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Agricultural and Environmental Chemistry
Synthesis of Novel 3,4-Chloroisothiazole-Based Imidazoles as Fungicides and Evaluation of Their Mode of Action Lai Chen, Bin Zhao, Zhijin Fan, Xiumei Liu, Qifan Wu, Hongpeng Li, and Haixia Wang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b02332 • Publication Date (Web): 18 Jun 2018 Downloaded from http://pubs.acs.org on June 19, 2018
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Journal of Agricultural and Food Chemistry
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Synthesis of Novel 3,4-Chloroisothiazole-Based Imidazoles as
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Fungicides and Evaluation of Their Mode of Action
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Lai Chen†, Bin Zhao*,†, Zhijin Fan*,†,‡, Xiumei Liu†, Qifan Wu†, Hongpeng Li†,
4
Haixia Wang†
5 6
†
State Key Laboratory of Elemento-Organic Chemistry, College of Chemistry, Nankai University, No. 94, Weijin Road, Nankai District, Tianjin 300071, P. R. China
7 8 9
‡
Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, No. 94, Weijin Road, Nankai District, Tianjin 300071, P. R. China.
10 11
*correspondence:
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Bin Zhao, Tel: +86-23499464, E-mail:
[email protected] 13
Zhijin Fan, Tel: +86-23499464, E-mail:
[email protected] ACS Paragon Plus Environment
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Abstract
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A molecular design approach was used in our laboratory to guide the development of
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imidazole-based fungicides.
17
studies
18
3,4-dichloroisothiazole-based imidazoles showed great potential.
19
compounds were then rationally designed, synthesized, characterized, and their
20
antifungal activities were evaluated.
21
as (R)-11, (R)-12, and (S)-11 have commendable, broad-spectrum antifungal activities
22
that are comparable to those of commercial products.
23
microscopy observations, the imidazole derivatives affect fungal cell wall formation
24
through the inhibition of the BcCYP51 expression system.
25
suggest that the mode of action of these imidazole compounds is similar to that of
26
tioconazole and imazalil.
27
not only practical but productive.
targeting
the
Based on homology modeling and molecular docking
cytochrome
P450-dependent
sterol
14α-demethylase, Several such
Bioassay results showed that compounds such
Based on Q-PCR testing and
These findings strongly
This report indicates that this molecular design strategy is
28 29
Keywords: BcCYP51, Fungicide, 3,4-Dichloroisothiazole, Imidazole, Mode of action
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Introduction
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Producing enough food for our growing population and keeping up with the
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increasingly strict safety regulations for consumers and the environment are a great
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challenge for modern agriculture.1-3
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effective tools for increasing both crop quality and quantity while reducing labor
35
costs.4 Smarter approaches are needed for identifying more effective agrochemicals
36
that can meet the increasing market demands and environment protection
37
requirements.5
38
Agrochemicals have been one of the most
Starting with heterocyclic compounds that have previously played important
39
roles in agrochemicals is a logical approach.6-9
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compounds have been widely used as antifungal agents (imazalil) for plant protection
41
and for the protection of animal health (tioconazole, econazole and miconazole)
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(Figure 1).10-12
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ergosterol biosynthesis, thus limiting fungal growth by affecting their cytochrome
44
P450 sterol 14α-demethylase.
45
efficiently binds with the pro-heme iron in the enzyme.13
46
Over a dozen imidazole-based
It is commonly believed that imidazole-based compounds can inhibit
Further studies have suggested that the imidazole ring
Heterocycles have also been widely used to improve biological activity in the
47
optimization of lead compounds in agrochemical studies.14,15
48
aromatic frameworks, such as 3,4-dichloroisothiazole, which contains N and S atoms,
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have potentially inducing plant defense responses, antifungal, antiviral and antitumor
50
activities.16,17
51
heterocyclic analogs such as isotianil and dichlobentiazox were successfully
Therefore, it could be a potential bioactive scaffold.
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Five-membered
In fact, several
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developed as novel fungicides for rice blast management by inducing plant defense
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responses.17,18
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We have designed and synthesized a series of novel 3,4-dichloroisothazoles to
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obtain novel fungicides for controlling plant pathogens such as Botrytis cinerea and to
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conveniently study their modes of action by homology modeling and molecular
57
docking using B. cinerea cytochrome P450-dependent sterol 14α-demethylase as the
58
target and by combining 2,4-dichlorobenzene imidazole fungicide lead compounds
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with 3,4-dichloroisothiazole, a substructure with systemic acquired resistance activity.
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Specific bioassays were used to verify the validity of our molecular design models
61
(Figure 1).
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Materials and Methods
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Equipment and materials: Melting points of the new compounds were
64
determined in an X-4 melting point apparatus (Beijing Tech Instruments Co., Beijing,
65
China).
66
at 400 MHz and 101 MHz (Bruker, Switzerland).
67
(HRMS) were obtained by using a 7.0T FTICR-MS instrument (Varian, Palo, Alto,
68
CA).
69
Tokyo, Japan).
70
spectrophotometer (Agilent, Australia).
71
341MC polarimeter (Perkin-Elmer, Norwalk, CT).
72 73
1
H NMR and 13C NMR spectra were taken on an Avance 400 spectrometer High-resolution mass spectra
Crystal structure was recorded on a Saturn 724 CCD diffractometer (Rigaku, UV spectra were determined with a Cary 5000 UV Optical rotations were measured on a
General Procedure for the Synthesis of Compound 2. synthesized as described by Chen et al.19
Compound 1 was
Compound 2 was prepared from
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corresponding compound 1.
To a stirred solution of compound 2 (0.60 g, 3.06 mmol)
75
in a solution of 33% hydrogen bromide in acetic acid (5 mL) was added pyridinium
76
tribromide (1.08 g, 3.37 mmol).20
77
h and then poured into ice-cold water; sodium bicarbonate was added until the
78
mixture became clear and colorless.
79
acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and
80
filtered.
81
gel column (203 mm × 26 mm) eluted with ethyl acetate/petroleum ether (b.p.
82
60-90 °C) (1:20, v/v) and afforded compound 2 in a 95% yield.
83
52-53 °C; 1H NMR (400 MHz, CDCl3) δ 4.47 (s, 2H, CH2);
84
CDCl3) δ 182.30 (s), 156.43 (s), 150.49 (s), 123.25 (s), 32.26 (s); HRMS (m/z) calcd
85
for C5H2BrCl2NOS (M-H)+: 271.8418, found 271.8344.
The mixture was stirred at room temperature for 3
The organic layer was extracted with ethyl
The solvent was evaporated, and the residue was then purified on a silica
2: white solid; m.p.: 13
C NMR (101 MHz,
86
General Procedure for the Synthesis of Compounds 3a and 3b.21
87
(+)-Diisopinocampheyl chloroborane (DIP-Cl) (3.40 mmol, dissolved in 2 mL of dry
88
heptane) was added to compound 2 (0.47 g, 1.70 mmol, dissolved in 15 mL of dry
89
tetrahydrofuran) under argon at -20 °C for 2 h.
90
room temperature.
91
the (+)-α-pinene was removed under a high vacuum at room temperature overnight.
92
The resulting viscous colorless oil was then purified on a silica gel column (203 mm
93
× 26 mm) eluted with ethyl acetate/petroleum ether (b.p. 60-90 °C) (1:20, v/v) and
94
afforded compound 3a in a 50-60% yield.
