1,2,4-oxadiazole-based bio-isosteres of benzamides: Synthesis, biological activity and toxicity to zebrafish embryo

Article abstract of DOI:10.3390/ijms22052367

To discover new compounds with broad spectrum and high activity, we designed a series of novel benzamides containing 1,2,4-oxadiazole moiety by bioisosterism, and 28 benzamides derivatives with antifungal activity were synthesized. These compounds were ev

Full text of DOI:10.3390/ijms22052367

International Journal of
Molecular Sciences
Article
1,2,4-Oxadiazole-Based Bio-Isosteres of Benzamides: Synthesis,
Biological Activity and Toxicity to Zebrafish Embryo
Sen Yang, Chao-Li Ren, Tian-Yang Ma, Wen-Qian Zou, Li Dai, Xiao-Yu Tian, Xing-Hai Liu and Cheng-Xia Tan *
College of Chemical Engineering, Zhejiang University of Technology, Hangzhou 310014, China;
y17764528124@163.com (S.Y.); 18708589843@163.com (C.-L.R.); m15755187338@163.com (T.-Y.M.);
zwqxyes@163.com (W.-Q.Z.); 15157357729@163.com (L.D.); t895745887@163.com (X.-Y.T.);
xhliu@zjut.edu.cn (X.-H.L.)
* Correspondence: tanchengxia@zjut.edu.cn; Tel.: +86-571-8832-0238; Fax: +86-571-8832-0238
Abstract: To discover new compounds with broad spectrum and high activity, we designed a
series of novel benzamides containing 1,2,4-oxadiazole moiety by bioisosterism, and 28 benzamides
derivatives with antifungal activity were synthesized. These compounds were evaluated against
four fungi: Botrytis cinereal, FusaHum graminearum, Marssonina mali, and Thanatephorus cucumeris. The
results indicated that most of the compounds displayed good fungicidal activities, especially against
Botrytis cinereal. For example, 10a (84.4%), 10d (83.6%), 10e (83.3%), 10f (83.1%), 10i (83.3%), and 10l
(83.6%) were better than pyraclostrobin (81.4%) at 100 mg/L. In addition, the acute toxicity of 10f to
zebrafish embryo was 20.58 mg/L, which was classified as a low-toxicity compound.
Keywords: benzamides; biological activity; 1,2,4-oxadiazole; synthesis; toxicity
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Citation: Yang, S.; Ren, C.-L.; Ma,
T.-Y.; Zou, W.-Q.; Dai, L.; Tian, X.-Y.;
1. Introduction
Liu, X.-H.; Tan, C.-X.
1,2,4-Oxadiazole-Based Bio-Isosteres
Chemical pesticides play a vital role in solving food problems. However, as peo-
ple’s environmental awareness gradually deepens, high-efficiency, low-toxicity, and en-
vironmentally friendly pesticides have become an inevitable trend in the creation of new
of Benzamides: Synthesis, Biological
Activity and Toxicity to Zebrafish
Embryo. Int. J. Mol. Sci. 2021, 22,
pesticides [
synthetic pesticides for their high-efficiency, various biological activities, and diversity
of possible substituents [ 25]. 1,2,4-Oxadiazole heterocycle, as an important kind of
the five-membered oxygen-nitrogen heterocycle, has exhibited a wide range of biological
activities in the field of pesticides, such as insecticidal [26 28], antifungal [29 30], and
1–3]. There is no doubt that heterocyclic structures are important feature in
2367. https://doi.org/10.3390/
ijms22052367
4
–
Academic Editor: Claudio Santi
–
,
herbicidal activities [31]. It has often been introduced as a synergist into the structure of
pesticides in order to improve the biological activities of the compounds. In addition, the
1,2,4-oxadiazole heterocycle, as a bio-isostere of the amide bond, has better hydrolytic and
metabolic properties.
Received: 17 January 2021
Accepted: 24 February 2021
Published: 27 February 2021
Diamide insecticides have attracted a lot of attention due to their novel mechanism
of action, high efficiency, and low toxicity [32–42]. Following Bayer’s report on the first
diamide insecticide flubendiamide, the use of chlorantraniliprole, cyantraniliprole, cy-
claniliprole, (Figure 1) and other products was launched successively. However, while this
type of insecticide showed excellent insecticidal effects, it gradually exposed potential risks
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with regard to jurisdictional claims in
published maps and institutional affil-
iations.
to environmental non-target organisms [43,44]. Broflanilide is a meta-diamide insecticide
developed by Mitsui Chemicals and co-developed with BASF SE. Because of its novel
mechanism of action, this product was expected to become a blockbuster product.
In view of these facts mentioned above, broflanilide was employed as the lead com-
Copyright:
© 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
pound in this study. According to the principle of bioisosterism [45,46], we searched for
the amide group of broflanilide in the 1,2,4-oxadiazole ring, replaced the benzene ring with
a pyridine structure containing a thioether derivative, and designed (Figure 2) and syn-
thesized (Scheme 1) a series of novel benzamides substituted with 1,2,4-oxadiazole. These
new compounds were confirmed by 1H NMR, 13C NMR, and HRMS, and their insecticidal
activities, fungicidal activities, and toxicity test of zebrafish embryo were studied.
Int. J. Mol. Sci. 2021, 22, 2367. https://doi.org/10.3390/ijms22052367
https://www.mdpi.com/journal/ijms

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Figure 1. Chemical structures of flubendiamide, chlorantraniliprole, cyantraniliprole, and cyclaniliprole.
Figure 2. Design strategy of target compounds.
Scheme 1. Synthetic route of target compounds. Reagents and conditions: (
KTB, DMF, 0–5 ◦C, rt; ( ) THF, OH-, reflux; ( ) CH3OH, H+, reflux; ( ) CuCN, L-proline, DMF, 100 ◦C; (
) THF, OH-, reflux; (
) SOCl2, reflux; Et3N, toluene, reflux; ( ) EDCI, Et3N, CH2Cl2, rt; (j
a
) DMS, K2CO3, acetone, reflux; (
) NH2OH
) mCPBA,
b
) CH3CH2SH,
c
d
e
f
•
HCl,
CH3CH2OH, rt; (
g
h
i
CH2Cl2, rt.

