Synthesis of Novel 3,4-Chloroisothiazole-Based Imidazoles as

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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

2

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

16

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

31

Producing enough food for our growing population and keeping up with the

32

increasingly strict safety regulations for consumers and the environment are a great

33

challenge for modern agriculture.1-3

34

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

40

compounds have been widely used as antifungal agents (imazalil) for plant protection

41

and for the protection of animal health (tioconazole, econazole and miconazole)

42

(Figure 1).10-12

43

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,

49

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.


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

55

obtain novel fungicides for controlling plant pathogens such as Botrytis cinerea and to

56

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

59

with 3,4-dichloroisothiazole, a substructure with systemic acquired resistance activity.

60

Specific bioassays were used to verify the validity of our molecular design models

61

(Figure 1).

62

Materials and Methods

63

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

105

(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.

106

Potassium

107

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.


4b: colorless oil; yield,

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118

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.

122

General Procedure for the Synthesis of Compounds 5a and 5b.

Potassium

123

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.

125

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%.

131

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),

133

4.39-4.31 (m, 2H, CH2);

134

138.39 (s), 128.50 (s), 120.76 (s), 116.63 (s), 68.20 (s), 50.83 (s); HRMS (m/z) calcd

135

for C8H7Cl2N3OS (M+H)+: 263.9687, found 263.9755.

136

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,


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.

144

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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162

found 304.0074.

163

Data for (R)-2: white solid; yield, 67%; m.p.: 49-50 °C; [ɑ]24 = +12.9 (c 0.67, D

164


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.

181


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


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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206

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


221


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


13

C NMR (101


Page 12 of 40

228

(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


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


234


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


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


244


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.


13

C NMR (101

Page 13 of 40


250

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


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

13

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




272

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


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.

13

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);


Page 14 of 40

Page 15 of 40


294

13

295

(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



Page 16 of 40

316

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.


Control

Page 17 of 40


338

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΄


The

Q-PCR was

and


and

Page 18 of 40

360

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


Intermediate epoxide 4,

Page 19 of 40


382

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.



Page 20 of 40

404

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


Page 21 of 40


426

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.


They


Page 22 of 40

448

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.


Page 23 of 40


470

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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489

modernagrochemicals. Pest Manage. Sci. 2010, 66, 10-27.

490

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7. Diao, X.; Han, Y. Y.; Liu, C. L. The fungicidal activity of tebuconazole enantiomers

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8. Wu, J. Q.; Zhang, S. S.; Gao, H.; Qi, Z.; Zhou, C. J.; Ji, W. W.; Liu, Y.; Chen, Y.; Li,

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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.



Page 30 of 40

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.


Page 31 of 40


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


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


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

Page 32 of 40

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


100±0


Figure graphics

Figure 1


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Figure 2



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


Page 36 of 40

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Figure 4



Figure 5


Page 38 of 40


Relative Gene Expression Level (Fold change)

Page 39 of 40

15

10

5

0 (S)-11

(R)-7

(R)-12 tioconazole imiazalil

Figure 6



Graphic for table of contents


Page 40 of 40

Synthesis of Novel 3,4-Chloroisothiazole-Based Imidazoles as

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