. Electroconvulsions were produced by an
electric stimulation generator (JTC‐1, Chengdu, China). The following
drugs were used in this study: pentylenetetrazole, 3‐MP, TSC, and
BIC (these drugs were obtained from Aladdin Industrial Inc.,
Shanghai, China), valproate sodium, carbamazepine, and phenytoin
(these drugs were obtained from Melongpharma, Dalian, China).
4‐(2‐(4‐Chlorobenzylthio)benzo[d]oxazol‐6‐yl)‐2H‐1,2,4‐triazol‐
3(4H)‐one (5j)
Mp 235–237°C, yield 63%. H‐NMR (CDCl3+DMSO‐d6, 300 MHz): δ
4.46 (s, 2H, SCH2), 6.79 (d, 2H, J = 8.4 Hz, benzyl‐H), 7.32 (d, 2H,
J = 8.4 Hz, benzyl‐H), 7.47 (dd, 1H, J1 = 8.5 Hz, J2 = 1.9 Hz, Ph‐H), 7.58
(d, 1H, J = 8.5 Hz, Ph‐H), 7.77 (d, 1H, J = 1.9 Hz, Ph‐H), 7.99 (s, 1H,
CH=N), 11.85 (s, 1H, CO‐NH). 13H‐NMR (CDCl3+DMSO‐d6, 75 MHz):
δ 165.7, 159.3, 153.6, 151.7, 140.6, 135.9, 130.4, 130.3, 127.8, 118.6,
118.5, 114.2, 104.5, 36.1. IR (KBr) cm−1: 3208 (N‐H), 1711 (C=O).
ESI‐HRMS calcd for C16H12ClN4O2S+ ([M+H]+): 359.0364; found:
359.0360.
4‐(2‐(2,4‐Dichlorobenzylthio)benzo[d]oxazol‐6‐yl)‐2H‐1,2,4‐triazol‐
3(4H)‐one (5k)
Mp 190–194°C, yield 70%. H‐NMR (CDCl3+DMSO‐d6, 300 MHz): δ
4.55 (s, 2H, SCH2), 7.17 (dd, 1H, J1 = 8.3 Hz, J2 = 1.5 Hz, benzyl‐H),
7.36 (d, 1H, J = 1.5 Hz, benzyl‐H), 7.45 (dd, 1H, J1 = 8.5 Hz, J2 = 1.7 Hz,
Ph‐H), 7.57 (d, 1H, J = 8.3 Hz, benzyl‐H), 7.58 (d, 1H, J = 8.5 Hz, Ph‐H),
7.77 (d, 1H, J = 1.7 Hz, Ph‐H), 7.93 (s, 1H, CH=N), 11.83 (s, 1H, CO‐
NH). 13H‐NMR (CDCl3+DMSO‐d6, 75 MHz): δ 165.1, 153.6, 151.9,
140.5, 135.7, 134.9, 134.4, 132.8, 132.4, 130.4, 129.4, 127.4, 118.7,
118.5, 104.6, 33.7. IR (KBr) cm−1: 3212 (N‐H), 1714 (C=O). ESI‐HRMS
calcd for C16H11Cl2N4O2S+ ([M+H]+): 392.9974; found: 392.9982.
|
4.2.2
Experimental method
The animal experiments, including the MES test, subcutaneous
pentylenetetrazole‐induced seizure test (sc‐PTZ), the rotarod test,
3‐MP‐induced seizure test, BIC‐induced seizures test, determination
of brain GABA concentrations by ELISA, and test for the effects of
TSC on the anti‐MES action of 5f were carried out according to the
previous report.[13]

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4.3
Docking studies
[4] B. Błaszczyk, W. Lasoń, S. J. Czuczwar, Pharmacol. Rep. 2015, 67,
426.
