Synthesis of Thiazolium-Labeled 1,3,4-Oxadiazole Thioethers as Prospective Antimicrobials: In Vitro and in Vivo Bioactivity and Mechanism of Action

Article abstract of DOI:10.1021/acs.jafc.9b03952

In this study, a type of thiazolium-labeled 1,3,4-oxadiazole thioether bridged by diverse alkyl chain lengths was constructed. The antimicrobial activity of the fabricated thioether toward plant pathogenic bacteria and fungi was then screened. Antibacterial evaluation indicated that title compounds possess specific characteristics that enable them to severely attack three phytopathogens, namely, Xanthomonas oryzae pv. oryzae, Ralstonia solanacearum, and Xanthomonas axonopodis pv. citri with minimal EC₅₀ values of 0.10, 3.27, and 3.50 μg/mL, respectively. Three-dimensional quantitative structure-activity relationship models were established to direct the following excogitation for exploring higher active drugs. The in vivo study against plant bacterial diseases further identified the prospective application of title compounds as alternative antibacterial agents. The proteomic technique, scanning electron microscopy patterns, and fluorescence spectrometry were exploited to investigate the antibacterial mechanism. Additionally, some target compounds performed superior inhibitory actions against three tested fungal strains. In view of their simple molecular architecture and highly efficient bioactivity, these substrates could be further explored as promising surrogates for fighting against plant microbial infections.

Full text of DOI:10.1021/acs.jafc.9b03952

Subscriber access provided by Miami University Libraries
Agricultural and Environmental Chemistry
Synthesis of Thiazolium-Labeled 1,3,4-Oxadiazole
Thioethers as Prospective Antimicrobials: In Vitro
and In Vivo Bioactivity and Mechanism of Action
Ming-Wei Wang, Huai-He Zhu, Peiyi Wang, Dan Zeng, Yuan-
Yuan Wu, Li-Wei Liu, Zhibing Wu, Zhong Li, and Song Yang
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03952 • Publication Date (Web): 28 Oct 2019
Downloaded from pubs.acs.org on November 2, 2019
Just Accepted
“Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted
online prior to technical editing, formatting for publication and author proofing. The American Chemical
Society provides “Just Accepted” as a service to the research community to expedite the dissemination
of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in
full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully
peer reviewed, but should not be considered the official version of record. They are citable by the
Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore,
the “Just Accepted” Web site may not include all articles that will be published in the journal. After
a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web
site and published as an ASAP article. Note that technical editing may introduce minor changes
to the manuscript text and/or graphics which could affect content, and all legal disclaimers and
ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or
consequences arising from the use of information contained in these “Just Accepted” manuscripts.
is published by the American Chemical Society. 1155 Sixteenth Street N.W.,
Washington, DC 20036
Published by American Chemical Society. Copyright © American Chemical Society.
However, no copyright claim is made to original U.S. Government works, or works
produced by employees of any Commonwealth realm Crown government in the
course of their duties.

Page 1 of 50
Journal of Agricultural and Food Chemistry
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
Synthesis of Thiazolium-Labeled 1,3,4-Oxadiazole Thioethers as Prospective
Antimicrobials: In Vitro and In Vivo Bioactivity and Mechanism of Action
†
a
† a
a
a
a
Ming-Wei Wang , Huai-He Zhu , Pei-Yi Wang* , Dan Zeng , Yuan-Yuan Wu ,
a
a
b
a,b
Li-Wei Liu , Zhi-Bing Wu , Zhong Li , Song Yang*
a
State Key Laboratory Breeding Base of Green Pesticide and Agricultural
Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering,
Ministry of Education, Center for R&D of Fine Chemicals of Guizhou University,
Guiyang, 550025, China.
b
1
1
1
1
1
1
1
College of Pharmacy, East China University of Science & Technology, Shanghai,
China 200237.
* Corresponding author.
E-mail: jhzx.msm@gmail.com (S. Yang), pywang888@126.com (P.-Y. Wang)
†
The two authors contribute equally to this work.
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 50
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
Abstract
In this study, a type of thiazolium-labeled 1,3,4-oxadiazole thioether bridged by
diverse alkyl chain lengths was constructed. The antimicrobial activity of the
fabricated thioether towards plant pathogenic bacteria and fungi was then screened.
Antibacterial evaluation indicated that title compounds possess specific characteristics
that enable it to severely attack three phytopathogens, namely, Xanthomonas oryzae
pv. oryzae, Ralstonia solanacearum, and Xanthomonas axonopodis pv. citri with
minimal EC50 of 0.10, 3.27, and 3.50 µg/mL, respectively. Three-dimensional
quantitative structure–activity relationship models were established to direct the
following excogitation for exploring higher active drugs. The in vivo study against
plant bacterial diseases further identified the prospective application of title
compounds as alternative antibacterial agents. The proteomic technique, scanning
electron microscopy patterns, and fluorescence spectrometry were exploited to
investigate the antibacterial mechanism. Additionally, some target compounds
performed superior inhibitory actions against three tested fungal strains. In view of
their simple molecular architecture and highly efficient bioactivity, these substrates
could be further explored as promising surrogates for fighting against plant microbial
infections.
Keywords
1,3,4-oxadiazole, thiazolium, antimicrobial, 3D-QSAR, proteomics, action
mechanism
2
ACS Paragon Plus Environment

Page 3 of 50
Journal of Agricultural and Food Chemistry
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1. Introduction
Despite the remarkable contribution of commercial antibiotic therapies to agriculture
in the past decade,1-2 microbial infections continue to cause global agricultural
production constraints because of the poor efficiency of existing commercial
antibiotic agents toward invasive phytopathogens and the emergence of a growing
number of multidrug-resistant microorganisms originating from various factors, such
as long-term usage of traditional bactericides bearing a single mode of action.3-7 In
addition, evidence shows that a large-scale outbreak of resistance can rapidly
accelerate within a relatively short period once resistant pathogenic races appear; this
condition further exacerbates the difficulty in managing this serious issue and elevates
the potential risks on human health.8-11 Thus, innovative structures to develop simple
and highly efficient antimicrobial drugs possessing unique modes of actions are better
alternatives to analogues of existing ones for the treatment of plant bacterial or fungal
diseases.
In drug design programs, one of the most efficient and promising approaches to
explore securely bioactive structures is based on existing pharmacological skeletons
from naturally occurring products, which have been verified to have a variety of
advantages, including inartificial structural features, well-balanced physicochemical
property, good biocompatibility, permissible environmental friendliness, low
cytotoxicity toward mammalian cells, and unique modes of action.12-17 Among these
native structural fragments, the thiazolium scaffold has been elaborately explored and
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 50
6
6
6
6
6
6
6
6
6
7
7
7
7
7
7
7
7
7
7
8
8
8
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
highlighted because it is a crucially active component of thiamine (vitamin B ) and
1
thiamin pyrophosphate (ThDP) and serves as a cofactor of certain enzymes or
multi-enzyme complexes, including pyruvate decarboxylase, α-ketoglutarate
dehydrogenase and transketolase, catalyzing several biochemical reactions in all
living organisms; in addition, this favorable building block offers diverse capabilities
in various aspects, such as improving the balance of the physicochemical and
amphiphilic properties of target molecules, allowing decoration with functional
groups, and reforming molecular pharmacological activities.18-20 Thus, comprehensive
investigations into this flexible motif are still being conducted, resulting in an array of
thiazolium-tailored compounds with excellent potential applications (Figure 1). For
instance, furazolium chloride containing a thiazolium moiety was discovered and
developed as a powerful antimicrobial agent against Proteus vulgaris, Pseudomonas
2
1
aeruginosa, Salmonella typhosa, and Staphylococcus aureus. Meanwhile, Kim et
2
2
al. evaluated the antibacterial effects of a type of cephalosporin derivative carrying a
thiazolium motif and found that these designed molecules exhibited potent
bioactivities against Gram-positive and Gram-negative microorganisms except P.
aeruginosa. In addition, three highly bioactive structures bearing bis-thiazolium
patterns exhibited in vitro antimalarial activities against the protozoan parasite
Plasmodium falciparum with IC values of 2.6 (R = -CH OH), 0.7 (R = -CH OCH ),
5
0
2
2
3
and 25.0 nM (R = -CH ); (R = -CH OH) is currently being clinically investigated by
3
2
Sanofi–Aventis and has entered phase II clinical trials on account of the promising
results in vivo study.23-25 Inspired by these results, the integration of a thiazolium
4
ACS Paragon Plus Environment

