Synthesis of 1,3,4-oxadiazole derivatives with anticonvulsant activity and their binding to the GABAA receptor

Article abstract of DOI:10.1016/j.ejmech.2020.112672

In this study, a series of 1,3,4-oxadiazole derivatives (5a-s, 10a-s, and 16a-d) were designed and synthesized using maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) models, to test the anticonvulsant activity of the target compounds

Full text of DOI:10.1016/j.ejmech.2020.112672

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Synthesis of 1,3,4-oxadiazole derivatives with anticonvulsant activity and their binding
to the GABA receptor
A
Shiben Wang, Hui Liu, Xuekun Wang, Kang Lei, Guangyong Li, Jun Li, Renmin Liu,
Zheshan Quan
PII:
S0223-5234(20)30644-9
DOI:
https://doi.org/10.1016/j.ejmech.2020.112672
EJMECH 112672
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 June 2020
Revised Date: 16 July 2020
Accepted Date: 16 July 2020
Please cite this article as: S. Wang, H. Liu, X. Wang, K. Lei, G. Li, J. Li, R. Liu, Z. Quan, Synthesis
of 1,3,4-oxadiazole derivatives with anticonvulsant activity and their binding to the GABA receptor,
A
European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112672.
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Synthesis of 1,3,4-oxadiazole derivatives with anticonvulsant activity
and their binding to the GABAA receptor
a
a
a
a
Shiben Wang a, *, Hui Liu b, Xuekun Wang , Kang Lei , Guangyong Li , Jun Li , Renmin Liu a, **,
Zheshan Quan c, ***
a College of pharmacy, Liaocheng University, Liaocheng, Shandong 252059, China.
b College of life sciences, Liaocheng University, Liaocheng, Shandong 252059, China
c College of Pharmacy, Yanbian University, Yanji, Jilin 133002, China
Corresponding Author:
* Dr. Shiben Wang, College of Pharmacy of Liaocheng University, 1 Hunan Road, Liaocheng,
252059 Shandong, China. Email: wangshiben110@163.com.

Abstract
In this study, a series of 1,3,4-oxadiazole derivatives (5a-s, 10a-s, and 16a-d) were designed and
synthesized using maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ) models,
to test the anticonvulsant activity of the target compounds in vivo. The neurotoxicity (NT) of the target
compounds was measured using the rotating rod (ROT) method. Seven compounds with potential
activity were selected to test the 50 % effective dose (ED50) and 50 % toxic dose (TD50).
Pharmacological
experiments
revealed
that
6-((5-(pentylthio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (5b) showed the best
anticonvulsant activity (MES, ED50 = 8.9 mg/kg; scPTZ, ED50 = 10.2 mg/kg), which was greater than
the activities of carbamazepine and ethosuximide. Compound 5b exhibited the most potent binding
affinity toward the GABAA receptor (IC50 = 0.11 μM) in the in vitro binding experiments. Compound
5b displayed significant anxiolytic activity at a low dose (1 mg/kg) in the elevated plus maze (EPM)
test. The GABA content in rat brains was also investigated, and the results showed that compound 5b
might have affected the GABA system. In our molecular docking experiment, compound 5b showed
significant interactions with residues present at the benzodiazepine binding site on the GABAA receptor.
The structure and physicochemical and pharmacokinetic properties of the target compound were
predicted using Discovery Studio 2019 and ChemBioDraw Ultra 14.0. Finally we demonstrated that
compound 5b mainly acted on GABAA receptor. Thus the present study has provided potential
candidates for further investigation in epilepsy.
Keywords: 1,3,4-Oxadiazole, Anticonvulsant, scPTZ, GABAA receptor, Molecular docking studies

1. Introduction
Epilepsy is a common disease of the nervous system characterized by recurrent seizures caused due
to abnormal electrical discharge from the brain neurons; it affects 1 % of the world’s population.
However, we are still unable to determine the pathogenesis of epilepsy at the cellular level [1].
Currently, epilepsy treatment mainly involves the use of drugs [2]. Statistics-based analyses suggest the
following: only 50 % of the patients are completely cured by the existing anticonvulsant drugs;
complete treatment in not observed in approximately 20 % of the patients who also show
treatment-related side effects; and the remaining 30‒40 % are intractable epilepsy patients who cannot
be cured by drugs [3]. Therefore, there is an urgent need to develop highly effective, low-dose
anticonvulsant drugs [4].
The development of new, targeted anticonvulsant drugs could serve as a very effective strategy in
epilepsy treatment. γ-Aminobutyric acid type A (GABAA) receptors are important targets of the central
nervous system (CNS) drugs, especially anticonvulsants [5,6]. The benzodiazepine sites (BZ-S) on the
GABAA receptor serve as important active sites, especially for the development of anticonvulsant drugs
[7]. Diazepam, clobazam, clonazepam, and triazolam are BZ drugs that exhibit a strong affinity for
BZ-S on the GABAA receptor. BZ drugs have common chemical structural features, i.e., hydrophobic
regions A and C, and a receptor region (B) at an appropriate distance from A and C (Fig. 1) [8].
Fig. 1. Structures of benzodiazepine agonists. A: aromatic ring, B: the coplanar proton-accepting group, C: the
out-of-plane aromatic ring
3,4-Dihydroquinolin-2(1H)-one is a promising compound in anticonvulsant drug research. Deng et al.
designed and synthesized a series of 3,4-dihydroquinolin-2(1H)-one derivatives, which showed robust
anticonvulsant activity [9]. In addition, five-membered azole-containing heterocyclic derivatives, such
as 1,3,4-oxadiazole [10] and 1,2,4-triazole [11], are widely used in anticonvulsant drug studies. Singh
et al. designed and synthesized a series of 1,3,4-oxadiazole derivatives, which showed good
anticonvulsant activity [12]. In this study, to identify compounds with similar structural characteristics

as BZs, we combined the structural fragments of 3,4-dihydroquinolin-2(1H)-one and 1,3,4-oxadiazole
to improve their efficacy against epilepsy; a series of compounds 5a-s were designed and synthesized.
In order to investigate the importance of the 3,4-dihydroquinolin-2(1H)-one structural fragment in
compounds 5a-s, a series of derivatives 10a-s were designed and synthesized by cutting at the 3 and 4
positions of 3,4-dihydroquinolin-2(1H)-one and by increasing the flexibility of the molecule for
investigating the effect of the ring opening on compound activity (Fig. 2). Pharmacological evaluation
of compounds 5a-s and 10a-s showed that the alkyl-chain‒substituted compounds have robust
anticonvulsant activity. Therefore, we used a fusion method to combine 1,3,4-oxadiazole and
1,2,4-triazole active fragments, and synthesized a series of alkyl-chain‒substituted compounds (16a-d)
to investigate the effect of the recombined compounds on activity (Fig. 2).
N
N
N
N
N
O
N
O
H
H
4H-1,2,4-triazole
3,4-dihydroquinolin-2(1
H)-one
1,3,4-oxadiazole
Combination
Combination
O
B
O
N
N
N
N
A
O
O
S
NH
S
R
N
R
O
C
N
16a-d
5a-s
N
Ring opening
N
N
O
N
N
H
N
O
Introduce triazole fragment
S
NH
R
O
10a-s
Fig. 2. Structures of 3,4-dihydroquinolin-2(1H)-one, 1,3,4-oxadiazole, and 4H-1,2,4-triazole, and target
compounds 5a-s, 10a-s, and 16a-d.
In this study, we used maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ)
mIn this study, we used the maximal electroshock (MES) and subcutaneous pentylenetetrazole (scPTZ)
methods to test in vivo the anticonvulsant activity of the target compound, and to investigate the
neurotoxicity of the target compound by the rotating rod (ROT) method. A radioreceptor binding assay
was performed to determine the possible mechanism of action of the compound. Potential compounds

that showed efficiency in both models were evaluated for their effect on the GABA content in rat brain.
The anxiolytic properties of the potent compounds were tested using the elevated plus maze (EPM)
assay. The molecular docking module in the computer software Discovery Studio 2019 (DS 2019) was
used to analyze the possible modes of action of the potential compounds and BZ active sites.
Simultaneously, DS 2019 and ChemBioDraw Ultra14.0 were used to generate the structure of the target
compounds; these software then used the same to predict the physicochemical and pharmacokinetic
properties of these compounds. To confirm the structure of the target compounds, we used
high-resolution mass spectrometry and 1H-nuclear and 13C-nuclear magnetic resonance.
2. Results and discussion
2.1. Chemistry
The various steps during the synthesis of target compounds 5a-s, 10a-s, and 16a-d are shown in
Schemes 1‒3. First, intermediates 2 and 7 were synthesized under similar reaction conditions. Under
reflux conditions, 6-hydroxy-3,4-dihydroquinolin-2(1H)-one/N-(4-hydroxyphenyl)acetamide was
reacted with BrCH2CO2CH3 to obtain intermediates 2 and 7. Then, intermediates 2 and 7 were reacted
with hydrazine hydrate using THF as a solvent, and intermediates 3 and 8 were obtained by reaction
under reflux conditions. Next, intermediates 4 and 9 were generated via the cyclization of intermediates
3 and 9 with CS2 under alkaline conditions. As this process resulted in an incomplete reaction, CS2 had
to be added after 5‒10 h to complete the reaction. Intermediates 2-4 and 7-9 were synthesized in
accordance with previously reported methods [13]. Intermediates 4 and 9 were reacted with bromides
to obtain target compounds 5a-s and 10a-s. In Scheme 3, 4-aminophenol was reacted with
HCONHNH2 and DMF-DMA to obtain intermediate 12 [14]. The methods used for the synthesis of
intermediate 13-15 and target compounds 16a-d were also similar to the method used for the synthesis
of compounds 2, 3, 4, and 5a-s. The structures of all target compound 5a-s, 10a-s, and 16a-d were
confirmed using the 1H, 13C, and high-resolution mass spectra (HRMS) techniques.