95
+38.9 (c 1.00, CH3OH); 1H NMR (400 MHz, CDCl3) δ 5.21 (dd, J = 7.8 Hz, 3.0 Hz,
The solution was stirred for 16 h at
The tetrahydrofuran and heptane were removed in vacuo, and
3a: white solid; m.p.: 57-58 °C; [ɑ]24 = D
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1H, CH), 3.82 (dd, J = 10.8, 3.0 Hz, 1H, CH2), 3.53 (dd, J = 10.8, 7.8 Hz, 1H, CH2);
97
13
98
HRMS (m/z) calcd for C5H4BrCl2NOS (M-H)+: 273.8574, found 273.8501.
C NMR (101 MHz, CDCl3) δ 163.23 (s), 148.55 (s), 118.08 (s), 69.06 (s), 36.29 (s);
The preparation of compound 3b followed the same steps as the above
99 100
procedure, except the reducing agent was (-)-DIP-Cl.
101
50-60%; m.p.: 57-58 °C; [ɑ]24 = -38.2 (c 0.90, CH3OH); 1H NMR (400 MHz, D
102
CDCl3) δ 5.28 (dd, J = 7.8, 3.0 Hz, 1H, OCH), 3.88 (dd, J = 10.8, 3.0 Hz, 1H, CH2),
103
3.60 (dd, J = 10.8, 7.8 Hz, 1H, CH2);
104
148.57 (s), 118.05 (s), 68.90 (s), 36.07 (s); HRMS (m/z) calcd for C5H4BrCl2NOS
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(M-H)+: 273.8574, found 273.8501.
13
3b: white solid; yield,
C NMR (101 MHz, CDCl3) δ 163.87 (s),
General Procedure for the Synthesis of Compounds 4a and 4b.
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Potassium
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carbonate (0.36 g, 2.62 mmol) was added to a solution of compound 3a (0.61 g, 2.19
108
mmol) in acetone.
109
filtered.
110
gel column (203 mm × 26 mm) eluted with ethyl acetate/petroleum ether (b.p.
111
60-90 °C) (1:20, v/v) and afforded compound 4a in a 50-60% yield.
112
[ɑ]24D = +23.3 (c 1.00, CH3OH); 1H NMR (400 MHz, CDCl3) δ 4.20 (dd, J = 3.9, 2.3
113
Hz, 1H, CH), 3.33 (dd, J = 5.3, 3.9 Hz, 1H, CH2), 2.96 (dd, J = 5.3, 2.3 Hz, 1H, CH2);
114
13
115
HRMS (m/z) calcd for C5H3Cl2NOS (M-H)+: 193.9312, found 193.9242.
116 117
The mixture was stirred at room temperature for 20 h and then
The solvent was evaporated, and the residue was then purified on a silica
4a: colorless oil;
C NMR (101 MHz, CDCl3) δ 161.30 (s), 148.41 (s), 120.77 (s), 52.10 (s), 47.36 (s);
The preparation of compound 4b followed the same steps as described above, except compound 3b was used as the starting material.
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4b: colorless oil; yield,
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50-60%; [ɑ]24 = -23.7 (c 1.00, CH3OH); 1H NMR (400 MHz, CDCl3) δ 4.11 (dd, J = D
119
3.9, 2.4 Hz, 1H, CH), 3.23 (dd, J = 5.3, 3.9 Hz, 1H, CH2), 2.87 (dd, J = 5.3, 2.4 Hz,
120
1H, CH2); 13C NMR (101 MHz, CDCl3) δ 161.26 (s), 148.55 (s), 120.85 (s), 52.19 (s),
121
47.44 (s); HRMS (m/z) calcd for C5H3Cl2NOS (M-H)+: 193.9312, found 193.9242.
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General Procedure for the Synthesis of Compounds 5a and 5b.
Potassium
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carbonate (0.36 g, 2.62 mmol) and imidazole (0.18 g, 2.62 mmol) were added to a
124
solution of compound 4a (0.42 g, 1.75 mmol) in 5 mL of N,N-dimethyl formamide.
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The mixture was stirred at room temperature for 20 h and then washed with water; the
126
organic layer was extracted with ethyl acetate, washed with saturated brine, dried over
127
anhydrous sodium sulfate, and filtered.
128
was then purified on a silica gel column (203 mm × 26 mm) eluted with ethyl
129
acetate/petroleum ether (b.p. 60-90 °C) (1:20, v/v) and afforded compound 5a in a
130
yield 80-86%.
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CH3OH); 1H NMR (400 MHz, DMSO-d6) δ 7.55 (s, 1H, imidazole-H), 7.09 (s, 1H,
132
imidazole-H), 6.88 (s, 1H, imidazole-H), 5.28 (dd, J = 7.1, 3.0 Hz, 1H, OCH),
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4.39-4.31 (m, 2H, CH2);
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138.39 (s), 128.50 (s), 120.76 (s), 116.63 (s), 68.20 (s), 50.83 (s); HRMS (m/z) calcd
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for C8H7Cl2N3OS (M+H)+: 263.9687, found 263.9755.
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The solvent was evaporated, and the residue
5a: colorless crystal; m.p.: 156-157 °C; [ɑ] 24 = +45.4 (c 1.00, D
13
C NMR (101 MHz, DMSO-d6) δ 167.84 (s), 147.21 (s),
The preparation of the compound 5b followed the same steps as described above,
137
except compound 4b was used as the starting material.
138
50-60%; m.p.: 156-157 °C; [ɑ]24 = -44.9 (c 1.00, CH3OH); 1H NMR (400 MHz, D
139
DMSO-d6) δ 7.53 (s, 1H, imidazole-H), 7.08 (s, 1H, imidazole-H), 6.86 (s, 1H,
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5b: colorless crystal; yield,
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imidazole-H), 5.25 (s, 1H, OCH), 4.40–4.32 (m, 1H, NCH2), 4.28 (dd, J = 14.3, 5.9
141
Hz, 1H, NCH2); 13C NMR (101 MHz, DMSO-d6) δ 167.02 (s), 147.56 (s), 137.44 (s),
142
127.70 (s), 121.07 (s), 116.94 (s), 67.93 (s), 50.75 (s); HRMS (m/z) calcd for
143
C8H7Cl2N3OS (M+H)+: 263.9687, found 263.9755.
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General Procedure for the Synthesis of Compounds (R)-1~12 and (S)-1, Sodium hydroxide (1 mol/L, 0.5 mL) and the desired amount of
145
(S)-11~12.
146
tetrabutylammonium bromide (TBAB) catalyst was added to a solution of R1CH2Br
147
(0.33 mmol) and compounds 5 (0.08 g, 0.30 mmol) in tetrahydrofuran.
148
was stirred at room temperature for 3 h.
149
was then purified on a silica gel column (203 mm × 26 mm) eluted with ethyl
150
acetate/petroleum ether (b.p. 60-90 °C) (1:20, v/v) and afforded compounds R and S
151
(Figure 3).
