Discovery of Novel Piperidinylthiazole Derivatives As Broad-Spectrum Fungicidal Candidates

Article abstract of DOI:10.1021/acs.jafc.8b06054

Oxathiapiprolin is one of the best active fungicides discovered for oomycetes control. To develop a fungicide candidate with a broad spectrum of activity, 22 new piperidinylthiazole derivatives were designed and synthesized. Compound 5l showed the best activity against Pseudoperonospora cubensis (Berk. et Curt.) Rostov and Phytophthora infestans in vivo with 100% and 80% of inhibition, respectively, at 1 mg/L, and 72.87% of field efficacy against P. cubensis at 1 g ai/667 m² validated these results. Compound 5i exhibited a broad spectrum of excellent activity against Sclerotinia sclerotiorum with EC₅₀ = 0.30 mg/L (>10 times more active than oxathiapiprolin and azoxystrobin in vitro), good activity against Botrytis cinerea, Cercospora arachidicola, and Gibberella zeae with EC₅₀ of 14.54, 5.57, and 14.03 mg/L in vitro and against P. cubensis and P. infestans with 60% and 30% inhibition rates, respectively, at 1 mg/L in vivo. Mode of action studies by RNA sequencing analysis discovered oxysterol-binding protein (OSBP), chitin synthase (CHS1), and (1,3)-β-glucan synthase (FKS2) as the potent target of 5i against S. sclerotiorum. Quenching studies validated that OSBP was the same target of both 5i and oxathiapiprolin; it was quenched by both of them. Our studies discovered isothiazole-containing piperidinylthiazole as an OSBP target-based novel lead for fungicide development.

Full text of DOI:10.1021/acs.jafc.8b06054

Subscriber access provided by Iowa State University | Library
Agricultural and Environmental Chemistry
Discovery of Novel Piperidinyl-thiazole Derivatives
as Broad-spectrum Fungicidal Candidate
Qifan Wu, Bin Zhao, Zhijin Fan, Xiaofeng Guo, Dong-yan Yang,
Nailou Zhang, Bin Yu, Shuang Zhou, Jiabao Zhao, and Fan Chen
J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06054 • Publication Date (Web): 14 Jan 2019
Downloaded from http://pubs.acs.org on January 14, 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 42
Journal of Agricultural and Food Chemistry
Discovery of Novel Piperidinyl-thiazole Derivatives as
Broad-spectrum Fungicidal Candidate
，
Qifan Wu†, Bin Zhao*,†, Zhijin Fan*,† ‡, Xiaofeng Guo†, Dongyan Yang†, Nailou
Zhang†, Bin Yu†, Shuang Zhou†, Jiabao Zhao†, Fan Chen†
†
State Key Laboratory of Elemento-Organic Chemistry, College of Chemistry, Nankai
University, Tianjin 300071, P. R. China.
‡ Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai
University, Tianjin 300071, P. R. China.
*
Address correspondence to these authors at State Key Laboratory of Elemento-
Organic Chemistry, College of Chemistry, Nankai University, No. 94, Weijin Road,
Nankai District, Tianjin 300071, P. R. China (telephone +86-23499464; Fax: +86 022-
23503620; e-mail: fanzj@nankai.edu.cn for Zhijin Fan or bdzhaobin@126.com for Bin
Zhao)
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 2 of 42
1
2
Abstract
Oxathiapiprolin is one of the best active fungicide discovered for oomycetes
control. To develop fungicide candidate with broad-spectrum of activity, 22 new
piperidinyl-thiazole derivatives were designed and synthesized. Compound 5l showed
the best activity against Pseudoperonospora cubensis (Berk.et Curt.) Rostov and
Phytophthora infestans in vivo with 100% and 80% of inhibition respectively at 1 mg/L,
and 72.87% of field efficacy against P. cubensis at 1 g ai/667m2 validated these results.
Compound 5i exhibited broad spectrum of excellent activity against Sclerotinia
sclerotiorum with EC50=0.30 mg/L (over 10-times more active than oxathiapiprolin and
azoxystrobin in vitro), good activity against Botrytis cinerea, Cercospora arachidicola,
Gibberella zeae with EC50 of 14.54 mg/L, 5.57 mg/L, 14.03 mg/L in vitro, and P.
cubensis, P. infestans with 60% and 30% inhibition rate respectively at 1 mg/L in vivo.
Mode of action studies by RNA sequencing analysis discovered Oxysterol-binding
protein (OSBP), Chitin synthase (CHS1) and (1,3)-β-Glucan Synthase (FKS2) as the
potent target of 5i against S. sclerotiorum. Quenching studies validated that, OSBP was
the same target of both 5i and oxathiapiprolin, it was quenched by both of them. Our
studies discovered isothiaole containing piperidinyl-thiazole as an OSBP target based
novel lead for fungicide development.
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
Keywords: piperidinyl-thiazole, 3,4-dichloroisothiazole, antifungal activity, broad-
21
22
spectrum, RNA sequencing.
ACS Paragon Plus Environment

Page 3 of 42
Journal of Agricultural and Food Chemistry
23 Introduction
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
In the last three decades, numerous highly-active fungicides such as metalaxyl,1-3
dimethomorph,4 ethaboxam5, 6 and oxathiapiprolin7, 8 have been successfully developed
for oomycete disease control with excellent activity and various mechanism or mode of
action all over the world. Oxathiapiprolin is a piperidinyl thiazole isoxazoline
11
fungicides9 with great activity on oomycetes.10,
The discovery procedure of
oxathiapiprolin (Figure 1) was based on the lead compound Tr1,12 which included in
Tripos Associates with certain degree of activity against oomycete. Piperidinyl-thiazole
is the key core structure of those compounds from Tripos Library, and it is also a key
active substructure of Tr1 and oxathiapiprolin. The active mechanism of
oxathiapiprolin is inhibiting a novel fungal oxysterol-binding protein (OSBP).12 P.
infestans oxysterol-binding protein (PiORP1, Protein ID: XP_002902250.1) is the
target protein of oxathiapiprolin, 13 its biological functions in oomycetes is unclear.8
Oxathiapiprolin exhibited excellent activity against Phytophthora nicotianae,
Phytophthora capsica and Peronospora belbahrii,10, 11, 14 its activity against other fungi
is uncertain.8
Ergosterol is one of the most common components in the fungi cell membrane
other than in oomycetes.15, 16 The ergosterol biosynthesis pathway is fungus specific. 17
ERG13 (HMG-CoA synthase) catalyzed acetoacetyl-CoA to HMG-CoA and HMG1
(HMG-CoA reductase) catalyzed HMG-CoA to mevalonate are two key consecutive
steps of ergosterol biosynthesis.18 The ERG11/CYP51 (Sterol 14α-demethylase) was
also an essential enzyme in the biosynthesis of ergosterol and the target enzyme of azole
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 4 of 42
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
fungicides that were widely used for the treatment of fungal infections.19 Mutation of
ERG6 also caused the decreased spore germination, higher susceptibility to stress
resistance, as well as reduced vacuolar transport capability. 20 Active novel isothiazole–
thiazole derivative (3-bromophenyl)(4-(4-(3,4-dichloroisothiazol-5-yl)thiazol-2-
yl)piperidin-1-yl)methanone against Pseudoperonospora cubensis (Berk. et Curt.)
Rostov might act at P. cubensis oxysterol-binding protein (PcORP1), the same target
as of oxathiapiprolin, was discovered.21 Therefore, fungicidal spectrum will be enlarged
if we design the target compounds by inhibiting both the activity of OSBP and synthesis
of ergosterol. As inspired by this hypothesis, oxathiapiprolin was artificially divided
into four parts and a series compounds shown in skeleton 1 were designed through
retaining the core skeleton of part B, while, part A was replaced with a heterocyclic ring
and then both part C and part D were replaced by R4. 3,4-Dichloroisothiazole as a
bioactive substructure of isotianil22 was used for replacement of heterocyclic part A in
skeleton 1. The molecular design route of target compounds was showed in Figure 2,
the detailed series of piperidinyl-thiazole derivatives were listed in Table 1, all the
synthesis procedures were conducted according to Figure 3.
61 Materials and Methods
62
63
64
65
66
Materials and Instruments. All reagents and extra-dry solvents were purchased
from J&K Scientific Ltd. (Beijing, China), Heowns Biochem LLC (Tianjin, China) or
Energy Chemical (Shanghai, China). All solvents of analytical reagent were purchased
from Tianjin Bohua Chemical Reagents Co., Ltd. (Tianjin, China). Melting points were
measured on an X-4 Digital Type Melting Point Tester (Gongyi, Chian) and were
ACS Paragon Plus Environment