R: 5a: C4H9; 5b: C5H11; 5c: C6H13; 5d: C7H15; 5e: C8H17; 5f: C9H19; 5g: CH2Ph; 5h: CH2Ph (o-Cl); 5i: CH2Ph
(m-Cl); 5j: CH2Ph (p-Cl); 5k: CH2Ph (o-F); 5l: CH2Ph (m-F); 5m: CH2Ph (p-F); 5n: CH2Ph (o-CF3); 5o: CH2Ph
(m-CF3); 5p: CH2Ph (p-CF3); 5q: CH2Ph (o-CH3); 5r: CH2Ph (m-CH3); 5s: CH2Ph (p-CH3).
Reagents and conditions: (i) BrCH2CO2CH3, K2CO3, CH3CN; Reflux, 6‒8 h; (ii) NH2NH2.H2O, THF, Reflux, 2 h;
(iii) NaOH, CS2, C2H5OH, 80 oC, 19‒21 h; (iv) RBr, K2CO3, Acetone, 65 oC, 10 h
Scheme 1. Synthesis of the target compounds 5a-s.
R: 10a: C4H9; 10b: C5H11; 10c: C6H13; 10d: C7H15; 10e: C8H17; 10f: C9H19; 10g: CH2Ph; 10h: CH2Ph (o-Cl); 10i:
CH2Ph (m-Cl); 10j: CH2Ph (p-Cl); 10k: CH2Ph (o-F); 10l: CH2Ph (m-F); 10m: CH2Ph (p-F); 10n: CH2Ph (o-CF3);
10o: CH2Ph (m-CF3); 10p: CH2Ph (p-CF3); 10q: CH2Ph (o-CH3); 10r: CH2Ph (m-CH3); 10s: CH2Ph (p-CH3).
Scheme 2. Synthesis of the target compounds 10a-s.
R: 16a: C4H9; 16b: C5H11; 16c: C6H13; 16d: C7H15.
Scheme 3. Synthesis of the target compounds 16a-d.
2.2. Pharmacology
2.2.1. Anticonvulsant activity

The target compounds 5a-s, 10a-s, and 16a-d were screened for anticonvulsant activity in three dose
groups (30, 100, and 300 mg/kg) using the MES and scPTZ tests [15,16]. Simultaneously, ROT was
used to test the neurotoxicity of the target compounds. Ethosuximide and carbamazepine were used as
positive control drugs in the scPTZ and MES model experiments, respectively. Diazepam was also
used as a positive control. Pharmacological experiments revealed that most of the target compounds
exhibited robust anticonvulsant activity in the two experimental models. The anticonvulsant activity
and neurotoxicity of all target compounds are shown in Table 1–3.
Table 1. Anticonvulsant activities of compounds 5a-s in the MES, scPTZ, and ROT tests.
0.5 h
0.5 h
Dose a
Dose
Compds
5a
Compds
5k
MESb
scPTZc
NTd
MES
scPTZ
NT
(mg/kg)
(mg/kg)
30
100
300
30
1/5 e
3/5
5/5
5/5
5/5
-
-
-
30
100
300
30
-
-
1/5
3/5
5/5
-
2/5
5/5
-
2/5
5/5
5/5
5/5
-
0/5
0/5
-
0/5
0/5
-
5b
5c
6d
5e
5f
5l
5m
5n
5o
5p
5q
5r
1/5
4/5
-
1/5
3/5
-
100
300
30
0/5
0/5
-
100
300
30
0/5
0/5
-
5/5
5/5
-
4/5
5/5
-
0/5
3/5
-
2/5
5/5
1/5
4/5
5/5
-
100
300
30
0/5
0/5
-
100
300
30
0/5
1/5
-
2/5
4/5
5/5
0/5
2/5
5/5
-
-
2/5
5/5
1/5
4/5
5/5
4/5
5/5
-
100
300
30
2/5
4/5
-
0/5
0/5
-
100
300
30
0/5
1/5
-
2/5
5/5
3/5
5/5
-
100
300
30
1/5
3/5
-
0/5
0/5
-
100
300
30
0/5
1/5
-
100
300
30
1/5
5/5
-
0/5
2/5
0/5
0/5
-
100
300
30
0/5
0/5
-
2/5
4/5
5/5
1/5
4/5
5/5
-
5g
5h
5i
2/5
5/5
-
100
300
30
2/5
5/5
-
1/5
4/5
-
0/5
0/5
-
100
300
30
0/5
0/5
-
1/5
5/5
100
300
30
2/5
5/5
1/5
4/5
5/5
-
0/5
3/5
-
0/5
0/5
-
100
300
30
0/5
0/5
-
-
-
5s
100
300
30
2/5
5/5
-
0/5
0/5
-
100
300
30
2/5
5/5
5/5
-
1/5
0/5
4/5
-
0/5
0/5
CBZf
ESM
DZP[16]
5j
3/5
-
0/5
5/5
100
300
1/5
4/5
0/5
1/5
0/5
0/5
100
2
5/5

a Doses of 30, 100, and 300 mg/kg were administrated intraperitoneally in mice. The animals were examined 0.5 h
b
c
d
after administration; Maximal electroshock; Subcutaneous pentylenetetrazole test; Neurotoxicity screening:
rotarod test; e No. of animals protected/No. of animals tested; (-): not tested at 30, 100, and 300 mg/kg;
f carbamazepine (CBZ), ethosuximide (ESM), diazepam (DZP).
The pharmacological data of the target compounds 5a-s MES, scPTZ, and NT are shown in Table 1.
As shown in Table 1, in the MES experimental model at the measurement time of 0.5 h, all compounds
—except for 5m—exhibited anticonvulsant activity at doses of 100 and 300 mg/kg; two compounds,
5a and 5b, showed the best anticonvulsant activity (protection rate, 5/5) at a dose of 30 mg/kg. In the
scPTZ experimental model at the measurement time of 0.5 h, all compounds showed anticonvulsant
activity at a dose of 300 mg/kg. At doses of 100 mg/kg—except for three compounds (5f, 5h, and 5j)
—all compounds showed anticonvulsant activity. However, at doses of 30 mg/kg, only five compounds
—5b-c, 5k, 5n, and 5p—showed anticonvulsant activity. In the neurotoxicity experimental model at
the measurement time of 0.5 h, only compound 5o showed neurotoxicity at 300 mg/kg.
Table 2. Anticonvulsant activities of compounds 10a-s in the MES, scPTZ, and ROT tests.
0.5 h
0.5 h
Dose
Dose
Compds
10a
Compds
10k
(mg/kg)
(mg/kg)
MES
scPTZ
NT
MES
scPTZ
NT
-
0/5
3/5
5/5
-
30
100
300
30
0/5
2/5
5/5
4/5
5/5
-
-
-
30
100
300
30
-
2/5
4/5
-
1/5
4/5
3/5
5/5
-
0/5
0/5
-
0/5
0/5
-
10b
10c
10d
10e
10f
10l
10m
10n
10o
10p
10q
10r
1/5
3/5
-
0/5
2/5
-
100
300
30
0/5
0/5
-
100
300
30
0/5
2/5
-
4/5
5/5
-
3/5
5/5
-
0/5
2/5
-
1/5
5/5
-
100
300
30
0/5
0/5
-
100
300
30
0/5
1/5
-
1/5
3/5
5/5
0/5
1/5
4/5
-
-
2/5
4/5
-
1/5
4/5
1/5
4/5
5/5
2/5
5/5
-
100
300
30
2/5
4/5
-
0/5
0/5
-
100
300
30
0/5
0/5
-
1/5
3/5
-
100
300
30
1/5
3/5
-
0/5
0/5
-
100
300
30
0/5
0/5
-
0/5
1/5
-
100
300
30
1/5
4/5
-
0/5
2/5
0/5
0/5
-
100
300
30
0/5
1/5
-
-
10g
10h
2/5
4/5
2/5
5/5
1/5
5/5
-
100
300
30
2/5
5/5
-
1/5
4/5
-
0/5
0/5
-
100
300
30
0/5
0/5
-
1/5
100
1/5
2/5
0/5
100
0/5

-
4/5
300
30
3/5
-
5/5
-
0/5
-
300
30
0/5
-
1/5
3/5
5/5
5/5
-
-
10i
10j
10s
100
300
30
3/5
5/5
-
0/5
3/5
-
0/5
0/5
-
100
300
30
2/5
0/5
5/5
-
0/5
0/5
CBZ
ESM
DZP
3/5
-
0/5
5/5
100
300
2/5
4/5
0/5
1/5
0/5
0/5
100
2
5/5
The pharmacological data of target compounds 10a-s are shown in Table 2. As shown in Table 2, in
the MES experimental model, all compounds—except for compounds 10m and 10p—exhibited
anticonvulsant activity at doses of 100 and 300 mg/kg, with five compounds, namely 10b-d and 10r-s
showing anticonvulsant activity at a dose of 30 mg/kg. In the scPTZ experimental model, all
compounds showed anticonvulsant activity at a dose of 300 mg/kg. At doses of 100 mg/kg, all
compounds—except for two compounds (10f and 10l)—showed anticonvulsant activity. However, at a
dose of 30 mg/kg, only two compounds (10o and 10p) showed anticonvulsant activity. In the
neurotoxicity experimental model, only compounds 10l-m and 10p showed neurotoxicity at
300 mg/kg.
The pharmacological data of target compounds 16a-d are shown in Table 3. As shown in Table 3, in
the MES experimental model, all compounds exhibited anticonvulsant activity at doses of 100 and
300 mg/kg, with three compounds (16a-c) showing anticonvulsant activity at a dose of 30 mg/kg;
compound 16b showed the best anticonvulsant activity (protection rate, 5/5). In the scPTZ
experimental model, all compounds displayed anticonvulsant activity at doses of 100 and 300 mg/kg.
At a dose of 30 mg/kg, two compounds—16b-c—showed anticonvulsant activity. In the neurotoxicity
experimental model, none of the four compounds showed neurotoxicity at doses of 30, 100, and
300 mg/kg.
Table 3. Anticonvulsant activities of compounds 16a-d in the MES, scPTZ, and ROT tests.
0.5 h
0.5 h
Dose
Dose
Compds
16a
Compds
(mg/kg)
(mg/kg)
MES
scPTZ
NT
MES
scPTZ
NT
-
-
0/5
3/5
5/5
-
0/5
-
30
100
300
30
1/5
4/5
5/5
5/5
5/5
-
-
-
300
30
0/5
2/5
5/5
1/5
3/5
3/5
5/5
-
0/5
0/5
-
16d
0/5
0/5
0/5
0/5
5/5
-
100
300
30
16b
16c
100
300
30
0/5
0/5
-
CBZ
ESM
DZP
-
5/5
-
3/5
-
100
2
3/5
5/5
3/5
5/5
5/5
-
-
100
0/5
-