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2. Results and Discussion
2.1. Synthesis of Target Compounds
The synthetic pathways to target compounds
9
and 10 are shown in Scheme 1. Inter-
mediate I was prepared from 3,6-dichloropyridinecarboxylic acid 1 as a starting material
via methylation, thioetherification, and hydrolysis reactions. During the thioetherification
reaction, the newly prepared potassium ethanethiolate should be slowly added to the DMF
solution of methyl 3,6-dichloropicolinate, and the temperature should be controlled at
◦
0–5 C to avoid the by-products (Scheme 2). As in our previous procedures [47], intermedi-
ate II was easily obtained. Methyl 2-chloro-5-(5-(6-chloro-3-(ethylthio) pyridin-2-yl)-1,2,4-
oxadiazol-3-yl) benzoate
and 6-chloro-3-(ethylthio)picolinoyl chloride that had been synthesized from intermediate
. Then, the compound was hydrolyzed and spliced with substituted anilines to give
a series of target compounds 9a 9n. Finally, compound was oxidized with mCPBA at
7 was synthesized by cyclization reaction from imtermediate II
I
7
–
9
room temperature to give target compound 10, which avoided the impurities of pyridine-
N-oxide. The characterization data for all synthesized compounds are provided in the
supporting information file (Figures S1–S39).
Scheme 2. Synthesis of compound 3 via thioetherification.
2.2. Spectrum Analysis of Target Compounds
All the target compounds were confirmed by 1H NMR, 13C NMR, and HRMS. In the
1H NMR spectra of 10a, the -NH proton signals were found at
δ
10.70 ppm. The signals
of CH of the benzene and pyridine rings were assigned at 8.61–7.13 ppm. The signals at
3.78 ppm and 1.26 ppm were -CH2 and -CH3 peak, respectively. In the 13C-NMR spectra
δ
of compound 10a, the appearances of signals at 166.81 and 163.79 ppm were assigned to
the carbons of the 1,2,4-oxadiazole ring. Finally, all of the novel benzamides substituted
with 1,2,4-oxadiazole exhibited a strong [M + H]+ peak in positive ion high-resolution
electrospray mass spectra (HR-ESI-MS) analysis.
2.3. Biological Activities of Target Compounds
In Table 1, The target compounds had weak death rates against Mythimna sepatara
(5–40%) at 500 mg/L, which were lower than the control drug broflanilide (100%). In
addition, all of the target compounds had good fungicidal activities at 100 mg/L. Overall,
the target compounds had better inhibitory activities against Botrytis cinereal than Fusarium
graminearum, Marssonina mali, and Thanatephorus cucumeris. In particular, the inhibitory
activity of compounds 10a (84.4%), 10d (83.6%), 10e (83.3%), 10f (83.1%), 10i (83.3%), and
10l (83.6%) were better than pyraclostrobin (81.4%), and 10b (80.8%), 10g (81.1%), 10h
(81.4%), and 10k (81.9%) were comparable to pyraclostrobin. At the same time, compounds
9e (57.7%), 9f (63.5%), 9g (65.9%), 9h (57.1%), and 9k (61.2%) also showed moderate
activities. For Fusarium graminearum and Marssonina mali, compounds 9a–9n displayed
weak inhibition, both of which were less than 25%. Some of compound 10 had good
activities (46.4–54.4%) but were inferior to pyraclostrobin. For Thanatephorus cucumeris,
compound
9 had no inhibitory activity, and some of compound 10 exhibited moderate
inhibitory activities (37.5–50.3%). From Table 2, we can see that compound 10f had good
inhibitory activity against Botrytis cinereal with EC50 of 14.44 µg/mL.

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Table 1. Insecticidal and fungicidal activities of title compounds 9 and 10.
Insecticidal
Activities (500 mg/L,
Death Rates %)
Fungicidal Activities (100 mg/L, Inhibition Rates %)
Compound
R
Botrytis
cinereal
Fusarium
graminearum
Marssonina
mali
Thanatephorus
Mythimna sepatara
cucumeris
9a
9b
9c
9d
9e
9f
9g
9h
9i
9j
9k
9l
9m
9n
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
H
10
5
20
0
30
25
15
30
10
0
48.2
25.0
48.2
18.5
57.7
63.5
65.9
57.1
23.5
19.7
61.2
20.9
5.3
17.7
84.4
80.8
22.9
83.6
83.3
83.1
81.1
81.4
83.3
31.8
81.9
83.6
22.7
17.9
nt
0
2.4
0.9
18.2
2.9
8.5
0
5.3
6.8
16.2
15.0
17.4
0
22.0
15.3
13.5
16.5
10.6
16.5
10.0
20.9
16.8
21.5
14.4
24.4
12.4
18.5
39.2
37.8
34.1
44.2
39.4
45.8
42.8
48.3
43.1
38.8
45.0
39.2
33.2
37.1
nt
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
44.2
41.9
37.5
44.7
50.3
44.2
0
46.1
44.4
0
2-CH3
4-CH3
4-t-Bu
2,4-di-CH3
3-CF3
3-Cl-2-CH3
2-F
4-F
2,6-di-F
2-Cl
5
0
5
3-Cl
4-Cl
4-Br
H
25
20
15
10
25
35
40
25
40
25
15
5
15
15
20
100
100
nt
4.7
25.0
29.4
53.1
52.5
46.4
43.6
50.0
51.4
54.4
23.2
50.3
48.1
13.2
11.8
nt
2-CH3
4-CH3
4-t-Bu
2,4-di-CH3
3-CF3
3-Cl-2-CH3
2-F
4-F
2,6-di-F
2-Cl
3-Cl
4-Cl
4-Br
10m
10n
0
Broflanilide
Chlorantraniliprole
Pyraclostrobin
nt
nt
100
nt
81.4
nt
100
nt
84.1
Note: nt = not tested. All the data were determined three times.
Table 2. EC50 of compounds 10f to Botrytis cinereal.
−1
)
Compound
10f
y = a + bx
r2
EC50/(µg mL
y = 1.5374x + 3.2171
0.9961
14.44
According to the insecticidal activity result, we speculated that the amide bridge
bond played a key role in maintaining insecticidal activity, and it was likely that it would
interact with the receptor through its hydrogen bond. Searching the amide bridge bond in
1,2,4-oxadiazole, we found that it lacked the corresponding hydrogen bond, and the group
bulk increased, which resulted in the blocking of the binding of the compounds to the
receptor and did not show a good insecticidal activity. It could be seen from Table 1 that the
fungicidal activities of compound 10 was significantly higher than compound 9, indicating
that the structure containing ethylsulfonyl was beneficial in increasing the activity. The
SAR of compound 10 in terms of fungicidal activities (Table 1) was that when there was
no substituent on the benzene ring, the activity against Botrytis cinereal was superior to
other compounds. In addition, by comparing the control efficacy of compounds 10k
10m 10c, and 10n, we found that they showed that the para-position of aniline-containing
substituents was not conducive to improving the activity.
, 10l,
,

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2.4. Toxicity to Zebrafish Embryo
According to the fungicidal activity result, we selected compounds 9f and 10f with
better activity to study the lethal and teratogenic effects exposure on zebrafish embryos
from 6 to 96 hpf (Figure 3). When the 9f concentration exceeded 2 mg/L, the mortality rate
increased sharply. At 10 mg/L, the mortality rate reached as high as 90%. The resulting
LC50 value for compound 9f was 5.26 mg/L (Figure 3A). Similarly, the mortality rate of
10f showed concentration-dependent curves (Figure 3) with a LC50 value of 20.58 mg/L
(Figure 3B). Moreover, 9f and 10f produced similar teratogenic and decreased hatching
effects on zebrafish embryos at 72 hpf (Figure 3C–F). At 72 hpf, the hatching rates of the
compounds 9f and 10f under 10 mg/L exposure were about 43% and 82%, respectively.
In addition, the malformation rate of 9f was significantly higher than 10f at the same
concentration.
Figure 3. Zebrafish embryo mortality, hatching rate, and malformation rates after exposure to
compounds 9f
(
A
,
C
,
E
)and 10f
(
B
,
D
,
F
). Note: “*” represents significant differences at ** p
≤
0.01 and
*** p ≤ 0.001 by one-way ANOVA followed by Dunnett’s test.
As the time and concentration increased, zebrafish embryos showed obvious devel-
opmental delay. At 76–96 hpf, a series of malformations appeared, such as delayed yolk
absorption, pericardial cyst, lack of melanin, yolk sac, and bent spine (Figure 4). Among