Page 5 of 42
Journal of Agricultural and Food Chemistry
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
uncorrected. Using tetramethylsilane (TMS) as an internal standard, 1H and 13C NMR
spectra were obtained in a 400 MHz Bruker AV400 spectrometer (Wisconsin, United
States of America) in chloroform-d (CDCl3) or dimethylsulfoxide-d6 (DMSO-d6).
High-resolution mass spectra (HRMS) were recorded with a 6520 Q-TOF LC/MS
instrument (State of California, United States of America). Crystal structures were
determined on a 007HF XtaLAB P200 diffractometer of Rigaku Corporation (Japan).
The thin-layer chromatography (TLC) was used to monitor the reaction progress by UV
analyzer ZF-type 1 (Hangzhou, China).
General Procedures for the Preparation of Intermediates 3. Starting material
23
1 was synthesized according to the description of references22,
from 3,4-
dichloroisothiazole-5-carboxylic acid by Weinreb amide24 formation and a Grignard
addition, and the succeeding reaction with pyridinium bromide perbromide.
Intermediate 2 was synthesized via t-butyloxy carbonyl protection and Lawson reaction
of piperidine-4-carboxamide.25
To a 100 mL round-bottomed flask, starting material 1 (2.25 g, 8.30 mmol),
intermediate 2 (2.21 g, 9.10 mmol) and tetrahydrofuran (THF) (50 mL) were added.
The mixture was heated for 3 h of refluxing. After filtration, the filter residue was
dissolved in the mixture of sodium hydroxide (NaOH) solution (1 mol/L, 50 mL) and
dichloromethane (CH2Cl2) (50 mL), then the organic layers were dried over anhydrous
sodium sulfate, and concentrated in vacuo to obtain crude product 3b 0.30 g (11.2%).
The filtrate was removed for the solvent in vacuo, and then added CH2Cl2, the mixture
was washed with water solution of NaOH (1 mol/L, 50 mL) and brine (50 mL)
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 6 of 42
89
90
successively, dried over anhydrous sodium sulfate, then concentrated in vacuo. The
residue was purified by column chromatography on silica gel with a mixture of ethyl
acetate /petroleum ether (60−90 °C fraction) (1:16, υ/υ) to give 1.46 g (yield 42%)
compound 3a.
91
92
93
A solution of compound 3a (1.46 g, 3.48 mmol) in dry CH2Cl2 (30 mL) under
nitrogen atmosphere cooled in ice water was added dropwise trifluoroacetic acid (9.92
g, 87.00 mmol). The reaction mixture was then stirred at room temperature for 2 h.
After the reaction was completed, the mixture was quenched with 1 mol/L water
solution of NaOH and was adjusted to pH value of 8 for extraction with CH2Cl2 (50
mL). The organic layers were washed using brine (50 mL) and dried over anhydrous
sodium sulfate, then concentrated under reduced pressure. The residue was purified by
column chromatography on silica gel with a mixture of CH2Cl2/CH3OH/Et3N
(6:1:0.001 (υ/υ/υ)) to give 1.02g (yield 92%) compound 3b.
94
95
96
97
98
99
100
101
102
103
104
105
106
107
108
109
110
Analytical data for compound 3a: White solid; m.p., 153−155 oC; 1H NMR (400
MHz, CDCl3) δ 8.04 (s, 1H), 4.21 (d, J = 10.7 Hz, 2H), 3.19 (t, J = 11.3 Hz, 1H), 2.93
(t, J = 12.1 Hz, 2H), 2.13 (d, J = 12.7 Hz, 2H), 1.77 (dd, J = 18.2, 5.6 Hz, 2H), 1.46 (d,
13
J = 22.8 Hz, 9H). C NMR (101 MHz, CDCl3) δ 175.31 (s), 155.10 (s), 154.66 (s),
148.73 (s), 143.35 (s), 116.74 (s), 79.75 (s), 43.40 (s), 40.38 (s), 32.19 (s), 28.44 (s).
HRMS (ESI) (M+H)+ calcd for C16H19Cl2N3O2S2: 420.0374, found: 420.0345; HRMS
(ESI) [M+Na]+ calcd for C16H19Cl2N3O2S2: 442.0194, found: 442.0183. Anal. Calcd for
C16H19Cl2N3O2S2: C, 45.72; H, 4.56; N, 10.00. Found: C, 45.45; H, 4.29; N, 10.13.
o
1
Analytical data for compound 3b: White solid; m.p., 97−99 C; H NMR (400
ACS Paragon Plus Environment