In the MES and scPTZ models, seven compounds (5b-c, 5p, 10b-c, and 16b-c) showing higher
activity at 30 mg/kg were selected, their 50 % effective doses (ED50s) and 50 % toxic doses (TD50s)
were tested, and their protection indexes (PIs, PI = TD50/ED50) were calculated. Simultaneously,
carbamazepine and ethosuximide, which are effective in the two models, were selected as control drugs.
The experimental results are shown in Table 4.
Table 4. Quantitative data of the target compound anticonvulsant activities.
ED50
(MES, mg/kg)a
ED50
TD50
(NT, mg/kg)b
PIc
Compds
(scPTZ, mg/kg)
MES scPTZ
8.9 (7.3-10.7)
10.4 (8.6-12.6)
23.1 (19.1-27.9)
22.6 (18.6-27.3)
20.9 (17.3-25.2)
18.7 (15.4-22.6)
26.6 (21.9-32.2)
11.8 (8.5-16.4)
-
10.2 (8.4-12.3)
17.9 (14.8-21.6)
27.6 (22.8-33.4)
28.1 (23.2-34.0)
26.8 (22.1-32.4)
27.9 (23.0-33.7)
28.4 (23.4-34.3)
-
575.4 (475.5-696.2)
465.7 (384.8-563.4)
376.2 (313.8-455.2)
388.6 (321.1-470.2)
420.1 (347.2-508.3)
421.3 (348.2-509.7)
385.4 (318.5-466.3)
76.1 (55.8-103.7)
64.6
44.7
16.2
17.2
20.1
22.5
14.4
6.4
56.4
26.0
13.6
13.8
15.6
15.1
13.5
-
5b
5c
5p
10b
10c
16b
16c
Carbamazepine
Ethosuximide
106.0 (93.0-121.0)
343.0 (311.0-378.0)
-
3.2
a ED50: median effective dose affording anticonvulsant protection in 50 % of animals.
bTD50: median toxic dose eliciting minimal neurological toxicity in 50 % of animals.
c PI: protective index (TD50/ED50).
- not tested.
Pharmacological data in Table 4 show that in the MES experimental model, compound 5b exhibited
the best anticonvulsant activity, with an ED50 of 8.9 mg/kg, and its anticonvulsant activity was higher
than that of carbamazepine (11.8 mg/kg). In the scPTZ experimental model, compound 5b again
showed the best anticonvulsant activity, with an ED50 of 10.2 mg/kg, and its anticonvulsant activity was
higher than that of ethosuximide (106.0 mg/kg). In the ROT rod model, compound 5b showed the
lowest neurotoxicity, much lower than that of carbamazepine and ethosuximide. In summary,
compound 5b showed higher efficacy and lower toxicity than all other compounds—and positive
control drugs—in addition to showing better safety (PIMES = 64.6, PIPTZ = 56.4) than carbamazepine
(PIMES = 6.4) and ethosuximide (PIPTZ = 3.2).
2.2.2. Radioreceptor binding assay
Seven potential compounds (5b-c, 5p, 10b-c, and 16b-c) were selected from the MES and scPTZ
models, and their binding affinities for the BZ active site of the GABAA receptor were determined in
order to study their possible effector mechanisms. The half-maximal inhibitory concentrations (IC50) of

the compounds were determined through this experiment. Pharmacological data are shown in Table 5.
As shown in Table 5, compound 5b exhibited high affinities for the BZ-S on the GABAA receptor
complex, and its IC50 value was 0.11 μM. Compound 5c also showed high affinity, and its IC50 value
was 0.88 μM.
Table 5. Affinities of compounds 5b-c, 5p, 11b-c, 16b-c, and diazepam for the benzodiazepine site of the
GABAA receptor.
IC50 (μM)
Compounds
0.11 ± 0.007
0.88 ± 0.09
5.28 ± 0.38
1.29 ± 0.11
4.12 ± 0.31
3.43 ± 0.19
9.31 ± 0.98
0.042 ± 0.0018
5b
5c
5p
10b
10c
16b
16c
Diazepam
2.2.3. GABA estimation
GABA is a CNS-inhibitory neurotransmitter and is closely related to the occurrence of epilepsy [17].
In order to investigate the effect of compound 5b on the GABA content in the brain of the experimental
animals, we measured the changes in the GABA content in the brain of Wistar rats after treatment with
compound 5b for 2 h and 7 days. As shown in Fig. 3, compound 5b and diazepam significantly
increased the GABA concentration in the rat brain after 2 h of treatment compared with that in the
control group. After 7 days of treatment, compound 5b and diazepam further increased the GABA
concentration in the rat brain. These results showed that compound 5b might affect the GABA system.

Fig. 3. Effect of compound 5b on the GABA system. Adult Wistar rats were divided into three groups of six each.
Compound 5b was administered at 30 mg/kg (i.p.), diazepam was administered at 10 mg/kg, and the GABA level
in the rat brain was evaluated as μg/100 mg of tissue. *Significant compared to control (0.01 <
P < 0.05), values
represent the mean ± SEM (n = 6).
2.2.4. Elevated plus maze experiments
The EPM results for compound 5b are shown in Fig. 4. At different doses, the percentage of time
spent in the open arms was different. At a dose of 1 mg/kg, compound 5b significantly (P < 0.05)
increased the time spent in the open-sided arms and showed the best anti-anxiety effect, and this
activity was similar to that of diazepam.
Fig. 4. Elevated plus maze tests for 5b (0.5, 1, 3, and 10 mg/kg), and diazepam (1 mg/kg). Data represent the mean
± SEM of the percentage of time spent on open arms relative to total time spent on all four arms. *P < 0.05,
significantly different from control (n = 10).
Flumazenil is a specific antagonist of the GABAA receptor BZ-binding site; it did not elicit any
response by itself in the EPM test. In the experiment, compound 5b was administered at a dose of
1 mg/kg (as the anti-anxiety effect peaks at a dose of 1 mg/kg), and flumazenil was administered at a
dose of 5 mg/kg (several doses of this antagonist were tested alone to identify the maximal dose
without any intrinsic action to be used in the blockade experiments). As shown in Fig. 5, pretreatment
of Wistar rats with flumazenil, resulted in the complete elimination of the anxiolytic-like effect of
compound 5b in the EPM test (reduced time percentage in open arms). These results suggest that
compound 5b could serve as an agonist of the BZ-binding site on the GABAA receptor.

Fig. 5. Effect of flumazenil on the anxiolytic activity of compound 5b. Adult Wistar rats were pretreated with
control or flumazenil (FMZ) 15 min before injection of compound 5b (i.p.). Bars indicate mean ± SEM of the
percentages of time spent in open arms evaluated in the plus maze assay using compound 5b. *P < 0.05,
significantly different from control (n = 10).
2.2.5. Structure-activity relationships
In this study, we combined 3,4-dihydroquinolin-2(1H)-one and 1,3,4-oxadiazole—showing
significant anticonvulsant effects—and designed and synthesized a series of target compounds 5a-s. In
order to investigate the importance of the 3,4-dihydroquinolin-2(1H)-one structural fragment in
compounds 5a-s, a series of derivatives 10a-s were designed and synthesized by cutting at the 3 and 4
positions of 3,4-dihydroquinolin-2(1H)-one and by increasing the flexibility of the molecule for
investigating the effect of the ring opening on compound activity. Pharmacological evaluation of
compounds 5a-s and 10a-s showed that the alkyl-chain‒substituted compounds exhibited robust
anticonvulsant activity. Based on these results, we used a fusion method to combine 1,3,4-oxadiazole
and 1,2,4-triazole active fragments, and synthesized four alkyl-chain‒substituted compounds (16a-d) to
investigate how the recombined compounds affect the activity. The target compounds were screened
using two models, MES and scPTZ. The compounds with better activity were studied further. Based on
the pharmacological results, the structure-activity relationships (SARs) of the target compounds were
analyzed (Fig. 6).