152
the target compounds (R)-1~12 and (S)-1, (S)-11~12 were as follows:
The solution
The solvent was evaporated, and the residue
The yields, physical properties, 1H NMR, 13C NMR, and HRMS data of
153
Data for (R)-1: white solid; yield, 89%; m.p.: 40-41 °C; [ɑ]24 = +6.5 (c 1.00, D
154
CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.42 (s, 1H, imidazole-H), 6.97 (s, 1H,
155
imidazole-H), 6.87 (s, 1H, imidazole-H), 5.72-5.58 (m, 1H, CH=CH2), 5.16 (s, 1H,
156
CH=CH2)5.13 (d, J = 3.9 Hz, 1H, CH=CH2), 4.83 (dd, J = 4.7, 2.4 Hz, 1H, OCH),
157
4.26 (dd, J = 5.9, 2.9 Hz, 1H, OCH2), 4.13 (dd, J = 14.5, 7.2 Hz, 1H, OCH2), 4.02 (dd,
158
J = 11.9 Hz, J = 2.9 Hz, 1H, NCH2), 3.86 (dd, J = 11.9, 5.9 Hz, 1H, NCH2); 13C NMR
159
(101 MHz, CDCl3) δ 161.82 (s), 148.56 (s), 137.79 (s), 132.49 (s), 129.43 (s), 119.79
160
(s), 119.26 (s), 118.86 (s), 75.20 (s), 71.91 (s), 50.35 (s); UV (CH3OH): λmax (log ε)
161
280 (2.67), 239 (2.00) nm; HRMS (m/z) calcd for C11H11Cl2N3OS (M+H)+: 304.0000,
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found 304.0074.
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Data for (R)-2: white solid; yield, 67%; m.p.: 49-50 °C; [ɑ]24 = +12.9 (c 0.67, D
164
CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.24 (s, 1H, imidazole-H), 6.79 (s, 1H,
165
imidazole-H), 6.69 (s, 1H, imidazole-H), 4.94 (dd, J = 6.6, 2.9 Hz, 1H, OCH), 4.14
166
(dd, J = 14.7, 2.8 Hz, 1H, OCH2), 4.06–3.96 (m, 2H, OCH2 and NCH2), 3.90 (dd, J =
167
16.1, 2.3 Hz, 1H, NCH2), 2.23 (t, J = 2.2 Hz, 1H, C≡CH);
168
CDCl3) δ 160.62 (s), 148.70 (s), 137.79 (s), 129.54 (s), 119.88 (s), 119.25 (s), 77.27
169
(s), 76.75 (s), 74.86 (s), 58.15 (s), 50.24 (s); UV (CH3OH): λmax (log ε) 277 (2.75),
170
242 (2.78), 239 (2.60) nm; HRMS (m/z) calcd for C11H9Cl2N3OS (M+H)+: 301.9843,
171
found 301.9919.
13
C NMR (101 MHz,
172
Data for (R)-3: white solid; yield, 67%; m.p.: 78-79 oC; [ɑ]24 = +37.5 (c 0.13, D
173
CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.42 (s, 1H, imidazole-H), 7.30-7.23 (s, 3H,
174
Ph-H), 7.11 (d, J = 2.9 Hz, 2H, Ph-H), 6.98 (s, 1H, imidazole-H), 6.83 (s, 1H,
175
imidazole-H), 4.86 (dd, J = 7.2, 2.0 Hz, 1H, OCH), 4.56 (d, J = 11.4, 1H, OCH2), 4.34
176
(d, J = 11.4, 1H, OCH2), 4.24 (dd, J = 14.6 Hz, 2.0 Hz, 1H, NCH2), 4.13 (dd, J = 14.4,
177
7.2 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 161.57 (s), 148.69 (s), 137.89 (s),
178
135.72 (s), 129.70 (s), 128.69 (s), 128.10 (s), 119.77 (s), 119.21 (s), 75.28 (s), 73.04
179
(s), 50.48 (s); UV (CH3OH): λmax (log ε) 279 (2.84), 242 (2.60) nm; HRMS (m/z)
180
calcd for C15H13Cl2N3OS (M+H)+: 354.0156 found 354.0227.
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Data for (R)-4: white solid; yield, 68%; m.p.: 56-57 oC; [ɑ]24 = +24.4 (c 0.50, D
182
CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.72 (s, 1H, imidazole-H), 7.37 (dd, J = 8.2,
183
5.5 Hz, 2H, Ph-H), 7.27 (s, 1H, imidazole-H), 7.24 (t, J = 8.6 Hz, 2H, Ph-H), 7.13 (s,
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1H, imidazole-H), 5.14 (dd, J = 7.5, 2.9 Hz, 1H, OCH), 4.81 (d, J = 11.4 Hz, 1H,
185
OCH2), 4.59 (d, J = 11.4 Hz, 1H, OCH2), 4.54 (dd, J = 14.6, 2.9 Hz, 1H, NCH2), 4.44
186
(dd, J = 14.6, 7.5 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 162.74 (d, J = 247.4
187
Hz), 161.34 (s), 148.78 (s), 137.85 (s), 131.56 (d, J = 3.1 Hz), 129.95 (d, J = 8.4 Hz),
188
129.71 (s), 119.66 (s), 119.33 (s), 115.70 (d, J = 21.7 Hz), 75.24 (s), 72.26 (s), 50.49
189
(s); UV (CH3OH): λmax (log ε) 277 (2.94), 242 (2.99) nm, 239 (2.96); HRMS (m/z)
190
calcd for C15H12Cl2FN3OS (M+H)+: 372.0062, found 372.0133.
191
Data for (R)-5: colorless crystal; yield, 85%; m.p.: 68-69 oC; [ɑ]24D = +60.5 (c
192
0.50, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.63 (s, 1H, imidazole-H), 7.43 (d, J =
193
7.3 Hz, 2H, Ph-H), 7.23 (d, J = 7.5 Hz, 2H, Ph-H), 7.18 (s, 1H, imidazole-H), 7.04 (s,
194
1H, imidazole-H), 5.06 (d, J = 7.0 Hz, 1H, OCH), 4.73 (d, J = 11.5 Hz, 1H, OCH2),
195
4.53–4.43 (m, 2H, OCH2 and NCH2), 4.35 (dd, J = 14.5, 7.3 Hz, 1H, NCH2);
196
NMR (101 MHz, CDCl3) δ 161.19 (s), 148.78 (s), 137.84 (s), 134.38 (s), 134.24 (s),
197
129.69 (s), 129.33 (s), 128.92 (s), 119.71 (s), 119.40 (s), 75.36 (s), 72.13 (s), 50.45 (s);
198
UV (CH3OH): λmax (log ε) 277 (2.95), 242 (2.97) nm; HRMS (m/z) calcd for
199
C15H12Cl3N3OS (M+3H)+: 389.9767, found 389.9813.
13
C
200
Data for (R)-6: colorless crystal; yield, 69%; m.p.: 81-82 oC; [ɑ]24D = +16.8 (c
201
1.00, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.54 (d, J = 7.7 Hz, 2H, Ph-H), 7.47 (s,
202
1H, imidazole-H), 7.23 (d, J = 7.5 Hz, 2H, Ph-H), 7.00 (s, 1H, imidazole-H), 6.86 (s,
203
1H, imidazole-H), 4.90 (d, J = 7.1 Hz, 1H, OCH), 4.63 (d, J = 11.9 Hz, 1H, OCH2),
204
4.42 (d, J = 11.9 Hz, 1H, OCH2), 4.31 (d, J = 14.5 Hz, 1H, NCH2), 4.19 (dd, J = 14.5,
205
7.4 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 160.86 (s), 148.90 (s), 139.76 (s),
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137.84 (s), 130.76 (s), 130.43 (s), 129.67 (s), 127.91 (s), 125.69 (q, J = 3.6 Hz),
207
119.69 (s), 119.58 (s), 75.76 (s), 72.07 (s), 50.48 (s); UV (CH3OH): λmax (log ε) 279
208
(2.78), 239 (2.52) nm; HRMS (m/z) calcd for C16H12Cl2F3N3OS (M+H)+: 422.0030,
209
found 422.0101.
