Synthesis and Biological Evaluation of Novel Fluorine-Containing

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Synthesis and Biological Evaluation of Novel Fluorine-Containing Stilbene Derivatives as Fungicidal agents against Phytopathogenic Fungi Weilin Jian, Daohang He, Pinggen Xi, and Xinwei Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b04367 • Publication Date (Web): 30 Oct 2015 Downloaded from http://pubs.acs.org on November 5, 2015

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Journal of Agricultural and Food Chemistry

Synthesis and Biological Evaluation of Novel Fluorine-Containing Stilbene Derivatives as Fungicidal agents against Phytopathogenic Fungi Weilin Jian,† Daohang He,*,† Pinggen Xi‡ and Xinwei Li† †

School of Chemistry and Chemical Engineering, South China University of

Technology, Guangzhou, Guangdong 510640, People’s Republic of China ‡

Guangdong Province Key Laboratory of Microbial Signals and Disease Control,

South China Agricultural University, Guangzhou, Guangdong 510642, People’s Republic of China

*

Corresponding author:

Phone/Fax: + 86-20- 8711 -0234. E-mail: [email protected] or [email protected].

1

ACS Paragon Plus Environment


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1

ABSTRACT

2

The rising development of resistance to conventional fungicides is driving the search

3

for new alternative candidates to control plant diseases. In this study, a series of new

4

fluorine-containing stilbene derivatives was synthesized on the basis of our previous

5

quantitative structure-activity relationship analysis results. Bioassays in vivo revealed

6

that the title compounds exhibited potent fungicidal activities against phytopathogenic

7

fungi (Colletotrichum lagenarium and Pseudoperonospora cubensis) from cucumber

8

plants. In comparison to the previous results, the introduction of a fluorine moiety

9

showed improved activities of some compounds against those fungi. Notably,

10

compounds 9 exhibited a comparable control efficacy against C. lagenarium (83.4 ±

11

1.3%) to that of commercial fungicide (82.7 ± 1.7%). For further understanding the

12

possible mode of action of the stilbene against C. lagenarium, the effects on hyphal

13

morphology, electrolyte leakage, and respiration of mycelial cell suspension were

14

studied.

15

morphology. The conductivity of mycelial suspension increased in the presence of

16

compound 9; whereas no significantly inhibitory effect on respiration was observed.

17

Taken together, the fungicidal mechanism of this stilbene is associated with its

18

membrane-disruption effect resulting in the increased membrane permeability. These

19

results would provide important clues for mechanistic study and derivatization of

20

stilbenes as alternative sources of fungicidal agents for plant disease control.

21

KEYWORDS: stilbene derivatives, fluorine, fungicidal activity, Colletotrichum

22

lagenarium, mode of action, electrolyte leakage

Microscopic

observation

showed

considerably

2


deformed

mycelial

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23

INTRODUCTION

24

Plant diseases have been recognized as a worldwide threat to crop production.1

25

Fungicide applications are, and will remain, essential for effective control of most

26

plant diseases in the agriculture sector, and have contributed greatly to crop yields

27

and quality benefits. Accordingly, more than 80% of the acres of most fruit and

28

vegetable crops grown in the U.S. are treated with fungicides every year, and the

29

benefit of fungicide use is estimated to boost farm income by nearly $13 billion

30

annually.2 In China, fungicides have also become an integral part of the sustainable

31

agriculture to ensure increased crop productions. However, the repetitive use of

32

conventional fungicides over many years has exerted a selective pressure on

33

pathogenic fungi, leading to the emerging fungicide-resistant biotypes.3-5 It has been

34

documented that many pathogens have evolved resistance within two years of a new

35

commercial fungicide introduction.6 According to the Fungicide Resistance Action

36

Committee (FRAC), the sustained supply of new and diverse types of biological

37

disease-control agents is highly beneficial both environmentally and to manage

38

resistance problems.6

39

In the past decades, the use of natural products as structural scaffolds has been

40

regarded as an effective strategy in the search for biologically active molecules.7-10

41

In line with such tendency, our group focused efforts on the design and synthesis of

42

bioactive molecules based on the trans-stilbene scaffold.11,12 Along with their broad

43

pharmacological properties claimed in medicine,13-16 our initial interest in stilbenes

44

arose from their pivotal roles in resistance mechanisms of certain plants against 3



45

fungal infections such as Botrytis cinerea,17,18 Uncinula necator,19,20 and

46

Plasmopara viticola.21 Nevertheless, research concerning the fungicidal activity of

47

synthetic stilbenes against plant pathogenic fungi still remains sparse in the literature.

48

Recently, we reported the synthesis and bioactivities of stilbene-derived compounds,

49

which exhibited potent fungicidal activities against phytopathogenic fungi in vivo.12

50

Three-dimensional quantitative structure-activity relationship (3D-QSAR) analysis

51

suggested that the bioactivities were associated with the electronic and steric

52

properties of substituents on stilbene ring.

53

Fluorinated organic compounds constitute an important family of commercial

54

agrochemicals.24-26 The introduction of fluorine-containing motifs have shown to be

55

an efficient tool in the quest for a modern crop protection product with optimal

56

efficacy, environmental safety, user friendliness, and economic viability.27

57

Furthermore, it is observed that oxadiazoles, if substituted with fluorine moieties,

58

could result in the formation of potent bioactive molecules.28 In this regard, fluorine

59

substituents used for the rational design of structural stilbene analogues could be

60

envisaged to gain improved biological and chemico-physical properties.