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them, yolk cyst was the most obvious. By comparing the lethal and teratogenic effects
of 9f and 10f exposure on zebrafish embryos, we were able to find that the toxicity of 9f
to zebrafish embryos was higher than that of 10f. Thus, we speculated that the structure
containing ethylsulfonyl was beneficial to reduce the toxicity to zebrafish embryos.
Figure 4. Zebrafish embryo malformation after exposure to compounds 9f and 10f. Note: PC, pericardial cyst; YS, yolk sac;
BS, bent spine.
3. Experimental Section
3.1. General Information
Melting points were determined using an X-4 apparatus (Taike, Beijing, China) and
the thermometer was uncorrected. 1H NMR and 13C NMR spectra were measured on
BRUKER Avance 500 MHz spectrometer (Bruker 500 MHz, Fallanden, Switzerland) using
CDCl3 or DMSO as the solvent. High-resolution electrospray mass spectra (HR-ESI–MS)
were determined using an UPLC H CLASS/QTOF G2 XS mass spectrometer (Waters,
Milford, CT, USA). All the reagents were analytical grade or synthesized in our laboratory.
Ethics statement: The Institutional Animal Care and Use Committee (IACUC) at
Wenzhou Medical University (SYXK 2019-0009, April 4, 2019 to April 4, 2024) approved
our study plan for proper use of zebrafish. All studies were carried out in strict accordance
with the guidelines of the IACUC. All dissection was performed on ice, and all efforts were
made to minimize suffering.
3.2. Synthesis
3.2.1. Synthesis of Intermediate I
Methyl 3,6-dichloropicolinate (2): To a stirred solution of 3,6-dichloropicolinic acid
(1) (0.10 mmol) in acetone (10 mL), we added DMS (1.3 g, 0.01 mol) and K2CO3 (1.7 g).
After stirring at 40 ◦C for 8 h, the mixture was cooled to room temperature and poured
into water, the precipitation was filtered and dried to give 1.8 g light yellow solid. Yield:
90.1%, m.p. 54–55 ◦C ([48], 53–54 ◦C); 1H NMR (500 MHz, Chloroform-d)
(d, J = 8.5 Hz, 1H), 7.42 (d, J = 8.5 Hz, 1H), 4.00 (s, 3H).
δ 7.77

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Methyl 6-chloro-3-(ethylthio) picolinate (
3
): KTB (1.4 g), DMF (30 mL◦), and ethyl
mercaptan (2.5 g, 0.04 mol) were added to a three-necked flask and reacted at 0 C for about
1 h to give CH3CH2SK. The CH3CH2SK was dropped into a stirred solution of compound
2
(3.7 g) in DMF (20 mL) at 0 ◦C. After stirring at room temperature for 2 h, the mixture
was quenched with water and extracted by EtOAc (100 mL). The extraction was dried
over anhydrous MgSO4 and filtered. The filtration was concentrated and separated by
◦
column chromatography to give 3.1 g yellow solid. Yield: 74.5%, m.p. 128–129 C; 1H NMR
(500 MHz, Chloroform-d)
(q, J = 7.5 Hz, 2H), 1.39 (t, J = 7.5 Hz, 3H).
Intermediate : 30% NaOH (5 mL) was added to a solution of compound 3 (3.3 g) in
δ 7.66 (d, J = 8.5 Hz, 1H), 7.41 (d, J = 8.5 Hz, 1H), 4.00 (s, 3H), 2.95
I
THF. The mixture was then refluxed for 3 h. Afterwards, the mixture was cooled to room
temperature and poured into water. Then, we adjusted the pH to 2–3, and 2.8 g white solid
◦
precipitate was obtained. Yield: 92.1%, m.p. 119–120 C; 1H NMR (500 MHz, Chloroform-d)
δ
10.76 (s, 1H), 7.71 (d, J = 8.5 Hz, 1H), 7.49 (d, J = 8.5 Hz, 1H), 2.97 (q, J = 7.5 Hz, 2H),
1.44 (t, 3H).
3.2.2. Synthesis of Intermediate II
The synthesis of intermediate II refers to our previous work.
3.2.3. Methyl 2-Chloro-5-(5-(6-Chloro-3-(Ethylthio) Pyridin-2-yl)-1,2,4-Oxadiazol-3-yl)
Benzoate (7)
The intermediate I (1.1 g, 5 mmol) and SOCl2 (20 mL) were added to a 100 mL flask
and the mixture was refluxed for 6 h. Then, SOCl2 was removed under reduced pressure
to give 6-chloro-3-(ethylthio)picolinoyl chloride.
To a solution of intermediate II (1.1 g, 5.5 mmol) and triethylamine (1.2 g, 12 mmol)
in toluene (100 mL), we added the prepared 6-chloro-3-(ethylthio)picolinoyl chloride
dropwise at 0 ◦C for 1 h. The mixture was then refluxed for 2 h. Afterwards, the mixture
was cooled to room temperature and washed by saturated sodium chloride solution
(100 mL
×
3). The organic layer was dried by Na2SO4 and removed to give yellow
◦
1
solid (1.4 g). Yield: 62.2%, m.p. 155–157 C; H NMR (500 MHz, Chloroform-d)
δ
8.63
(d, J = 2.0 Hz, 1H), 8.35–8.20 (m, 1H), 7.89 (d, J = 8.5 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H), 7.54
(d, J = 8.5 Hz, 1H), 3.87 (s, 3H), 3.17 (q, J = 7.0 Hz, 2H), 1.32 (t, J = 7.5 Hz, 3H); HRMS calcd
for C17H14Cl2N3O3S [M + H]+ 410.0127, found 410.0126.
3.2.4. 2-Chloro-5-(5-(6-Chloro-3-(Ethylthio) Pyridin-2-yl)-1,2,4-Oxadiazol-3-yl) Benzoic
Acid (8)
We added 30% NaOH (5 mL) to a solution of compound 7 (0.8 g, 2.0 mmol) in THF. The
mixture was then refluxed for 2 h. Afterwards, the mixture was cooled to room temperature
and the solvent was removed. Then, we adjusted the pH to 2–3, and white solid precipitate
was obtained (0.7 g). Yield: 90.8%, m.p. 225–227 ◦C; 1H NMR (500 MHz, Chloroform-d)
δ
13.47 (s, 1H), 8.55 (d, J = 2.0 Hz, 1H), 8.36 (d, J = 8.5 Hz, 1H), 8.26–8.19 (m, 1H), 7.91 (d,
J = 8.5 Hz, 1H), 7.80 (d, J = 8.0 Hz, 1H), 3.16 (q, J = 7.5 Hz, 2H), 1.30 (t, J = 7.0 Hz, 3H);
HRMS calcd for C16H12Cl2N3O3S [M + H]+ 395.9971, found 395.9973.
3.2.5. Synthesis of Target Compound 9
To a solution of compound
8
(1.0 g, 2.0 mmol), EDCI (0.1 g) a◦nd triethylamine (0.2 g)
in DCM (100 mL), we added the substituted aniline (3.0 mmol) at 0 C, and the mixture was
stirred for 8 h to give compound 9 by the method of column chromatography separation.
2-Chloro-5-(5-(6-chloro-3-(ethylthio)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-phenylbenzamide
(
9a): White solid, yield 73.4%, m.p. 243–245 ◦C; 1H NMR (500 MHz, DMSO-d6) 1H NMR
(500 MHz, Chloroform-d)
δ 10.70 (s, 1H), 8.23–8.18 (m, 2H), 8.13 (d, J = 9.0 Hz, 1H),
7.88–7.84 (m, 1H), 7.79 (d, J = 8.5 Hz, 1H), 7.73 (d, J = 8.0 Hz, 2H), 7.38 (t, J = 8.0 Hz, 2H), 7.14
(t, J = 7.0 Hz, 1H), 3.15 (q, J = 7.5 Hz, 2H), 1.30 (t, J = 7.0 Hz, 3H); 13C NMR (500 MHz,
DMSO-d6)
δ 172.43, 167.11, 164.10, 145.88, 138.71, 138.64, 138.22, 138.13, 137.88, 135.87,