210
Data for (R)-7: colorless crystal; yield, 64%; m.p.: 44-45 oC; [ɑ]24 = +8.5 (c 0.40, D
211
CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.38 (s, 1H, imidazole-H), 7.25 (d, J = 7.0
212
Hz, 2H, Ph-H), 7.02 (d, J = 7.2 Hz, 2H, Ph-H), 6.92 (s, 1H, imidazole-H), 6.79 (s, 1H,
213
imidazole-H), 4.83 (d, J = 6.9 Hz, 1H, OCH), 4.48 (d, J = 11.1 Hz, 1H, OCH2), 4.28
214
(d, J = 11.1 Hz, 1H, OCH2), 4.19 (d, J = 14.2 Hz, 1H, NCH2), 4.08 (dd, J = 14.2, 6.9
215
Hz, 1H, NCH2), 1.20 (s, 9H, t-Bu-H);
216
151.70 (s), 148.62 (s), 137.86 (s), 132.72 (s), 129.63 (s), 127.99 (s), 125.67 (s), 119.73
217
(s), 119.08 (s), 75.35 (s), 72.98 (s), 50.49 (s), 31.30 (s); UV (CH3OH): λmax (log ε) 279
218
(2.70), 242 (2.29) nm; HRMS (m/z) calcd for C19H21Cl2N3OS (M+H)+: 410.0782,
219
found 410.0862.
13
C NMR (101 MHz, CDCl3) δ 161.79 (s),
220
Data for (R)-8: white solid; yield, 91%; m.p.: 88-89 oC; [ɑ]24 = +29.0 (c 0.90, D
221
CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.48 (s, 1H, imidazole-H), 7.38 (d, J = 1.8
222
Hz, 1H, Ph-H), 7.25 (dd, J = 8.3, 1.9 Hz, 1H, Ph-H), 7.18 (d, J = 8.3 Hz, 1H, Ph-H),
223
7.04 (s, 1H, imidazole-H), 6.91 (s, 1H, imidazole-H), 5.01 (dd, J = 7.1, 3.0 Hz, 1H,
224
OCH), 4.66 (d, J = 12.2 Hz, 1H, OCH2), 4.54 (d, J = 12.2 Hz, 1H, OCH2), 4.37 (dd, J
225
= 14.6, 3.0 Hz, 1H, NCH2), 4.25 (dd, J = 14.6, 7.1 Hz, 1H, NCH2);
226
MHz, CDCl3) δ 160.92 (s), 148.74 (s), 137.81 (s), 134.96 (s), 134.07 (s), 132.26 (s),
227
130.43 (s), 129.76 (s), 129.44 (s), 127.50 (s), 119.63 (s), 119.33 (s), 76.23 (s), 69.58
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(s), 50.36 (s); UV (CH3OH): λmax (log ε) 278 (2.72), 242 (2.56) nm; HRMS (m/z)
229
calcd for C15H11Cl4N3OS (M+H)+: 421.9377, found 421.9451.
230
Data for (R)-9: colorless crystal; yield, 61%; m.p.: 64-65 oC; [ɑ]24D = +53.2 (c
231
0.50, CH3OH); 1H NMR (400 MHz, CDCl3) δ 8.14 (d, J = 1.3 Hz, 1H, pyridine-H),
232
7.44–7.35 (m, 2H, pyridine-H and imidazole-H), 7.23 (d, J = 8.3 Hz, 1H, pyridine-H),
233
6.96 (s, 1H, imidazole-H), 6.84 (s, 1H, imidazole-H), 4.89 (dd, J = 7.5, 2.8 Hz, 1H,
234
OCH), 4.55 (d, J = 11.9 Hz, 1H, OCH2), 4.34 (d, J = 11.9 Hz, 1H, OCH2), 4.29 (dd, J
235
= 14.6, 2.8 Hz,1H, NCH2), 4.17 (dd, J = 14.6, 7.5 Hz, 1H, NCH2);
236
MHz, CDCl3) δ 160.49 (s), 151.63 (s), 148.95 (s), 148.89 (s), 138.39 (s), 137.78 (s),
237
130.44 (s), 129.79 (s), 124.56 (s), 119.74 (s), 119.65 (s), 75.89 (s), 69.53 (s), 50.39 (s);
238
UV (CH3OH): λmax (log ε) 281 (2.25), 239 (1.42) nm; HRMS (m/z) calcd for
239
C14H11Cl3N4OS (M+H)+: 388.9719, found 388.9790.
13
C NMR (101
240
Data for (R)-10: white solid; yield, 92%; m.p.: 147-149 oC; [ɑ]24 = +15.7 (c 0.66, D
241
CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.77 (d, J = 8.6 Hz, 1H, Ar-H), 7.67 (d, J =
242
1.8 Hz, 1H, Ar-H), 7.44 (s, 1H, imidazole-H), 7.35 (dd, J = 9.7, 2.8 Hz, 2H, Ar-H ),
243
7.03 (s, 1H, imidazole-H), 6.87 (s, 1H, imidazole-H), 4.98 (dd, J = 7.2, 3.1 Hz, 1H,
244
OCH), 4.81 (d, J = 12.2 Hz, 1H, OCH2), 4.63 (d, J = 12.2 Hz, 1H, OCH2), 4.29 (dd, J
245
= 14.6, 3.1 Hz, 1H, NCH2), 4.19 (dd, J = 14.6, 7.2 Hz, 1H, NCH2);
246
MHz, CDCl3) δ 160.97 (s), 148.71 (s), 138.73 (s), 138.63 (s), 137.62 (s), 130.90 (s),
247
130.41 (s), 129.44 (s), 128.45 (s), 125.34 (s), 123.91 (s), 121.48 (s), 119.74 (s), 119.43
248
(s), 75.18 (s), 66.61 (s), 50.42 (s); UV (CH3OH): λmax (log ε) 278 (2.79), 249 (2.52)
249
nm; HRMS (m/z) calcd for C17H12Cl3N3OS2 (M+H)+: 443.9487, found 443.9559.
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Data for (R)-11: white solid; yield, 44%; m.p.: 106-107 oC; [ɑ]24D = +4.0 (c 1.00,
251
CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.42 (s, 1H, imidazole-H), 7.04–6.95 (m, 2H,
252
thiophene-H and imidazole-H), 6.82 (s, 1H, imidazole-H), 6.70 (d, J = 5.3 Hz, 1H,
253
thiophene-H), 4.84 (d, J = 7.3 Hz, 1H, OCH), 4.49 (d, J = 11.8 Hz, 1H, OCH2), 4.37
254
(d, J = 11.8 Hz, 1H, OCH2), 4.22 (d, J = 14.2 Hz, 1H, NCH2), 4.11 (dd, J = 14.2, 7.3
255
Hz, 1H, NCH2);
256
132.95 (s), 129.67 (s), 129.23 (s), 127.46 (s), 123.76 (s), 119.61 (s), 119.21 (s), 75.38
257
(s), 65.03 (s), 50.43 (s); UV (CH3OH): λmax (log ε) 278 (2.99) nm; HRMS (m/z) calcd
258
for C13H10Cl3N3OS2 (M+H)+: 393.9331, found 393.9403.