Fig. 6. SARs of target compounds 5a-s, 10a-s, and 16a-d.
The pharmacological results for compounds 5a-s showed that among the alkyl chain
C4-C9-substituted compounds, the C5-substituted compound showed the best anticonvulsant activity.
When the alkyl chain length was >C5, the anticonvulsant activity of the target compound did not
improve and was lower than that of the C5-substituted compound, and the C5-substituted compound
showed the maximum activity peak. For the benzyl-substituted derivatives, the ortho (o), meta (m), or
para (p) positions of electron-donating (CH3) and electron-withdrawing groups (Cl, F, and CF3) on the
benzene ring affected the anticonvulsant activity of the compound. The activities of derivatives with
different electronic groups on the benzene ring were in the following order: m-Cl > o-Cl > p-Cl, o-F >
m-F > p-F, p-CF3 > m-CF3 > o-CF3, o-CH3 > m-CH3 > p-CH3. Overall, the anticonvulsant activity of
the alkyl-chain‒substituted compounds was better than that of the benzyl-substituted derivatives.
Pharmacological studies on compounds 10a-s showed that among the alkyl chain C4-C9-substituted
compounds, the C5-substituted compounds showed the best anticonvulsant activity. When the length of
the alkyl chain increased beyond C5, the anticonvulsant activity of the target compound did not increase
and was lower than that of the C5-substituted compound; the C5-substituted compound showed the
maximum activity peak. For the benzyl-substituted derivatives, the activities of derivatives with
different electronic groups on the benzene ring followed the order, m-Cl > p-Cl > o-Cl, o-F > m-F > p-F,
o-CF3 > m-CF3 > p-CF3, m-CH3 > p-CH3 > o-CH3. The anticonvulsant activity of the
alkyl-chain‒substituted was better than that of the benzyl-substituted derivatives. Pharmacological
studies on compounds 16a-d revealed that among the alkyl chain C4-C7-substituted compounds, the
C5-substituted compounds showed the best anticonvulsant activity. Moreover, the C5-substituted
compounds showed maximum peak activity.
Next, the 3,4-dihydroquinolin-2(1H)-one structural fragment of the target compounds 5a-s was

opened at the 3,4 position (compound 10a-s was obtained). The target compounds 16a-d—obtained via
a combination of 1,3,4-oxadiazole and 1,2,4-triazole active fragments—showed an overall decrease in
the anticonvulsant activity compared with compounds 5a-s. The experimental results showed that the
3,4-dihydroquinolin-2(1H)-one structure had an important role in the design of the target compounds,
and significantly improved their anticonvulsant activity. Among the target compounds 5a-s, 10a-s, and
16a-d, the C5-substituted derivatives showed the best anticonvulsant activity among all the
alkyl-chain‒substituted compounds. It could be speculated that C5 exhibit an appropriate lipid-water
partition coefficient and are more likely to enter the brain tissue to exert an anticonvulsant effect.
Moreover, the anticonvulsant activity of the compounds with alkyl-chain‒substituted derivatives was
better than that of those with benzyl-substituted derivatives. It could be speculated that the
alkyl-chain‒substituted derivatives might have an important effect on determining the anticonvulsant
activity of the compounds, and that they would allow an easier entry into the brain tissue through the
blood-brain barrier and exert anticonvulsant effects.
2.2.6. Docking study
In recent years, molecular docking—a theoretical simulation method for studying intermolecular
interactions and predicting the binding mode and affinity—has become an important technology in the
field of computer-aided drug study. The most active compound—5b—diazepam (positive control), and
the BZ-S of the GABAA receptor (PDB Code: 6HUP) were selected for molecular docking studies. The
molecular docking module in the DS 2019 software was used for docking analysis. The molecular
docking results are shown in Fig. 7.
Diazepam
5b
Fig. 7. Compound 5b and diazepam show interactions with residues present at the BZ-binding site of the GABAA

receptor.
Molecular docking studies (Fig. 7) showed that the main amino acids around the BZ-S—that
interacted with compound 5b and diazepam—were TYR58, PHE77, TYR160, VAL203, SER205,
SER206, THR 207, and TYR210. Diazepam could form the following bonds: non-classical hydrogen
bonds via TYR160 and SER205; pi-pi stacks via TYR58 and PHE77; and pi-alkyl interactions via
TYR160 and TYR210. Docking revealed that the interaction force of compound 5b with the amino
acids around the BZ-S was higher than that of diazepam; it revealed hydrogen bond formation with
SER205 and TYR160; non-classical hydrogen bonding with TYR160, SER205, and SER207; pi-pi
stacking with TYR58, PHE77, TYR160, and TYR210; and pi-alkyl interaction with VAL 203. The
results of docking for compound 5b and diazepam superimposed in the BZ-binding pocket of the
GABAA receptor are shown in Fig. 8. By analyzing the docking results of compound 5b and diazepam,
it could be speculated that the interaction between compound 5b and the GABAA receptor was similar
to that between diazepam and this receptor.
Fig. 8. Compound 5b (brown) and diazepam (yellow) superimposed in the BZ-binding pocket of the GABAA
receptor.
2.2.7. Prediction of physicochemical and pharmacokinetic properties
DS 2019 was used to predict the physicochemical properties of the target compounds. The target
compound drug-likeness parameters (molecular weight, CLogP, number of hydrogen donors, number
of acceptors, number of rotatable bonds, and the polar surface area) have been provided in the
supporting information file.
In order to ensure that the compound exhibits robust pharmacokinetic properties, the ADME/T
module in the DS 2019 software package was used to predict the pharmacokinetic properties of the

target compounds 5a-s, 10a-s and 16a-d (i.e. absorption (ABS), blood-brain barrier permeability) to
ensure easy penetration of the compounds through the blood-brain barrier (BBB), and to avoid low
solubility and low oral bioavailability. The prediction revealed that all target compounds exhibit robust
ABS and favorable BBB permeability (Fig. 9).
Fig. 9. ADME/T value prediction for target compounds 5a-s, 10a-s, and 16a-d.
3. Conclusions
In this study, three series of 1,3,4-oxadiazole derivatives were designed, synthesized, and evaluated
for their anticonvulsant activity. Most of the compounds showed significant anticonvulsant effects at
different
doses.
Compound
6-((5-(pentylthio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (5b), showed the best
anticonvulsant activity in the MES and scPTZ models (among all compounds); it also showed
significantly better safety than the controls carbamazepine and ethosuximide. The radioreceptor
binding assay revealed compound 5b to be the most suitable compound, with an IC50 of 0.11 μM and a
high binding affinity toward GABAA receptors. The EPM experiment revealed that compound 5b
exhibited a significant anti-anxiety effect at a dose of 1.0 mg/kg. Molecular docking revealed that
compound 5b exhibited a significant interaction with the amino acid residues around the BZ-S of the
GABAA receptors. Physicochemical and pharmacokinetic property prediction of the target
compounds—using the DS 2019 and ChemDraw 14.0 software—revealed that all compounds had good
physicochemical properties, and exhibited robust BBB permeability and ABS. These properties are
very critical for the compounds to be explored as potential drug candidates for further investigations.
4. Experimental
4.1. Chemistry

1
The H and 13C spectrum data were measured using a Bruker AV 300 or 400 nuclear magnetic
resonance spectrometer (Bruker, Switzerland). The target compound was measured by dissolving in
DMSOd6 or CDCl3, and tetramethylsilane was selected as an internal standard. The abbreviations used
in the spectral data are as follows: s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet. An X-4
binocular microscope melting point apparatus (Shanghai, China) was used to determine the melting
point of the compound. HRMS was determined using a mass spectrometer (Bruker Daltonik, Germany).
[3H]flunitrazepam (85.2 Ci/mmol) was purchased from Perkin-Elmer (Waltham, USA). The solvent
and reagent used were of analytical or chemical grades (Aladdin, China Reagent Network, Shenyang
Chemical Reagent Factory, Sigma-Aldrich). The silica gel used for thin-layer plates and column
chromatography was produced by the Qingdao Ocean Chemical Factory (Qingdao, China).
4.1.1. Synthesis of intermediate 2
Quinolinone (10.0 mmol), K2CO3 (11.0 mmol) and 30 ml CH3CN were added to a 100 ml
single-necked round bottom flask, and the reaction was stirred at 70 oC. After 0.5 h, methyl
bromoacetate (10.5 mmol) was added dropwise to the reaction, and the reaction was continued for 6 h.
The reaction was detected by TLC, and after the reaction was completed, the solvent was vacuum dried
under reduced pressure to obtain a crude solid; water (30 ml) was added to the round bottom flask
containing the crude solid, which was then stirred for 10 min, filtered with suction, washed with water
3 times, dried, and recrystallized with ethanol to obtain intermediate 2.
4.1.2. Synthesis of intermediate 3
A reaction of intermediate 2 (10.0 mmol), hydrazine hydrate (11.0 mmol), and 30 ml THF was
stirred at reflux for 6 h in a 100 ml single-necked round bottom flask. The reaction was detected by
TLC. After the reaction was completed, the excess THF was evaporated under reduced pressure in
vacuo to obtain a solid residue. CH3OH (10 ml) was added to the residue and stirred at room
temperature for 5 min; this was followed by suction filtration, washing with cooled CH3OH (4 ml *3
times), and drying to obtain intermediate 3.
4.1.3. Synthesis of intermediate 4
Intermediate 3 (10.0 mmol), NaOH (15.0 mmol), CS2 (20.0 mmol), and 30 ml EtOH were added to a
100 ml single-necked round bottom flask, the reaction was stirred at reflux for 12 h, then CS2 (5.0
mmol) was added to continue the reaction for 7 h. The reaction was detected by TLC. After the reaction
was completed, 10 % HCl was added to adjust the pH to 4‒5, until a large amount of white solids was