Int. J. Mol. Sci. 2021, 22, 2367
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133.66, 131.22, 129.60, 129.01, 127.81, 127.29, 124.30, 119.89, 25.53, 13.36; HRMS calcd for
C22H17Cl2N4O2S [M + H]+ 471.0444, found 471.0447.
2-Chloro-5-(5-(6-chloro-3-(ethylthio)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(o-tolyl)benzamide
(
9b): White solid, yield 77.4%, m.p. 230–232 ◦C; 1H NMR (500 MHz, DMSO-d6)
δ 10.19 (s,
1H), 8.26 (d, J = 2.0 Hz, 1H), 8.20 (dd, J = 8.5, 2.0 Hz, 1H), 8.14 (d, J = 9.0 Hz, 1H), 7.86 (d,
J = 8.5 Hz, 1H), 7.80 (d, J = 8.5 Hz, 1H), 7.49 (d, J = 7.5 Hz, 1H), 7.29 (d, J = 7.5 Hz, 1H),
7.25 (t, J = 6.5 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H), 3.17 (q, J = 7.0 Hz, 2H), 2.30 (s, 3H), 1.32
(t, J = 7.5 Hz, 3H); 13C NMR (500 MHz, DMSO-d6) 13C NMR (126 MHz, DMSO-d6)
δ 172.45,
166.07, 163.92, 145.90, 138.67, 137.96, 137.61, 136.24, 133.69, 133.53, 133.38, 132.91, 131.23,
130.94, 129.56, 129.38, 127.83, 127.29, 125.25, 119.91, 25.55, 20.64, 13.38; HRMS calcd for
C23H19Cl2N4O2S [M + H]+ 485.0600, found 485.0600.
2-Chloro-5-(5-(6-chloro-3-(ethylthio)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(p-tolyl)benzamide
(
9c): White solid, yield 74.3%, m.p. 251–253 ◦C; 1H NMR (500 MHz, DMSO-d6)
δ 10.61 (s,
1H), 8.19 (m, 2H), 8.13 (d, J = 9.0 Hz, 1H), 7.85 (d, J = 8.5 Hz, 1H), 7.79 (d, J = 9.0 Hz, 1H),
7.61 (d, J = 8.0 Hz, 2H), 7.18 (d, J = 8.0 Hz, 2H), 3.16 (d, J = 7.5 Hz, 2H), 2.29 (s, 3H), 1.30
(t, J = 7.5 Hz, 3H); 13C NMR (500 MHz, DMSO-d6)
δ 172.46, 167.15, 163.96, 145.90,
138.63, 138.29, 137.98, 137.57, 136.26, 133.71, 133.41, 131.25, 129.58, 129.41, 127.85, 127.32,
125.02, 119.93, 25.56, 20.67, 13.39; HRMS calcd for C23H19Cl2N4O2S [M + H]+ 485.0600,
found 485.0606.
N-(4-(tert-Butyl) phenyl)-2-chloro-5-(5-(6-chloro-3-(ethylthio) pyridin-2-yl)-1,2,4-oxadiazol-
◦
3-yl) benzamide (9d): White solid, yield 78.4%,. m.p. 243–244 C; 1H NMR (500 MHz, DMSO-
d6)
J = 8.0 Hz, 1H), 7.79 (d, J = 8.5 Hz, 1H), 7.64 (d, J = 8.5 Hz, 2H), 7.39 (d, J = 8.5 Hz, 2H), 3.16
(q, J = 7.5 Hz, 2H), 1.34–1.26 (m, 12H); 13C NMR (500 MHz, DMSO-d6)
172.46, 167.40,
δ 10.63 (s, 1H), 8.23–8.17 (m, 2H), 8.14 (d, J = 9.0 Hz, 1H), 7.85 (d,
δ
167.19, 138.54, 138.11, 135.64, 133.72, 133.30, 133.04, 133.00, 131.21, 127.88, 127.53, 126.84,
126.17, 124.98, 25.64, 20.70, 18.01, 13.34; HRMS calcd for C26H25Cl2N4O2S [M + H]+
527.1070, found 527.1071.
2-Chloro-5-(5-(6-chloro-3-(ethylthio)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(2,4-dimethylphenyl)
benzamide (9e): Grey solid, yield 69.2%, m.p. 255–256 ◦C; 1H NMR (500 MHz, DMSO-d6)
δ
10.10 (s, 1H), 8.24 (d, J = 2.0 Hz, 1H), 8.19 (dd, J = 8.0, 2.0 Hz, 1H), 8.14 (d, J = 8.5 Hz, 1H),
7.83 (dd, J = 24.0, 8.5 Hz, 2H), 7.34 (d, J = 8.0 Hz, 1H), 7.10 (s, 1H), 7.05 (d, J = 8.0 Hz, 1H),
3.17 (q, J = 7.0 Hz, 2H), 2.28 (d, J = 6.0 Hz, 6H), 1.32 (t, J = 7.5 Hz, 3H); 13C NMR (500 MHz,
DMSO-d6)
δ 172.46, 167.40, 167.19, 138.54, 138.11, 135.64, 133.72, 133.30, 133.04, 131.21,
127.88, 127.53, 126.84, 126.17, 25.64, 20.70, 18.01, 13.34; HRMS calcd for C24H21Cl2N4O2S
[M + H]+ 499.0757, found 499.0763.
2-Chloro-5-(5-(6-chloro-3-(ethylthio)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(3-(trifluoromethyl)
◦
1
phenyl)benzamide (9f): White solid, yield 77.8%, m.p. 211–214 C; H NMR (500 MHz,
DMSO-d6) 11.06 (s, 1H), 8.27 (d, J = 2.0 Hz, 1H), 8.25–8.21 (m, 2H), 8.14 (d, J = 9.0 Hz,
1H), 7.92 (d, J = 8.0 Hz, 1H), 7.89 (d, J = 8.5 Hz, 1H), 7.80 (d, J = 9.0 Hz, 1H), 7.64 (t,
J = 8.0 Hz, 1H), 7.51 (d, J = 8.0 Hz, 1H), 3.16 (q, J = 7.5 Hz, 2H), 1.30 (t, J = 7.0 Hz, 3H); 13
NMR (500 MHz, DMSO-d6) 172.48, 167.10, 164.59, 145.91, 139.49, 138.68, 138.63, 138.59,
δ
C
δ
138.28, 137.58, 137.35, 133.70, 131.35, 131.29, 130.37, 127.88, 127.81, 125.11, 123.53, 120.68,
116.00, 25.57, 13.36; HRMS calcd for C23H16Cl2F3N4O2S [M + H]+ 539.0318, found 539.0322.
2-Chloro-N-(3-chloro-2-methylphenyl)-5-(5-(6-chloro-3-(ethylthio)
oxadiazol-3-yl) benzamide (9g): Yellow solid, yield 79.7%. m.p. 225–226 C; H NMR
(500 MHz, DMSO-d6) 10.44 (s, 1H), 8.29 (d, J = 1.5 Hz, 1H), 8.21 (dd, J = 8.0, 1.5 Hz,
pyridin-2-yl)-1,2,4-
◦
1
δ
1H), 8.14 (d, J = 9.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 9.0 Hz, 1H), 7.47 (d,
J = 8.0 Hz, 1H), 7.39 (d, J = 8.0 Hz, 1H), 7.29 (t, J = 8.0 Hz, 1H), 3.16 (q, J = 7.5 Hz, 2H), 2.35
(s, 3H), 1.32 (t, J = 7.5 Hz, 3H); 13C NMR (500 MHz, DMSO-d6)
δ 172.39, 167.05, 164.53,
145.85, 138.65, 138.24, 137.61, 137.10, 134.00, 133.62, 131.53, 131.19, 129.61, 127.77, 127.40,
127.18, 127.14, 127.08, 125.45, 124.97, 25.54, 15.30, 13.26; HRMS calcd for C23H18Cl3N4O2S
[M + H]+ 519.0211, found 519.0211.
2-Chloro-5-(5-(6-chloro-3-(ethylthio)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(2-fluorophenyl)
benzamide (9h): White solid, yield 66.5%, m.p. 213–217 ◦C; 1H NMR (500 MHz, DMSO-d6)