61

In agreement with the bioactive potency, previous evidence has revealed that

62

stilbenes may alter fungal morphogenesis;22,23 however, their mechanisms of action

63

behind the fungicidal activities, particularly against phytopathogenic fungi, are yet to

64

be fully understood. As a whole, further insights into fungicidal mechanisms of

65

synthetic stilbenes would be of great help in the development of novel crop

66

protection agents. 4


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The aim of this research was to design and synthesize novel fluorine-containing

68

stilbene derivatives on the basis of the 3D-QSAR analysis results. Further in vivo

69

bioassay was performed to evaluate the fungicidal activities against phytopathogenic

70

fungi (Colletotrichum lagenarium and Pseudoperonospora cubensis) from cucumber

71

plants, and their structure-activity relationships (SARs). To elucidate the possible

72

mechanism of action of stilbenes against C. lagenarium, the effects on hyphal

73

morphology, electrolyte leakage, and respiration of mycelia cell suspension were

74

studied.

75

MATERIALS AND METHODS

76

Chemicals and Instruments. All chemicals and reagents were commercially

77

available and used without further purification. All solvents were dried and

78

redistilled prior to use. Melting points were determined on a SGW X-4 microscope

79

melting point apparatus (Shanghai Instrument Physical Optics Instrument Co. Ltd.,

80

Shanghai, China) and were uncorrected. 1H and

81

(NMR) spectra were recorded in CDCl3 or DMSO-d6 on an Avance 600 MHz NMR

82

spectrometer (Bruker, Karlsruhe, Germany) using tetramethylsilane (TMS) as an

83

internal standard. High resolution mass spectra (HRMS) were obtained with a maXis

84

Impact electrospray ionization (ESI) spectrometer (Bruker). The purity of the

85

compounds was confirmed by thin-layer chromatography (TLC) on silica gel

86

“G”-coated glass plates, and spots were visualized under ultraviolet (UV) irradiation.

87

Pathogens and Cultures. The strains of C. lagenarium was provided by

88

Guangdong Province Key Laboratory of Microbial Signals and Disease Control,

13

C nuclear magnetic resonance

5



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89

South China Agricultural University (Guangdong, China), and maintained on the

90

potato dextrose agar (PDA) medium (extract of 200 g of boiled potatoes, 20 g of

91

dextrose, and 20 g of agar in 1 L of distilled water) at 4 °C. P. cubensis sporangia

92

were obtained by washing off the leaves of highly infected cucumber plants that

93

were kept overnight (above 80% relative humidity). All of the spore suspensions

94

were adjusted to the desired concentrations prior to use.

95

General Synthetic Procedures for Title Compounds 2-13. The target

96

compounds 2-13 (Figure 1) were synthesized according to our previously reported

97

procedures.12,29 To a stirred solution of the aromatic aldehyde (2.5 mmol) and

98

intermediate 1 (1 g, 2.5 mmol) in anhydrous THF (15 mL) under nitrogen

99

atmosphere was added dropwise a solution of t-BuOK (0.42 g, 3.75 mmol) in 5 mL

100

of ethanol. The resulting mixture was stirred overnight at room temperature and then

101

filtered and washed with ethanol. The residue was recrystallized from

102

ethanol/DMSO to afford the corresponding stilbene derivatives. The data for target

103

compounds 2-13 is shown below. (E)-2-(4-fluorophenyl)-5-(4-(4-methoxystyryl)phenyl)-1,3,4-oxadiazole

104

2:

105

yellow-green solid; yield, 68.9%; melting point (mp), 195–196 °C; 1H NMR (600

106

MHz, CDCl3) δ 8.19 – 8.14 (m, 2H, C6H4 2,6-H), 8.10 (d, J = 7.9 Hz, 2H, C6H4

107

2,6-H), 7.64 (d, J = 7.9 Hz, 2H, C6H4 3,5-H), 7.50 (d, J = 8.2 Hz, 2H, C6H4 2,6-H),

108

7.25 (t, J = 8.2 Hz, 2H, C6H4 3,5-H), 7.21 (d, J = 16.4 Hz, 1H, CH═CH), 7.02 (d, J =

109

16.1 Hz, 1H, CH═CH), 6.94 (d, J = 8.3 Hz, 2H, C6H4 3,5-H), 3.86 (s, 3H, OCH3);

110

13

C NMR (151 MHz, CDCl3) δ 165.63, 164.60, 163.95, 163.63, 159.87, 141.23, 6


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111

130.68, 129.53, 129.22, 129.16, 128.10, 127.24, 126.73, 125.25, 122.06, 120.38,

112

120.36, 119.47, 116.48, 116.34, 114.29, 55.34; HRMS (ESI), m/z calcd for

113

C23H17FN2NaO2 [M + Na]+ 395.1166; found, 395.1168.