Page 5 of 50
Journal of Agricultural and Food Chemistry
8
8
8
8
8
8
8
9
9
9
9
9
9
9
9
9
9
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
motif in a target molecule may lead to improved biological activities for its versatile
functions of this valuable moiety.
Meanwhile, 1,3,4-oxadiazole skeleton that acts as another versatile building
block has been extensively studied and developed for its dramatic behavior in
reforming the bioactivity.26-30 It can serve as a desirable surrogate for carboxylic
acids, esters, and amides, which are always accompanied by augmented biological
silhouettes.31-33 A certain amount of designed frameworks owning 1,3,4-oxadiazole
moieties are in the commercialization stage or have been launched into the market.
3
4
35
For example, zibotentan (anticancer agent) and furamizole (antibiotic agent) are in
3
6
37
the late stage of clinical trials, whereas raltegravir and fenadiazole that serve as
antiretroviral and hypnotic drugs, respectively, have been successfully exploited to
manage HIV infection and insomnia. In our previous works, we demonstrated that
1,3,4-oxadiazole thioether/sulfoxide/sulfone derivatives possess superior bioactivities
against plant bacterial diseases; two sulfone candidates (5-(4-fluorophenyl or
2,4-dichlorophenyl)-2-(methylsulfonyl)-1,3,4-oxadiazole) are in the novel pesticide
registration stage.33,38 Such diverse range of applications motivates us to explore and
develop 1,3,4-oxadiazole-labeled derivatives as prospective antimicrobial surrogates.
As another key fragment, the silhouette of 2,4-dichlorophenyl is always
observable in existing pesticide chemicals,39-41 especially for antibiotic structures such
as triarimole, diniconazole, and hexaconazole.42,43 Therefore, the fusion of these
bioactive substructures of thiazolium, 1,3,4-oxadiazole, and 2,4-dichlorophenyl in a
single molecule may probably promote the discovery of excellent substrates as
1
00
01
02
03
04
1
1
1
1
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 50
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
antimicrobial indicators against plant microbial diseases (Figure 2). In our previous
works, we have evaluated the antibacterial functions of various pyridinium-tailored
derivatives.44,45 However, pyridinium-tailored derivatives displayed significant
phytotoxicity (Figure S1a-S1b, supporting information). To explore novel frameworks
with low phytotoxicity and highly efficient antimicrobial ability, herein, a series of
5-(2,4-dichlorophenyl)-1,3,4-oxadiazole thioethers bearing natural thiazolium
scaffolds linked by different alkyl chain lengths46,47 was initially fabricated. The
antimicrobial effects of these thioethers against three invasive and widespread
phytopathogens, namely, Xanthomonas oryzae pv. oryzae (Xoo), Ralstonia
solanacearum (R. solanacearum), and Xanthomonas axonopodis pv. citri (Xac); as
well as three virulent phytopathogenic fungi, namely, including Gibberella zeae (G.
zeae), Fusarium oxysporum (F. oxysporum), and Phytophthora cinnamomi (P.
cinnamomi), were evaluated. Subsequently, the substituents at the 5-position of
1,3,4-oxadiazole toward bioactivity were also investigated. On the basis of the
obtained molecular structures and collected bioassay data, three-dimensional
quantitative structure–activity relationship (3D-QSAR) models were established to
direct the following excogitation for exploring higher active drugs. An in vivo study
against plant bacterial diseases was performed to identify the prospective application
of these compounds as alternative antibacterial agents. Meanwhile, the proteomic
technique, scanning electron microscopy (SEM), and fluorescence spectrometry were
exploited to investigate the antimicrobial mechanism of the thioethers.
2. Materials and methods
6
ACS Paragon Plus Environment

Page 7 of 50
Journal of Agricultural and Food Chemistry
1
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
2.1 Instruments and Chemicals
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
Instruments: NMR, JEOL-ECX-500 and Bruker Biospin AG-400 apparatuses;
Centrifuge, Centrifuge 5424 R, eppendorf; spectrometer (UV-Vis), Fluoromax-4
Spectrofluorometer, HORIBA Scientific; High resolution mass spectrometer,
UItiMate 3000, Thermo SCIENTIFIC; Melting point apparatus, SGW® X-4B,
Shanghai Yidian Physical Optical Instrument Co., Ltd; High performance liquid
chromatography (HPLC), 1290 Infinity Ⅱ, Agilent Technologies; Scanning electron
microscope, FEI Nova NanoSEM 450. All the chemicals (≥98%) used for reaction
were purchased from Energy Chemical of Saen Chemical Technology (Shanghai) Co.,
Ltd. The solvents including Ethyl acetate (99.5%), Dichloromethane (99.5%),
Methanol (99.5%), Petroleum ether (99.5%), N,N-Dimethylformamide (99.5%) and
Acetonitrile (AR) were purchased from Tianjin Fuyu Chemical Co., Ltd; Glucose (≥
99.5%) and Beef Extract (Reagent Grade) were purchased from Sangon Biotech
(Shanghai) Co., Ltd; Peptone (test reagent LR) was purchased from Shanghai
Bio-way technology Co., Ltd; Yeast extract (AR) was purchased from Beijing
Solarbio Science & Technology Co., Ltd.
2.2 Experimental section
In vitro and in vivo antibacterial testing, in vitro antifungal testing, label-free
quantitative proteomics analysis, PI uptake assay, scanning electron microscopy
(SEM), synthesis for intermediates and title compounds, see supplementary data.
3. Results and Discussion
To explore the integration of key fragments of thiazolium, 1,3,4-oxadiazole, and
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 50
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
69
70
2,4-dichlorophenyl toward bioactivity, we initially constructed thiazolium-tailored
5-(2,4-dichlorophenyl)-1,3,4-oxadiazole thioethers bridged by diverse alkyl chain
lengths (Figure 3). Intermediate 4 owning a 1,3,4-oxadiazole pattern was obatined as
2
described in our previous approach, which was carried out via two-step reactions
with dibromo-substituted alkyls and thiazole (or 4-methylthiazole) to obtain the target
compounds A and B . The achieved molecules were screened to test their
n
n
antibacterial potency against Xoo, R. solanacearum, and Xac through a turbidimeter
4
8
test. The agricultural antibiotic agents thiodiazole copper (TC) and bismerthiazol
(BT) were co-assayed as reference drugs. The screening results (Table 1) revealed
that these reconstructed compounds displayed good to excellent antibacterial
efficiency against the three tested strains and were relatively superior to BT and TC.
The title compounds exerted selectivity and specificity against Xoo with EC within
5
0
4.84 µg/mL to 0.23 µg/mL. Notably, the antibiotic competence increased from A to
6
A and B to B with prolonging alkyl chain lengths, thereby suggesting that the
1
2
6
12
increment of molecular hydrophobicity was beneficial to the bioactivity. In
comparison with the substituent group and its thiazolium pattern toward bioactivity,
the methyl group displayed a slightly ameliorative tendency, as illustrated by A (4.84
6
µg/mL) < B (3.12 µg/mL), A (3.04 µg/mL) < B (1.87 µg/mL), and A (0.24
6
8
8
12
µg/mL) < B12 (0.23 µg/mL). For anti-R. solanacearum and anti-Xac activities, a
plausible posture was observed: the inhibition effect initially increased and then
decreased with the fine-tuning of the length of alkyl tailors. Thus, compounds A8
(25.51 and 4.78 µg/mL) and B (29.80 and 3.80 µg/mL) exerted the most laudable
8
8
ACS Paragon Plus Environment