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1H NMR (500 MHz, Chloroform-d)
δ
10.56 (s, 1H), 8.21 (m, 2H), 8.14 (d, J = 8.5 Hz, 1H),
7.91–7.82 (m, 2H), 7.80 (d, J = 8.5 Hz, 1H), 7.36–7.21 (m, 3H), 3.16 (q, J = 7.5 Hz, 2H), 1.31 (t,
J = 7.0 Hz, 3H); 13C NMR (500 MHz, DMSO-d6) 13C NMR (126 MHz, DMSO-d6)
δ
172.42,
164.54, 158.63, 145.88, 143.26, 139.54, 138.66, 138.23, 137.37, 136.91, 132.59, 131.18, 129.68,
127.80, 127.49, 125.78, 125.19, 124.60 (d, J = 10 Hz), 123.80, 25.55, 13.31; HRMS calcd for
C22H16Cl2FN4O2S [M + H]+ 489.0350, found 489.0355.
2-Chloro-5-(5-(6-chloro-3-(ethylthio)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(4-fluorophenyl)
benzamide (9i): Yellow solid, yield 61.6%, m.p. 268–270 ◦C; 1H NMR (500 MHz, DMSO-d6)
δ
10.76 (s, 1H), 8.24–8.18 (m, 2H), 8.13 (d, J = 9.0 Hz, 1H), 7.89–7.83 (m, 1H), 7.82–7.72 (m,
3H), 7.22 (t, J = 9.0 Hz, 2H), 3.16 (q, J = 7.5 Hz, 2H), 1.30 (t, J = 7.5 Hz, 3H); 13C NMR
(500 MHz, DMSO-d6) δ 172.41, 167.06, 163.96, 145.85, 138.61, 138.19, 137.70, 137.57, 133.64,
131.20, 129.64, 127.77, 127.28, 124.99, 121.74, 121.68, 115.67, 115.49, 25.50, 13.33; HRMS calcd
for C22H16Cl2FN4O2S [M + H]+ 489.0350, found 489.0350.
2-Chloro-5-(5-(6-chloro-3-(ethylthio)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(2,6-difluorophenyl)
◦
1
benzamide (9j): Grey solid, yield 57.8%, m.p. 258–261 C; H NMR (500 MHz, DMSO-
d6) 10.57 (s, 1H), 8.27–8.18 (m, 2H), 8.14 (d, J = 8.5 Hz, 1H), 7.90–7.76 (m, 3H), 7.39
(t, J = 8.5 Hz, 1H), 7.16 (t, J = 8.5 Hz, 1H), 3.16 (q, J = 7.5 Hz, 2H), 1.31 (t, J = 7.5 Hz, 3H); 13
NMR (500 MHz, DMSO-d6) 172.46, 167.78, 167.11, 158.28, 152.27, 146.15, 145.92, 138.69,
δ
C
δ
138.25, 137.62, 137.22, 133.78, 131.25, 129.81, 127.84, 127.51, 117.00, 116.01, 111.58, 25.59,
13.33; HRMS calcd for C22H15Cl2F2N4O2S [M + H]+ 507.0255, found 507.0258.
2-Chloro-5-(5-(6-chloro-3-(ethylthio)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(2-chlorophenyl)
benzamide (9k): White solid, yield 75.0%, m.p. 207–208 ◦C; 1H NMR (500 MHz, DMSO-d6)
δ
10.48 (s, 1H), 8.30 (s, 1H), 8.21 (d, J = 8.5 Hz, 1H), 8.14 (d, J = 8.5 Hz, 1H), 7.86 (d,
J = 8.5 Hz, 1H), 7.80 (d, J = 8.5 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.58 (d, J = 8.0 Hz, 1H),
7.42 (t, J = 7.0 Hz, 1H), 7.32 (t, J = 7.5 Hz, 1H), 3.16 (q, J = 7.0 Hz, 2H), 1.32 (t, J = 7.5 Hz,
3H); 13C NMR (500 MHz, DMSO-d6)
δ 172.45, 167.07, 164.37, 145.87, 140.12, 138.25, 137.55,
137.44, 133.65, 133.32, 131.24, 130.76, 129.93, 129.85, 127.78, 127.29, 125.06, 119.40, 119.32,
118.33, 25.54, 13.35; HRMS calcd for C22H16Cl3N4O2S [M + H]+ 505.0054, found 505.0055.
2-Chloro-5-(5-(6-chloro-3-(ethylthio)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(3-chlorophenyl)
benzamide (9l): White solid, yield 75.4%, m.p. 232–234 ◦C; 1H NMR (500 MHz, DMSO-d6)
δ
10.90 (s, 1H), 8.26–8.20 (m, 2H), 8.14 (d, J = 8.5 Hz, 1H), 7.94 (t, J = 1.5 Hz, 1H), 7.87
(d, J = 8.5 Hz, 1H), 7.80 (d, J = 9.0 Hz, 1H), 7.60 (d, J = 8.0 Hz, 1H), 7.42 (t,
J = 8.0 Hz, 1H), 7.24–7.18 (m, 1H), 3.16 (q, J = 7.0 Hz, 2H), 1.30 (t, J = 7.0 Hz, 3H); 13C NMR
(126 MHz, DMSO-d6) 13C NMR (126 MHz, Chloroform-d)
δ 177.49, 172.13, 169.39, 150.90,
145.27, 143.38, 142.55, 138.73, 138.41, 136.35, 136.28, 135.80, 134.93, 132.82, 132.51, 130.17,
129.06, 124.45, 124.38, 123.35, 30.60, 18.41; HRMS calcd for C22H16Cl3N4O2S [M + H]+
505.0054, found 505.0055.
2-Chloro-5-(5-(6-chloro-3-(ethylthio)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(4-chlorophenyl)
benzamide (9m): White solid, yield 77.3%, m.p. 242–243 ◦C; 1H NMR (500 MHz, DMSO-d6)
δ
10.84 (s, 1H), 8.24–8.18 (m, 2H), 8.11 (d, J = 9.0 Hz, 1H), 7.85 (d, J = 8.0 Hz, 1H), 7.77 (m,
3H), 7.43 (d, J = 9.0 Hz, 2H), 3.14 (q, J = 7.5 Hz, 2H), 1.29 (t, J = 7.5 Hz, 3H); 13C NMR
(126 MHz, DMSO-d6) δ 172.42, 167.07, 164.17, 145.86, 138.55, 138.25, 137.66, 137.57, 137.52,
133.65, 131.22, 129.75, 128.92, 127.92, 127.78, 127.33, 125.04, 121.45, 25.54, 13.33; HRMS calcd
for C22H16Cl3N4O2S [M + H]+ 505.0054, found 505.0055.
N-(4-Bromophenyl)-2-chloro-5-(5-(6-chloro-3-(ethylthio)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)
benzamide (9n): Yellow solid, yield 78.8%, m.p. 267–269 ◦C; 1H NMR (500 MHz, DMSO-d6)
δ
10.84 (s, 1H), 8.25–8.19 (m, 2H), 8.13 (d, J = 8.5 Hz, 1H), 7.87 (d, J = 8.0 Hz, 1H), 7.79
(d, J = 9.0 Hz, 1H), 7.72 (d, J = 9. Hz, 2H), 7.57 (d, J = 8.5 Hz, 2H), 3.16 (q, J = 7.5 Hz, 2H),
1.30 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, DMSO-d6)
172.43, 167.08, 164.20, 145.88,
δ
138.60, 138.24, 138.08, 137.57, 133.64, 131.84, 131.24, 129.77, 127.80, 127.33, 125.04, 121.83,
115.99, 25.55, 13.35; HRMS calcd for C22H16BrCl2N4O2S [M + H]+ 548.9549, found 548.9554.