114

(E)-2-(4-fluorophenyl)-5-(4-(3,4,5-trimethoxystyryl)phenyl)-1,3,4-oxadiazole 3:

115

light green solid; yield, 80.5%; mp, 164–166 °C; 1H NMR (600 MHz, CDCl3) δ 8.15

116

(dd, J = 8.4, 5.3 Hz, 2H, C6H4 2,6-H), 8.11 (d, J = 8.1 Hz, 2H, C6H4 2,6-H), 7.65 (d,

117

J = 8.3 Hz, 2H, C6H4 3,5-H), 7.24 (t, J = 8.5 Hz, 2H, C6H4 3,5-H), 7.17 (d, J = 16.2

118

Hz, 1H, CH═CH), 7.04 (d, J = 16.3 Hz, 1H, CH═CH), 6.78 (s, 2H, C6H2 2,6-H),

119

3.94 (s, 6H, OCH3), 3.90 (s, 3H, OCH3);

120

164.50, 163.96, 163.68, 153.51, 140.73, 138.71, 132.39, 131.07, 129.22, 129.16,

121

127.26, 127.15, 126.91, 126.80, 122.44, 120.33, 120.31, 116.48, 116.34, 104.08,

122

63.76, 60.96, 56.20; HRMS (ESI), m/z calcd for C25H22FN2O4 [M + H]+ 433.1558;

123

found, 433.1558.

13

C NMR (151 MHz, CDCl3) δ 165.64,

124

(E)-2-(4-(3-chlorostyryl)phenyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole 4: light

125

green solid; yield, 90.3%; mp, 208–209 °C; 1H NMR (600 MHz, CDCl3) δ 8.16 (brs,

126

2H, C6H4 2,6-H), 8.13 (d, J = 7.0 Hz, 2H, C6H4 2,6-H), 7.66 (d, J = 7.2 Hz, 2H, C6H4

127

3,5-H), 7.54 (s, 1H, C6H4 2-H), 7.41 (d, J = 6.6 Hz, 1H, C6H4 6-H), 7.32 (t, J = 7.5

128

Hz, 1H, C6H4 5-H), 7.29 (s, 1H, C6H4 4-H), 7.25 (t, J = 7.6 Hz, 2H, C6H4 3,5-H),

129

7.15 (s, 2H, CH═CH);

130

163.75, 140.25, 138.61, 134.81, 129.99, 129.57, 129.25, 129.19, 128.80, 128.14,

131

127.31, 127.18, 126.55, 125.02, 122.92, 120.29, 120.28, 116.51, 116.37; HRMS

132

(ESI), m/z calcd for C22H15ClFN2O [M + H]+ 377.0851; found, 377.0848.

13

C NMR (151 MHz, CDCl3) δ 165.67, 164.42, 163.99,

7



Page 8 of 33

133

(E)-2-(4-(4-chlorostyryl)phenyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole 5: light

134

green solid; yield, 82.6%; mp, 188–189 °C; 1H NMR (600 MHz, CDCl3) δ 8.15 (brs,

135

2H, C6H4 2,6-H), 8.10 (d, J = 6.7 Hz, 2H, C6H4 2,6-H), 7.64 (d, J = 6.8 Hz, 2H, C6H4

136

2,6-H), 7.47 (d, J = 6.7 Hz, 2H, C6H4 3,5-H), 7.36 (d, J = 7.1 Hz, 2H, C6H4 3,5-H),

137

7.24 (brs, 2H, C6H4 3,5-H), 7.17 (d, J = 16.3 Hz, 1H, CH═CH), 7.09 (d, J = 16.1 Hz,

138

1H, CH═CH);

139

140.41, 135.21, 133.92, 129.69, 129.21, 129.16, 128.98, 127.94, 127.91, 127.27,

140

127.05, 126.99, 122.71, 120.28, 120.26, 116.49, 116.34; HRMS (ESI), m/z calcd for

141

C22H15ClFN2O [M + H]+ 377.0851; found, 377.0851.

142

13

C NMR (151 MHz, CDCl3) δ 165.64, 164.42, 163.96, 163.69,

(E)-2-(4-(3,4-dichlorostyryl)phenyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole

6:

143

yellow-green solid; yield, 61.7%; mp, 191–192 °C; 1H NMR (600 MHz, CDCl3) δ

144

8.15 (dd, J = 8.6, 5.3 Hz, 2H, C6H4 2,6-H), 8.11 (d, J = 8.2 Hz, 2H, C6H4 2,6-H),

145

7.64 (d, J = 8.3 Hz, 2H, C6H4 3,5-H), 7.61 (d, J = 1.6 Hz, 1H, C6H3 2-H), 7.44 (d, J

146

= 8.3 Hz, 1H, C6H3 5-H), 7.35 (dd, J = 8.3, 1.7 Hz, 1H, C6H3 6-H), 7.24 (t, J = 8.5

147

Hz, 2H, C6H4 3,5-H), 7.10 (s, 2H, CH═CH); 13C NMR (151 MHz, CDCl3) δ 165.68,

148

164.34, 164.00, 163.76, 139.93, 136.85, 133.01, 131.89, 130.69, 129.23, 129.17,

149

128.56, 128.42, 128.32, 127.31, 127.20, 125.84, 123.08, 120.26, 120.24, 116.51,

150

116.36; HRMS (ESI), m/z calcd for C22H13Cl2FN2NaO [M + Na]+ 433.0281; found,

151

433.0282.