precipitated; the reaction mixture was then suction filtered and washed 3 times with water to obtain the
white solids, and dried to obtain intermediate 4.
4.1.4. Synthesis of target compounds 5a-s
Intermediate 4 (10.0 mmol), K2CO3 (11.0 mmol) and 30 ml of acetone were reacted in a 100 ml
single-necked round bottom flask by stirring at 65 oC for 1 h, and then bromine (10.5 mmol) was added
to continue the reaction for 9 h. The reaction was detected by TLC. After the reaction was completed,
the solvent was evaporated to dryness in vacuo to obtain a white solid; to this white solid, 30 ml of
water was added, and the mixture was stirred at room temperature for 10 min, filtered with suction,
washed with water 3 times, dried, purified by column chromatography (CH3OH : CH2Cl2 = 1: 100) to
obtain target compounds 5a-s.
6-((5-(Butylthio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one（5a）
1
Yield: 56 %. Mp: 186-187 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 8.91 (s, 1H, CONH), 6.75-6.84
(m, 3H, Ar-H), 5.18 (s, 2H, OCH2), 3.26 (t, 2H, J = 7.5 Hz, SCH2), 2.94 (t, 2H, J = 7.5 Hz, COCH2),
2.61 (t, 2H, J = 7.5 Hz, COCH2CH2), 1.41-1.83 (m, 4H, CH2), 0.94 (t, 3H, J = 7.5 Hz, CH3). 13C NMR
(75 MHz, CDCl3) δ: 171.59, 163.08, 153.31, 132.11, 125.21, 116.32, 115.03, 113.73, 90.32, 60.26,
32.19, 31.05, 30.40, 25.55, 21.68, 13.45. ESI-HRMS calcd for C16H20N3O3S+ ([M+H]+): 334.1220;
found: 334.1215.
6-((5-(Pentylthio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one（5b）
1
Yield: 47 %. Mp: 208-210 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 8.86 (s, 1H, CONH), 6.75-6.84
(m, 3H, Ar-H), 5.18 (s, 2H, OCH2), 3.25 (t, 2H, J = 7.5 Hz, SCH2), 2.94 (t, 2H, J = 7.5 Hz, COCH2),
2.62 (t, 2H, J = 7.5 Hz, COCH2CH2), 1.31-1.85 (m, 6H, CH2), 0.91 (t, 3H, J = 7.5 Hz, CH3). 13C NMR
(75 MHz, CDCl3) δ: 171.54, 163.06, 153.32, 132.12, 125.20, 116.30, 115.05, 113.75, 90.33, 60.28,
32.47, 30.62, 30.40, 28.75, 25.55, 22.04, 13.83. ESI-HRMS calcd for C17H22N3O3S+ ([M+H]+):
348.1376; found: 348.1371.
6-((5-(Hexylthio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (5c)
1
Yield: 51 %. Mp: 205-206 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 9.00 (s, 1H, CONH), 6.76-6.84
(m, 3H, Ar-H), 5.18 (s, 2H, OCH2), 3.25 (t, 2H, J = 7.5 Hz, SCH2), 2.94 (t, 2H, J = 7.5 Hz, COCH2),
2.61 (t, 2H, J = 7.5 Hz, COCH2CH2), 1.28-1.85 (m, 8H, CH2), 0.89 (t, 3H, J = 6.0 Hz, CH3). 13C NMR
(75 MHz, CDCl3) δ: 171.62, 163.06, 153.32, 132.13, 125.19, 116.34, 115.02, 113.75, 90.33, 60.27,
32.50, 31.10, 30.39, 29.02, 28.18, 25.54, 22.41, 13.93. ESI-HRMS calcd for C18H24N3O3S+ ([M+H]+):
362.1533; found: 362.1539.
6-((5-(Heptylthio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (5d)
1
Yield: 44 %. Mp: 198-199 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 8.92 (s, 1H, CONH), 6.75-6.84

(m, 3H, Ar-H), 5.18 (s, 2H, OCH2), 3.25 (t, 2H, J = 7.5 Hz, SCH2), 2.95 (t, 2H, J = 7.5 Hz, COCH2),
2.62 (t, 2H, J = 7.5 Hz, COCH2CH2), 1.29-1.85 (m, 10H, CH2), 0.88 (t, 3H, J = 6.0 Hz, CH3). 13C NMR
(75 MHz, CDCl3) δ: 171.56, 163.08, 153.32, 132.13, 125.20, 116.31, 115.04, 113.76, 90.33, 60.28,
32.50, 31.56, 30.40, 29.06, 28.60, 28.48, 25.55, 22.49, 14.00. ESI-HRMS calcd for C19H26N3O3S+
([M+H]+): 376.1689; found: 376.1694.
6-((5-(Octylthio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (5e)
1
Yield: 49 %. Mp: 199-201 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 8.64 (s, 1H, CONH), 6.74-6.85
(m, 3H, Ar-H), 5.18 (s, 2H, OCH2), 3.26 (t, 2H, J = 7.5 Hz, SCH2), 2.95 (t, 2H, J = 7.5 Hz, COCH2),
2.62 (t, 2H, J = 7.5 Hz, COCH2CH2), 1.28-1.83 (m, 12H, CH2), 0.88 (t, 3H, J = 7.5 Hz, CH3). 13C NMR
(75 MHz, CDCl3) δ: 171.40, 163.87, 153.33, 132.09, 125.22, 116.24, 115.07, 113.75, 90.34, 60.28,
32.51, 31.68, 30.41, 29.04, 28.90, 28.52, 25.56, 22.56, 14.03. ESI-HRMS calcd for C20H28N3O3S+
([M+H]+): 390.1846; found: 390.1851.
6-((5-(Nonylthio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (5f)
1
Yield: 47 %. Mp: 194-195 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 8.62 (s, 1H, CONH), 6.73-6.85
(m, 3H, Ar-H), 5.18 (s, 2H, OCH2), 3.25 (t, 2H, J = 7.5 Hz, SCH2), 2.94 (t, 2H, J = 7.5 Hz, COCH2),
2.62 (t, 2H, J = 7.5 Hz, COCH2CH2), 1.27-1.83 (m, 14H, CH2), 0.88 (t, 3H, J = 6.0 Hz, CH3). 13C NMR
(75 MHz, CDCl3) δ: 171.36, 163.06, 153.35, 132.11, 125.24, 116.24, 115.10, 113.78, 90.34, 60.31,
32.53, 31.77, 30.42, 29.34, 29.15, 29.07, 28.95, 28.53, 25.58, 22.60, 14.05. ESI-HRMS calcd for
C21H30N3O3S+ ([M+H]+): 404.2002; found: 404.2008,
6-((5-(Benzylthio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (5g)
1
Yield: 48 %. Mp: 198-199 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 8.83 (s, 1H, CONH), 6.74-7.43
(m, 8H, Ar-H), 5.18 (s, 2H, OCH2), 4.48 (s, 2H, SCH2), 2.94 (t, 2H, J = 7.5 Hz, COCH2), 2.61 (t, 2H, J
= 7.5 Hz, COCH2CH2). 13C NMR (75 MHz, CDCl3) δ: 171.54, 165.66, 163.29, 153.26, 135.15, 132.12,
129.09, 129.09, 128.78, 128.78, 128.14, 125.23, 116.31, 115.02, 113.72, 60.24, 36.67, 30.41, 25.55.
ESI-HRMS calcd for C19H18N3O3S+ ([M+H]+): 368.1063; found: 368.1067.
6-((5-((2-Chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (5h)
1
Yield: 53 %. Mp: 188-189 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 8.29 (s, 1H, CONH), 6.73-7.56
(m, 7H, Ar-H), 5.17 (s, 2H, OCH2), 4.58 (s, 2H, SCH2), 2.94 (t, 2H, J = 7.5 Hz, COCH2), 2.61 (t, 2H, J
= 7.5 Hz, COCH2CH2). 13C NMR (75 MHz, CDCl3) δ: 171.45, 165.67, 163.37, 153.26, 134.32, 133.33,
132.12, 131.43, 129.76, 129.67, 127.03, 125.23, 116.27, 115.03, 113.71, 60.23, 34.48, 30.42, 25.56.
ESI-HRMS calcd for C19H17ClN3O3S+ ([M+H]+): 402.0674; found: 402.0677.
6-((5-((3-Chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (5i)
1
Yield: 62 %. Mp: 232-234 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 8.60 (s, 1H, CONH), 6.72-7.42
(m, 7H, Ar-H), 5.17 (s, 2H, OCH2), 4.43 (s, 2H, SCH2), 2.94 (t, 2H, J = 7.5 Hz, COCH2), 2.61 (t, 2H, J

= 7.5 Hz, COCH2CH2). 13C NMR (75 MHz, CDCl3) δ: 171.27, 165.19, 163.42, 153.27, 137.34, 134.52,
132.16, 129.98, 129.10, 128.33, 127.25, 125.26, 116.21, 115.12, 113.75, 60.29, 35.93, 30.40, 25.57.
ESI-HRMS calcd for C19H17ClN3O3S+ ([M+H]+): 402.0674; found: 402.0679.
6-((5-((4-Chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (5j)
1
Yield: 50 %. Mp: 264-265 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.84 (s, 1H, CONH), 7.35-7.45
(dd, 4H, J = 12.0 Hz, J = 12.0 Hz, Ar-H), 6.67-6.83 (m, 3H, Ar-H), 5.17 (s, 2H, OCH2), 4.43 (s, 2H,
SCH2), 2.94 (t, 2H, J = 7.5 Hz, COCH2), 2.62 (t, 2H, J = 7.5 Hz, COCH2CH2). 13C NMR (75 MHz,
CDCl3) δ: 171.42, 165.30, 163.41, 153.25, 134.03, 133.89, 132.16, 130.44, 130.44, 128.90, 128.90,
125.25, 116.26, 115.04, 113.69, 60.23, 35.88, 30.39, 25.55. ESI-HRMS calcd for C19H17ClN3O3S+
([M+H]+): 402.0674; found: 402.0679.
6-((5-((2-Fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (5k)
1
Yield: 46 %. Mp: 222-223 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 8.67 (s, 1H, CONH), 6.76-7.49
(m, 7H, Ar-H), 5.18 (s, 2H, OCH2), 4.51 (s, 2H, SCH2), 2.94 (t, 2H, J = 7.5 Hz, COCH2), 2.62 (t, 2H, J
= 7.5 Hz, COCH2CH2). 13C NMR (75 MHz, CDCl3) δ: 171.37, 165.49, 163.39, 153.30, 132.19, 131.31,
130.19, 130.08, 125.25, 124.31, 116.25, 115.75, 115.47, 115.11, 113.79, 60.31, 30.44, 30.06, 25.58.
ESI-HRMS calcd for C19H17FN3O3S+ ([M+H]+): 386.0969; found: 386.0977.
6-((5-((3-Fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (5l)
1
Yield: 42 %. Mp: 244-245 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 8.90 (s, 1H, CONH), 6.77-7.30
(m, 7H, Ar-H), 5.18 (s, 2H, OCH2), 4.46 (s, 2H, SCH2), 2.94 (t, 2H, J = 7.5 Hz, COCH2), 2.62 (t, 2H, J
= 7.5 Hz, COCH2CH2). 13C NMR (75 MHz, CDCl3) δ: 170.86, 167.54, 163.51, 153.42, 132.44, 130.26,
130.15, 125.28, 124.60, 116.06, 115.77, 115.39, 115.18, 114.91, 114.06, 60.58, 36.21, 30.40, 25.63.
ESI-HRMS calcd for C19H17FN3O3S+ ([M+H]+): 386.0969; found: 386.0975.
6-((5-((4-Fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (5m)
1
Yield: 47 %. Mp: 228-230 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 8.04 (s, 1H, CONH), 6.69-7.42
(m, 7H, Ar-H), 5.17 (s, 2H, OCH2), 4.45 (s, 2H, SCH2), 2.94 (t, 2H, J = 7.5 Hz, COCH2), 2.62 (t, 2H, J
= 7.5 Hz, COCH2CH2). 13C NMR (75 MHz, CDCl3) δ: 171.28, 164.85, 160.80, 153.28, 132.15, 130.91,
130.91, 130.80, 130.80, 125.29, 116.21, 115.85, 115.57, 115.10, 113.72, 60.27, 35.91, 30.43, 25.58.
ESI-HRMS calcd for C19H17FN3O3S+ ([M+H]+): 386.0969; found: 386.0975.
6-((5-((2-Trifluoromethylbenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-o
ne (5n)
1
Yield: 44 %. Mp: 202-203 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 8.39 (s, 1H, CONH), 6.72-7.76
(m, 7H, Ar-H), 5.19 (s, 2H, OCH2), 4.69 (s, 2H, SCH2), 2.95 (t, 2H, J = 7.5 Hz, COCH2), 2.62 (t, 2H, J
= 7.5 Hz, COCH2CH2). 13C NMR (75 MHz, CDCl3) δ: 171.20, 166.04, 163.52, 153.28, 134.03, 132.33,
132.14, 131.97, 128.38, 126.51, 126.43, 126.36, 125.28, 116.17, 115.13, 113.74, 60.27, 33.12, 30.41,