Page 7 of 42
Journal of Agricultural and Food Chemistry
111
MHz, DMSO-d6) δ 8.45 (s, 1H), 4.41 (s, 1H), 4.01 (s, 1H), 3.45 – 2.84 (m, 3H), 2.62
(d, J = 10.3 Hz, 1H), 1.98 (s, 2H), 1.70 – 1.42 (m, 2H). 13C NMR (101 MHz, CDCl3) δ
176.51 (s), 155.26 (s), 148.73 (s), 143.20 (s), 116.66 (s), 46.25 (s), 40.86 (s), 33.66 (s).
HRMS (ESI) [M+H]+ calcd for C11H11Cl2N3S2: 319.9849, found: 319.9850. Anal. Calcd
for C11H11Cl2N3S2: C, 41.26; H, 3.46; N, 13.12. Found: C, 40.75; H, 3.57; N, 13.23.
General Procedure for the Preparation of the Target Compound 4. To a
solution of compound 3b (0.12 g, 0.38 mmol) in 20 mL methanol was added 0.94 mL
4 mol/L hydrochloric acid solution in 1,4-dioxane, the mixture was stirred for 3 h at
room temperature. After stop of the the reaction, the solvent in the reaction mixture was
removed at a reduced pressure, and the residue was dried at 60 oC for 12 h under vacuum
to obtain 0.12 g (92%) compound 4.
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
Analytical data for compound 4: White solid; m.p., >200 oC; 1H NMR (400 MHz,
DMSO-d6) δ 9.54 (s, 1H), 9.32 (s, 1H), 8.50 (d, J = 16.0 Hz, 1H), 3.35 (m, 2H), 3.05
13
(m, 2H), 2.53 (d, J = 14.0 Hz, 1H), 2.24 (s, 2H), 2.04 (t, J = 11.8 Hz, 2H). C NMR
(101 MHz, DMSO-d6) δ 175.30 (s), 155.55 (s), 148.38 (s), 142.10 (s), 120.05 (s), 42.85
(s), 37.28 (s), 28.76 (s). HRMS (ESI) [M+H]+ calcd for C11H12Cl3N3S2: 319.9849,
found: 319.9849.
General Procedures for the Preparation of the Target Compounds 5a−5t.
Compounds 5a−5t were synthesized by the reaction of piperidine with corresponding
acids. The mixture of corresponding acid (0.40 mmol), 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCI) (0.45 mmol), and 1-hydroxybenzotriazole
(HOBt) (0.40 mmol) was stirred in dry CH2Cl2 (10 mL) in an ice-water bath for 1 h
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 8 of 42
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
150
151
152
153
154
under nitrogen atmosphere. Then compound 3b (0.38 mmol) was dissolved in 10 mL
CH2Cl2, and added to the reaction system, then Et3N (0.45 mmol) was added. The
reaction mixture was then stirred at room temperature overnight, after stopping the
reaction, the mixture washed with saturated sodium bicarbonate (20 mL) and brine (20
mL) respectively, dried over anhydrous sodium sulfate, and then concentrated under
reduced pressure. The residue was purified by column chromatography on silica gel
with a mixture of ethyl acetate /petroleum ether (60−90 °C fraction) (1:9−1:3, υ/υ) to
give compounds 5a−5t (59%−100%).
Analytical data for compound 5a: White solid; yield, 82%; m.p., 141−143 oC; 1H
NMR (400 MHz, DMSO-d6) δ 8.52 (s, 1H), 8.21 (s, 1H), 8.02 (d, J = 12.8 Hz, 2H),
7.96 – 7.81 (m, 2H), 4.60 (s, 1H), 3.83 (s, 1H), 3.43 (d, J = 47.0 Hz, 2H), 3.04 (d, J =
10.2 Hz, 1H), 2.17 (d, J = 42.0 Hz, 2H), 1.70 (s, 2H). 13C NMR (101 MHz, CDCl3) δ
173.08 (s), 160.18 (s), 153.88 (s), 147.81 (s), 142.45 (s), 140.41 (s), 137.79 (s),
131.37−130.20 (m), 128.35 (d, J = 39.3 Hz), 125.51 (s), 125.02 (s), 123.79 (s), 121.08
(s), 119.72 (s), 119.34 (s), 117.03 (s), 115.95 (s), 45.92 (s), 40.48 (s), 38.92 (s), 31.24
(s), 30.66 (s). HRMS (ESI) [M+H]+ calcd for C23H15Cl2F6N5OS2: 626.0077, found:
626.0071.
o
Analytical data for compound 5f: White powder; yield, 72%; m.p., 109−111 C;
1H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1H), 7.36 (d, J = 7.1 Hz, 1H), 7.07 (dd, J =
13.9, 8.1 Hz, 3H), 4.46 (d, J = 13.1 Hz, 1H), 4.06 (d, J = 13.2 Hz, 1H), 3.80 (s, 2H),
3.37 (s, 1H), 3.19 (t, J = 12.5 Hz, 1H), 2.78 (t, J = 12.0 Hz, 1H), 2.06 (t, J = 11.0 Hz,
2H), 1.53 (d, J = 12.0 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 174.42 (s), 168.94 (s),
ACS Paragon Plus Environment