152

(E)-2-(4-(2,4-dichlorostyryl)phenyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole

7:

153

light green solid; yield, 76.7%; mp, 212–213 °C; 1H NMR (600 MHz, CDCl3) δ 8.15

154

(brs, 2H, C6H4 2,6-H), 8.12 (d, J = 7.4 Hz, 2H, C6H4 2,6-H), 7.68 (d, J = 7.4 Hz, 2H, 8


Page 9 of 33


155

C6H4 3,5-H), 7.64 (d, J = 8.1 Hz, 1H, C6H3 6-H), 7.55 (d, J = 16.2 Hz, 1H, CH═CH),

156

7.43 (s, 1H, C6H3 3-H), 7.28 (s, 1H, C6H3 5-H), 7.24 (t, J = 7.8 Hz, 2H, C6H4 3,5-H),

157

7.09 (d, J = 16.2 Hz, 1H, CH═CH); 13C NMR (151 MHz, CDCl3) δ 165.66, 164.35,

158

163.98, 163.76, 140.12, 134.19, 134.17, 133.44, 130.34, 129.71, 129.24, 129.19,

159

127.43, 127.39, 127.31, 127.28, 125.86, 123.13, 120.25, 120.24, 116.51, 116.36;

160

HRMS (ESI), m/z calcd for C22H13Cl2FN2NaO [M + Na]+ 433.0281; found,

161

433.0291.

162

(E)-2-(4-(4-bromostyryl)phenyl)-5-(4-fluorophenyl)-1,3,4-oxadiazole 8: light

163

green solid; yield, 76.7%; mp, 213–214 °C; 1H NMR (400 MHz, CDCl3) δ 8.17 (dd,

164

J = 8.6, 5.4 Hz, 2H, C6H4 2,6-H), 8.13 (d, J = 8.3 Hz, 2H, C6H4 2,6-H), 7.67 (d, J =

165

8.3 Hz, 2H, C6H4 2,6-H), 7.53 (d, J = 8.4 Hz, 2H, C6H4 3,5-H), 7.43 (d, J = 8.4 Hz,

166

2H, C6H4 3,5-H), 7.25 (d, J = 8.6 Hz, 2H, C6H4 3,5-H), 7.19 (d, J = 16.4 Hz, 1H,

167

CH═CH), 7.14 (d, J = 16.3 Hz, 1H, CH═CH);

168

165.66, 164.46, 163.75, 163.27, 135.68, 131.95, 129.81, 129.25, 129.24, 129.22,

169

129.19, 128.21, 128.11, 127.32, 127.09, 122.81, 122.11, 120.76, 120.33, 120.31,

170

116.51, 116.36. HRMS (ESI), m/z calcd for C22H14BrFN2NaO [M + Na]+ 443.0166;

171

found, 443.0168.

172

13

C NMR (151 MHz, CDCl3) δ

(E)-2-(4-fluorophenyl)-5-(4-(4-fluorostyryl)phenyl)-1,3,4-oxadiazole

9:

light

173

green solid; yield, 78.0%; mp, 192–194 °C; 1H NMR (600 MHz, CDCl3) δ 8.17 –

174

8.13 (m, 2H, C6H4 2,6-H), 8.10 (d, J = 8.4 Hz, 2H, C6H4 2,6-H), 7.64 (d, J = 8.3 Hz,

175

2H, C6H4 3,5-H), 7.54 – 7.50 (m, 2H, C6H4 2,6-H), 7.26 – 7.21 (m, 2H, C6H4 3,5-H),

176

7.19 (d, J = 16.3 Hz, 1H, CH═CH), 7.11 – 7.07 (m, 2H, C6H4 3,5-H), 7.05 (d, J = 9



13

Page 10 of 33

177

16.3 Hz, 1H, CH═CH);

C NMR (151 MHz, CDCl3) δ 165.64, 164.47, 163.96,

178

163.68, 140.63, 132.91, 129.83, 129.22, 129.16, 128.37, 128.31, 127.27, 127.16,

179

127.15, 126.95, 122.54, 120.31, 120.29, 116.50, 116.35, 115.87, 115.73; HRMS

180

(ESI), m/z calcd for C22H15F2N2O2 [M + H]+ 361.1147; found, 361.1146.

181

(E)-2-(4-fluorophenyl)-5-(4-(3-nitrostyryl)phenyl)-1,3,4-oxadiazole 10: yellow

182

solid; yield, 73.5%; mp, 225–226 °C; 1H NMR (600 MHz, CDCl3) δ 8.41 (s, 1H,

183

C6H4 2-H), 8.18 (d, J = 5.3 Hz, 1H, C6H4 6-H), 8.16 (m, 4H, C6H4 2,6-H), 7.84 (d, J

184

= 7.7 Hz, 1H, C6H4 5-H), 7.70 (d, J = 8.3 Hz, 2H, C6H4 3,5-H), 7.57 (t, J = 7.9 Hz,

185

1H, C6H4 4-H), 7.28 (s, 2H, CH═CH), 7.25 (t, J = 8.6 Hz, 2H, C6H4 3,5-H);

186

NMR (151 MHz, CDCl3) δ 165.70, 164.31, 164.02, 163.82, 148.82, 139.64, 138.54,

187

132.46, 130.42, 129.72, 129.26, 129.20, 128.39, 127.38, 123.40, 122.60, 121.12,

188

120.24, 120.22, 116.54, 116.39; HRMS (ESI), m/z calcd for C22H14FN3NaO3 [M +

189

Na]+ 410.0911; found, 410.0914.

