Discovery of Novel Piperidinyl-thiazole Derivatives as Broad-spectrum

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Agricultural and Environmental Chemistry

Discovery of Novel Piperidinyl-thiazole Derivatives as Broad-spectrum Fungicidal Candidate Qifan Wu, Bin Zhao, Zhijin Fan, Xiaofeng Guo, Dong-yan Yang, Nailou Zhang, Bin Yu, Shuang Zhou, Jiabao Zhao, and Fan Chen J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b06054 • Publication Date (Web): 14 Jan 2019 Downloaded from http://pubs.acs.org on January 14, 2019

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Journal of Agricultural and Food Chemistry

Discovery of Novel Piperidinyl-thiazole Derivatives as Broad-spectrum Fungicidal Candidate Qifan Wu†, Bin Zhao*,†, Zhijin Fan*,†，‡, Xiaofeng Guo†, Dongyan Yang†, Nailou Zhang†, Bin Yu†, Shuang Zhou†, Jiabao Zhao†, Fan Chen†

†

State Key Laboratory of Elemento-Organic Chemistry, College of Chemistry, Nankai University, Tianjin 300071, P. R. China.

‡

Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, P. R. China.

*

Address correspondence to these authors at State Key Laboratory of Elemento-

Organic Chemistry, College of Chemistry, Nankai University, No. 94, Weijin Road, Nankai District, Tianjin 300071, P. R. China (telephone +86-23499464; Fax: +86 02223503620; e-mail: [email protected] for Zhijin Fan or [email protected] for Bin Zhao)

ACS Paragon Plus Environment


1

Abstract

2

Oxathiapiprolin is one of the best active fungicide discovered for oomycetes

3

control. To develop fungicide candidate with broad-spectrum of activity, 22 new

4

piperidinyl-thiazole derivatives were designed and synthesized. Compound 5l showed

5

the best activity against Pseudoperonospora cubensis (Berk.et Curt.) Rostov and

6

Phytophthora infestans in vivo with 100% and 80% of inhibition respectively at 1 mg/L,

7

and 72.87% of field efficacy against P. cubensis at 1 g ai/667m2 validated these results.

8

Compound 5i exhibited broad spectrum of excellent activity against Sclerotinia

9

sclerotiorum with EC50=0.30 mg/L (over 10-times more active than oxathiapiprolin and

10

azoxystrobin in vitro), good activity against Botrytis cinerea, Cercospora arachidicola,

11

Gibberella zeae with EC50 of 14.54 mg/L, 5.57 mg/L, 14.03 mg/L in vitro, and P.

12

cubensis, P. infestans with 60% and 30% inhibition rate respectively at 1 mg/L in vivo.

13

Mode of action studies by RNA sequencing analysis discovered Oxysterol-binding

14

protein (OSBP), Chitin synthase (CHS1) and (1,3)-β-Glucan Synthase (FKS2) as the

15

potent target of 5i against S. sclerotiorum. Quenching studies validated that, OSBP was

16

the same target of both 5i and oxathiapiprolin, it was quenched by both of them. Our

17

studies discovered isothiaole containing piperidinyl-thiazole as an OSBP target based

18

novel lead for fungicide development.

19 20

Keywords: piperidinyl-thiazole, 3,4-dichloroisothiazole, antifungal activity, broad-

21

spectrum, RNA sequencing.

22


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23

Introduction

24

In the last three decades, numerous highly-active fungicides such as metalaxyl,1-3

25

dimethomorph,4 ethaboxam5, 6 and oxathiapiprolin7, 8 have been successfully developed

26

for oomycete disease control with excellent activity and various mechanism or mode of

27

action all over the world. Oxathiapiprolin is a piperidinyl thiazole isoxazoline

28

fungicides9 with great activity on oomycetes.10,

29

oxathiapiprolin (Figure 1) was based on the lead compound Tr1,12 which included in

30

Tripos Associates with certain degree of activity against oomycete. Piperidinyl-thiazole

31

is the key core structure of those compounds from Tripos Library, and it is also a key

32

active substructure of Tr1 and oxathiapiprolin. The active mechanism of

33

oxathiapiprolin is inhibiting a novel fungal oxysterol-binding protein (OSBP).12 P.