Page 9 of 50
Journal of Agricultural and Food Chemistry
1
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
91
92
antibacterial performance. These findings indicate that compounds A12 and B12
exhibited exclusive and promising potency against Xoo. Therefore, further
optimization and manipulation of the molecular structures were carried out.
As compounds A and B showed excellent potential as antibacterial indicators,
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
12
the substituents at 5-postion of 1,3,4-oxadiazole toward bioactivity were investigated.
Thiazolium-tailored 1,3,4-oxadiazole thioethers with various substitutional units at the
5-position and a providential dodecyl tail were synthesized accordingly (Figure 4). In
general, these target compounds C and D (n = 1–10) were fabricated following the
n
n
synthetic
protocols
of
A ,
n
in
which
the
key
intermediate
5-(2,4-dichlorophenyl)-1,3,4-oxadiazole-2-thiol was changed into an array of
5-substituted-1,3,4-oxadiazole-2-thiol. The obtained structures were characterized by
NMR, HRMS, and HPLC (supporting information). The crystal structures of
compound D further confirmed the accurate molecular frameworks (Figure 5). A
7
squint in the screening result (Table 2) suggested that this type of compound did
possess specific and selective competences for severely attacking pathogen Xoo and
provided EC within 0.10 µg/mL to 1.68 µg/mL for compounds C and D (n = 1–
5
0
n
n
10), indicating that the integration of these valuable building blocks could promote
the discovery of highly efficient surrogates. The antibacterial efficacy (for compounds
C and D ) was almost maintained after removing the 4-Cl from compounds A and
1
1
12
B , whereas the antibiotic capacity (for compounds C and D ) declined by twofold
1
2
2
2
after the replacement of 2,4-diCl into 4-Cl. Hence, 4-Cl on the benzene ring was
slightly negative toward anti-Xoo activity. Further breaking the molecular rigidity and
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 50
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
13
14
introducing a methylene group between the benzene motif and 1,3,4-oxadiazole (for
compounds C and D ) still did not improve the inhibitory effect in comparison with
3
3
that for compounds C and D . The electron-withdrawing groups had an insignificant
2
2
influence on bioactivity, as indicated by comparing EC values of D (4-CF , 0.33
5
0
5
3
µg/mL) and D (4-NO , 0.34 µg/mL). Ameliorative antibacterial ability was observed
6
2
by introducing a methyl group at the 2-position of benzene ring and led to EC of
5
0
0.15 and 0.13 µg/mL for compounds D and C , respectively. A superior antibacterial
8
8
candidate (C ) bearing 4-fluoro-3-methylphenyl at 5-position of 1,3,4-oxadiazole
1
0
was discovered with EC50 of 0.10 µg/mL. For anti-R. solanacearum activity, the
antibiotic potency was dramatically elevated by approximately ninefold after
changing 2,4-diCl and substituting it into 2-Cl on the benzene ring. Thus, the EC50
values varied from 79.71 µg/mL (A ) to 8.45 µg/mL (C ) and from 31.28 µg/mL (B )
1
2
1
12
to 3.27 µg/mL (D ). This result suggested that 4-Cl on the benzene ring might block
1
other interactions with bacterial receptors. An opposite pattern for the inhibitory effect
was observed after shifting the chlorine atom to the 4-position of the benzene ring (C2,
21.72 µg/mL; D , 11.37 µg/mL). Such observation revealed that the location of the
2
substituents had a significant action toward bioactivity. Notably, the pharmaceutical
effect was gravely knocked down after inserting a methylene group (C and D ) to
3
3
break the planar pattern of the benzene ring and 1,3,4-oxadiazole core in comparison
with those of compounds C and D . Compound C bearing a 4-CF group (39.94
2
2
5
3
µg/mL) showed better activity than that of compound C owning a 4-NO group
6
2
(85.27 µg/mL). However, opposite results were obtained for compounds D and D6
5
1
0
ACS Paragon Plus Environment