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3.2.6. Synthesis of Target Compound 10
To a stirred solution of compound
9
(1.0 mmol) in DCM (20 mL), we added mCPBA
(0.5 g, 3.0 mmol). After stirring at room temperature for 3 h, the mixture was poured into
water and the pH was adjusted to 7–8 with NaHCO3. The organic layer was dried by
Na2SO4 and removed under reduced pressure to give compound 10.
2-Chloro-5-(5-(6-chloro-3-(ethylsulfonyl)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-phenylbenzamide
(
10a): Pink solid, yield 83.7%, m.p. 263–267 ◦C; 1H NMR (500 MHz, DMSO-d6)
δ 10.70 (s,
1H), 8.61 (d, J = 8.5 Hz, 1H), 8.23 (d, J = 2.5 Hz, 1H), 8.20 (m, 2H), 7.85 (d, J = 8.5 Hz, 1H),
7.74 (d, J = 8.0 Hz, 2H), 7.37 (t, J = 8.0 Hz, 2H), 7.13 (t, J = 7.5 Hz, 1H), 3.78 (q, J = 7.5 Hz,
2H), 1.26 (t, J = 7.5 Hz, 3H); 13C NMR (500 MHz, DMSO-d6)
δ 171.62, 166.81, 163.79, 154.63,
142.95, 142.22, 138.64, 137.80, 135.62, 133.90, 131.16, 129.50, 128.82, 127.36, 124.27, 124.07,
119.71, 50.37, 6.64; HRMS calcd for C22H17Cl2N4O4S [M + H]+ 503.0342, found 503.0347.
2-Chloro-5-(5-(6-chloro-3-(ethylsulfonyl)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(o-tolyl)benzamide
(
10b): White solid, yield 77.4%, m.p. 257–259 ◦C; 1H NMR (500 MHz, DMSO-d6)
δ 10.18
(s, 1H), 8.62 (d, J = 9.0 Hz, 1H), 8.26 (d, J = 2.0 Hz, 1H), 8.20 (d, J = 8.5 Hz, 2H), 7.85
(d, J = 8.5 Hz, 1H), 7.49 (d, J = 7.5 Hz, 1H), 7.31–7.20 (m, 2H), 7.18 (td, J = 7.5, 1.5 Hz, 1H),
3.79 (q, J = 7.5 Hz, 2H), 2.31 (s, 3H), 1.27 (t, J = 7.5 Hz, 3H); 13C NMR (500 MHz, DMSO-d6)
δ
171.64, 166.85, 164.14, 154.64, 142.98, 142.23, 137.97, 135.64, 135.47, 133.89, 132.94, 131.14,
130.61, 130.44, 129.37, 128.79, 127.38, 126.07, 124.24, 50.37, 39.93, 17.90, 6.63; HRMS calcd
for C23H19Cl2N4O4S [M + H]+ 517.0499, found 517.0500.
2-Chloro-5-(5-(6-chloro-3-(ethylsulfonyl)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(p-tolyl)benzamide
◦
1
(
10c): White solid, yield 84.3%, m.p. 260–261 C; H NMR (500 MHz, DMSO-d6) δ
10.59 (s, 1H), 8.61 (d, J = 8.5 Hz, 1H), 8.26–8.15 (m, 3H), 7.84 (d, J = 8.5 Hz, 1H), 7.61
(d, J = 8.5 Hz, 2H), 7.17 (d, J = 8.0 Hz, 2H), 3.77 (q, J = 7.5 Hz, 2H), 2.28 (s, 3H), 1.26
(t, J = 7.0 Hz, 3H); 13C NMR (500 MHz, DMSO-d6) 13C NMR (126 MHz, DMSO-d6)
δ 171.61,
166.81, 163.60, 154.63, 142.94, 142.20, 137.85, 136.11, 135.60, 133.90, 133.14, 131.16, 129.45,
129.18, 128.78, 127.32, 124.22, 119.72, 50.36, 20.46, 6.61; HRMS calcd for C23H19Cl2N4O4S
[M + H]+ 517.0499, found 517.0505.
N-(4-(tert-Butyl)phenyl)-2-chloro-5-(5-(6-chloro-3-(ethylsulfonyl)pyridin-2-yl)-1,2,4-oxadiazol-
◦
3-yl)benzamide (10d): Grey yield, yield 79.2%, m.p. 268–270 C; 1H NMR (500 MHz, DMSO-
d6)
(d, J = 8.5 Hz, 2H), 7.39 (d, J = 8.5 Hz, 2H), 3.78 (q, J = 7.5 Hz, 2H), 1.33–1.27 (m, 12H); 13
NMR (500 MHz, DMSO-d6) 176.85, 172.05, 168.89, 159.88, 151.78, 148.21, 147.45, 143.08,
δ 10.61 (s, 1H), 8.62 (d, J = 8.5 Hz, 1H), 8.24–8.12 (m, 3H), 7.85 (d, J = 9.0 Hz, 1H), 7.64
C
δ
141.26, 140.83, 139.14, 136.39, 134.64, 134.06, 132.50, 130.66, 129.46, 124.77, 55.61, 39.28, 36.36,
11.87; HRMS calcd for C26H25Cl2N4O4S [M + H]+ 559.0968, found 559.0971.
2-Chloro-5-(5-(6-chloro-3-(ethylsulfonyl)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(2,4-dimethylphenyl)
◦
benzamide (10e): White solid, yield 78.4%,. m.p. 246–248 C; 1H NMR (500 MHz, DMSO-d6)
δ
10.09 (s, 1H), 8.61 (d, J = 8.5 Hz, 1H), 8.24 (d, J = 2.0 Hz, 1H), 8.22–8.17 (m, 2H), 7.84
(d, J = 8.5 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.08 (s, 1H), 7.04 (dd, J = 8.0, 2.0 Hz, 1H), 3.79
(q, J = 7.0 Hz, 2H), 2.27 (d, J = 7.5 Hz, 6H), 1.27 (t, J = 7.5 Hz, 3H); 13C NMR (500 MHz,
DMSO-d6)
δ 171.62, 166.84, 164.16, 154.64, 142.96, 142.21, 138.02, 135.62, 135.34, 133.87,
132.84, 132.81, 131.12, 130.92, 129.32, 128.78, 127.34, 126.57, 125.93, 124.20, 50.37, 20.49, 17.81,
6.62; HRMS calcd for C24H21Cl2N4O4S [M + H]+ 531.0655, found 531.0660.
2-Chloro-5-(5-(6-chloro-3-(ethylsulfonyl)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(3-(trifluoromethyl)
◦
phenyl)benzamide (10f): Yellow solid, yield 76.8%, m.p. 214–217 C; H NMR (500 MHz,
DMSO-d6)
δ 11.07 (s, 1H), 8.61 (d, J = 8.5 Hz, 1H), 8.30 (d, J = 2.0 Hz, 1H), 8.26–8.17 (m,
3H), 7.92 (d, J = 8.5 Hz, 1H), 7.87 (d, J = 8.5 Hz, 1H), 7.62 (t, J = 8.0 Hz, 1H), 7.50 (d,
J = 7.5 Hz, 1H), 3.78 (q, J = 7.5 Hz, 2H), 1.26 (t, J = 7.0 Hz, 3H); 13C NMR (500 MHz, DMSO-d
) δ 171.64, 166.77, 164.23, 154.62, 142.95, 142.20, 139.37, 137.23, 135.61, 133.90, 131.23, 130.13,
6
129.80, 129.54 (d, J = 32.1 Hz), 128.79, 127.49, 125.10, 124.32, 123.30, 120.44 (d), 115.78
(d, J = 4.0 Hz), 50.37, 6.62; HRMS calcd for C23H16Cl2F3N4O4S [M + H]+ 571.0216, found
571.0222.
2-Chloro-N-(3-chloro-2-methylphenyl)-5-(5-(6-chloro-3-(ethylsulfonyl)pyridin-2-yl)-1,2,4-
◦
1
oxadiazol-3-yl)benzamide (10g): Yellow solid, yield 69.1%, m.p. 255–256 C; H NMR