13

C

190

(E)-2-(4-fluorophenyl)-5-(4-(4-nitrostyryl)phenyl)-1,3,4-oxadiazole 11: yellow

191

solid; yield, 80.6%; mp, 210–211 °C; 1H NMR (600 MHz, CDCl3) δ 8.25 (d, J = 7.0

192

Hz, 2H, C6H4 3,5-H), 8.16 (brs, 2H, C6H4 2,6-H), 8.15 (brs, 2H, C6H4 2,6-H), 7.71 (d,

193

J = 6.8 Hz, 2H, C6H4 2,6-H), 7.68 (d, J = 7.4 Hz, 2H, C6H4 3,5-H), 7.31 (d, J = 16.2

194

Hz, 1H, CH═CH), 7.29 (d, J = 16.4 Hz, 1H, CH═CH), 7.25 (d, J = 7.8 Hz, 2H, C6H4

195

3,5-H);

196

143.09, 139.51, 131.83, 129.26, 129.20, 128.53, 127.55, 127.38, 127.17, 124.19,

197

123.65, 120.21, 120.19, 116.53, 116.38; HRMS (ESI), m/z calcd for C22H15FN3O3

198

[M + H]+ 388.0192; found, 388.0192.

13

C NMR (151 MHz, CDCl3) δ 165.70, 164.24, 164.02, 163.85, 147.21,

10


Page 11 of 33


199

(E)-4-(4-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)styryl)-N,N-dimethylaniline

200

12: yellow solid; yield, 63.8%; mp, 246–247 °C; 1H NMR (600 MHz, CDCl3) δ 8.17

201

(brs, 2H, C6H4 2,6-H), 8.09 (brs, 2H, C6H4 2,6-H), 7.63 (brs, 2H, C6H4 2,6-H), 7.47

202

(brs, 2H, C6H4 3,5-H), 7.32 – 7.22 (m, 2H, C6H4 3,5-H), 7.20 (d, J = 16.3 Hz, 1H,

203

CH═CH), 6.96 (d, J = 16.3 Hz, 1H, CH═CH), 6.76 (brs, 2H, C6H4 3,5-H), 3.03 (s,

204

6H, CH3); 13C NMR (151 MHz, CDCl3) δ 165.60, 164.73, 163.93, 163.54, 142.51,

205

141.82, 131.30, 129.19, 129.14, 128.02, 127.21, 126.43, 121.48, 120.45, 116.45,

206

116.31, 112.47, 41.04, 40.46; HRMS (ESI), m/z calcd for C24H21FN3O [M + H]+

207

386.1666; found, 386.1663.

208

Sodium

(E)-2-(4-(5-(4-fluorophenyl)-1,3,4-oxadiazol-2-yl)styryl)benzene

209

sulfonate 13: light yellow solid; yield, 68.5%; mp > 300 °C; 1H NMR (600 MHz,

210

DMSO-d6) δ 8.46 (d, J = 16.5 Hz, 1H, CH═CH), 8.21 (dd, J = 8.7, 5.4 Hz, 2H, C6H4

211

3,5-H), 8.15 (d, J = 8.3 Hz, 2H, C6H4 2,6-H), 7.85 (dd, J = 7.7, 1.1 Hz, 1H, C6H4

212

3-H), 7.82 (d, J = 7.7 Hz, 1H, C6H4 6-H), 7.75 (d, J = 8.3 Hz, 2H, C6H4 3,5-H), 7.49

213

(t, J = 8.8 Hz, 2H, C6H4 2,6-H), 7.38 (t, J = 7.4 Hz, 1H, C6H4 5-H), 7.28 (t, J = 7.5

214

Hz, 1H, C6H4 4-H), 7.23 (d, J = 16.5 Hz, 1H, CH═CH);

215

DMSO-d6) δ 165.45, 164.49, 163.79, 163.64, 146.63, 141.85, 134.26, 131.22, 129.91,

216

129.84, 129.34, 127.69, 127.57, 127.55, 127.46, 125.95, 122.22, 120.57, 120.55,

217

117.20, 117.05; HRMS (ESI), m/z calcd for C22H14FN2Na2O4S [M + Na]+ 467.0448;

218

found, 467.0449.

13

C NMR (151 MHz,

219

In Vivo Bioassays. The fungicidal activities of the title compounds against P.

220

cubensis, and C. lagenarium in vivo at 400 µg/mL were evaluated as described 11



221

previously.12 Cucumber plants were used as the host for inoculations of fungal

222

pathogens, and were cultivated in plastic pots in a growth room. All of the

223

compounds dissolved in dimethylformamide (DMF) and distilled water (containing

224

0.1% Tween 80) at a tested concentration of 400 µg/mL were sprayed over the plant.

225

The cucumber plants were inoculated by a spore suspension of P. cubensis (1×105

226

spores/mL) or C. lagenarium (1×106 spores/mL) before the solution on leaves were

227

air-dried. Two commercial fungicides, 80% Mancozeb WP and 80% Carbendazim

228

WP were evaluated as positive controls against the fungi at the same condition.