Page 11 of 50
Journal of Agricultural and Food Chemistry
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
15
with EC50 of 43.89 and 7.59 µg/mL, respectively. Hence, the type of
electron-withdrawing groups also played a crucial role in judging the final activity of
a molecule. Compounds D –D possessing electron-donating groups (2-CH , 3-OCH ,
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
8
10
3
3
4-F-3-CH ) presented agreeable powers against R. solanacearum with EC of 6.66,
3
50
7.39, and 6.38 µg/mL, respectively. As for the screening results, compound Cn
bearing the thiazolium moiety exerted lower antibacterial performance than
compound Dn owning 4-methylthiazol-3-ium parts; however, compound C5
performed slightly better than D . In terms of anti-Xac activity, all compounds exerted
5
acceptable activities with EC within 3.50 µg/mL to 17.21 µg/mL. Compound D
7
5
0
without any substitutional group on the benzene ring displayed the strongest bacterial
growth inhibition effect. This type of compound clearly possessed comprehensive
inhibitory actions against the tested bacterial strains, particularly for the pathogen Xoo.
Hence, the fusion of these favorable fragments of thiazolium and 1,3,4-oxadiazole in
a molecule could yield highly efficient antimicrobial alternatives.
As these compounds demonstrated powerful antibacterial potentials and
promising applications for developing anti-Xoo drugs, 3D-QSAR models employing
comparative molecular field analysis (CoMFA) and comparative molecular similarity
index analysis (CoMSIA) were established to elaborately expound the structure–
activity relationship and direct the future excogitation of the molecular structure. By
using SYBYL-X 2.0 software, we systematically assembled the 3D structures of 28
molecules with the “Sketch Molecule” function and then aligned them on the basis of
the common substructure and conformation of compound C , which exhibited the
1
0
1
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 50
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
greatest antibacterial potency toward Xoo. The alignment pattern is shown in Figure 6.
To fabricate the 3D-QSAR models, we denoted the antibacterial activity of all the
target compounds as pEC (Table 3). Twenty of the target molecules were selected as
5
0
the training set for CoMFA and CoMSIA, while the left molecules were employed as
the testing set. The predicted pEC of all the molecules are listed in Table 3. The
5
0
predicted results were similar to the corresponding experimental data. The
observation of linear correlations for the experimental and predicted pEC in CoMFA
5
0
and CoMSIA models manifested the credibly predictive competence of these models
(Figure 7). The obtained statistical parameters are illustrated in Table 4. The internal
2
validations of the cross-validated q value (> 0.5) and non-cross-validated coefficient
r2 value (> 0.8) are normally considered as a reference for the predictive capacity of
2
2
3D-QSAR models. The corresponding q and r values for the CoMFA and CoMSIA
models were (0.766, 0.951) and (0.792, 0.976), respectively, thereby satisfying the
performance demands. Moreover, the related standard errors of estimate (SEEs) and
F-values were (0.123, 54.632) and (0.098, 54.786), respectively. In the CoMFA
model, the contributions for steric and electrostatic fields were 59.0 and 41.0%,
respectively, indicating that the bioactivity slightly depended on the steric
interactions. In the CoMSIA model, the steric, electrostatic, hydrophobic, H-bond
donor, and H-bond acceptor contributions were 13.5%, 52.7%, 33.3%, 0.0%, and
0.5%, respectively, indicating that electrostatic interactions contributed significantly
to the anti-Xoo activity. The fabricated contour maps for the CoMFA models were
dispalyed in Figure 8. The yellow contours around the benzene ring and thiazolium
1
2
ACS Paragon Plus Environment

Page 13 of 50
Journal of Agricultural and Food Chemistry
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
59
scaffold in the CoMFA steric field suggested that the large groups at these positions
were unfavorable for the bioactivity (Figure 8A), illustrated by comparing EC of C
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
79
80
5
0
4
(4-F, 0.28 µg/mL) and C (4-CF , 0.39 µg/mL), C (thiazolium, 0.15 µg/mL) and D
5
3
9
9
(4-methylthiazol-3-ium, 0.45 µg/mL) bearing the large groups for the latters,
respectively. For the CoMFA electrostatic field (Figure 8B), red polyhedra with
electron-donating groups increased the activity, displaying by the screening results of
compounds C (3-OCH , 0.15 µg/mL), C (H, 0.44 µg/mL), D (3-OCH , 0.45
9
3
7
9
3
µg/mL), and D7 (H, 1.68 µg/mL). By contrast, the blue pattern with
electron-withdrawing groups at this area enhanced the bioactivity in accordance with
the observation of a blue color surrounding the thiazolium skeleton. Thus, this
fragment was indeed devoted to the bioactivity. The CoMSIA contour maps are
presented in Figure 9. The steric field pattern (Figure 9A) revealed that the bulky
groups at the 3-position of the benzene ring located at the green region could improve
the antibacterial power, as revealed by comparing EC50 of C10 (4-F-3-CH , 0.10
3
µg/mL) and C (4-F, 0.28 µg/mL). For the electrostatic field map (Figure 9B), the
4
blue area suggested that the introduction of electron-withdrawing groups improved
the bioactivity, which further verified the strong electron-withdrawing nature of the
thiazolium scaffolds powerfully contributing to the antibacterial competence. On the
contrary, the red portion located at the 2-position indicated that an electron-donating
group was preferred for the bioactivity, as verified from the bioassay result of
compounds C (2-CH , 0.15 µg/mL) and C (2-Cl, 0.20 µg/mL). The hydrophobic
8
3
1
field pattern (Figure 9C) showed that integrating the hydrophobic group at the yellow
1
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 50
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
2
3
3
3
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
01
02
region or the hydrophilic group at the gray contour would enhance the bioactivity. A
red pattern was observed from the H-bond acceptor field map (Figure 9D), and it
suggested that the reception of hydrogen bonding in this area could ameliorate the
antibacterial efficiency. Given the study’s results and structural insights, this model
provides an insight for the rational design of fresh bioactive compounds as
antibacterial surrogates.
In vivo trials against bacterial blight on rice were performed to identify the
prospective application of the developed compounds as agricultural agents. As
illustrated in Table 5, Figure 10 and Figure S1c-S1d, the highly active compound C10
presented excellent in vivo curative activity with low phytotoxicity, providing a
control efficiency of 48.5% at 200 μg/mL; these results were relatively superior to BT
(32.8%) and TC (39.2%). This compound’s protection effect was also remarkable and
provided a relevant control rate of 51.5%. This outcome manifested that the designed
molecules were capable of fighting against plant bacterial diseases.
To understand the regulatory role and antibacterial mechanism triggered by the
thiazolium-labeled 1,3,4-oxadiazole thioethers, we derived a label-free quantitative
proteomic profile by treating Xoo with hyperactive compound C . The quality of the
1
0
obtained result was assessed and confirmed from Figure S2. Meanwhile, the enriched
peptides were monitored by LC–MS/MS and were subsequently analyzed and
4
9
quantified. As a result, 2353 proteins were detected in the control and treatment
samples, with 2107 proteins (89.5%) holding quantitative information (MS-identified
information and Table S1). At the filtering conditions of fold changes >1.5, p<0.05,
1
4
ACS Paragon Plus Environment