Page 9 of 42
Journal of Agricultural and Food Chemistry
155
164.22 (s), 161.77 (s), 154.96 (s), 148.83 (s), 143.44 (s), 137.32 (d, J = 7.9 Hz), 130.24
(d, J = 8.2 Hz), 124.35 (s), 116.86 (s), 115.69 (d, J = 21.7 Hz), 113.93 (d, J = 20.9 Hz),
45.75 (s), 41.58 (s), 40.67 (s), 40.01 (s), 32.28 (s), 31.85 (s). HRMS (ESI) [M+H]+ calcd
for C19H16Cl2FN3OS2:456.0174, found: 456.0171.
156
157
158
159
160
161
162
163
164
165
166
167
168
169
170
171
172
173
174
175
176
o
Analytical data for compound 5g: White solid; yield, 100%; m.p., >200 C; 1H
NMR (400 MHz, CDCl3) δ 8.07 (s, 1H), 5.86 (s, 1H), 4.90 (d, J = 11.7 Hz, 2H), 4.60
(d, J = 13.4 Hz, 1H), 4.09 (d, J = 13.7 Hz, 1H), 3.40−3.19 (m, 2H), 2.89 (m, 1H), 2.23
13
(m, 7H), 1.89−1.64 (m, 3H). C NMR (101 MHz, CDCl3) δ 174.37 (s), 165.25 (s),
154.97 (s), 148.84 (s), 148.13 (s), 143.46 (s), 140.73 (s), 116.89 (s), 105.81 (s), 50.89
(s), 44.84 (s), 41.92 (s), 40.00 (s), 32.31 (s), 31.84 (s), 13.48 (s), 11.17 (s). HRMS (ESI)
[M+H]+ calcd for C18H19Cl2N5OS2: 456.0486 found: 456.0485.
Analytical data for compound 5h: White powder; yield, 70%; m.p., 127−129 oC;
1H NMR (400 MHz, CDCl3) δ 8.37 – 8.01 (m, 1H), 4.82 (d, J = 51.1 Hz, 1H), 4.08 (d,
J = 52.0 Hz, 1H), 3.59 – 3.18 (m, 2H), 3.11 – 2.75 (m, 1H), 2.59 – 2.16 (m, 4H), 1.90
(d, J = 49.0 Hz, 2H), 1.20 (t, J = 28.1 Hz, 1H), 0.87 – 0.54 (m, 2H), 0.48 – 0.11 (m,
13
2H). C NMR (101 MHz, CDCl3) δ 174.74 (s), 171.09 (s), 155.02 (s), 148.78 (s),
143.40 (s), 116.86 (s), 45.32 (s), 41.21 (s), 40.29 (s), 38.62 (s), 32.71 (s), 32.02 (s), 7.34
(s), 4.55 (s). HRMS (ESI) [M+H]+ calcd for C16H17Cl2N3OS2: 402.0268, found:
402.0264.
o
Analytical data for compound 5i: White powder; yield, 85%; m.p., 166−168 C;
1H NMR (400 MHz, DMSO-d6) δ 8.47 (d, J = 6.8 Hz, 1H), 7.50 (s, 1H), 7.29 (s, 3H),
4.54 (s, 1H), 3.63 (s, 1H), 3.42 (s, 1H), 3.23 (s, 1H), 2.99 (s, 1H), 2.11 (d, J = 52.2 Hz,
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 10 of 42
13
177
178
179
180
181
182
183
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
2H), 1.72 (s, 2H). C NMR (101 MHz, CDCl3) δ 174.39 (s), 169.00 (s), 163.82 (s),
161.36 (s), 154.95 (s), 148.85 (s), 143.48 (s), 137.86 (s), 130.38 (s), 122.59 (s), 116.93
(s), 114.16 (s), 47.15 (s), 41.84 (s), 40.22 (s), 32.54 (s), 32.03 (s). HRMS (ESI) [M+H]+
calcd for C18H14Cl2FN3OS2: 442.0017, found: 442.0008.
o
Analytical data for compound 5q: White crystal; yield, 65%; m.p., 155−157 C;
1H NMR (400 MHz, DMSO-d6) δ 8.49 (s, 1H), 4.41 (d, J = 30.6 Hz, 2H), 3.42 (d, J =
9.5 Hz, 1H), 3.29 (d, J = 10.4 Hz, 1H), 2.79 (d, J = 10.2 Hz, 1H), 2.27 – 1.98 (m, 3H),
13
1.62 (d, J = 57.9 Hz, 2H), 0.74 (d, J = 5.5 Hz, 4H). C NMR (101 MHz, CDCl3) δ
174.85 (s), 171.92 (s), 155.04 (s), 148.76 (s), 143.37 (s), 116.86 (s), 45.18 (s), 41.89 (s),
40.46 (s), 32.40 (d, J = 77.0 Hz), 11.05 (s), 7.40 (s). HRMS (ESI) [M+H]+ calcd for
C15H15Cl2N3OS2: 388.0112, found: 388.0105.
Analytical data for compound 5t: White powder; yield, 100%; m.p., 168−170 oC;
1H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1H), 8.08 (s, 1H), 7.68 (s, 1H), 4.32 (s, 2H),
3.86 (s, 3H), 3.41 (d, J = 8.3 Hz, 1H), 3.08 (s, 2H), 2.11 (s, 2H), 1.66 (s, 2H). 13C NMR
(101 MHz, CDCl3) δ 173.56 (s), 162.66 (s), 153.96 (s), 147.80 (s), 142.41 (s), 137.73
(s), 131.34 (s), 116.15 (s), 115.88 (s), 39.32 (s), 38.16 (s), 31.35 (s), 28.67 (s). HRMS
(ESI) [M+H]+ calcd for C16H15Cl2N5OS2: 428.0173, found: 428.0167.
In Vitro Fungicidal Activity Test
Fungicidal activities of the compounds 3−5 against Alternaria solani (A. s),
Botrytis cinerea (B. c), Cercospora arachidicola (C. a), Gibberella zeae (G. z),
Physalospora piricola (P. p), Pellicularia sasakii (P. s), Rhizoctonia cerealis (R. c) and
Sclerotinia sclerotiorum (S. s) were tested in vitro at a concentration of 50 mg/L using
ACS Paragon Plus Environment

Page 11 of 42
Journal of Agricultural and Food Chemistry
199
the mycelium growth inhibition method.23 The target fungus was plated on a potato
dextrose agar (PDA) culture plate and precultured at 25±1°C for 5 days. And then the
fungi cake with the same diameter of 4 mm were removed for inoculation on the center
of culture plate with medicine and water by inoculation needle under sterilized
condition. Each experiment was repeated for three times. The diameter of fungi spread
was measured after 5 days. The average growth diameter was obtained by calculating
the average of three replicates. Growth inhibition was then calculated with a formula
as: Efficacy (%) = [(CK0 - PT1)/(CK0 - 4)] × 100%; CK0 means the average growth
diameter of fungi cake cultured on a plate with water; PT1 means the average growth
diameter of fungi cake cultured on a plate with fungicide. And based on the results of
in vitro antifungal activities, those compounds with inhibition above 70% in vitro at a
concentration of 50 mg/L were further measured for their median effective
concentration (EC50).23
200
201
202
203
204
205
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
In Vivo Fungicidal Activity Test
The protective activities of target compounds 3−5 against Pseudoperonospora
cubensis (Berk. et Curt.) Rostov (P. cubensis), Phytophthora infestans (P. infestans),
Sphaerotheca fuliginea (S. fuliginea) and Puccinia sorghi Schwat (P. sorghi Schwat)
causing disease in cucumber, tomato, potato and corn respectively were determined
according to the ref. 26 in Shenyang Research Institute of Chemical Industry (Shenyang,
China) at a concentration of 100 mg/L by pot bioassay. Those compounds with
inhibition over 90% were further tested at lower concentrations.
Field Efficacy Trials of Compound 5l
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 12 of 42
221
222
223
224
225
226
227
228
229
230
According to the good inhibitory activity against P. cubensis in vivo, efficacy of
compound 5l was further evaluated by cucumber field experiment for fungicidal
activity validation. Compound 5l was prepared as 5.32 % emulsifiable concentrate, and
diluted to 0.5 g ai/667m2, 1 g ai/667m2 and 2 g ai/667m2 with water for application. In
the early onset of P. cubensis, the cucumbers were uniformly sprayed with the
compound 5l. Equal amount of water was used as the blank and oxathiapiprolin (10 %,
flowable agent) was used as positive control. The disease indexes (DI) and control
efficacies were calculated according to the disease level of the investigated leaves. DI
was caculated by formula as DI=Σ(A×B)×100/(C×9), and the control efficacy (CE) was
evaluated by formula as CE(%)=[1－CK0×pt1/(CK1×pt0)]×100, among of them, A was
the number of diseased leaves, B was the corresponding disease level, C was the total
number of leaves investigated, CK0 indicated the disease index of the blank before the
pesticide application, CK1 indicated the disease index of the blank after the pesticide
application, pt0 was the disease index of the pesticide treatment group before compound
application, pt1 indicated the disease index of the pesticide treatment group after
compound application. The statistical analysis of control effects was established via
reverse arcsine transformation and variance analysis. Moreover, the significant
difference between each treatment was compared by LRS method.27
231
232
233
234
235
236
237
238
239
240
241
242
The Modeling and Docking Analysis Between Compounds and PiORP1
Modeling the structure of P. infestans oxysterol-binding protein (PiORP1, Protein
ID: XP_002902250.1) was conducted by YASARA program28 with default parameters.
The ligand binding center of PiORP1 was identified by Autodock Tools.29,30 The
ACS Paragon Plus Environment