229

Distilled water without compounds or commercial fungicides treatments was set as a

230

blank control. In all cases, the final concentration of DMF was < 0.1% (v/v). Three

231

replicates were used per treatment, and the experiment was repeated twice.

232

The fungicidal activity was evaluated according to the National Research &

233

Development Center for Pesticide. The relative control efficacy of compounds

234

compared to the blank assay was calculated via the following equation:

235

I (%) = [(C−T)/C] ×100%

236

Where I is the relative control efficacy, C and T is the average disease index of the

237

blank control and treated plants respectively.

238

Effect on Hyphal Morphology of C. lagenarium.30 To elucidate the effect on

239

hyphal morphology changes with the most active compound 9, the mycelia of C.

240

lagenarium taken from stilbene-treated medium were placed on the slides and

241

observed under a light microscope. A sample processed similarly with 0.01% of

242

DMF was set as the control. 12


Page 12 of 33

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243

Electrolyte Leakage. Mycelial discs (7 mm in diameter) from 1-week-old

244

cultures of C. lagenarium were incubated in potato dextrose broth (PDB) for 4 d in a

245

ZWY 103B constant temperature cyclotron oscillator (LABWIT Scientific Pty Ltd.,

246

Shanghai, China). Mycelia were harvested from the medium and washed three times

247

with sterile distilled water and subsequently filtered and weighed. Stock solution of

248

compound 9 was diluted with sterile distilled water to the concentrations of 50 or

249

100 µg/mL. Control contained 0.01% DMF at the same concentration as treatments.

250

The conductivity of the solution containing 1 g of fresh mycelia was measured using

251

a DDS-307 conductivity meter (Shanghai INESA Scientific Instrument Co. Ltd.,

252

Shanghai, China) at 0 (J0), 1, 4, 8, 12, 16, 24, 30, 36, 48, 60 h (J1). The final

253

conductivity (J2) was determined after mycelia were boiled and cooled to room

254

temperature. Each experiment was repeated twice with three replicates per treatment.

255

Relative permeability (P) was calculated according to the formula:31,32

256

P (%) = [(J1- J0)]/[(J2- J0)] ×100%

257

Respiration Measurement. The effect on mycelial respiration of C. lagenarium

258

was determined according to Yan et al.33 Briefly, mycelial plugs (7 mm in diameter)

259

were placed in 250-mL flasks containing 100 mL of PDB and cultivated as described

260

above. The mycelia were then washed three times with 50 mM potassium phosphate

261

buffer (pH 7.2) and resuspended in 0.1 M phosphate buffer (pH 7.2), containing 2%

262

(w/v) glucose (50 mg fresh weight of mycelia mL-1). Then kresoxim-methyl or

263

compound 9 was added into the mycelia suspension to obtain final concentrations of

264

100 or 10 µg/mL. Control was treated with 0.01% of DMF at the same concentration 13



265

as treatments. All treatments were carried out at least in triplicate. Oxygen

266

consumption of mycelial cultures was determined with a JPB-607A dissolved

267

oxygen meter (Shanghai INESA Scientific Instrument Co. Ltd., Shanghai, China).

268

The inhibition rate of respiration (IR) was calculated as: IR (%) = (R0-R1)/R0 ×100%

269 270

where R0 and R1 (expressed as µmol of O2/min/g of mycelia) are the ratios of

271

mycelial oxygen uptake pre- and post-addition of fungicides. Results were analyzed

272

statistically using Data Processing System (DPS, version 7.05).

273

RESULTS AND DISCUSSION

274

Synthesis. The target compounds 2-13 were efficiently synthesized by reaction

275

of phosphite ester 1 with aldehydes via Wittig-Horner reaction under mild conditions

276

(Figure 1). The Wittig–Horner reaction is an important synthetic route for the

277

formation of the oleﬁn functional group, which plays an important role in bioactivity

278

of stilbenes.34 The doublet of CH═CH with a coupling constant (16.1–16.5 Hz) in

279

the 1H NMR spectra of title compounds confirmed the trans-structure of stilbene.29

280

All of the spectroscopic and analytical data were consistent with the assigned

281

structures.

282

Fungicidal Activity and SAR Analysis. The fungicidal activities of the

283

synthesized compounds were evaluated in vivo against two plant pathogenic fungi

284

using pot culture test. To systematically summarize the SARs of stilbenes, three

285

compounds (14, 15, 16), which showed potent activity in our previous study were

286

evaluated at the same concentration of 400 µg/mL, and the results are presented in 14


Page 14 of 33

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287

Table 1. As expected, some of the newly synthesized compounds showed promising

288

fungicidal activities against the tested fungi. For example, compounds 3, 9, 10, 11,

289

13 exhibited relatively high fungicidal potency against C. lagenarium. In particular,

290

the activity of compound 9 was 83.4 ± 1.3%, which was comparable to that of 80%

291

Carbendazim WP (82.7 ± 1.7%). It is worth noting that the sulfated stilbene, 13, also

292

exhibited good control efficacy (71.2 ± 3.1%) against C. lagenarium. Compounds 2,

293

11, 12 showed a significant inhibition effect against P. cubensis with the control

294

efficacy of 66.0 ± 1.8, 65.1 ± 4.6, and 70.2 ± 3.7%, respectively, and were found to

295

be almost the same activity level as that of 80% Mancozeb WP (72.5 ± 4.7%). In

296

addition, the morphological changes of C. lagenarium treated with compound 9 were

297

observed under a light microscope (Figure 2B). Microscopic observation revealed

298

the considerably abnormal mycelial morphology compared to the control (Figure

299

2A), which might result in hyphal lysis. This assumption was further confirmed by

300

the membrane permeability assay.