Page 15 of 50
Journal of Agricultural and Food Chemistry
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
03
314 proteins were differentially expressed in response to C stimulation. Among the
1
0
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
314 proteins, 161 and 153 proteins were upregulated and downregulated, respectively
(Figures 11a and 11b). GO categories divided into BP, CC, and MF were used to
clarify the biological functions.50-52 As shown in Figure 12, the BP analysis suggested
that proteins differently expressed were associated with single-organism process,
metabolic process, cellular process, localization, biological regulation, signaling,
response to stimulus, cellular component organization or biogenesis, and locomotion.
The proteins involved in the single-organism process, cellular process, and metabolic
process were significantly enriched (Figure 12a). In the CC category (Figure 12b),
proteins experiencing great changes were located in the macromolecular complex,
cell, membrane, organelle, extracellular region, virion, and nucleoid. In the MF
analysis (Figure 12c), varying levels of proteins involved in molecular transducer
activity, catalytic activity, binding, structural molecular activity, and antioxidant
activity were dramatically expressed. In addition, these changed proteins mainly
located in the cytoplasm from the subcellular structure location pattern (Figure S3).
This result showed that C could regulate and disturb many aspects of physiological
1
0
processes and functions of pathogen Xoo.
To elucidate the bacterial pathways affected by compound C , we performed a
1
0
KEGG analysis. The result showed that “ribosome,” “biotin metabolism,”
“cyanoamino acid metabolism,” “other glycan degradation,” and “RNA polymerase
biogenesis” were the distinct pathways enriched. In particular, “ribosome,” which
plays a significant role in protein translation, was the most prominent pathway
1
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 50
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
involving a considerable amount of these differentially expressed proteins (Figure
S4).53-55 As illustrated in Figure S5, a variety of prominent proteins were
downregulated, and they included the large subunit ribosomal protein L1 (rp1A),
protein L4 (rp1D), protein L5 (rp1E), protein L6 (rp1F), protein L10 (rp1J), protein
L11 (rp1K), protein L17 (rp1Q), and protein L27 (rpmA); as well as the small subunit
ribosomal protein S1 (rpsA), protein S5 (rpsE), and protein S13 (rpsM). These
proteins were significant components of the ribosome participating in various actions
in the protein translation program. However, their distinct downregulated expression
triggered by compound C10 would significantly disrupt the pathways of ribosome
assembly and the subsequent synthesis of a mass of functional proteins that are
essential for normal life, finally leading to bacterial death. To further understand the
significance and extent of these differentially expressed proteins in the ribosome
pathway, we generated a protein–protein interaction network using STRING database
version 10.5. As shown in Figure S6, the formed interaction relationship containing
11 down-regulated proteins indicated that these proteins serving as significant factors
could affect one another and consequently regulate and disorganize various
physiological processes triggered by C stimulation.
1
0
To study the morphological changes before and after treatment of Xoo with
compound C , we obtained SEM images via a concentration-dependent manner. The
1
0
Xoo morphology was switched from well-shaped (Figure 13a) to partially broken or
distorted after treatment of Xoo with C (10 µg/mL) (Figure 13b). Further elevating
1
0
the drug dose to 25 µg/mL resulted in completely broken bacteria with large leakage
1
6
ACS Paragon Plus Environment

Page 17 of 50
Journal of Agricultural and Food Chemistry
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
47
holes (Figure 13c). This outcome indicated that thiazolium-labeled 1,3,4-oxadiazole
thioethers possessed the special ability to damage the cell membrane by the
synergistic effects of hydrophilic and hydrophobic parts. To examine the changes of
membrane permeability, we used a typical dye propidium iodide (PI) without
fluorescence. This dye can provide significant red fluorescence after binding with
DNA, but it cannot thread the intact membrane of a living pathogen.10,56 As shown in
Figure 13d, an enhanced fluorescent intensity at 598 nm was observed with increasing
drug dosages of C , thereby demonstrating that bacterial membrane permeability
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
67
68
1
0
gradually increased and formed a PI-DNA complex to create fluorescence. This
outcome was in accordance with the observed SEM images. The results of
quantitative proteomics, SEM images, and fluorescence spectrum confirmed that title
molecules significantly affected the diverse physiological processes of pathogens and
consequently resulted in bacterial death.
The antifungal ability of the target molecules against an array of fungal strains,
namely, G. zeae, F. oxysporum, and P. cinnamomic, was examined via the poison
plate technique.57,58 Meanwhile, the agricultural antifungal agents vitavax (VT),
prochloraz (PC), hymexazol (HM), and carbendazim (CB) were used as the reference
drugs. The preliminary screening results (Table 6) revealed that most target
compounds exhibited superior potency against three fungal strains relative to VT.
Moreover, some of them exhibited better toxic effects than HM at 50 µg/mL. All title
molecules exhibited moderate activity against G. zeae with the inhibition rates within
38.8%-57.9%, which were lower than that for HM (61.8%). A scene for inhibition
1
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 50
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
89
90
rates against F. oxysporum indicated that most of the title compounds performed
admirable growth suppression effects in comparison with HM (56.4%) and VT
(15.7%). Specifically, compounds A , A , B , B , and D afforded excellent
8
12
8
12
10
inhibition values of 80.5%, 73.1%, 72.1%, 77.8%, and 71.2%, respectively. For
anti-P. cinnamomi activity, compounds A , A , B , B , B , C , C , C , C , D , D ,
8
10
8
10
12
3
7
8
10
6
7
and D exerted powerful antibiotic performance in comparison with HM (50.5%),
1
0
with the inhibition rates exceeding 61.1%; D , in particular, afforded the highest rate
1
0
of 71.6%. These investigations indicated that these compounds could be considered as
novel compounds in the exploration of antifungal drugs.
In summary, a class of thiazolium-labeled 1,3,4-oxadiazole thioethers was
constructed. Antibacterial results showed that compounds C , D , and D could
1
0
1
7
severely attack three tested phytopathogens, including Xoo, R. solanacearum and Xac,
with EC50 of 0.10, 3.27, and 3.50 µg/mL, respectively. Subsequently, 3D-QSAR
models against Xoo were established to direct the following excogitation for
discovering higher active drugs. In vivo study showed that C presented excellent
1
0
curative and protective activities (48.5% and 51.5%, 200 µg/mL) against bacterial
blight. Quantitative proteomic profiles indicated that 314 proteins (161 and 153
proteins were upregulated and downregulated, respectively) were observed to be
differentially expressed in response to C stimulation, suggesting that this kind of
1
0
compounds could regulate and disturb many aspects of physiological processes and
functions of pathogens. Scanning electron microscopy images and fluorescence
spectrum further confirmed this outcome. Moreover, some target compounds
1
8
ACS Paragon Plus Environment