34

infestans oxysterol-binding protein (PiORP1, Protein ID: XP_002902250.1) is the

35

target protein of oxathiapiprolin, 13 its biological functions in oomycetes is unclear.8

36

Oxathiapiprolin exhibited excellent activity against Phytophthora nicotianae,

37

Phytophthora capsica and Peronospora belbahrii, 10, 11, 14 its activity against other fungi

38

is uncertain.8

11

The discovery procedure of

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Ergosterol is one of the most common components in the fungi cell membrane

40

other than in oomycetes.15, 16 The ergosterol biosynthesis pathway is fungus specific. 17

41

ERG13 (HMG-CoA synthase) catalyzed acetoacetyl-CoA to HMG-CoA and HMG1

42

(HMG-CoA reductase) catalyzed HMG-CoA to mevalonate are two key consecutive

43

steps of ergosterol biosynthesis.18 The ERG11/CYP51 (Sterol 14α-demethylase) was

44

also an essential enzyme in the biosynthesis of ergosterol and the target enzyme of azole



45

fungicides that were widely used for the treatment of fungal infections.19 Mutation of

46

ERG6 also caused the decreased spore germination, higher susceptibility to stress

47

resistance, as well as reduced vacuolar transport capability. 20 Active novel isothiazole–

48

thiazole

49

yl)piperidin-1-yl)methanone against Pseudoperonospora cubensis (Berk. et Curt.)

50

Rostov might act at P. cubensis oxysterol-binding protein (PcORP1), the same target

51

as of oxathiapiprolin, was discovered.21 Therefore, fungicidal spectrum will be enlarged

52

if we design the target compounds by inhibiting both the activity of OSBP and synthesis

53

of ergosterol. As inspired by this hypothesis, oxathiapiprolin was artificially divided

54

into four parts and a series compounds shown in skeleton 1 were designed through

55

retaining the core skeleton of part B, while, part A was replaced with a heterocyclic ring

56

and then both part C and part D were replaced by R4. 3,4-Dichloroisothiazole as a

57

bioactive substructure of isotianil22 was used for replacement of heterocyclic part A in

58

skeleton 1. The molecular design route of target compounds was showed in Figure 2,

59

the detailed series of piperidinyl-thiazole derivatives were listed in Table 1, all the

60

synthesis procedures were conducted according to Figure 3.

61

Materials and Methods

derivative

(3-bromophenyl)(4-(4-(3,4-dichloroisothiazol-5-yl)thiazol-2-

62

Materials and Instruments. All reagents and extra-dry solvents were purchased

63

from J&K Scientific Ltd. (Beijing, China), Heowns Biochem LLC (Tianjin, China) or

64

Energy Chemical (Shanghai, China). All solvents of analytical reagent were purchased

65

from Tianjin Bohua Chemical Reagents Co., Ltd. (Tianjin, China). Melting points were

66

measured on an X-4 Digital Type Melting Point Tester (Gongyi, Chian) and were


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uncorrected. Using tetramethylsilane (TMS) as an internal standard, 1H and 13C NMR

68

spectra were obtained in a 400 MHz Bruker AV400 spectrometer (Wisconsin, United

69

States of America) in chloroform-d (CDCl3) or dimethylsulfoxide-d6 (DMSO-d6).

70

High-resolution mass spectra (HRMS) were recorded with a 6520 Q-TOF LC/MS

71

instrument (State of California, United States of America). Crystal structures were

72

determined on a 007HF XtaLAB P200 diffractometer of Rigaku Corporation (Japan).

73

The thin-layer chromatography (TLC) was used to monitor the reaction progress by UV

74

analyzer ZF-type 1 (Hangzhou, China).

75

General Procedures for the Preparation of Intermediates 3. Starting material

76

1 was synthesized according to the description of references22,

23

77

dichloroisothiazole-5-carboxylic acid by Weinreb amide24 formation and a Grignard

78

addition, and the succeeding reaction with pyridinium bromide perbromide.

79

Intermediate 2 was synthesized via t-butyloxy carbonyl protection and Lawson reaction

80

of piperidine-4-carboxamide.25

from 3,4-

81

To a 100 mL round-bottomed flask, starting material 1 (2.25 g, 8.30 mmol),

82

intermediate 2 (2.21 g, 9.10 mmol) and tetrahydrofuran (THF) (50 mL) were added.

83

The mixture was heated for 3 h of refluxing. After ﬁltration, the filter residue was

84

dissolved in the mixture of sodium hydroxide (NaOH) solution (1 mol/L, 50 mL) and

85

dichloromethane (CH2Cl2) (50 mL), then the organic layers were dried over anhydrous

86

sodium sulfate, and concentrated in vacuo to obtain crude product 3b 0.30 g (11.2%).

87

The filtrate was removed for the solvent in vacuo, and then added CH2Cl2, the mixture

88

was washed with water solution of NaOH (1 mol/L, 50 mL) and brine (50 mL)



89

successively, dried over anhydrous sodium sulfate, then concentrated in vacuo. The

90

residue was purified by column chromatography on silica gel with a mixture of ethyl

91

acetate /petroleum ether (60−90 °C fraction) (1:16, υ/υ) to give 1.46 g (yield 42%)

92

compound 3a.