13
C NMR (101 MHz, CDCl3) δ 161.30 (s), 148.68 (s), 137.79 (s),
259
Data for (R)-12: colorless crystal; yield, 99%; m.p.: 100-101 oC; [ɑ]24D = +48.3 (c
260
0.40, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.71 (t, J = 7.9 Hz, 3H,), 7.47 (s, 1H,
261
imidazole-H), 7.45–7.36 (m, 3H, naphthalene-H), 7.15 (d, J = 8.4 Hz, 1H,
262
naphthalene-H), 6.95 (s, 1H, imidazole-H), 6.79 (s, 1H, imidazole-H), 4.86 (dd, J =
263
7.2, 3.0 Hz, 1H, OCH), 4.68 (d, J = 11.7 Hz, 1H, OCH2), 4.44 (d, J = 11.7 Hz, 1H,
264
OCH2), 4.17 (dd, J = 14.6, 3.0 Hz, 1H, NCH2), 4.08 (dd, J = 14.6, 7.2 Hz, 1H, NCH2);
265
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266
(s), 133.07 (s), 129.47 (s), 128.74 (s), 128.01 (s), 127.75 (s), 127.20 (s), 126.48 (s),
267
125.47 (s), 119.83 (s), 119.31 (s), 75.02 (s), 67.16 (s), 50.47 (s); UV (CH3OH): λmax
268
(log ε) 277 (2.74), 243 (2.98) nm; HRMS (m/z) calcd for C19H15Cl2N3OS (M+H)+:
269
404.0313, found 404.0388.
C NMR (101 MHz, CDCl3) δ 161.51 (s), 148.72 (s), 137.86 (s), 133.21 (s), 133.14
270
Data for (S)-1: white solid; yield, 90%; m.p.: 40-41 °C; [ɑ]24 = -6.1 (c 0.90, D
271
CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.50 (s, 1H, imidazole-H), 7.05 (s, 1H,
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imidazole-H), 6.95 (s, 1H, imidazole-H), 5.71 (m, 1H, CH=CH2), 5.24 (s, 1H,
273
CH=CH2), 5.21 (d, J = 3.7 Hz, 1H, CH=CH2), 4.91 (d, J = 5.8 Hz, 1H, OCH), 4.35 (d,
274
J = 14.5 Hz, 1H, OCH2), 4.21 (dd, J = 14.5, 7.1 Hz, 1H, OCH2), 4.10 (d, J = 11.8, 1H,
275
NCH2), 3.94 (dd, J = 11.8, 5.8 Hz, 1H, NCH2); 13C NMR (101 MHz, CDCl3) δ 161.82
276
(s), 148.56 (s), 137.79 (s), 132.49 (s), 129.43 (s), 119.79 (s), 119.26 (s), 118.86 (s),
277
75.20 (s), 71.91 (s), 50.35 (s); UV (CH3OH): λmax (log ε) 280 (2.57), 239 (1.77) nm;
278
HRMS (m/z) calcd for C11H11Cl2N3OS (M+H)+: 304.0000, found 304.0074.
279
Data for (S)-11: white solid; yield, 37%; m.p.: 106-107 oC; [ɑ]24D = -4.3 (c 1.00,
280
CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.39 (s, 1H, imidazole-H), 7.01 (d, J = 5.6
281
Hz, 1H, thiophene-H), 6.96 (s, 1H, imidazole-H), 6.82 (s, 1H, imidazole-H), 6.70 (d, J
282
= 5.7 Hz, 1H, thiophene-H), 4.84 (dd, J = 7.3, 4.7 Hz, 1H, OCH), 4.48 (d, J = 11.9 Hz,
283
1H, OCH2), 4.37 (d, J = 11.9 Hz, 1H, OCH2), 4.21 (dd, J = 14.6, 4.7 Hz, 1H, NCH2),
284
4.11 (dd, J = 14.6, 7.3 Hz, 1H, NCH2);
285
148.70 (s), 137.80 (s), 132.94 (s), 129.70 (s), 129.25 (s), 127.45 (s), 123.75 (s), 119.59
286
(s), 119.22 (s), 75.40 (s), 65.05 (s), 50.46 (s); UV (CH3OH): λmax (log ε) 277 (2.96)
287
nm; HRMS (m/z) calcd for C13H10Cl3N3OS2 (M+H)+: 393.9331, found 393.9403.
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C NMR (101 MHz, CDCl3) δ 161.29 (s),
288
Data for (S)-12: colorless crystal; yield, 91%; m.p.: 100-101 oC; [ɑ]24D = -49.0 (c
289
0.40, CH3OH); 1H NMR (400 MHz, CDCl3) δ 7.83 (t, J = 8.9 Hz, 3H, naphthalene-H),
290
7.59 (s, 1H, imidazole-H), 7.55-7.48 (m, 3H, naphthalene-H), 7.27-7.25 (s, 1H,
291
naphthalene-H), 7.07 (s, 1H, imidazole-H), 6.91 (s, 1H, imidazole-H), 4.97 (dd, J =
292
7.2, 2.7 Hz, 1H, OCH), 4.81 (d, J = 11.7 Hz, 1H, OCH2), 4.57 (d, J = 11.7 Hz, 1H,
293
OCH2), 4.31 (dd, J = 14.5, 2.7 Hz, 1H, NCH2), 4.22 (dd, J = 14.6, 7.4 Hz, 1H, NCH2);
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(s), 129.66 (s), 128.75 (s), 128.00 (s), 127.75 (s), 127.24 (s), 126.48 (s), 125.45 (s),
296
119.77 (s), 119.34 (s), 75.10 (s), 59.19 (s), 50.57 (s); UV (CH3OH): λmax (log ε) 278
297
(2.61), 243 (2.86) nm; HRMS (m/z) calcd for C19H15Cl2N3OS (M+H)+: 404.0313,
298
found 404.0388.
299
Crystal Structure Determination of Compound (S)-12
300
C NMR (101 MHz, CDCl3) δ 161.48 (s), 148.78 (s), 137.89 (s), 133.26 (s), 133.10
A crystal of compound (S)-12 was obtained from methanol (Figure 4). All
301
measurements were made on a Saturn 724 CCD diffractometer (Rigaku, Tokyo, Japan)
302
with Mo Kα radiation (λ = 0.71073 Å).
303
collection. A total of 9788 integrated reflections were collected, and of those 4280
304
were unique in the range of 1.771° ≤ θ ≤ 27.934° with Rint = 0.0489 and Rsigma =
305
0.0457, and 3637 with I >2σ(I) were used in the subsequent refinements.
306
structure was solved by using the Olex2 software and the ShelXL-97 program
307
according to reported methods.22
308
anisotropically by full-matrix least-squares to give the final values of R=0.0446 and
309
wR = 0.1185 (w = 1/[σ2((Fo2)+(0.0741P)2+0.2676P], where P = (Fo2+2Fc2)/3 with
310
(∆/σ)max = 0.998 and S = 1.009. The hydrogen atoms were added according to
311
theoretical models. The X-ray crystal structure data of (S)-12 was submitted to
312
Cambridge Crystallographic Data Centre (CCDC 1838941).
313
Molecular Docking
The crystal was kept at 113 K during data
The
All non-hydrogen atoms were refined
314
The protein sequence of the cytochrome P450 14α-demethylase enzyme from B.
315
cinerea (BcCYP51) was obtained from the National Center for Biotechnology
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Information (NCBI).