The homology model of the diazepam‐bound GABAA receptor devel-
oped by Ernst et al. was retrieved from the Supporting Information of
their published paper.[18] The three‐dimensional structures of 5f was
constructed using Chem3D Ultra 12.0 software (Chemical Structure
Drawing Standard; Cambridge Soft Corporation [2010]), then it was
energetically minimized by using MMFF94 with 5000 iterations and
minimum RMS gradient of 0.10. For protein preparation, the hydrogen
atoms were added, and water and impurities were removed. The 3D
structure of 5f was placed during the molecular docking procedure.
Types of interactions of the docked protein with 5f were analyzed after
the end of molecular docking. Compound would retain 10 poses, and
were ranked and selected by CDOCKER_INTERACTION_ENERGY. The
lowest energy conformation of ligand‐enzyme complex was evaluated
by Discovery Studio 4.5 Client.
[5] P. Ferrer, E. Ballarín, M. Sabaté, X. Vidal, M. Rottenkolber, J. Amelio,
J. Hasford, S. Schmiedl, L. Ibáñez, Neuroepidemiology 2014, 42, 107.
[6] M. Mula, Future Med. Chem. 2011, 3, 177.
[7] P. V. Ramachandran, A. Shekhar, Future Med. Chem. 2011, 3, 139.
[8] A. Ayati, S. Emami, A. Foroumadi, Eur. J. Med. Chem. 2016, 109, 380.
[9] M. X. Song, X. Q. Deng, J. Enzyme Inhib. Med. Chem. 2018, 33, 453.
[10] M. X. Song, B. Q. Rao, B. B. Cheng, Y. Wu, H. Zeng, Y. G. Luo, X. Q.
Deng, CNS Neurol. Disord. Drug Target. 2017, 16, 187.
[11] T. Akbarzadeh, S. A. Tabatabai, M. J. Khoshnoud, B. Shafaghi, A.
Shafiee, Bioorg. Med. Chem. 2003, 11, 769.
[12] M. Faizi, S. Dabirian, H. Tajali, F. Ahmadi, E. R. Zavareh, S.
Shahhosseini, S. A. Tabatabai, Bioorg. Med. Chem. 2015, 23, 480.
[13] M. X. Song, Z. Y. Wang, S. H. He, S. W. Yu, S. L. Chen, D. F. Guo, W. H.
Zhao, X. Q. Deng, Molecules 2018, 23, 756.
[14] E. W. Crick, I. Osorio, M. Frei, A. P. Mayer, C. E. Lunte, Eur. J. Pharm.
Sci. 2014, 57, 25.
[15] C. Rubio, M. Rubio‐Osornio, S. Retana‐Marquez, M. Lopez, V.
Custodio, C. Paz, Cent. Nerv. Syst. Agents Med. Chem. 2010, 10, 298.
[16] G. S. Collins, Biochem. Pharmacol. 1973, 22, 101.
[17] M. Mohammadi‐Khanaposhtani, M. Shabani, M. Faizi, I. Aghaei,
R. Jahani, Z. Sharafi, N. Shamsaei, M. Zafarghandi, M. Mahdavi,
T. Akbarzadeh, S. Emami, T. Akbarzadeh, S. Emami, A. Shafieem,
A. Foroumadi, Eur. J. Med. Chem. 2016, 112, 91.
ACKNOWLEDGMENT
This work was supported by the National Natural Science Foundation
of China (No. 21562028) and the Doctoral Foundation of Jinggang-
shan University (No. JZB1316).
[18] L. Richter, C. de Graaf, W. Sieghart, Z. Varagic, M. Mörzinger, I. J. P.
de Esch, G. F. Ecker, M. Ernst, Nat. Chem. Biol. 2012, 8, 455.
CONFLICT OF INTERESTS
SUPPORTING INFORMATION
The authors declare that there are no conflict of interests.
Additional supporting information may be found online in the
Supporting Information section.
ORCID
Xian‐Qing Deng
http://orcid.org/0000-0001-8180-610X
How to cite this article: Song M‐X, Huang Y, Wang S,
Wang Z‐T, Deng X‐Q. Design, synthesis, and evaluation of
anticonvulsant activities of benzoxazole derivatives
containing the 1,2,4‐triazolone moiety. Arch Pharm Chem Life
Sci. 2019;e1800313.
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