Page 19 of 50
Journal of Agricultural and Food Chemistry
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
4
4
4
91
performed superior inhibitory actions against three tested fungal strains. Given their
simple molecular architecture and highly efficient bioactivity, these substrates could
be further developed as promising surrogates for fighting against microbial infections.
Supporting Information
92
93
94
95
96
97
98
99
00
01
02
03
04
05
06
07
08
09
10
11
12
Supporting Information including detailed experimental section, Tabel S1, Figures
1
13
S1-S6, experimental characterization data of title compounds, H NMR, C NMR,
19F NMR, HRMS spectra and HPLC analysis associated with this article can be
found, in the online version.
Funding Sources
We thank funds from China’s NNSF (21877021, 21702037, 31860516, 21662009),
R.P. Ministry of Education (20135201110005, 213033A), and Guizhou PS&TP (LH
[2017]7259, [2012]6012, [2017]5788).
Conflict of interest
The authors declare no competing financial interest.
References
(1) Morel, C.; Stermitz, F. R.; Tegos, G.; Lewis, K. Isoflavones as potentiators of
antibacterial activity. J. Agric. Food Chem. 2003, 51, 5677-5679.
(2) Wang, P. Y.; Chen, L.; Zhou, J.; Fang, H. S.; Wu, Z. B.; Song, B. A.; Yang,
S. Synthesis and bioactivities of 1-aryl-4-hydroxy-1H-pyrrol-2(5H)-one derivatives
bearing 1,3,4-oxadiazole moiety. J. Saudi Chem. Soc. 2017, 21, 315-323.
(3) Levy, S. B.; Marshall, B. Antibacterial resistance worldwide: causes,
challenges and responses. Nat. Med. 2004, 10, S122-S129.
1
9
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 50
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
(4) Hsieh, P. C.; Siegel, S. A.; Rogers, B.; Davis, D.; Lewis, K. Bacteria lacking
a multidrug pump: A sensitive tool for drug discovery. Proc. Nat. Acad. Sci. USA
1998, 95, 6602-6606.
(5) Li, P.; Hu, D. Y.; Xie, D. D.; Chen, J. X.; Jin, L. H.; Song, B. A. Design,
synthesis, and evaluation of new sulfone derivatives containing a 1,3,4-oxadiazole
moiety as active antibacterial agents. J. Agric. Food Chem. 2018, 66, 3093-3100.
(6) Tejero, R.; Gutierrez, B.; Lopez, D.; Lopez-Fabal, F.; Gomez-Garces, J. L.;
Fernandez-Garcia, M. Copolymers of acrylonitrile with quaternizable thiazole and
triazole side-chain methacrylates as potent antimicrobial and hemocompatible
systems. Acta Biomater. 2015, 25, 86-96.
(7) Davies, J.; Davies, D. Origins and evolution of antibiotic resistance.
Microbiol. Mol. Biol. Rev. 2010, 74, 417-433.
(8) Dai, X. M.; Zhao, Y.; Li, J. S.; Li, S.; Lei, R. D.; Chen, X. L.; Zhang, X. E.;
Li, C. X. Thiazolium-derivative functionalized silver nanocomposites for suppressing
bacterial resistance and eradicating biofilms. New J. Chem. 2018, 42, 1316-1325.
(9) Li, P.; Tian, P. Y.; Chen, Y. Z.; Song, X. P.; Xue, W.; Jin, L. H.; Hu, D. Y.;
Yang, S.; Song, B. A. Novel bisthioether derivatives containing a 1,3,4-oxadiazole
moiety: design, synthesis, antibacterial and nematocidal activities. Pest Manag. Sci.
2018, 74, 844-852.
(10)Zhou, J.; Tao, Q. Q.; Wang, P. Y.; Shao, W. B.; Wu, Z. B.; Li, Z.; Yang, S.
Antimicrobial evaluation and action mechanism of pyridinium-decorated
1,4-pentadien-3-one derivatives. Bioorg. Med. Chem. Lett. 2018, 28, 1742-1746.
2
0
ACS Paragon Plus Environment

Page 21 of 50
Journal of Agricultural and Food Chemistry
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
35
(11)He, H. F.; Wang, W.; Zhou, Y.; Xia, Q.; Ren, Y. L.; Feng, J. T.; Peng, H.; He,
H. W.; Feng, L. L. Rational design, synthesis and biological evaluation of
1,3,4-oxadiazole pyrimidine derivatives as novel pyruvate dehydrogenase complex E1
inhibitors. Bioorg. Med. Chem. 2016, 24, 1879-1888.
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
(12)Kim, M. K.; Choi, G. J.; Lee, H. S. Fungicidal property of Curcuma longa L.
rhizome-derived curcumin against phytopathogenic fungi in a greenhouse. J. Agric.
Food Chem. 2003, 51, 1578-1581.
(13)Widsten, P.; Cruz, C. D.; Fletcher, G. C.; Pajak, M. A.; McGhie, T. K.
Tannins and Extracts of Fruit Byproducts: Antibacterial Activity against Foodborne
Bacteria and Antioxidant Capacity. J. Agric. Food Chem. 2014, 62, 11146-11156.
(14)Rodrigues, T.; Reker, D.; Schneider, P.; Schneider, G. Counting on natural
products for drug design. Nat. Chem. 2016, 8, 531-541.
(15)Li, H. N.; Sun, B. J.; Wang, M. Y.; Hu, X.; Gao, X.; Xu, S. T.; Xu, Y. N.; Xu,
J. Y.; Hua, H. M.; Li, D. H. Bioactive enmein-type 6,7-seco-ent-kaurane diterpenoids:
natural products, synthetic derivatives and apoptosis related mechanism. Arch.
Pharm. Res. 2018, 41, 1051-1061.
(16)Newman, D. J.; Cragg, G. M. Natural products as sources of new drugs from
1981 to 2014. J. Nat. Prod. 2016, 79, 629-661.
(17)Bauer, A.; Bronstrupt, M. Industrial natural product chemistry for drug
discovery and development. Nat. Prod. Rep. 2014, 31, 35-60.
(18)Zhou, Y.; Zhang, S. S.; He, H. W.; Jiang, W.; Hou, L. F.; Xie, D.; Cai, M.;
Peng, H.; Feng, L. L. Design and synthesis of highly selective pyruvate
2
1
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 50
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
57
58
59
60
61
62
63
64
65
66
67
68
69
70
71
72
73
74
75
76
77
78
dehydrogenase complex E1 inhibitors as bactericides. Bioorg. Med. Chem. 2018, 26,
84-95.
(19)Arjunan, P.; Sax, M.; Brunskill, A.; Chandrasekhar, K.; Nemeria, N.; Zhang,
S.; Jordan, F.; Furey, W. A thiamin-bound, pre-decarboxylation reaction intermediate
analogue in the pyruvate dehydrogenase E1 subunit induces large scale
disorder-to-order transformations in the enzyme and reveals novel structural features
in the covalently bound adduct. J. Biol. Chem. 2006, 281, 15296-15303.
(20)Klaus, A.; Pfirrmann, T.; Glomb, M. A. Transketolase A from E-coli
Significantly Suppresses Protein Glycation by Glycolaldehyde and Glyoxal in Vitro.
J. Agric. Food Chem. 2017, 65, 8196-8202.
(21)Snyder, H. R. J.; Benjamin, L. E. Nitrofuryl heterocytes. IX. Some
derivatives
and
analogs
of
6,7-dihydro-3-(5-nitro-2-furyl)-5H-imidazo[2,1-b]thiazolium chloride. J. Med. Chem.
1970, 13, 164-165.
(22)Kim, S. H.; Son, H.; Nam, G.; Chi, D. Y.; Kim, J. H. Synthesis and in vitro
antibacterial activity of 3-[N-methyl-N-(3-methyl-1,3-thiazolium-2-yl)aminolmethyl
cephalosporin derivatives. Bioorg. Med. Chem. Lett. 2000, 10, 1143-1145.
(23)Ben Mamoun, C.; Prigge, S. T.; Vial, H. Targeting the Lipid Metabolic
Pathways for the Treatment of Malaria. Drug Dev. Res. 2010, 71, 44-55.
(24)Hamze, A.; Rubi, E.; Arnal, P.; Boisbrun, M.; Carcel, C.; Salom-Roig, X.;
Maynadier, M.; Wein, S.; Vial, H.; Calas, M. Mono- and bis-thiazolium salts have
potent antimalarial activity. J. Med. Chem. 2005, 48, 3639-3643.
2
2
ACS Paragon Plus Environment