Page 13 of 42
Journal of Agricultural and Food Chemistry
243
structures of ligands were optimized with the ligand minimization protocol. The
molecular docking analysis was performed by the Autodock Tools with default values.
The optimal structure in complex was selected according the visual inspection and
docking score. The structures of complexes were showed by Pymol.31
244
245
246
247
248
249
250
251
252
253
254
255
256
257
258
259
260
261
262
263
264
Fluorescence Quenching Analysis of Oxysterol-binding Protein
Full-length yeast oxysterol-binding protein (Osh4p) was cloned into pGEX-4T-3
vector to code for glutathione S-transferase (GST)-fused constructs, which was gifted
by Prof. Guillaume Drin (CNRS, France), the methods of protein induction and
purification for Osh4p were conducted as document description.32 Fluorescence
quenching analysis was preformed using F-4500 fluorescence spectrophotometer
(Hitachi, Tokyo, Japan). Fluorescence emission spectra were recorded at a wavelength
o
of 290-520 nm, and an excitation wavelength of 275 nm at 4 C, with emission band
width setting at 10 nm and medium sensitive. Oxathiapiprolin and 5i with 0 to 9 mg/L
was added to Osh4p for fluorescence determination. The highest concentration of N,N-
Dimethylformamide (DMF) in Osh4p solution was used as blank, the highest
concentration of oxathiapiprolin or 5i in PBS were also determined separately.
RNA Sequencing and Data Analysis
S. sclerotiorum, which grew in PDA at 25 oC for 1 week, was treated by compound
5i (0.1mg/L) for 36 h and the same amount of DMF was used as control. Total RNAs
of samples were extracted using TRIzol Reagent (Invitrogen, USA). cDNA Libraries
were constructed using a Truseq stranded mRNA kit (Illumina, San Diego, USA).
Sequencing was conducted on an Illumina MiSeq sequencing system using the HiSeq
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 14 of 42
265
266
267
268
269
270
271
272
273
274
4000 SBS Kit (Illumina, San Diego, USA) following the manufacturer’s instructions.
S.
sclerotiorum
transcripts
available
in
the
database
(http://www.broadinstitute.org/annotation/genome/sclerotinia_sclerotiorum/MultiHo
me.html) were used as a reference for mapping the short reads. The count data were
normalized to generate effective library sizes using the scaling method Trimmed Means
of Means values (TMM). Statistical analysis was performed using a generalized linear
model linked to the negative binomial distribution performed using the EdgeR package.
The different expression genes were identified with false discovery rate (FDR)≤0.05
and |log2[fold change]|＞1. The Gene ontology (GO) terms and kyoto encyclopedia of
genes and genomes (KEGG) pathway for each gene were extracted.
275 Results and Discussion
276
277
278
279
280
281
282
283
284
285
286
Chemistry
The target compounds 5a−5t were synthesized by a condensation reaction from
piperidine and different acids in the presence of Et3N in dry CH2Cl2 using EDCI and
HOBt as condensation reagent and had favorable yields. The crystal data and structure
refinement of compound 5p, which was cultured from the mixture of CH2Cl2 and ethyl
acetate, were shown in Figure 4. The key intermediate 3a was obtained by condensation
of an α-halocarboxylates (2-bromo-1-(3,4-dichloroisothiazol-5-yl) ethan-1-one) and
thioamides (tert-butyl 4-carbamothioylpiperidine-1-carboxylate),33 and the succeeding
reaction of the key intermediate 3a with hydrogen halide formed compound 3b. α-
Halocarboxylates 1 was synthesized by the reaction of 1-(3,4-dichloroisothiazol-5-
yl)ethan-1-one with pyridinium tribromide in the presence of hydrogen bromide–acetic
ACS Paragon Plus Environment

Page 15 of 42
Journal of Agricultural and Food Chemistry
287
acid.23 Thioamide 2 was synthesized via the reaction between the -NH-Boc protected
hexahydroisonicotinamide and Lawesson's Reagent.
288
289
290
291
292
293
294
295
296
297
298
299
300
301
302
303
304
305
306
307
308
In Vitro Antifungal Activity
Table 2 showed the in vitro activity of compounds 3−5 against A. solani, B. cinerea,
C. arachidicola, G. azeae, P. piricola, P. sasakii, R. cerealis and S. sclerotiorum at 50
mg/L. The commercial fungicides oxathiapiprolin, isotianil and azoxystrobin were used
as positive controls. All the compounds exhibited various degrees of inhibitory
activities against fungi tested. Most compounds exhibited good activity against S.
sclerotiorum at 50 mg/L in vitro. Particularly, compounds 3b, 4, 5i and 5k showed
outstanding activity (100%), which was higher than those of positive controls
oxathiapiprolin (90%) and isotianil (45%).
Based on the results of antifungal activities in vitro, the compounds with inhibition
over 70% were further determined for their EC50 (Table 3). Compound 3b, with EC50
of 5.99−11.44 mg/L against B. cinerea, S. sclerotiorum and R. cerealis, exhibited
broad-spectrum of fungicidal activity. In particular, compound 5i strongly inhibited the
growth of B. cinerea, C. arachidicola and S. sclerotiorum with EC50 of 14.54 mg/L,
5.57 mg/L and 0.30 mg/L respectively, it was much better than those of positive control
oxathiapiprolin with corresponding EC50 of 35.91 mg/L (2.45 times), 79.20 mg/L
(14.22 times) and 5.98 mg/L (19.90 times) against B. cinerea, C. arachidicola and S.
sclerotiorum respectively, and at the same level as that of the positive control
azoxystrobin with the EC50 of 15.11 mg/L against B. cinerea,and more active (13.47
times) than the positive control azoxystrobin with the EC50 of 4.04 mg/L against S.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 16 of 42
309
310
311
312
313
314
315
316
317
318
319
320
321
322
323
324
325
326
327
328
329
330
sclerotiorum. The activity of compound 5i was more than 10 times than these of both
positive controls oxathiapiprolin and azoxystrobin against S. sclerotiorum.
In Vivo Antifungal Activity
The protective activities of pot bioassay for the compounds 3−5 against P. cubensis,
P. infestans, S. fuliginea and P. sorghi Schw were detected in Shenyang Research
Institute of Chemical Industry (Shenyang, China), the results were listed in Table 4 and
Table 5. The commercial fungicides oxathiapiprolin and isotianil were used as the lead
compounds and positive controls, the commercial fungicides ethirimol and
azoxystrobin were also used as positive controls for S. fuliginea and P. sorghi. Most
target compounds, commercial fungicides oxathiapiprolin and isotianil had no
inhibitory activity against S. fuliginea and P. sorghi, only compounds 3a, 5q and 5r
showed 20%, 20% and 30% in vivo inhibition against S. fuliginea at a concentration of
100 mg/L respectively. However, most of the compounds exhibited excellent inhibitory
activity in vivo against P. cubensis and P. infestans at 100 mg/L. Among them,
compounds 5c, 5h, 5l and 5r exhibited better activities (>80% inhibition at 1 mg/L)
than isotianil (70% inhibition at 1 mg/L) against P. cubensis. And 5l also exhibit great
activity against P. infestans at 1 mg/L (80% inhibition) in vivo.
The results of in vitro (Table 3) and in vivo (Table 4 and Table 5) indicate that the
methylene between N-formylpiperidine and D ring can’t increase the activity, and the
D ring doesn’t necessarily need to be a pyrazole ring. However, when the D ring is an
ordinary substituted benzene ring, the compounds have better fungicidal activity.
Further, if the substituent on the benzene ring contains an F atom capable of forming
ACS Paragon Plus Environment