The 3D structure of BcCYP51 was established based on the
317
crystal structure of the Saccharomyces cerevisiae CYP51 (PDB, 4 LXJ) by
318
homologous modeling using Modeler 9.13 software.23
319
evaluated by using Procheck,24 Verify3d,25 and Errat procedures.26
320
three evaluation systems, the modeled structure of BcCYP51 met all the criteria.
321
The docking of the receptor (BcCYP51) and ligands (designed target compounds,
322
tioconazole, and imazalil) was analyzed using the AutoDockVina program.27 We
323
followed the format used by Pymol and Ligpolt to show the docking results.28
324
Fungicidal Activity
The modeled structure was Under these
325
The fungicidal activities of the test compounds against Alternaria solani,
326
Botrytis cinerea, Rhizoctonia cerealis, Cercospora arachidicola, Pellicularia sasakii,
327
Gibberella zeae, Sclerotinia sclerotiorum, Physalospora piricola, Phytophthora
328
infestans (Mont) de Bary were evaluated in vitro at 50 µg/mL according to established
329
procedures.29
330
concentration, were further evaluated, and their median effective concentration (EC50)
331
values were established following reported procedures.29
Any compounds that exhibited 90% or better inhibition at this
332
In addition, the disease preventive activity of the selected compounds against B.
333
cinerea on cucumber were also tested at 100 µg/mL using the following procedure.30
334
Briefly, the selected compound (10 mg) was dissolved in 0.5 mL of N,N-dimethyl
335
formamide and then diluted with distilled water containing 0.1% Tween 80 to a final
336
concentration of 100 µg/mL.
337
plants and allowed to runoff.
This test solution was sprayed onto the cucumber Then, the plants were allowed to dry for 2 h.
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plants were sprayed with a blank solution without any test compound.
339
the cucumbers were inoculated with B. cinerea spores.
340
recorded after inoculation.
341
diseased control plants compared with that in the healthy control plants, wherein the
342
disease control is set as 100 and the healthy control as 0.
343
were used as the positive controls in this disease prevention experiment.
344
Microscopy Observations
345
Optical Microscopy.
After 24 h,
Diseased plants were
The results are relative to the percentage of disease in the
Imazalil and tioconazole
(R)-12 was selected as a representative test compound in
346
this study.
347
then the spore germination, mycelium growth, and degree of infection of B. cinerea
348
were observed.
349
The B. cinerea spores were treated with 1 µg/mL (R)-12 for 24 h, and
These effects were observed by using an optical microscope.
Transmission Electron Microscopy (TEM).
Ultrastructures on the growing
350
mycelium were prepared (24 h post treatment) for TEM analysis according to the
351
standard protocols.31
352
microscope (Hitachi, Tokyo, Japan) at an accelerating voltage of 60 kV.
353
RNA Extraction and Q-PCR Analysis
354
The effect was examined with an H7650 transmission electron
The B. cinerea was treated with compounds (S)-11, (R)-7 and (R)-12 at 1 µg/mL
355
for 24 h.
Imazalil and tioconazole were used as positive controls in this study.
356
RNA extract was then isolated by using an E.Z.N.A. fungal RNA Kit (Omega,
357
Norcross, GA), and mRNA was reverse transcribed into cDNA.
358
performed with Top Green Q-PCR Super Mix (TransStart, Beijing, China).
359
BcCYP51
(5΄-TATGTGGCAGTTGATGCG-3΄
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5΄-TCTGATGGAGAGGGAGTTTG-3΄)
tubulin
361
(5΄-TCTCCGTCAAGAGTGGGTTG-3΄
362
5΄-ACTGTGGCTACAGGGTACATT-3΄) were used for the fluorescence quantitative
363
PCR as references.32
364
Results and Discussion
365
Molecular Design and Chemistry
and
366
The affinity values determined in the docking analysis demonstrated that
367
tioconazole, (R)-7, and (R)-12 had the highest affinities between the ligands and
368
BcCYP51, suggesting these three compounds may have good antifungal activities.
369
Their docking models, performed by Pymol tools, are shown in Figure 2.
370
H-bonding was predicted between the compounds and the protein, and all tested
371
compounds were surrounded by residues (e.g., Phe130, Leu125, Val124, and Tyr122)
372
due to van der Waals interactions.
373
to the Fe2+ of the heme of BcCYP51 (Figure 2B).
374
at R1 in (R)-12 formed a π–π stacking interaction with Tyr122, which effectively
375
enhanced its binding strength.
376
fungicide, as it can block sterol biosynthesis by targeting the BcCYP51 site.
No
Moreover, a nitrogen atom in imidazole can bind In particular, the naphthalene ring
These results suggested (R)-12 would be a good
377
A synthesis of (R)-12 and its analogs was designed as shown in Figure 3.
378
Starting material 1 was synthesized according to a reported procedure.19
379
Intermediate 2 was prepared by α-bromination of the acetyl group of compound 1 in
380
90-95% yield.
381
tetrahydrofuran to give compounds 3 in 50-60% yield.21
Intermediate 2 reacted with (+)-DIP-Cl or (-)-DIP-Cl in anhydrous
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obtained by removing hydrogen bromide from compounds 3 using potassium
383
carbonate in acetone, underwent subsequent nucleophilic addition with imidazole in
384
N,N-dimethyl formamide to give compounds 5 in 40-50% yield.
385
(R)-1~12, (S)-1, and (S)-11~12 were obtained by etherification of 5 and R1CH2Br in
386
36-99% yield.
387
Fungicidal Activity
388
Target compounds
To validate our efficient molecular design method, an in vitro fungicidal activity
389
study on several target compounds was carried out.
Their antifungal activities were
390
evaluated at 50 µg/mL, and the results are shown in Table 1.
391
(R)-5 showed completed growth inhibition of R. cerealis, P. sasakii, and P. piricola,
392
which is the same as the positive controls, imazalil and tioconazole.
393
has a 4-butylbenzyl moiety at the R1 position, also showed 100% inhibition of R.
394
cerealis, and S. sclerotiorum, and this analog showed better inhibition of C.
395
arachidicola (100% inhibition) than that of the positive controls, imazalil (50%) and
396
tioconazole (88%).
397
position, showed 100% inhibition of B. cinerea, R. cerealis, and P. sasakii.
398
with a 1-naphthyl moiety at the R1 position, seemed to have a broader spectrum of
399
activity; it provided 100% inhibition of B. cinerea, R. cerealis, P. sasakii, S.
400
sclerotiorum, and P. piricola.
401
cerealis growth, but they did not show this level of inhibition against other species.
402
(R)-8 exhibited total inhibition of S. sclerotiorum and R. cerealis but did not inhibit
403
other species. These results indicated that the introduction of 2-chlorothiophene-3-yl
Our data indicated that
(R)-7, which
(R)-11, which has a 2-chlorothiophen-3-yl moiety at the R1 (R)-12,
(R)-3, (R)-4, and (R)-6 could also completely inhibit R.
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and 1-naphthyl moieties at the R1 showed good and broader activities, respectively,
405
against 5-6 different kinds of the plant fungi tested.
406
that our molecular design targeting specific enzymes could lead to potent fungicide
407
candidates.
408
the most potent fungicides, and this speculation was verified by the antifungal
409
bioassays.
410
structures; therefore, all the target compounds synthesized were the R isomers.
411
were interested to see if there was any significant difference in activities between the
412
different isomers; thus, we chose two active compounds (R)-11 and (R)-12) and
413
synthesized their S isomers for comparison.