301

Previously, we have found that the introduction of electron-withdrawing groups

302

(e.g. F, Br, Cl, and NO2) on meta and/or para position of stilbenes are favorable to

303

the improvement of activity. The similar structure-activity trends were further

304

confirmed in the present study. One possible reason for this phenomenon is that

305

electron-withdrawing substituents at such positions of stilbene ring are essential for

306

the formation of charge transfer complexes, which play important roles in contact

307

and affinity with (membrane) proteins.35 In comparison to the previous results, some

308

of the newly synthesized compounds showed remarkably improved activities against 15



309

those fungi. For example, compounds 2 and 9 had superiority fungicidal activities

310

than those of compounds 14 and 15, respectively. However, some exceptions such as

311

compound 4 (compared to compound 16) could also be observed. These results

312

suggested that the presence of fluorinated moiety at the 5-position of oxadiazole ring

313

may be essential for enhancing fungicidal potency.

314

Effect on Membrane Permeability of C. lagenarium. Cell membrane is of

315

fundamental importance for micro-organisms to maintain a homeostatic environment,

316

and could be cellular targets for many bioactive agents.33,36 To examine the

317

membrane-disruption effects of compound 9 on mycelial cells, the relative

318

permeability rate of C. lagenarium was determined. Mycelial cell membrane

319

permeability was expressed as the relative electrolyte leakage. As shown in Figure 3,

320

the relative permeability rate of mycelia treated with compound 9 (50 or 100 µg/mL)

321

increased gradually during incubation, being much higher than that of the control.

322

These results indicated that compound 9 disturbed mycelial cell membrane system

323

and subsequently induced electrolyte leakage from the cells, and thus resulting in the

324

increased membrane permeability. It is interesting to note that some recognized

325

antifungal agents, including triazole and imidazole compounds, can inhibit cell

326

growth by interrupting ergosterol biosynthesis pathway that may induce cell

327

membrane damage and morphological changes.37

328

The above observation is in good agreement with the previous result described

329

for resveratrol, which caused a disruption of the cell wall associated with leakage of

330

cellular contents in B. cinerea conidia.17 More specifically, a recent study also 16


Page 16 of 33

Page 17 of 33


331

indicated that polyphenolic stilbenes may act on the cell membrane by upsetting

332

osmotic pressure, as shown by the fact that zoospore rupture was greatly reduced

333

after the addition of glucose (an osmotic stabilizer) prior to treatment with

334

stilbenes.38 Considering the data so far reported, the fungicidal mechanism of

335

stilbenes may result, at least in part, from its action on the cell membrane leading to

336

disruption of cell growth and proliferation. Nevertheless, the fungicidal potency of

337

stilbenes cannot be ascribed solely to the electrolyte leakage because a complete

338

cessation of respiration could be synchronously observed.39 Thus, we next examined

339

the effects of stilbenes on mycelial respiration.

340

Effect on Mycelial Respiration. An oxygen consumption test for mycelia of C.

341

lagenarium was determined using the respiration inhibitor kresoxim-methyl as a

342

positive control, and the results are listed in Table 2. In the presence of

343

kresoxim-methyl, the mycelial respiration was strongly inhibited with an inhibition

344

rate of 72.2 ± 3.1%, while compound 9 seemed not to affect the oxygen consumption

345

of mycelia at 10 µg/mL compared to the control. At the higher concentration of 100

346

µg/mL, no significant effect (p > 0.05) on oxygen consumption was observed. These

347

results suggested that compound 9 did not disturb the energy generation system of C.

348

lagenarium.

349

The effects of stilbenes on respiration have led to varying results. Previously,

350

Pezet and Pont39 reported that pterostilbene could interfere in mitochondrial

351

respiration processes, as supported by the fact that respiration of the conidia of B.

352

cinerea was totally inhibited 10 min after treatment with the phytoalexins. Moreover, 17



353

the different effects were found to be related with the variances, such as positions

354

and/or electronic character, of substituents on stilbene ring;40 notably those with

355

hydroxyl groups tend to be essential for the target of actions involving in the rotary

356

mechanism of F1-ATPase inhibition.41 However, the recent study mentioned above

357

has found that the addition of ATP, an energy supplier, was not able to reduce the

358

inhibition of stilbenes on zoospore release and zoospore rupture, which are

359

connected to the impairment of energy generation system.38 Another study on 2-furyl

360

derivatives of resveratrol showed that, at low concentration, the compound did not

361

affect the respiratory chain of B. cinerea conidia while at high concentration it might

362

act as an uncoupler, but this effect seemed not so obvious.42

363

It should be noted that several fungi could utilize the alternative oxidase (AOX)

364

that enables respiration to continue in the presence of inhibitors, yet the requirement

365

for such induction and regulation of AOX is poorly understood.43 On the basis of

366

these findings, we conclude that the inhibitory effect on respiration may be

367

somewhat organism- and stilbene structure-dependent, and the fungicidal activity

368

and target of actions could be modulated through structural modifications. Notably,

369

those with multi-site activity including actions on cell membrane and the effect on

370

respiration may be expected. Further mechanistic insights will reveal additional

371

information on modes of action of different stilbene-type compounds.