Page 17 of 42
Journal of Agricultural and Food Chemistry
331
an H bond, the activity of the compounds can be further increased.
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
350
351
352
Analysis of Field Efficacy Trials
A large number of cucumber leaves had black mold layer in the blank, and their
DI values increased from 1.72 to 19.81. While the conditions of cucumber leaves treated
with the different compounds were well controlled, and the DI values were between
3.86 and 6.47. Efficacies of compound 5l against P. cubensis in the cucumber field were
shown in Table 6. The efficacy of high dosage (2 g ai/667m2) of 5l application showed
no significant difference as compared with positive control oxathiapiprolin with
medium (1 g ai/667m2) and low (0.5 g ai/667m2) dosage application, which was lower
than that of the high dosage (2 g ai/667m2) application of oxathiapiprolin. There were
no obvious efficacy differences between the high and middle dosages application of 5l,
and both of them were evidently higher than the corresponding efficacy of the low dose
application. To control P. cubensis in field, compound 5l was recommended at the
dosage of 1 g ai/667m2 to 2 g ai/667m2 once a week for 3 times.
Docking Analysis
Docking analysis was employed to elucidate the binding modes and affinities between
compounds and protein. The results (Figure 5A, 5B, 5D) showed that 5i and 5l could
bind with the active center of PiORP1, the thiazole ring in 5i, 5l and oxathiapiprolin all
had a hydrogen bond with PiORP1 on Asn835. Since the crystal structure of OSBP
from oomycetes has not been elucidated yet, we have constructed the structure of
PiORP1 by homology modeling base on 5H2D.34 The substrate of 5H2D was ergosterol,
so the binding mode of ergosterol to OSBP was docked, although ergosterol was not
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 18 of 42
353
354
355
356
357
358
359
360
361
362
363
364
365
366
367
368
369
370
371
372
373
374
present in oomycetes. As shown in Figure 5E, ergosterol could bind to OSBP with
hydrophobic interaction. As were known, no hydrogen bond on Asn835 was a key
binding amino acid34,35 in the transport of steroids by oxidized sterol-binding proteins
and hydrogen bonding force was stronger than hydrophobic force. As shown in Figure
5C and 4F, 5i and 5l also had a same combination mode with oxathiapiprolin except
for 5-methyl-3-(trifluoromethyl)-1H-pyrazol-1-yl. In summary, we indicated that the
target compounds 5i and 5l had suitable binding positions in its potent target PiORP1,
therefore, 5i and 5l might have a same mechanism with oxathiapiprolin. 5-Methyl-3-
(trifluoromethyl)-1H-pyrazol-1-yl was also an important group of oxathiapiprolin,
which led the higher activity of oxathiapiprolin. Compounds 5i and 5l contained no F
atoms in their structures as compared with oxathiapiprolin, both methyl and
trifluoromethyl can be introduced into 5i and 5l on the same ring for future comparative
study.
RNA Sequencing Analysis
Because of the good fungicidal activity in vitro and in vivo, compound 5i showed
very different novel chemical structure and the potent similar target at OSBP as
compared with the positive control oxathiapiprolin, to validate this novel mode of
action of the target compounds, RNA sequencing was conducted to identify the
differential expression genes (DGEs) affected by compound 5i. The results indicated
that, totally 3080 DGEs with fold changes >1.50 or <0.67 (p < 0.05) were identified.
The volcano plots for those 3080 DGEs were illustrated in Figure 6A. GO annotations
were performed on the 3080 DGEs which classified into molecular function, cell
ACS Paragon Plus Environment

Page 19 of 42
Journal of Agricultural and Food Chemistry
375
component, and biological process (Figure 6B). The results showed that 20 functional
groups had been annotated with those DGEs, including 8 biological processes, 8
cellular components and 4 molecular functions. In the cell components, the DGEs were
mainly involved in membrane, membrane part, cell and cell part. In the biological
processes, the DGEs were mostly proteins associated with single-organism process,
cellular process and metabolic process. With regard to the molecular functions, the
DGEs were mainly involved in protein binding and catalytic activity, which indicated
that 5i might act at binding protein which played an important role in the structure of
membrane or cell.
376
377
378
379
380
381
382
383
384
385
386
387
388
389
390
391
392
393
394
395
396
The DGEs after treatment by 5i were showed in Table S2, out of those
differentially expressed genes identified being relevant to lipid metabolism. Four genes
belong to ergosterol biosynthesis (erg13, hmg1, erg11/cyp51 and erg6) were shown in
Figure 7. All those key enzymes were downregulated when treated by 5i. We speculate
that the OSBP related protein was inhibited, and thus caused large number of sterols
unable to be transported. When the OSBP was inhibited, the transport of ergosterol and
many other cellular processes including vesicular trafficking, and signaling could be
affected, the germ-tube emergence and mycelium growth were inhibited.36 Under the
feedback regulation mechanism of sterol synthesis,37 the gene expression in sterol
synthesis were inhibited. In all eukaryotic organisms, steroid as a critical components
of cell membranes were required for the regulation of membrane fluidity and
permeability.38 In addition, 1,3-β-glucan and chitin are important component of cell
wall. FKS2 (SS1G_10788), alternative subunits of the glucan synthase complex, taking
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 20 of 42
397
398
399
400
401
402
403
404
405
406
responsibility for 1,3-β-glucan chains synthesis, was also downregulated. The chitin
was a long-chain polymer of N-acetylglucosamine and derivative of glucose. It was a
primary component of cell walls in fungi. The gene expression of Chitin synthase (chs1,
SS1G_04961) was also downregulated. These results indicate that compound 5i not
only potentially acted on OSBP but also very close related with CHS1 or FKS2.
5i Can Cause Fluorescence Quenching of Oxysterol-binding Protein
Fluorescence quenching of oxysterol-binding protein (Osh4p) by 5i and
oxathiapiprolin were conducted to validate combination between compound and the
protein. As shown in Figure 8A, with the concentration increase of oxathiapiprolin, the
fluorescence of Osh4p protein gradually quenched; and the same phenomenon occurred
in the mixture of 5i and Osh4p (Figure 8B), and the 1 ‰ DMF in Osh4p did not affect
the basic shape of the fluorescence spectrum of Osh4p, 5i and oxathiapiprolin in PBS
were not detected at emission spectra wavelength of 290-500 nm with an excitation
wavelength of 275 nm (Figure 8C), this indicated that compound-protein mixtures were
not affect by DMF. It was shown that, 5i had the same mode of action as that of
oxathiapiprolin, they both were quenched by binding with OSBP. Target and lead
discovery and validation are often very important basis for novel pesticide development,
novel target can be discovered from the active lead compound,39 the novel lead
compound also can be found from the target or mode of action studies. 40 Our studies
discovered a kind of novel fungicide lead compound targeted at OSBP.
407
408
409
410
411
412
413
414
415
416
417
418
ABBREVIATIONS USED
OSBP, oxysterol-binding protein; TMS, tetramethylsilane; CDCl3, chloroform-d;
ACS Paragon Plus Environment