Page 23 of 50
Journal of Agricultural and Food Chemistry
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
5
79
(25)Nicolas, O.; Margout, D.; Taudon, N.; Wein, S.; Calas, M.; Vial, H. J.;
Bressolle, F. M. M. Pharmacological properties of a new antimalarial bisthiazolium
salt, T3, and a corresponding prodrug, TE3. Antimicrob. Agents Ch. 2005, 49,
3631-3639.
80
81
82
83
84
85
86
87
88
89
90
91
92
93
94
95
96
97
98
99
00
(26)Cui, Z. N.; Shi, Y. X.; Zhang, L.; Ling, Y.; Li, B. J.; Nishida, Y.; Yang, X. L.
Synthesis and fungicidal activity of novel 2,5-disubstituted-1,3,4-oxadiazole
derivatives. J. Agric. Food Chem. 2012, 60, 11649-11656.
(27)Behalo, M. S. An efficient one-pot catalyzed synthesis of
2,5-disubstituted-1,3,4-oxadiazoles and evaluation of their antimicrobial activities.
RSC Adv. 2016, 6, 103132-103136.
(28)Wet-osot, S.; Phakhodee, W.; Pattarawarapan, M. Application of
N-acylbenzotriazoles in the synthesis of 5-substituted 2-ethoxy-1,3,4-oxadiazoles as
building blocks toward 3,5-disubstituted 1,3,4-oxadiazol-2(3H)-ones. J. Org. Chem.
2017, 82, 9923-9929.
(29)Tao, Q. Q.; Liu, L. W.; Wang, P. Y.; Long, Q. S.; Zhao, Y. L.; Jin, L. H.; Xu,
W. M.; Chen, Y.; Li, Z.; Yang, S. Synthesis and In Vitro and In Vivo Biological
Activity Evaluation and Quantitative Proteome Profiling of Oxadiazoles Bearing
Flexible Heterocyclic Patterns. J. Agric. Food Chem. 2019, 67, 7626-7639.
(30)Du, Q. R.; Li, D. D.; Pi, Y. Z.; Li, J. R.; Sun, J.; Fang, F.; Zhong, W. Q.;
Gong, H. B.; Zhu, H. L. Novel 1,3,4-oxadiazole thioether derivatives targeting
thymidylate synthase as dual anticancer/antimicrobial agents. Bioorg. Med. Chem.
2013, 21, 2286-2297.
2
3
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 50
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
(31)Desai, N. C.; Kotadiya, G. M.; Trivedi, A. R.; Khedkar, V. M.; Jha, P. C.
Design, synthesis, and biological evaluation of novel fluorinated pyrazole
encompassing pyridyl 1,3,4-oxadiazole motifs. Med. Chem. Res. 2016, 25,
2698-2717.
(32)Manjunatha, K.; Poojary, B.; Lobo, P. L.; Fernandes, J.; Kumari, N. S.
Synthesis and biological evaluation of some 1,3,4-oxadiazole derivatives. Eur. J.
Med. Chem. 2010, 45, 5225-5233.
(33)Xu, W. M.; Han, F. F.; He, M.; Hu, D. Y.; He, J.; Yang, S.; Song, B. A.
Inhibition of tobacco bacterial wilt with sulfone derivatives containing an
1,3,4-oxadiazole moiety. J. Agric. Food Chem. 2012, 60, 1036-1041.
(34)Fizazi, K. S.; Higano, C. S.; Nelson, J. B.; Gleave, M.; Miller, K.; Morris, T.;
Nathan, F. E.; McIntosh, S.; Pemberton, K.; Moul, J. W. Phase III, randomized,
placebo-controlled study of docetaxel in combination with zibotentan in patients with
metastatic castration-resistant prostate cancer. J. Clin. Oncol. 2013, 31, 1740-1748.
(35)Khan, P. R.; Durgaprasad, M.; Reddy, S. G.; Reddy, G. R.; Hussein, I. A.;
Reddy, B. V. S. IBX/KI promoted synthesis of 2,5-disubstituted 1,3,4-oxadiazoles.
Lett. Org. Chem. 2018, 15, 64-69.
(36)McMahon, D.; Jones, J.; Wiegand, A.; Gange, S. J.; Kearney, M.; Palmer, S.;
McNulty, S.; Metcalf, J. A.; Acosta, E.; Rehm, C.; Coffin, J. M.; Mellors, J. W.;
Maldarelli, F. Short-course raltegravir intensification does not reduce persistent
low-level viremia in patients with HIV-1 suppression during receipt of combination
antiretroviral therapy. Clin. Infect. Dis. 2010, 50, 912-919.
2
4
ACS Paragon Plus Environment

Page 25 of 50
Journal of Agricultural and Food Chemistry
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
23
(37)Sarshira, E. M.; Hamada, N. M.; Moghazi, Y. M.; Abdelrahman, M. M.
Synthesis and biological evaluation of some heterocyclic compounds from salicylic
acid hydrazide[1]. J. Heterocylic Chem. 2016, 53, 1970-1982.
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
(38)Li, P.; Yin, J.; Xu, W. M.; Wu, J.; He, M.; Hu, D. Y.; Yang, S.; Song, B. A.
Synthesis, Antibacterial Activities, and 3D-QSAR of Sulfone Derivatives Containing
1,3,4-Oxadiazole Moiety. Chem. Biol. Drug Des. 2013, 82, 546–556.
(39)Arnoldi, A.; Carzaniga, R.; Morini, G.; Merlini, L.; Farina, G. Synthesis,
fungicidal activity, and QSAR of a series of 2-dichlorophenyl-3-triazolylpropyl
ethers. J. Agric. Food Chem. 2000, 48, 2547-2555.
(40)Friggeri, L.; Hargrove, T. Y.; Wawrzak, Z.; Blobaum, A. L.; Rachakonda,
G.; Lindsley, C. W.; Villalta, F.; Nes, W. D.; Botta, M.; Guengerich, F. P.;
Lepesheva, G. I. Sterol 14 alpha-Demethylase Structure-Based Design of VNI
((R)-N-(1-(2,4-Dichlorophenyl)-2-(1H-imidazol-1-yl)ethyl)-4-(5-phenyl-1,3,4-oxadia
zol-2-yl)benzamide)) Derivatives To Target Fungal Infections: Synthesis, Biological
Evaluation, and Crystallographic Analysis. J. Med. Chem. 2018, 61, 5679-5691.
(41)Arnoldi, A.; Dallavalle, S.; Merlini, L.; Musso, L.; Farina, G.; Moretti, M.;
Jayasinghe, L. Synthesis and antifungal activity of a series of N-substituted
[2-(2,4-dichlorophenyl)-3-(1,2,4-triazol-1-yl)]propylamines. J. Agric. Food Chem.
2007, 55, 8187-8192.
(42)Amjadi, M.; Jalili, R. Molecularly imprinted mesoporous silica embedded
with carbon dots and semiconductor quantum dots as a ratiometric fluorescent sensor
for diniconazole. Biosens. Bioelectron. 2017, 96, 121-126.
2
5
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 50
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
(43)Mustafa, I. F.; Hussein, M. Z.; Saifullah, B.; Idris, A.; Hilmi, N. H. Z.;
Fakurazi, S. Synthesis of (Hexaconazole-Zinc/Aluminum-Layered Double Hydroxide
Nanocomposite) Fungicide Nanodelivery System for Controlling Ganoderma Disease
in Oil Palm. J. Agric. Food Chem. 2018, 66, 806-813.
(44)Wang, P. Y.; Fang, H. S.; Shao, W. B.; Zhou, J.; Chen, Z.; Song, B. A.;
Yang, S. Synthesis and biological evaluation of pyridinium-functionalized carbazole
derivatives as promising antibacterial agents. Bioorg. Med. Chem. Lett. 2017, 27,
4294-4297.
(45)Wang, P. Y.; Zhou, L.; Zhou, J.; Wu, Z. B.; Xue, W.; Song, B. A.; Yang, S.
Synthesis
and
antibacterial
activity
of
pyridinium-tailored
2,5-substituted-1,3,4-oxadiazole thioether/sulfoxide/sulfone derivatives. Bioorg. Med.
Chem. Lett. 2016, 26, 1214-1217.
(46)Fang, C.; Kong, L. L.; Ge, Q.; Zhang, W.; Zhou, X. J.; Zhang, L.; Wang, X.
P. Antibacterial activities of N-alkyl imidazolium-based poly(ionic liquid)
nanoparticles. Polym. Chem. 2019, 10, 209-218.
(47)Chien, H. W.; Chen, Y. Y.; Chen, Y. L.; Cheng, C. H.; Lin, J. C.; Studies of
PET nonwovens modified by novel antimicrobials configured with both N-halamine
and dual quaternary ammonium with different alkyl chain length. RSC Adv. 2019, 9,
7257-7265.
(48)Song, X. P.; Li, P.; Li, M. W.; Yang, A. M.; Yu, L.; Luo, L. Z.; Hu, D. Y.;
Song, B. A. Synthesis and investigation of the antibacterial activity and action
mechanism of 1,3,4-oxadiazole thioether derivatives. Pestic. Biochem. Phys. 2018,
2
6
ACS Paragon Plus Environment