25.58. ESI-HRMS calcd for C20H17F3N3O3S+ ([M+H]+): 436.0937; found: 436.0945.
6-((5-((3-Trifluoromethylbenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-o
ne (5o)
1
Yield: 51 %. Mp: 212-213 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 8.66 (s, 1H, CONH), 6.73-7.69
(m, 7H, Ar-H), 5.17 (s, 2H, OCH2), 4.51 (s, 2H, SCH2), 2.94 (t, 2H, J = 7.5 Hz, COCH2), 2.62 (t, 2H, J
= 7.5 Hz, COCH2CH2). 13C NMR (75 MHz, CDCl3) δ: 171.44, 165.09, 163.50, 153.26, 145.02, 136.49,
132.54, 132.16, 129.27, 125.85, 125.28, 125.00, 116.28, 115.06, 113.70, 90.34, 60.25, 35.94, 30.39,
25.56. ESI-HRMS calcd for C20H17F3N3O3S+ ([M+H]+): 436.0937; found: 436.0944.
6-((5-((4-Trifluoromethylbenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-o
ne (5p)
1
Yield: 57 %. Mp: 224-226 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 9.10 (s, 1H, CONH), 7.53-7.59
(dd, 4H, J = 12.0 Hz, J = 12.0 Hz, Ar-H), 6.75-6.82 (m, 3H, Ar-H), 5.17 (s, 2H, OCH2), 4.50 (s, 2H,
SCH2), 2.93 (t, 2H, J = 7.5 Hz, COCH2), 2.62 (t, 2H, J = 7.5 Hz, COCH2CH2). 13C NMR (75 MHz,
CDCl3) δ: 171.64, 165.04, 163.56, 153.26, 139.57, 132.27, 129.45, 129.45, 129.45, 125.71, 125.66,
125.25, 122.04, 116.36, 115.03, 113.70, 60.26, 35.86, 30.39, 25.55. ESI-HRMS calcd for
C20H17F3N3O3S+ ([M+H]+): 436.0937; found: 436.0944.
6-((5-((2-Methylbenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (5q)
1
Yield: 47 %. Mp: 178-180 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 8.61 (s, 1H, CONH), 6.78-7.35
(m, 7H, Ar-H), 5.18 (s, 2H, OCH2), 4.52 (s, 2H, SCH2), 2.94 (t, 2H, J = 7.5 Hz, COCH2), 2.62 (t, 2H, J
= 7.5 Hz, COCH2CH2), 2.42 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3) δ: 171.34, 165.76, 163.23,
153.30, 137.05, 132.65, 132.15, 130.72, 130.21, 128.58, 126.36, 125.25, 116.23, 115.09, 113.77, 60.29,
35.03, 30.41, 25.57, 19.13. ESI-HRMS calcd for C20H20N3O3S+ ([M+H]+): 382.1220; found: 382.1225.
6-((5-((3-Methylbenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (5r)
1
Yield: 51 %. Mp: 197-199 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 9.01 (s, 1H, CONH), 6.75-7.22
(m, 7H, Ar-H), 5.17 (s, 2H, OCH2), 4.45 (s, 2H, SCH2), 2.93 (t, 2H, J = 7.5 Hz, COCH2), 2.62 (t, 2H, J
= 7.5 Hz, COCH2CH2), 2.33 (s, 3H, CH3). 13C NMR (75 MHz, CDCl3) δ: 171.66, 165.68, 163.24,
153.29, 138.57, 134.98, 132.18, 129.77, 128.93, 128.68, 126.12, 125.22, 116.36, 115.03, 113.75, 60.28,
36.70, 30.42, 25.56, 21.30. ESI-HRMS calcd for C20H20N3O3S+ ([M+H]+): 382.1220; found: 382.1226.
6-((5-((4-Methylbenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)-3,4-dihydroquinolin-2(1H)-one (5s)
1
Yield: 41 %. Mp: 185-186 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 8.11 (s, 1H, CONH), 7.12-7.32
(dd, 4H, J = 12.0 Hz, J = 12.0 Hz, Ar-H), 6.69-6.84 (m, 3H, Ar-H), 5.18 (s, 2H, OCH2), 4.45 (s, 2H,
SCH2), 2.92 (t, 2H, J = 7.5 Hz, COCH2), 2.62 (t, 2H, J = 7.5 Hz, COCH2CH2), 2.33 (s, 3H, CH3). 13C
NMR (75 MHz, CDCl3) δ: 171.47, 165.78, 163.16, 153.28, 138.00, 132.14, 132.05, 129.45, 129.45,
128.99, 128.99, 125.22, 116.28, 115.05, 113.75, 60.27, 36.51, 30.42, 25.56, 21.12. ESI-HRMS calcd

for C20H20N3O3S+ ([M+H]+): 382.1220; found: 382.1224.
4.1.5. Synthesis of intermediates 7, 8, and 9, and target compounds 10a-s
The method used for the synthesis of intermediates 7, 8, and 9, and target compounds 10a-s is
similar to the one used for the synthesis of compounds 2, 3, 4, and 5a-s.
N-(4-((5-(Butylthio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10a)
1
Yield: 50 %. Mp: 177-178 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.60 (s, 1H, CONH), 6.91-7.60
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 5.17 (s, 2H, OCH2), 3.24 (t, 2H, J = 7.5 Hz, SCH2), 2.14 (s, 3H,
COCH3), 1.42-1.79 (m, 4H, CH2), 0.93 (t, 3H, J = 7.5 Hz, CH3). 13C NMR (75 MHz, CDCl3) δ: 168.38,
166.40, 163.13, 154.08, 132.51, 121.79, 121.79, 115.28, 115.28, 60.11, 32.22, 31.09, 24.26, 21.66,
13.42. ESI-HRMS calcd for C15H20N3O3S+ ([M+H]+): 322.1220; found: 322.1224.
N-(4-((5-(Pentylthio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10b)
1
Yield: 52 %. Mp: 199-201 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.66 (s, 1H, CONH), 6.91-7.44
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 5.17 (s, 2H, OCH2), 3.23 (t, 2H, J = 7.5 Hz, SCH2), 2.14 (s, 3H,
COCH3), 1.30-1.84 (m, 6H, CH2), 0.90 (t, 3H, J = 7.5 Hz, CH3). 13C NMR (75 MHz, CDCl3) δ: 168.44,
166.43, 163.11, 154.05, 132.49, 121.79, 121.79, 115.23, 115.23, 60.07, 32.48, 30.63, 28.77, 24.29,
22.05, 13.86. ESI-HRMS calcd for C16H22N3O3S+ ([M+H]+): 336.1376; found: 336.1383.
N-(4-((5-(Hexylthio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10c)
1
Yield: 53 %. Mp: 196-197 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.56 (s, 1H, CONH), 6.92-7.44
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 5.18 (s, 2H, OCH2), 3.24 (t, 2H, J = 7.5 Hz, SCH2), 2.14 (s, 3H,
COCH3), 1.27-1.83 (m, 8H, CH2), 0.88 (t, 3H, J = 6.0 Hz, CH3). 13C NMR (75 MHz, CDCl3) δ: 168.38,
166.44, 163.12, 154.08, 132.46, 121.79, 121.79, 115.26, 115.26, 60.09, 32.52, 31.10, 29.04, 28.19,
24.30, 22.42, 13.94. ESI-HRMS calcd for C17H24N3O3S+ ([M+H]+): 350.1533; found: 350.1537.
N-(4-((5-(Heptylthio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10d)
1
Yield: 51 %. Mp: 189-190 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.68 (s, 1H, CONH), 6.91-7.44
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 5.17 (s, 2H, OCH2), 3.23 (t, 2H, J = 7.5 Hz, SCH2), 2.14 (s, 3H,
COCH3), 1.27-1.83 (m, 10H, CH2), 0.87 (t, 3H, J = 6.0 Hz, CH3). 13C NMR (75 MHz, CDCl3) δ:
168.43, 166.43, 163.11, 154.04, 132.49, 121.78, 121.78, 115.22, 115.22, 60.06, 32.50, 31.55, 29.06,
28.60, 28.46, 24.26, 22.49, 14.00. ESI-HRMS calcd for C18H26N3O3S+ ([M+H]+): 364.1689; found:
364.1696.
N-(4-((5-(Octylthio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10e)
1
Yield: 46 %. Mp: 190-191 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.60 (s, 1H, CONH), 6.92-7.44
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 5.17 (s, 2H, OCH2), 3.23 (t, 2H, J = 7.5 Hz, SCH2), 2.14 (s, 3H,
COCH3), 1.19-1.81 (m, 12H, CH2), 0.87 (t, 3H, J = 6.0 Hz, CH3). 13C NMR (75 MHz, CDCl3) δ:
168.38, 166.41, 163.09, 154.06, 132.47, 121.77, 121.77, 115.24, 115.24, 60.08, 32.51, 31.68, 29.05,