414
isomer of (R)-1 for comparison because (R)-1 showed no fungicidal activity.
415
results of the in vitro antifungal evaluation showed that the S isomers exhibited
416
fungicidal activities similar to the corresponding R isomers, while the (S)-1 isomer
417
showed poor antifungal activity.
418
The bioassay data suggested
The molecular docking analysis predicted (R)-7 and (R)-12 would be
In this study, we also noticed that there was a chiral carbon atom in these We
We also decided to construct the S The
To better compare the antifungal activities of these active compounds, their EC50
419
values were calculated and are shown in Table 2.
The result indicated that (R)-12
420
showed excellent activities against B. cinerea, R. cerealis, P. sasakii, S. sclerotiorum,
421
and P. piricola with EC50 values of 3.14, 0.31, 2.84, 0.33, and 0.64 µg/mL,
422
respectively.
423
(R)-11~12 against R. cerealis were in general better than that of the positive control
424
tioconazole.
425
EC50 value of 0.02 µg/mL, which was 10 times better than that of imazalil and 80
In the single species evaluation, the EC50 values of (R)-6~8 and
It is worth pointing out that (R)-7 showed the best inhibition with an
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times better than that of tioconazole.
In addition, (S)-11 was twice as active against
427
B. cinerea, R. cerealis, and S. sclerotiorum than was tioconazole and 6 times more
428
active against C. arachidicola than was imazalil.
429
control used in this study, exhibited good fungicidal activity with EC50 values of 0.06
430
µg/mL against R. cerealis and 15.11 µg/mL against B. cinerea.17
Azoxystrobin, another positive
431
In summary, (R)-12 and (S)-11 exhibited excellent and relatively broad-spectrum
432
fungicidal activities. Their fungicidal activities were in accord with the predictions of
433
the molecular docking studies. The results of the evaluation of the in vivo protective activities are listed in Table
434 435
3.
The data suggested that any compounds with a 2-chlorothiophen-3-yl moiety at
436
R1 showed better activity than compounds without that substituent, and (S)-11
437
provided complete protection at 100 µg/mL as did imazalil and tioconazole; however,
438
the spatial arrangement of substituents on tioconazole and (S)-11 is different (Figure
439
2).
440
target compounds showed fungicidal activity against several fungi species.
441
Morphology and Ultrastructure Transformation: Effects of (R)-12 on B. cinerea
The data also suggested that by introducing a 3,4-dichloroisothiazole ring, the
442
Optical microscopy observations of B. cinerea treated with 1 µg/mL (R)-12
443
revealed that the mycelium growth, spore germination and infection were
444
significantly affected by the treatment (Figures 5A and D).
445
Transmission electron microscopy (TEM) observation of the B. cinerea cell
446
structure indicated that after exposure to 1 µg/mL (R)-12 for 24 h, most of the cell
447
structure was damaged, and cell wall formation was seriously affected.
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become thinner, and wrinkled cell walls were observed (Figures 5E and F).
449
results showed that (R)-12 affected cell wall formation in B. cinerea.
450
comparison, in untreated cells, the cell walls and various organelles grew normally.
451
Good adhesion between the cytoplasmic membrane and the outer wall as well as
452
normal, functional cytoplasm could clearly be observed (Figures 5B and C).
453
Effects of BcCYP51 Gene Expression
454
These For
To further explore the mode of action of these compounds, (R)-7, (R)-12, and
455
(S)-11 were chosen as representatives for this study.
The CYP51 gene displayed a
456
feedback regulation of sterol biosynthesis.33
457
CYP51 as the target would affect the CYP51 expression level.
458
shown in Figure 6, showed that the BcCYP51 expression level was significantly
459
affected, and the cytochrome P450-dependent sterol 14α-demethylase was inhibited
460
by these three compounds.
461
the strongest effect as a sterol biosynthesis inhibitor.
Any antifungal compounds with The Q-PCR results,
Among these representative compounds, (R)-12 showed
462
Proposed mode of action: Based on the result of this gene express study plus
463
the microcopy observations and the Q-PCR data, it is reasonable to conclude that the
464
mode of action of (R)-12 and its analogs is similar to that of tioconazole and imazalil;
465
they function as sterol biosynthesis inhibitors and target 14α-demethylase.
466 467
Abbreviations Used
468
EC50: median effective concentration; TBAB: tetrabutylammonium bromide; DIP-Cl:
469
diisopinocampheyl chloroborane; TEM: Transmission electron microscopy.
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Acknowledgment
471
This work was supported in part by the National Key Research & Development Plan
472
(No. 2017YFD0200900), the National Natural Science Foundation of China (No.
473
31571991), the Fundamental Research Funds for the Central Universities (No.
474
020/63171311), and the China Postdoctoral Science Foundation (2017M611156).
475
The authors also thank Dr Jiaxing Huang for providing pyrazole compounds
476
(synthesized in the Key Technologies R&D program of China, 2015BAK45B01,
477
CAU) for model reaction.
478 479
Supporting Information
480
1
481
Score of molecular docking; Amino acid sequence homology of CYP51. This material
482
is available free of charge via the Internet at http: //pubs.acs.org.
H NMR, 13C NMR and UV spectra for the target compounds; Crystal data of (S)-12;
483 484
Conflict of interest
485
The authors declare no competing financial interest.
486 487
References
488
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FIGURE CAPTIONS Figure 1. Design of target compounds. Figure 2. (A) Docking model display in Pymol; Tioconazole, (S)-11, (R)-12, and (R)-7. (B) Binding model in Ligplot. Figure 3. General synthetic procedure for the target compounds including its isomers R and S. Figure 4. Crystal structure for compound (S)-12 by X-ray diffraction determination. Figure 5. (A, D) Optical microscopy observations of mycelium and spore germination of B. cinerea. (B, C) Transmission electron microscopy observations of cell structure of B. cinerea. Sections of B. cinerea cell were grown in absence of (R)-12 (control). (E, F) Sections of B. cinerea cell were grown containing 1 µg/mL (R)-12.
C, cytoplasm; CW, cell wall; ehy, empty hyphae; N: nucleus.
Figure 6. Gene expression of BcCYP51 influenced by active compounds.
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Table 1. In Vitro Fungicidal Activity of Compounds (R)-1~12 and (S)-1, (S)-11~12. Compd.