Page 21 of 42
Journal of Agricultural and Food Chemistry
419
DMSO-d6, dimethyl sulfoxide-d6; HRMS, high-resolution mass spectra; TLC, thin-
420
421
422
423
424
425
426
427
428
429
430
431
432
layer chromatography; THF, tetrahydrofuran; NaOH, sodium hydroxide;
CH2Cl2,
dichloromethane; EDCI, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride; HOBt, 1-Hydroxybenzotriazole; Et3N, Triethylamine; PDA, potato
dextrose agar; EC50, median effective concentration; DI, disease index; CE, control
efficacy; DMF, N,N-Dimethylformamide; TMM, Trimmed Means of Means; FDR,
false discovery rate; KEGG, kyoto encyclopedia of genes and genomes; GO, Gene
ontology; DGEs, differential expression genes.
ACKNOWLEDGMENT
The authors thank research team of Prof. Guillaume Drin for their help to provide us
the Osh4p prokaryotic expression vector. This work was supported in part by the
National Natural Science Foundation of China (No. 31571991 and No. 31872007), the
International Science & Technology Cooperation Program of China (No.
2014DFR41030), the Tianjin Natural Science Foundation (No. 18JCZDJC33500), and
the China Postdoctoral Science Foundation (No. 2017M611156).
433
434
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 22 of 42
435
436
Reference
1. Almughrabi, K. I.; Peters, R. D.; Platt, H. W.; Moreau, G.; Vikram, A.; Poirier, R.;
Macdonald, I. In-furrow applications of metalaxyl and phosphite for control of pink
rot (Phytophthora erythroseptica) of potato in new brunswick, Canada. Plant Dis.
2007, 91, 1305−1309.
437
438
439
440
441
442
443
444
445
446
447
448
449
450
451
452
453
454
455
456
457
2. Silva, P. V.; Vélez, M. L.; Otaño, D. H.; Nuñez, C.; Greslebin, A. G. Action of fosetyl‐
Al and metalaxyl against Phytophthora austrocedri. Forest Pathol. 2016, 46, 54–66.
3. Chen, F.; Zhou, Q.; Xi, J.; Li, D. L.; Schnabel, G.; Zhan, J. Analysis of RPA190
revealed multiple positively selected mutations associated with metalaxyl resistance
in Phytophthora infestans. Pest Manage. Sci. 2018, 74, 1916−1924.
4. Jackson, K. L.; Yin, J.; Ji, P. Sensitivity of Phytophthora capsici on vegetable crops
in georgia to mandipropamid, dimethomorph, and cyazofamid. Plant Dis. 2012, 96,
1337−1342.
5. Shin, K. H.; Kim, D. S.; Chun, S. J.; Park, E. W. Improvement in fungicidal activity
of ethaboxam by a non-ionic surfactant, polyoxyethylene cetyl ether. Plant Pathol.
J. 2006, 22, 303−308.
6. Kim, D. S.; Chun, S. J.; Jeon, J. J.; Lee, S. W.; Joe, G. H. Synthesis and fungicidal
activity of ethaboxam against oomycetes. Pest Manage. Sci. 2004, 60, 1007−1012.
7. Miao, J.; Dong, X.; Lin, D.; Wang, Q.; Liu, P.; Chen, F.; Du, Y.; Liu, X. Activity of
the novel fungicide oxathiapiprolin against plant-pathogenic oomycetes. Pest
Manage. Sci. 2016, 72, 1572−1577.
8. Bittner, R. J.; Mila, A. L. Efficacy and timing of application of oxathiapiprolin
against black shank of flue-cured tobacco. Crop Prot. 2017, 93, 9−18.
ACS Paragon Plus Environment

Page 23 of 42
Journal of Agricultural and Food Chemistry
458
9. Pasteris, R. J.; Hanagan, M. A., Fungicidal azocyclic amides. In US,
WO/2013/0261154, 2014.
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
10. Ji, P.; Csinos, A. S.; Hickman, L. L.; Hargett, U. Efficacy and application methods
of oxathiapiprolin for management of black shank on tobacco. Plant Dis. 2014, 98,
1551−1554.
11. Ji, P.; Csinos, A. S. Effect of oxathiapiprolin on asexual life stages of Phytophthora
capsici and disease development on vegetables. Ann. Appl. Biol. 2015, 166,
229−235.
12. Pasteris, R. J.; Hanagan, M. A.; Bisaha, J. J.; Finkelstein, B. L.; Hoffman, L. E.;
Gregory, V.; Andreassi, J. L.; Sweigard, J. A.; Klyashchitsky, B. A.; Henry, Y. T.
Discovery of oxathiapiprolin, a new oomycete fungicide that targets an oxysterol
binding protein. Bioorg. Med. Chem. 2015, 24, 354−361.
13. Miao, J. Q.; Li, X. H.; Lin, D.; Liu, X. L.; Tyler, B. M. Oxysterol-binding protein-
related protein 2 is not essential for Phytophthora sojae based on CRISPR/Cas9
deletions. Environ. Microbiol. Rep. 2018, 10, 293−298.
14. Wyenandt, C. A.; Simon, J. E.; Pyne, R. M.; Homa, K.; McGrath, M. T.; Zhang,
S.; Raid, R. N.; Ma, L. J.; Wick, R.; Guo, L.; Madeiras, A. Basil downy mildew
(Peronospora belbahrii): discoveries and challenges relative to its control.
Phytopathology. 2015, 105, 885−849.
15. Fan, J.; Urban, M.; Parker, J. E.; Brewer, H. C.; Kelly, S. L.; Hammond-Kosack,
K. E.; Fraaije, B. A.; Liu, X.; Cools, H. J. Characterization of the sterol 14α-
demethylases of fusarium graminearum identifies a novel genus-specific CYP51
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 24 of 42
480
481
482
483
484
485
486
487
488
489
490
491
492
493
494
495
496
497
498
499
500
501
function. New Phytol. 2013, 198, 821−835.
16. Patterson, G. W.; Nes, W. D. Physiology and biochemistry of sterols. In American
Oil Chemists' Society, 1991.
17. Parks, L. W.; Casey, W. M. Physiological implications of sterol biosynthesis in
yeast. Annu. Rev. Microbiol. 1995, 49, 95−116.
18. Oh, Y.; Donofrio, N.; Pan, H.; Coughlan, S.; Brown, D. E.; Meng, S.; Mitchell, T.;
Dean, R. A. Transcriptome analysis reveals new insight into appressorium
formation and function in the rice blast fungus magnaporthe oryzae. Genome Biol.
2008, 9, 1−24.
19. Nakayama, H.; Nakayama, N.; Arisawa, M.; Aoki, Y. In vitro and in vivo effects
of 14α-demethylase (ERG11) depletion in candida glabrata. Antimicrob. Agents
Chemother. 2001, 45, 3037−3045.
20. Kaneshiro, E. S.; Johnston, L. Q.; Nkinin, S. W.; Romero, B. I.; Giner, J. L. Sterols
of saccharomyces cerevisiae erg6 knockout mutant expressing the pneumocystis
carinii S‐adenosylmethionine: Sterol C‐24 methyltransferase. J. Eukaryotic
Microbiol. 2015, 62, 298−306.
21. Wu, Q. F.; Zhao, B.; Fan, Z. J.; Zhao, J. B.; Guo, X. F.; Yang, D. Y.; Zhang, N. L.;
Yu, B.; Kalinina, T.; Glukhareva, T. Design, synthesis and fungicidal activity of
isothiazole–thiazole derivatives. RSC Adv., 2018, 8, 39593-39601.
22. Chen, L.; Guo, X. F.; Fan, Z. J.; Zhang, N. L.; Zhu, Y. J.; Zhang, Z. M.; Khazhieva,
I.; Yurievich, M. Y.; Belskaya, N. P.; Bakulev, V. A. Synthesis and fungicidal
activity of 3,4-dichloroisothiazole based strobilurins as potent fungicide candidates.
ACS Paragon Plus Environment