28.89, 28.51, 25.30, 24.25, 22.55, 14.02. ESI-HRMS calcd for C19H28N3O3S+ ([M+H]+): 378.1846;
found: 378.1841.
N-(4-((5-(Nonylthio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10f)
1
Yield: 46 %. Mp: 185-186 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.56 (s, 1H, CONH), 6.92-7.44
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 5.17 (s, 2H, OCH2), 3.24 (t, 2H, J = 7.5 Hz, SCH2), 2.15 (s, 3H,
COCH3), 1.26-1.83 (m, 14H, CH2), 0.87 (t, 3H, J = 6.0 Hz, CH3). 13C NMR (75 MHz, CDCl3) δ:
168.36, 166.42, 163.09, 154.07, 132.43, 121.77, 121.77, 115.24, 115.24, 60.07, 32.51, 31.76, 29.33,
29.14, 29.06, 28.94, 28.52, 24.29, 22.60, 14.06. ESI-HRMS calcd for C20H30N3O3S+ ([M+H]+):
392.2002; found: 392.2007.
N-(4-((5-(Benzylthio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10g)
1
Yield: 40 %. Mp: 205-206 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.54 (s, 1H, CONH), 6.91-7.43
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 7.28-7.39 (m, 5H, Ar-H), 5.17 (s, 2H, OCH2), 4.46 (s, 2H,
SCH2), 2.14 (s, 3H, COCH3). 13C NMR (75 MHz, CDCl3) δ: 168.33, 165.66, 163.33, 154.07, 135.16,
132.51, 129.06, 129.06, 128.78, 128.78, 128.12, 121.79, 121.79, 115.28, 115.28, 60.10, 36.73, 24.29.
ESI-HRMS calcd for C18H18N3O3S+ ([M+H]+): 356.1063; found: 356.1067.
N-(4-((5-((2-Chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10h)
1
Yield: 43 %. Mp: 223-225 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.49 (s, 1H, CONH), 6.91-7.43
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 7.17-7.56 (m, 4H, Ar-H), 5.17 (s, 2H, OCH2), 4.57 (s, 2H,
SCH2), 2.14 (s, 3H, COCH3). 13C NMR (75 MHz, CDCl3) δ: 168.30, 165.60, 163.44, 154.04, 134.30,
133.31, 132.47, 131.37, 129.75, 129.66, 127.05, 121.77, 121.77, 115.24, 115.24, 60.06, 34.51, 24.29.
ESI-HRMS calcd for C18H17ClN3O3S+ ([M+H]+): 390.0674; found: 390.0678.
N-(4-((5-((3-Chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10i)
1
Yield: 49 %. Mp: 281-283 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.14 (s, 1H, CONH), 6.94-7.43
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 7.25-7.44 (m, 4H, Ar-H), 5.19 (s, 2H, OCH2), 4.43 (s, 2H,
SCH2), 2.17 (s, 3H, COCH3). 13C NMR (75 MHz, CDCl3) δ: 168.35, 165.20, 163.46, 154.03, 137.31,
134.49, 132.45, 130.00, 129.10, 128.32, 127.26, 121.80, 121.80, 115.25, 115.25, 60.05, 35.93, 24.29.
ESI-HRMS calcd for C18H17ClN3O3S+ ([M+H]+): 390.0674; found: 390.0679.
N-(4-((5-((4-Chlorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10j)
1
Yield: 47 %. Mp: 199-201 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.56 (s, 1H, CONH), 6.90-7.43
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 7.25-7.36 (dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 5.17 (s, 2H,
OCH2), 4.41 (s, 2H, SCH2), 2.14 (s, 3H, COCH3). 13C NMR (75 MHz, CDCl3) δ: 168.38, 165.26,
163.45, 154.00, 133.99, 133.88, 132.47, 130.42, 130.42, 128.90, 128.90, 121.81, 121.81, 115.22,
115.22, 60.01, 35.89, 24.29. ESI-HRMS calcd for C18H17ClN3O3S+ ([M+H]+): 390.0674; found:
390.0677.

N-(4-((5-((2-Fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10k)
1
Yield: 52 %. Mp: 249-251 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.79 (s, 1H, CONH), 6.89-7.43
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 7.02-7.48 (m, 4H, Ar-H), 5.16 (s, 2H, OCH2), 4.48 (s, 2H,
SCH2), 2.13 (s, 3H, COCH3). 13C NMR (75 MHz, CDCl3) δ: 168.51, 165.44, 163.50, 162.53, 153.99,
132.59, 131.25, 130.08, 124.27, 122.63, 121.81, 121.81, 115.73, 115.21, 115.21, 60.06, 30.02, 24.22.
ESI-HRMS calcd for C18H17FN3O3S+ ([M+H]+): 374.0969; found: 374.0962.
N-(4-((5-((3-Fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10l)
1
Yield: 58 %. Mp: 235-236 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.75 (s, 1H, CONH), 6.89-7.43
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 6.94-7.31 (m, 4H, Ar-H), 5.16 (s, 2H, OCH2), 4.43 (s, 2H,
SCH2), 2.13 (s, 3H, COCH3). 13C NMR (75 MHz, CDCl3) δ: 168.53, 163.45, 154.00, 137.75, 132.54,
130.36, 130.24, 124.76, 121.82, 121.82, 116.16, 115.87, 115.18, 115.18, 115.00, 60.02, 36.02, 24.27.
ESI-HRMS calcd for C18H17FN3O3S+ ([M+H]+): 374.0969; found: 374.0965.
N-(4-((5-((4-Fluorobenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10m)
1
Yield: 56 %. Mp: 219-221 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.56 (s, 1H, CONH), 6.91-7.43
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 6.91-7.38 (m, 4H, Ar-H), 5.17 (s, 2H, OCH2), 4.43 (s, 2H,
SCH2), 2.14 (s, 3H, COCH3). 13C NMR (75 MHz, CDCl3) δ: 168.38, 164.03, 160.76, 153.99, 132.48,
131.10, 131.06, 130.88, 130.88, 121.80, 121.80, 115.54, 115.54, 115.21, 115.21, 77.42, 76.99, 76.57,
60.02, 35.89, 24.29. ESI-HRMS calcd for C18H17FN3O3S+ ([M+H]+): 374.0969; found: 374.0964.
N-(4-((5-((2-Trifluoromethylbenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10n)
1
Yield: 45 %. Mp: 193-194 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.76 (s, 1H, CONH), 6.90-7.44
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 7.38-7.71 (m, 4H, Ar-H), 5.18 (s, 2H, OCH2), 4.66 (s, 2H,
SCH2), 2.13 (s, 3H, COCH3). 13C NMR (75 MHz, CDCl3) δ: 168.51, 165.60, 163.57, 153.98, 133.96,
132.57, 132.37, 131.92, 128.39, 128.10, 126.41, 126.34, 121.82, 121.82, 115.20, 115.20, 60.04, 33.15,
24.23. ESI-HRMS calcd for C19H17F3N3O3S+ ([M+H]+): 424.0937; found: 424.0941.
N-(4-((5-((3-Trifluoromethylbenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10o)
1
Yield: 49 %. Mp: 203-204 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.68 (s, 1H, CONH), 6.91-7.43
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 7.40-7.64 (m, 4H, Ar-H), 5.17 (s, 2H, OCH2), 4.50 (s, 2H,
SCH2), 2.14 (s, 3H, COCH3). 13C NMR (75 MHz, CDCl3) δ: 168.42, 166.10, 165.07, 163.53, 153.97,
136.46, 132.50, 129.27, 125.81, 125.76, 124.97, 124.92, 121.81, 121.81, 115.19, 115.19, 60.00, 35.93,
24.28. ESI-HRMS calcd for C19H17F3N3O3S+ ([M+H]+): 424.0937; found: 424.0942.
N-(4-((5-((4-Trifluoromethylbenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10p)
1
Yield: 41 %. Mp: 213-214 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.12 (s, 1H, CONH), 6.93-7.44
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 7.53-7.60 (dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 5.19 (s, 2H,
OCH2), 4.50 (s, 2H, SCH2), 2.17 (s, 3H, COCH3). 13C NMR (75 MHz, CDCl3) δ: 167.83, 164.02,