372

A new fungicide has to be effective, and, in the resistance context, it should

373

work against strains that are resistant to existing fungicides.6 In this study, some of

374

the fluorine-containing stilbenes showed potent activities against phytopathogenic 18


Page 18 of 33

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375

fungi comparable to those of commercial fungicides, and therefore can be considered

376

as alternative sources of fungicidal agents for plant disease control. The primary

377

mode of action study would provide important clues for further understanding the

378

fungicidal mechanism of stilbene-derived compounds. Current investigations are in

379

progress to identify whether the induced physiology alterations (morphological

380

changes, increases in membrane permeability) are related with cell wall associated

381

enzymes.

382 383

ASSOCIATED CONTENT

384

Supporting Information Available:

385

Synthetic procedures and analytical data for intermediates 1. This material is

386

available free of charge via the Internet at http://pubs.acs.org.

387 388

Notes

389

The authors declare no competing financial interest.

390 391

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392

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525 526

Funding

527

This work was financially supported by the Fundamental Research Funds for the

528

Central Universities (No.2012ZM0035), and the University-Industry Cooperation

529

Research Program of Zhaoqing city, China (No. 2013C005).

26


Page 26 of 33

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FIGURE CAPTIONS Figure 1. General synthetic route for the target compounds 2-13, and chemical structures of compounds 14-16 in our previous work. 2 (R=4-OCH3); 3 (R=3,4,5-tri-OCH3); 4 (R=3-Cl); 5 (R=4-Cl); 6 (R=3,4-di-Cl); 7 (R=2,4-di-Cl); 8 (R=4-Br); 9 (R=4-F); 10 (R=3-NO2); 11 (R=4-NO2); 12 (R=4-N,N-dimethyl); 13 (R=2-SO3Na).

14

16

; 15

;

.

Figure 2. Microscopic observation of hyphal morphology of (A) C. lagenarium from the control and (B) cultures treated with compound 9 (100 µg/mL) showing deformed mycelia of C. lagenarium. Arrows indicate hyphal lysis. Figure 3. Effect of compound 9 on the membrane permeability of C. lagenarium. Each point represents the mean of three independent experiments ± SD.

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TABLES Table 1. In Vivo Fungicidal Activities of Testing Compounds 2-16 at 400 µg/mL control efficacy (%)a Compd.

R C. lagenarium

P. cubensis

2

4-OCH3

61.3 ± 3.0

66.0 ± 1.8

3

3,4,5-tri-OCH3

68.1 ± 1.3

34.5 ± 4.2

4

3-Cl

64.3 ± 4.1

52.4 ± 5.6

5

4-Cl

54.1 ± 0.7

53.5 ± 1.1

6

3,4-di-Cl

60.8 ± 4.4

34.8 ± 6.3

7

2,4-di-Cl

66.7 ± 4.7

51.5 ± 1.7

8

4-Br

58.9 ± 1.6

60.4 ± 5.9

9

4-F

83.4 ± 1.3

54.6 ± 3.6

10

3-NO2

75.9 ± 3.1

54.0 ± 1.9

11

4-NO2

70.8 ± 3.6

65.1 ± 4.6

12

4-N,N-dimethyl

38.1 ± 5.7

70.2 ± 3.7

13

2-SO3Na

71.2 ± 3.1

40.0 ± 7.1

14b

61.3 ± 3.0

42.1 ± 3.3

15b

73.1 ± 2.6

61.8 ± 0.6

16b

54.6 ± 4.2

46.0 ± 6.5

Fungicidesc

82.7 ± 1.7A

72.5 ± 4.7B

a

Values represent means of three independent replicates ± standard deviation (SD).

b

Compounds in our previous study used for comparison of fungicidal activity.

c

Control fungicides: A, 80% Carbendazim WP; B, 80% Mancozeb WP.

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Table 2. Inhibition of Respiration in Mycelia of C. lagenarium by Compound 9a concentration

R0 (µmol

R1 (µmol

inhibition rateb

(µg/mL)

O2/g/min)

O2/g/min)

IR (%)

10

27.71 ± 0.36

25.42 ± 1.57

8.3 ± 5.7 b

100

27.71 ± 0.36

23.46 ± 1.92

15.8 ± 6.1 b

kresoxim-methyl

100

27.71 ± 0.36

7.71 ± 0.95

72.2 ± 3.1 a

DMF

0

27.71 ± 0.36

25.63 ± 1.25

7.5 ± 3.5 b

inhibitors

compd. 9

a

Rate of oxygen consumption was determined from 50 mg fresh weight of mycelia

mL-1 at room temperature. b

Values represent means of three independent replicates ± SD. Different letters within

a column indicate statistically significant differences between the means (p < 0.05).

29



FIGURES

Figure 1

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Figure 2

31



Figure 3

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Table of Contents (TOC) Graphic:

(3.2×2.07 in.)

33


Synthesis and Biological Evaluation of Novel Fluorine-Containing

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