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(500 MHz, DMSO-d6)
δ
10.44 (s, 1H), 8.62 (d, J = 8.5 Hz, 1H), 8.29 (d, J = 2.0 Hz, 2H),
8.21 (m, 1H), 7.86 (d, J = 8.5 Hz, 1H), 7.46 (d, J = 8.0 Hz, 1H), 7.38 (d, J = 7.5 Hz, 1H),
7.28 (t, J = 8.0 Hz, 1H), 3.79 (q, J = 7.0 Hz, 2H), 2.34 (s, 3H), 1.27 (t, J = 7.5 Hz, 3H); 13
NMR (500 MHz, DMSO-d6) 13C NMR (126 MHz, DMSO-d6)
171.64, 166.81, 164.30, 154.63,
C
δ
142.97, 142.21, 137.60, 137.00, 135.63, 133.88, 131.45, 131.17, 129.55, 128.81, 128.78, 127.40,
127.01, 126.95, 125.34, 124.28, 50.36, 15.19, 6.64; HRMS calcd for C23H18Cl3N4O4S [M + H]+
551.0109, found 551.0108.
2-Chloro-5-(5-(6-chloro-3-(ethylsulfonyl)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(2-fluorophenyl)
◦
benzamide (10h): Brown solid, yield 73.7%, m.p. 253–257 C; 1H NMR (500 MHz, DMSO-d6)
δ
10.58 (s, 1H), 8.61 (d, J = 8.5 Hz, 1H), 8.27–8.11 (m, 3H), 7.94–7.81 (m, 2H), 7.38–7.17
(m, 3H), 3.78 (q, J = 7.0 Hz, 2H), 1.26 (t, J = 7.0 Hz, 3H); 13C NMR (500 MHz, DMSO-d6)
13C NMR (126 MHz, DMSO-d6)
171.62, 166.80, 166.07, 154.64, 142.94, 142.19, 137.26,
δ
135.60, 133.97, 133.31, 132.69, 131.16, 130.64, 129.60, 128.77, 127.87, 127.51, 125.62, 124.45,
115.80 (d, J = 77.5 Hz), 50.37, 6.58; HRMS calcd for C22H16Cl2FN4O4S [M + H]+ 521.0248,
found 521.0255.
2-Chloro-5-(5-(6-chloro-3-(ethylsulfonyl)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(2-fluorophenyl)
◦
benzamide (10i): Yellow solid, yield 71.6%, m.p. 229–231 C; 1H NMR (500 MHz, DMSO-d6)
δ
10.75 (s, 1H), 8.61 (d, J = 8.5 Hz, 1H), 8.24 (d, J = 2.0 Hz, 1H), 8.23–8.18 (m, 2H), 7.85
(d, J = 8.5 Hz, 1H), 7.78–7.71 (m, 2H), 7.22 (t, J = 9.0 Hz, 2H), 3.78 (q, J = 7.0 Hz, 2H),
1.26 (t, J = 7.5 Hz, 3H); 13C NMR (500 MHz, DMSO-d6) 13C NMR (126 MHz, DMSO-d6)
δ
171.62, 166.79, 163.71, 157.51, 154.63, 142.94, 142.20, 137.61, 135.60, 134.97, 133.90, 131.18,
129.58, 128.78, 127.37, 124.27, 121.60, 115.43 (d, J = 88.0 Hz), 50.37, 6.62; HRMS calcd for
C22H16Cl2FN4O4S [M + H]+ 521.0248, found 521.0251.
2-Chloro-5-(5-(6-chloro-3-(ethylsulfonyl)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(2,6-difluorophenyl)
◦
benzamide (10j): Brown solid, yield 72.8%, m.p. 237–239 C; 1H NMR (500 MHz, DMSO-d6)
δ
10.56 (s, 1H), 8.61 (d, J = 8.5 Hz, 1H), 8.25 (m, 1H), 8.21 (d, J = 8.5 Hz, 2H), 7.89–7.82 (m,
2H), 7.42–7.34 (m, 1H), 7.19–7.12 (m, 1H), 3.78 (q, J = 7.5 Hz, 2H), 1.27 (t, J = 7.5 Hz, 3H);
13C NMR (500 MHz, DMSO-d6)
171.63, 166.79, 164.35, 154.64, 142.94, 142.18, 137.08, 135.61,
δ
133.97, 131.19, 129.68, 128.79, 127.52, 127.17 (d, J = 7.5 Hz), 124.17, 111.40 (d, J = 12.5 Hz),
111.22, 104.60, 104.40 (d, J = 9.0 Hz), 104.19, 50.36, 6.58; HRMS calcd for C22H15Cl2F2N4O4S
[M + H]+ 539.0154, found 539.0159.
2-Chloro-5-(5-(6-chloro-3-(ethylsulfonyl)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(2-chlorophenyl)
benzamide (10k): White solid, yield 82.0%, m.p. 209–212 ◦C; 1H NMR (500 MHz, DMSO-
d6)
δ 10.50 (s, 1H), 8.62 (d, J = 8.0 Hz, 1H), 8.34–8.14 (m, 3H), 7.93–7.82 (m, 1H), 7.73
(d, J = 7.0 Hz, 1H), 7.58 (d, J = 7.5 Hz, 1H), 7.48–7.38 (m, 1H), 7.36–7.29 (m, 1H), 3.86–3.73
(m, 2H), 1.28 (t, J = 7.0 Hz, 3H); 13C NMR (500 MHz, DMSO-d6) 13C NMR (126 MHz,
DMSO-d6)
δ 171.62, 166.79, 164.38, 154.66, 142.94, 142.18, 135.58, 134.07, 134.00, 131.23,
129.68, 129.63, 128.79, 128.77, 127.87, 127.84, 127.73, 127.59, 127.48, 124.15, 50.38, 6.61; HRMS
calcd for C22H16Cl3N4O4S [M + H]+ 536.9952, found 536.9958.
2-Chloro-5-(5-(6-chloro-3-(ethylsulfonyl)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(2-chlorophenyl)
benzamide (10l): Yellow solid, yield 85.4%, m.p. 217–218 ◦C; 1H NMR (500 MHz, DMSO-
d6)
δ 10.91 (s, 1H), 8.62 (d, J = 8.5 Hz, 1H), 8.31–8.17 (m, 3H), 7.97–7.84 (m, 2H), 7.60 (d,
J = 7.5 Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H), 7.21 (d, J = 7.5 Hz, 1H), 3.79 (q, J = 7.0 Hz, 2H),
1.26 (t, J = 7.0 Hz, 3H); 13C NMR (126 MHz, DMSO-d6) 13C NMR (126 MHz, DMSO-d6)
δ
171.63, 166.77, 164.09, 154.64, 142.93, 142.18, 139.97, 137.32, 135.58, 133.87, 133.15, 131.23,
130.58, 129.75, 128.79, 127.40, 124.30, 123.85, 119.19, 118.15, 50.37, 6.62; HRMS calcd for
C22H16Cl3N4O4S [M + H]+ 536.9952, found 536.9959.
2-Chloro-5-(5-(6-chloro-3-(ethylsulfonyl)pyridin-2-yl)-1,2,4-oxadiazol-3-yl)-N-(2-chlorophenyl)
◦
benzamide (10m): White solid, yield 84.3%, m.p. 233–236 C; 1H NMR (500 MHz, DMSO-d6)
13C NMR (126 MHz, DMSO-d6)
137.32, 135.58, 133.87, 133.15, 131.23, 130.58, 129.75, 128.79, 127.40, 124.30, 123.85, 119.19,
118.15, 50.37, 6.62; 13C NMR (126 MHz, DMSO-d6) 13C NMR (126 MHz, DMSO-d6)
171.62,
166.78, 163.88, 154.64, 142.94, 142.19, 137.53, 137.47, 135.59, 133.88, 131.20, 129.66, 128.78,
δ 171.63, 166.77, 164.09, 154.64, 142.93, 142.18, 139.97,
δ