Fungicidal activity (%) ± SD at 50 µg/mL A. sa
B. c
R. c
C. a
P. s
G. z
S. s
P. p
P. i
(R)-1
14.3±1.1
33.5±0.6
86.2±1.3
19.6±0.4
49.4±1.6
12.5±1.3
67.5±1.3
56.2±1.2
18.2±0.5
(R)-2
36.2±0.8
19.7±1.9
50.0±0.9
40.5±1.0
36.1±0.7
18.8±0.5
57.9±0.0
31.8±0.2
39.0±0.2
(R)-3
53.8±1.0
53.2±0.5
100±0.0
48.9±0.3
68.3±1.2
20.0±0.3
80.5±2.2
78.0±1.4
39.7±2.0
(R)-4
22.3±1.2
68.6±0.8
100±0.0
39.5±1.1
72.8±0.0
40.5±0.4
87.6±0.1
70.0±0.0
78.2±0.9
(R)-5
58.8±0.0
38.9±0.6
100±0.0
87.4±0.3
100±0.0
33.5±0.9
78.2±0.0
100±0.0
19.3±0.0
(R)-6
65.0±1.1
82.3±0.0
100±0.0
68.5±0.6
80.5±0.8
19.0±1.2
83.0±1.4
82.0±0.0
59.4±0.6
(R)-7
65.6±0.2
49.6±1.0
100±0.0
100±0.0
88.0±0.3
46.2±1.3
100±0.0
80.5±0.4
85.1±0.0
(R)-8
55.6±2.0
79.6±0.7
100±0.0
78.4±0.4
68.5±0.0
30.0±0.5
100±0.0
66.5±1.8
84.1±0.7
(R)-9
6.4±1.0
24.2±1.5
63.2±2.3
28.1±0.6
30.9±1.0
57.4±0.1
36.2±0.2
19.4±0.6
26.5±0.3
(R)-10
8.8±0.0
26.4±2.0
46.1±0.3
33.1±0.8
10.0±2.0
20.2±1.5
11.7±0.6
25.0±0.0
2.4±0.9
(R)-11
73.8±0.0
100±0.0
100±0.0
96.5±0.3
100±0.0
76.6±0.4
92.2±0.0
85.2±0.5
50.4±0.3
(R)-12
73.0±1.5
100±0.0
100±0.0
90.1±0.0
100±0.0
25.3±0.7
100±0.0
100±0.0
32.6±2.0
(S)-1
20.0±1.0
32.1±1.2
50.4±0.0
22.6±0.7
50.2±0.3
24.5±0.6
73.6±1.2
60.0±1.8
40.0±0.1
(S)-11
72.3±2.1
100±0.0
100±0.0
92.5±1.2
100±0.0
71.2±0.1
100±0.0
80.2±0.7
64.7±0.9
(S)-12
71.5±1.0
65.1±0.8
100±0.0
63.5±0.0
88.2±0.6
70.5±1.0
100±0.0
88.0±0.1
35.7±2.1
imazalil
100±0.0
100±0.0
100±0.0
50.4±1.0
100±0.0
100±0.0
100±0.0
100±0.0
100±0.0
tioconazole
80.0±0.9
100±0.0
100±0.0
88.2±0.6
100±0.0
52.6±4.6
100±0.0
100±0.0
66.1±0.9
a.
A. s: Alternaria solani; B. c: Botrytis cinerea; R. c: Rhizoctonia cerealis; C. a: Cercospora
arachidicola; P. s: Pellicularia sasakii; G. z: Gibberella zeae; S. s: Sclerotinia sclerotiorum; P. p: Physalospora piricola; P. i: Phytophthora infestans (Mont) de Bary.
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Table 2. The EC50 Values of Compounds R and S. Fungi B. cinerea
R. cerealis
C. arachidicola
P. sasakii
S. sclerotiorum
Compd. (R)-11 (R)-12 (S)-11 imazalil tioconazole azoxystrobina (R)-3 (R)-4 (R)-5 (R)-6 (R)-7 (R)-8 (R)-11 (R)-12 (S)-11 (S)-12 imazalil tioconazole azoxystrobina (R)-7 (R)-11 (R)-12 (S)-11 imazalil tioconazole azoxystrobinb (R)-5 (R)-11 (R)-12 (S)-11 imazalil tioconazole azoxystrobina (R)-7 (R)-8 (R)-11 (R)-12 (S)-11 (S)-12 imazalil tioconazole
Regression equation y=3.9260+1. 6416x y=4.2479+1.5338x y=4.7762+0.7986x y=5.4712+0.5906x y=2.6388+3.2756x y=4.1911+0.6860x y=4.1403+1.0779x y=3.0235+3.7172x y=4.6254+1.0493x y=4.9686+1.2068x y=7.2408+1.2685x y=4.3870+2.8551x y=5.4039+1.3027x y=5.7183+1.3754x y=5.1814+0.9896x y=4.5270+1.2212x y=7.9444+5.2994x y=4.5563+1.8667x y=5.3684+0.2997x y=3.7609+1.9061x y=3.7960+1.0199x y=3.7445+1.6143x y=3.5401+1.7117x y=3.7188+0.7647x y=4.8716+1.0333x y=4.8911+0.2738x y=3.1646+1.9175x y=4.1336+1.2902x y=4.2363+1.7104x y=4.4979+1.1234x y=4.3188+0.8985x y=3.7985+2.5757x y=4.6174+0.6765x y=3.7355+1.6334x y=3.8383+1.8955x y=4.1308+1.4602x y=5.6634+1.3847x y=5.3552+1.5789x y=5.4824+1.7045x y=5.0936+1.2175x y=4.4859+1.4506 x
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R2 0.9927 0.9785 0.9716 0.9848 0.9869 0.9549 0.9993 0.9479 0.9905 0.9976 0.9983 0.9930 0.9847 0.9974 0.9877 0.9936 0.9633 0.9463 0.9785 0.9956 0.9864 0.9370 0.9896 0.9977 0.9863 0.9987 0.9913 0.9899 0.9949 0.9954 0.9559 0.9387 0.9235 0.9967 0.9913 0.9904 0.9854 0.9979 0.9885 0.9979 0.9939
EC50 (µg/mL) 4.65 3.14 1.96 0.18 5.26 15.11 6.44 3.40 2.36 1.11 0.02 1.63 0.50 0.31 0.67 2.56 0.29 1.76 0.06 4.76 16.21 6.10 7.44 47.92 1.36 2.50 9.16 5.08 2.84 2.90 6.07 3.06 3.68 6.52 4.21 3.99 0.33 0.63 0.57 0.87 2.28
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P. piricola
a
azoxystrobina (R)-5 (R)-12 imazalil tioconazole azoxystrobin
y=4.6795+0.5283x y=4.5048+1.1843x y=5.2487+1.2850x y=5.2813+1.0448x y=2.0174+4.3348x y=4.7465+0.2720x
Azoxystrobin, the data cited from the reference.17
0.8375 0.9879 0.9895 0.9581 0.9144 0.9896
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4.04 2.62 0.64 0.57 5.09 14.48
b
Azoxystrobin, the data cited from the
reference.19 All these data were determined by our group at the same conditions as in this study.
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Table 3. In Vivo Fungicidal Activity of Compounds (R)-1~12 and (S)-1, (S)-11~12 Against Botrytis cinerea. Fungicidal activity (%) ± SD at 100 µg/mL Compd. B. cinerea Compd. B. cinerea Compd.
B. cinerea
(R)-1
50±2
(R)-7
20±2
(S)-1
50±2
(R)-2
30±3
(R)-8
70±1
(S)-11
100±0
(R)-3
40±1
(R)-9
55±2
(S)-12
60±3
(R)-4
60±2
(R)-10
40±1
imazalil
100±0
(R)-5
10±0
(R)-11
95±4 tioconazole
(R)-6
0±0
(R)-12
60±0
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Figure graphics
Figure 1
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Figure 2
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a
Reagents and conditions: (i) pyridinium tribromide, 33% wt HBr in HOAc, r.t.; (ii)
(+)-DIP-Cl, THF, -20 oC, 16 h; (iii) (-)-DIP-Cl, THF, -20 oC, 16 h; (iv) K2CO3, acetone, 20 h; (v) imidazole, K2CO3, DMF, 24 h; (vi) 1 mol/L NaOH, R1CH2Br, TBAB, THF, 2 h.
Figure 3
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Figure 4
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Figure 5
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Relative Gene Expression Level (Fold change)
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15
10
5
0 (S)-11
(R)-7
(R)-12 tioconazole imiazalil
Figure 6
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Graphic for table of contents
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