163.97, 152.78, 141.58, 133.74, 129.76, 129.76, 127.99, 125.91, 125.38, 122.30, 120.39, 120.39,
115.01, 115.01, 59.65, 35.03, 23.80. ESI-HRMS calcd for C19H17F3N3O3S+ ([M+H]+): 424.0937; found:
424.0945.
N-(4-((5-((2-Methylbenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10q)
1
Yield: 48 %. Mp: 169-170 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.67 (s, 1H, CONH), 6.92-7.44
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 7.14-7.36 (m, 4H, Ar-H), 5.18 (s, 2H, OCH2), 4.50 (s, 2H,
SCH2), 2.42 (s, 3H, ArCH3), 2.14 (s, 3H, COCH3). 13C NMR (75 MHz, CDCl3) δ: 168.46, 165.75,
163.30, 154.05, 137.05, 132.62, 132.53, 130.72, 130.18, 128.58, 126.36, 121.83, 121.83, 115.25,
115.25, 60.08, 35.05, 24.24, 19.12. ESI-HRMS calcd for C19H20N3O3S+ ([M+H]+): 370.1220; found:
370.1226.
N-(4-((5-((3-Methylbenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10r)
1
Yield: 46 %. Mp: 181-182 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.58 (s, 1H, CONH), 6.91-7.44
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 7.09-7.27 (m, 4H, Ar-H), 5.17 (s, 2H, OCH2), 4.44 (s, 2H,
SCH2), 2.33 (s, 3H, ArCH3), 2.14 (s, 3H, COCH3). 13C NMR (75 MHz, CDCl3) δ: 168.38, 165.76,
163.30, 154.07, 138.56, 134.97, 132.52, 129.74, 128.92, 128.67, 126.09, 121.80, 121.80, 115.28,
115.28, 60.11, 36.74, 24.28, 21.27. ESI-HRMS calcd for C19H20N3O3S+ ([M+H]+): 370.1220; found:
370.1227.
N-(4-((5-((4-Methylbenzyl)thio)-1,3,4-oxadiazol-2-yl)methoxy)phenyl)acetamide (10s)
1
Yield: 51 %. Mp: 199-201 oC. H-NMR (CDCl3, 300 MHz, ppm) δ: 7.51 (s, 1H, CONH), 6.92-7.44
(dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 7.11-7.30 (dd, 4H, J = 9.0 Hz, J = 9.0 Hz, Ar-H), 5.17 (s, 2H,
OCH2), 4.44 (s, 2H, SCH2), 2.33 (s, 3H, ArCH3), 2.15 (s, 3H, COCH3). 13C NMR (75 MHz, CDCl3) δ:
168.32, 165.80, 163.31, 154.08, 137.99, 132.50, 132.05, 129.46, 128.98, 121.78, 121.78, 115.29,
115.29, 77.42, 76.99, 76.57, 60.11, 36.56, 24.30, 21.11. ESI-HRMS calcd for C19H20N3O3S+ ([M+H]+):
370.1220; found: 370.1225.
4.1.6. Synthesis of intermediate 12
Dimethoxy-N,N-dimethylmethanamine (DMF-DMA, 30 mmol), formyl hydrazide (30 mmol), and
CH3CN (40 ml) were added to a 100 ml single-necked round bottom flask, and the reaction was stirred
under reflux for 1 h. Then, 4-hydroxyaniline (15 mmol) and acetic acid (3 ml) were added to the
reaction, and the temperature was raised to 120 oC and the reaction was continued in reflux for 10‒16 h.
The reaction was tracked using TLC. After the reaction was completed, the solvent was vacuum dried
under reduced pressure to obtain the crude product. Recrystallization using ethanol resulted in the
production of a light brown solid.
4.1.7. Synthesis of intermediates 13, 14, and 15, and target compounds 16a-d

The method used for the synthesis of intermediates 13, 14, and 15, and target compounds 16a-d is
also similar to that used for the synthesis of compounds 2, 3, 4, and 5a-s.
2-((4-(4H-1,2,4-triazol-4-yl)phenoxy)methyl)-5-(butylthio)-1,3,4-oxadiazole (16a)
o
1
Yield: 54 %. Mp: 189-191 C. H-NMR (CDCl3, 400 MHz, ppm) δ: 8.42 (s, 2H, Triazole-H),
7.17-7.37 (dd, 4H, J = 8.0 Hz, J = 8.0 Hz, Ar-H), 5.30 (s, 2H, OCH2), 3.28 (t, 2H, J = 8.0 Hz, SCH2),
1.43-1.84 (m, 4H, CH2), 0.95 (t, 3H, J = 6.0 Hz, CH3). 13C NMR (100 MHz, CDCl3) δ: 166.81, 162.45,
157.70, 141.76, 141.76, 128.14, 124.20, 124.20, 116.35, 116.35, 59.93, 32.27, 31.08, 21.75, 13.52, 0.02.
ESI-HRMS calcd for C15H18N5O2S+ ([M+H]+): 332.1176; found: 332.1181.
2-((4-(4H-1,2,4-triazol-4-yl)phenoxy)methyl)-5-(pentylthio)-1,3,4-oxadiazole (16b)
o
1
Yield: 48 %. Mp: 215-216 C. H-NMR (CDCl3, 400 MHz, ppm) δ: 8.43 (s, 2H, Triazole-H),
7.16-7.36 (dd, 4H, J = 8.0 Hz, J = 8.0 Hz, Ar-H), 5.30 (s, 2H, OCH2), 3.27 (t, 2H, J = 8.0 Hz, SCH2),
1.25-1.82 (m, 6H, CH2), 0.91 (t, 3H, J = 8.0 Hz, CH3). 13C NMR (100 MHz, CDCl3) δ: 166.85, 162.43,
157.73, 141.78, 141.78, 128.12, 124.22, 124.22, 116.36, 116.36, 59.93, 32.55, 30.70, 28.79, 22.12,
13.93. ESI-HRMS calcd for C16H20N5O2S+ ([M+H]+): 346.1332; found: 346.1339.
2-((4-(4H-1,2,4-triazol-4-yl)phenoxy)methyl)-5-(hexylthio)-1,3,4-oxadiazole (16c)
o
1
Yield: 54 %. Mp: 203-204 C. H-NMR (CDCl3, 400 MHz, ppm) δ: 8.43 (s, 2H, Triazole-H),
7.17-7.37 (dd, 4H, J = 8.0 Hz, J = 8.0 Hz, Ar-H), 5.30 (s, 2H, OCH2), 3.27 (t, 2H, J = 8.0 Hz, SCH2),
1.64-1.83 (m, 8H, CH2), 0.88 (t, 3H, J = 8.0 Hz, CH3). 13C NMR (100 MHz, CDCl3) δ: 167.64, 162.44,
157.70, 141.76, 128.13, 124.18, 124.18, 116.35, 116.35, 59.93, 32.57, 31.15, 30.28, 28.25, 22.47, 14.01.
ESI-HRMS calcd for C17H22N5O2S+ ([M+H]+): 360.1489; found: 360.1482.
2-((4-(4H-1,2,4-triazol-4-yl)phenoxy)methyl)-5-(heptylthio)-1,3,4-oxadiazole (16d)
o
1
Yield: 56 %. Mp: 201-202 C. H-NMR (CDCl3, 400 MHz, ppm) δ: 8.43 (s, 2H, Triazole-H),
7.17-7.37 (dd, 4H, J = 8.0 Hz, J = 8.0 Hz, Ar-H), 5.30 (s, 2H, OCH2), 3.27 (t, 2H, J = 8.0 Hz, SCH2),
1.29-1.85 (m, 10H, CH2), 0.88 (t, 3H, J = 8.0 Hz, CH3). 13C NMR (100 MHz, CDCl3) δ: 166.81, 162.44,
157.70, 141.75, 141.75, 128.13, 124.19, 124.19, 116.35, 116.35, 59.94, 32.57, 31.61, 29.08, 28.67,
28.54, 22.56, 14.08. ESI-HRMS calcd for C18H24N5O2S+ ([M+H]+): 374.1645; found: 374.1650.
4.2. Pharmacology
4.2.1. Animals and treatment
Kunming mice (18–22 g) and Wistar rats (280–300 g) were used for the experiment. All animals
used in the experiment have been approved by the local ethics committee (No. 20181009). All
experimental animals were free to move, eat, and drink before the experiment. We dissolved the target
compound in solution of 0.9% NaCl or dimethyl sulfoxide, and the target compound was administered

via the intraperitoneal or subcutaneous injection route. The pharmacological experimental procedure
was performed according to the method described in the Antiepileptic Drug Development Program in
the National Institutes of Health (NIH) [18-20]. Seven compounds with potential activity were
measured for ED50 and TD50 values. GABA content determination and [3H]Flunitrazepam binding
assay in the animal brain were conducted in accordance with previously reported methods, with a slight
modification [6,21,22]; the anxiolytic properties of potent compound was tested by elevated plus maze
(EPM) method, and data were analyzed by Graph Pad Prism 5.0 software. DS 2019 or ChemBioDraw
Ultra 14.0 was used to predict the physicochemical and pharmacokinetic properties of the target
compound.
4.2.1. Animals and treatment
Kunming mice (18–22 g) and Wistar rats (280–300 g) were used for the experiments. All animal
experiments were approved by the local ethics committee (No. 20181009). All experimental animals
were free to move, and had ad libitum access to food and water before the initiation of the experiments.
The target compound was dissolved in 0.9 % NaCl or dimethyl sulfoxide, and administered via the
intraperitoneal or subcutaneous injection route. The pharmacological experiment was performed
according to the method proposed by the National Institutes of Health (NIH; Antiepileptic Drug
Development Program) [18-20]. The ED50 and TD50 values of seven compounds with potential activity
were measured. GABA content determination and [3H]Flunitrazepam binding assay in the animal brain
were performed as per previously reported methods, with a slight modification [6,21,22]; the anxiolytic
properties of the potent compound were tested using the EPM assay, and data were analyzed using
Graph Pad Prism 5.0 software. DS 2019 and ChemBioDraw Ultra 14.0 were used to predict the
physicochemical and pharmacokinetic properties of the target compounds.
4.2.2. Anticonvulsant activity evaluation
Anticonvulsant activity and neurotoxicity tests were conducted in accordance with previously
reported methods; MES and scPTZ models were used for anticonvulsant activity screening [23-25],
and the ROT method was used for neurotoxicity testing [26]. Based on the preliminary anticonvulsant
activity—and results of the toxicity tests—seven compounds (5b-c, 5p, 6b-c, and 16b-c) with potential
activity and low toxicity were selected, and their ED50s and TD50s were determined.
4.2.3. [3H]Flunitrazepam binding assays
Based on preliminary anticonvulsant activity—and on the results of the toxicity tests—seven