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128.75, 127.74, 127.39, 124.28, 121.29, 50.37, 6.62; HRMS calcd for C22H16Cl3N4O4S [M + H]+
536.9952, found 521.0251.
N-(4-Bromophenyl)-2-chloro-5-(5-(6-chloro-3-(ethylsulfonyl)pyridin-2-yl)-1,2,4-oxadiazol-3-
◦
yl)benzamide (10n): Yellow solid, yield 78.3%, m.p. 263–265 C; 1H NMR (500 MHz, DMSO-
d6)
(d, J = 8.5 Hz, 2H), 7.56 (d, J = 8.5 Hz, 2H), 3.77 (q, J = 7.5 Hz, 2H), 1.26 (t, J = 7.5 Hz, 3H);
13C NMR (126 MHz, DMSO-d6) 13C NMR (126 MHz, DMSO-d6)
171.61, 166.77, 163.91,
δ 10.83 (s, 1H), 8.61 (d, J = 8.5 Hz, 1H), 8.33–8.11 (m, 3H), 7.85 (d, J = 8.0 Hz, 1H), 7.71
δ
154.65, 142.93, 142.18, 137.93, 137.44, 135.58, 133.87, 131.66, 131.21, 129.67, 128.78, 127.38,
124.27, 121.67, 115.81, 50.38, 6.61; HRMS calcd for C22H16BrCl2N4O4S [M + H]+ 580.9447,
found 580.9449.
3.3. Biological Activity and Toxicity Determination
The insecticidal and fungicidal activities were investigated in the National Pesticide
Engineering Research Centre, Nankai University, according to references [49
,50], and the
results of the activity test are shown in Table 1.
Through acute exposure, we assessed the toxicity of compounds 9f and 10f on ze-
brafish embryo. According to the preliminary exposure experiments, a series of gradient
concentrations of compounds 9f and 10f were set on the basis of mortality rates in the
range of 10–95%. LC50 values for zebrafish embryos exposed to compound 9f or 10f from 6
to 96 hpf: control (0 mg/L of 9f), 1, 5, 10 mg/L of 9f; control (0 mg/L of 10f), 5, 10 mg/L
of 10f. The LC50 (median lethal concentration) values were computed by the Boltzmann
equation [51,52]. The observational indexes included 96 hpf mortality rate, 72 hpf hatching
rate, and 96 hpf malformation rate.
4. Conclusions
In conclusion, a series of novel benzamides containing 1,2,4-oxadiazole moiety were
designed by bioisosterism and were synthesized easily via thioetherification, cyclization,
aminolysis, and oxidation reactions. Their structures were confirmed by 1H NMR, 13
C
NMR, and HRMS. The bioassay results showed that some of the title compounds displayed
excellent fungicidal activities against Botrytis cinereal at 100 mg/L. For example, 10a (84.4%),
10d (83.6%), 10e (83.3%), 10f (83.1%), 10i (83.3%), and 10l (83.6%) were better than the
control fungicide pyraclostrobin (81.4%). In addition, the acute toxicity of 10f to zebrafish
embryo was 20.58 mg/L, which was classified as a low toxicity compound. Therefore,
these compounds could potentially be the lead compounds for further study.
Supplementary Materials: The following are available online at https://www.mdpi.com/1422-006
7/22/5/2367/s1.
Author Contributions: S.Y., X.-Y.T., T.-Y.M., L.D., C.-L.R., and W.-Q.Z. carried out experimental
work; S.Y. prepared the manuscript; C.-X.T. designed the material and supervised the project; and
X.-H.L. and C.-X.T. revised the paper. All authors have read and agreed to the published version of
the manuscript.
Funding: This work was funded by the Wynca Group and Siga Co. Ltd. R&D Program, grant
numbers KYY-HX-20180412 and KYY-HX-20180737.
Data Availability Statement: Samples of the compounds are not available from the authors.
Acknowledgments: We acknowledge Hui-Li Wang for support with the toxicity determination.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
HRMS (High Resolution Mass Spectrometer), DMF (N,N-Dimethylformamide), mCPBA (3-Chloroperbenzoic
acid), DMS (Dimethyl sulfate), rt (Room temperature), KTB (Potassium t-butoxide), THF (Tetrahydro-
furan), EtOAc (Ethyl acetate), EDCI (1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride),
SAR (Structure activity relationships).

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