Page 25 of 42
Journal of Agricultural and Food Chemistry
502
RSC Adv. 2017, 7, 3145−3151.
503
504
505
506
507
508
509
510
511
512
513
514
515
516
517
518
519
520
521
522
523
23. Chen, L.; Zhao, B.; Fan, Z.; Liu, X.; Wu, Q.; Li, H.; Wang, H. Synthesis of novel
3,4-chloroisothiazole-based imidazoles as fungicides and evaluation of their mode
of action. J. Agric. Food Chem. 2018, 66, 7319−7327.
24. Shimizu, T.; Osako, K.; Nakata, T. Efficient method for preparation of N-methoxy-
N-methyl amides by reaction of lactones or esters with Me2AlCl-MeONHMe·HCl.
Tetrahedron Lett. 1997, 38, 2685−2688.
25. Bellale, E.; Naik, M.; V, V. B.; Ambady, A.; Narayan, A.; Ravishankar, S.;
Ramachandran, V.; Kaur, P.; Mclaughlin, R.; Whiteaker, J. Diarylthiazole: An
antimycobacterial scaffold potentially targeting PrrB-PrrAtwo-component system.
J. Med. Chem. 2012, 57, 6572−6582.
26. Guan, A.; Wang, M.; Yang, J.; Wang, L.; Xie, Y.; Lan, J.; Liu, C. The discovery of
a new fungicide candidate through lead optimization of pyrimidinamine
derivatives and their activity against cucumber downy mildew. J. Agric. Food
Chem. 2017, 65, 10829−10835.
27. Chen, L.; Zhu, Y. J.; Fan, Z. J.; Guo, X. F.; Zhang, Z. M.; Xu, J. H.; Song, Y. Q.;
Yurievich, M. Y.; Belskaya, N. P.; Bakulev, V. A. Synthesis of 1,2,3-thiadiazole
and thiazole based strobilurins as potent fungicide candidates. J. Agric. Food
Chem. 2017, 65, 745−751.
28. Land, H.; Humble, M. S. YASARA: A tool to obtain structural guidance in
biocatalytic investigations. Methods Mol. Biol. 2018, 1685, 43−67.
29. Muzio, E. D.; Toti, D.; Polticelli, F. DockingApp: a user friendly interface for
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 26 of 42
524
525
526
527
528
529
530
531
532
533
534
535
536
537
538
539
540
541
542
543
544
545
facilitated docking simulations with AutoDock Vina. J. Comput.-Aided Mol. Des.
2017, 31, 1−6.
30. Morris, G. M.; Goodsell, D. S.; Huey, R.; Olson, A. J. Distributed automated
docking of flexible ligands to proteins: parallel applications of AutoDock 2.4. J.
Mol. Recognit. 1996, 10, 293−304.
31. Seeliger, D.; de Groot, B. L. Ligand docking and binding site analysis with PyMOL
and Autodock/Vina. J. Comput.-Aided Mol. Des. 2010, 24, 417−422.
32. Von Filseck, J. M.; Vanni, S.; Mesmin, B.; Antonny, B.; Drin, G. A
phosphatidylinositol-4-phosphate powered exchange mechanism to create a lipid
gradient between membranes. Nat. Commun. 2015, 6, 6671.
33. Kazzouli, S. D. E.; Berteina-Raboin, S.; Mouaddib, A.; Guillaumet, G. Solid
support synthesis of 2,4-disubstituted thiazoles and aminothiazoles. Tetrahedron
Lett. 2002, 43, 3193−3196.
34. Manik, M. K.; Yang, H.; Tong, J.; Im, Y. J. Structure of yeast OSBP-related
protein Osh1 reveals key determinants for lipid transport and protein targeting at
the nucleus-vacuole junction. Structure 2017, 25, 617−629.
35. Chung, J.; Torta, F.; Masai, K.; Lucast, L.; Czapla, H.; Tanner, L.
B.; Narayanaswamy, P.; Wenk, M. R.; Nakatsu, F.; De Camilli, P.
PI4P/phosphatidylserine countertransport at ORP5- and ORP8-mediated ER–
plasma membrane contacts. Science 2015, 349, 428-432.
36. Raychaudhuri, S.; Prinz, W. A. The diverse functions of oxysterol-binding proteins.
Annu. Rev. Cell Dev. Biol. 2010, 26, 157−177.
ACS Paragon Plus Environment

Page 27 of 42
Journal of Agricultural and Food Chemistry
546
37. Brown, M. S.; Goldstein, J. L. Multivalent feedback regulation of HMG CoA
reductase, a control mechanism coordinating isoprenoid synthesis and cell growth.
J. Lipid Res. 1980, 21, 505−517.
547
548
549
550
551
552
553
554
555
556
557
558
559
560
38. Lepesheva, G. I.; Waterman, M. R., Sterol 14alpha-demethylase cytochrome P450
(CYP51), a P450 in all biological kingdoms. Biochim. Biophys. Acta-Gen. Subj.
2007, 1770, 467−477.
39. Zhao, B.; Fan, S. J.; Fan, Z. J.; Wang, H. X.; Zhang, N. L.; Guo, X. F.; Yang, D.
Y.; Wu, Q. F.; Yu, B.; Zhou, S. Discovery of pyruvate kinase as a novel target of
new fungicide candidate 3-(4-methyl-1,2,3-thiadiazolyl)-6-trichloromethyl-
[1,2,4]-triazolo-[3,4-b][1,3,4]- thiadizole. J. Agric. Food Chem. 2018, 66, 12439-
12452.
40. Zhu, Y. J.; Wu, Q. F.; Fan, Z. J.; Huo, J. Q.; Zhang, J. L.; Zhao, B.; Chen, L.; Qian,
X. L.; Ma, D. J.; Wang, D. W. Synthesis, bioactivity and mode of action of 5A5B6C
tricyclic spirolactones as novel antiviral lead compounds. Pest Manag. Sci. 2019,
75, 292-301.
ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry
Page 28 of 42
Figure 1. Discovery process of oxathiapiprolin
ACS Paragon Plus Environment

Page 29 of 42
Journal of Agricultural and Food Chemistry
Figure 2. Design process of the target compounds
ACS Paragon Plus Environment