Page 27 of 50
Journal of Agricultural and Food Chemistry
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
5
67
147, 11-19.
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
(49)Wang, Y. M.; Gupta, R.; Song, W.; Huh, H. H.; Lee, S. E.; Wu, J. N.;
Agrawal, G. K.; Rakwal, R.; Kang, K. Y.; Park, S. R.; Kim, S. T. Label-free
quantitative secretome analysis of Xanthomonas oryzae pv. oryzae highlights the
involvement of a novel cysteine protease in its pathogenicity. J. Proteomics 2017,
169, 202-214.
(50)Chen, J.; Shi, J.; Yu, L.; Liu, D. Y.; Gan, X. H.; Song, B. A.; Hu, D. Y.
Design, Synthesis, Antiviral Bioactivity, and Defense Mechanisms of Novel
Dithioacetal Derivatives Bearing a Strobilurin Moiety. J. Agric. Food Chem. 2018,
66, 5335-5345.
(51)Chen, X. L.; Xie, X.; Wu, L. Y.; Liu, C. Y.; Zeng, L. R.; Zhou, X. P.; Luo, F.;
Wang, G. L.; Liu, W. D. Proteomic Analysis of Ubiquitinated Proteins in Rice (Oryza
sativa) After Treatment With Pathogen-Associated Molecular Pattern (PAMP)
Elicitors. Front. Plant Sci. 2018, 9, DOI: 10.3389/fpls.2018.01064.
(52)Zhao, Y. L.; Huang, X.; Liu, L. W.; Wang, P. Y.; Long, Q. S.; Tao, Q. Q.; Li,
Z.; Yang, S. Identification of Racemic and Chiral Carbazole Derivatives Containing
an Isopropanolamine Linker as Prospective Surrogates against Plant Pathogenic
Bacteria: In Vitro and In Vivo Assays and Quantitative Proteomics. J. Agric. Food
Chem. 2019, 67, 7512-7525.
(53)Wilson, D. N. Ribosome-targeting antibiotics and mechanisms of bacterial
resistance. Nat. Rev. Microbiol. 2014, 12, 35-48.
(54)Lai, W. J. C.; Ermolenko, D. N. Ensemble and single-molecule FRET studies
2
7
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 50
5
5
5
5
5
5
5
5
5
5
5
6
6
89
90
91
92
93
94
95
96
97
98
99
00
01
of protein synthesis. Methods 2018, 137, 37-48.
(55)de Loubresse, N. G.; Prokhorova, I.; Holtkamp, W.; Rodnina, M. V.;
Yusupova, G.; Yusupov, M. Structural basis for the inhibition of the eukaryotic
ribosome. Nature 2014, 513, 517-522.
(56)Kong, J.; Zhang, Y.; Ju, J.; Xie, Y. F.; Guo, Y. H.; Cheng, Y. L.; Qian, H.;
Quek, S. Y.; Yao, W. R. Antifungal effects of thymol and salicylic acid on cell
membrane and mitochondria of Rhizopus stolonifer and their application in
postharvest preservation of tomatoes. Food Chem. 2019, 285, 380-388.
(57)Chattapadhyay,
T.
K.;
Dureja,
P.
Antifungal
activity
of
4-methyl-6-alkyl-2H-pyran-2-ones. J. Agric. Food Chem. 2006, 54, 2129-2133.
(58)Heise, T.; Schmidt, F.; Knebel, C.; Rieke, S.; Haider, W.; Geburek, I.;
Niemann, L.; Marx-Stoelting, P. Hepatotoxic combination effects of three azole
fungicides in a broad dose range. Arch. Toxicol. 2018, 92, 859-872.
2
8
ACS Paragon Plus Environment

Page 29 of 50
Journal of Agricultural and Food Chemistry
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
02
Figure captions
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
Figure 1. Some bioactive structures containing thiazolium, 1,3,4-oxadiazole, or
2,4-dichlorophenyl scaffolds.
Figure 2. Design strategy for target molecules.
Figure 3. Synthetic route for A and B (n = 6, 8, 10, 12).
n
n
Figure 4. Synthetic route for C and D (n = 1–10).
n
n
Figure 5. Crystal structures of compound D7.
Figure 6. Molecular alignment of all target compounds.
Figure 7. Plots of predicted pEC versus experimental pEC against Xoo. Predicted
5
0
50
pEC values were calculated for A) CoMFA model and B) CoMSIA model.
5
0
Figure 8. CoMFA contour maps with compound C inside the fields: A) steric fields,
1
0
with green and yellow polyhedra indicating the regions where steric bulk would
enhance and reduce the activity; B) electrostatic fields, with blue and red polyhedra
indicating the regions where positive and negative charges would enhance activity.
Figure 9. CoMSIA contour maps with compound C10 inside the fields: A) steric
fields, with green and yellow polyhedra indicating the regions where steric bulk
would enhance and reduce the activity; B) electrostatic fields, with blue and red
polyhedra indicating the regions where positive and negative charges would enhance
activity; C) hydrophobic fields, with yellow and grey polyhedra indicating the regions
where hydrophobicity and hydrophilicity would enhance activity; D) H-bond acceptor
fields, with red contours indicating the regions where H-bond acceptor groups would
enhance activity.
2
9
ACS Paragon Plus Environment