93

A solution of compound 3a (1.46 g, 3.48 mmol) in dry CH2Cl2 (30 mL) under

94

nitrogen atmosphere cooled in ice water was added dropwise trifluoroacetic acid (9.92

95

g, 87.00 mmol). The reaction mixture was then stirred at room temperature for 2 h.

96

After the reaction was completed, the mixture was quenched with 1 mol/L water

97

solution of NaOH and was adjusted to pH value of 8 for extraction with CH2Cl2 (50

98

mL). The organic layers were washed using brine (50 mL) and dried over anhydrous

99

sodium sulfate, then concentrated under reduced pressure. The residue was purified by

100

column chromatography on silica gel with a mixture of CH2Cl2/CH3OH/Et3N

101

(6:1:0.001 (υ/υ/υ)) to give 1.02g (yield 92%) compound 3b.

102

Analytical data for compound 3a: White solid; m.p., 153−155 oC; 1H NMR (400

103

MHz, CDCl3) δ 8.04 (s, 1H), 4.21 (d, J = 10.7 Hz, 2H), 3.19 (t, J = 11.3 Hz, 1H), 2.93

104

(t, J = 12.1 Hz, 2H), 2.13 (d, J = 12.7 Hz, 2H), 1.77 (dd, J = 18.2, 5.6 Hz, 2H), 1.46 (d,

105

J = 22.8 Hz, 9H).

106

148.73 (s), 143.35 (s), 116.74 (s), 79.75 (s), 43.40 (s), 40.38 (s), 32.19 (s), 28.44 (s).

107

HRMS (ESI) (M+H)+ calcd for C16H19Cl2N3O2S2: 420.0374, found: 420.0345; HRMS

108

(ESI) [M+Na]+ calcd for C16H19Cl2N3O2S2: 442.0194, found: 442.0183. Anal. Calcd for

109

C16H19Cl2N3O2S2: C, 45.72; H, 4.56; N, 10.00. Found: C, 45.45; H, 4.29; N, 10.13.

110

13

C NMR (101 MHz, CDCl3) δ 175.31 (s), 155.10 (s), 154.66 (s),

Analytical data for compound 3b: White solid; m.p., 97−99 oC; 1H NMR (400


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MHz, DMSO-d6) δ 8.45 (s, 1H), 4.41 (s, 1H), 4.01 (s, 1H), 3.45 – 2.84 (m, 3H), 2.62

112

(d, J = 10.3 Hz, 1H), 1.98 (s, 2H), 1.70 – 1.42 (m, 2H). 13C NMR (101 MHz, CDCl3) δ

113

176.51 (s), 155.26 (s), 148.73 (s), 143.20 (s), 116.66 (s), 46.25 (s), 40.86 (s), 33.66 (s).

114

HRMS (ESI) [M+H]+ calcd for C11H11Cl2N3S2: 319.9849, found: 319.9850. Anal. Calcd

115

for C11H11Cl2N3S2: C, 41.26; H, 3.46; N, 13.12. Found: C, 40.75; H, 3.57; N, 13.23.

116

General Procedure for the Preparation of the Target Compound 4. To a

117

solution of compound 3b (0.12 g, 0.38 mmol) in 20 mL methanol was added 0.94 mL

118

4 mol/L hydrochloric acid solution in 1,4-dioxane, the mixture was stirred for 3 h at

119

room temperature. After stop of the the reaction, the solvent in the reaction mixture was

120

removed at a reduced pressure, and the residue was dried at 60 oC for 12 h under vacuum

121

to obtain 0.12 g (92%) compound 4.

122

Analytical data for compound 4: White solid; m.p., >200 oC; 1H NMR (400 MHz,

123

DMSO-d6) δ 9.54 (s, 1H), 9.32 (s, 1H), 8.50 (d, J = 16.0 Hz, 1H), 3.35 (m, 2H), 3.05

124

(m, 2H), 2.53 (d, J = 14.0 Hz, 1H), 2.24 (s, 2H), 2.04 (t, J = 11.8 Hz, 2H). 13C NMR

125

(101 MHz, DMSO-d6) δ 175.30 (s), 155.55 (s), 148.38 (s), 142.10 (s), 120.05 (s), 42.85

126

(s), 37.28 (s), 28.76 (s). HRMS (ESI) [M+H]+ calcd for C11H12Cl3N3S2: 319.9849,

127

found: 319.9849.

128

General Procedures for the Preparation of the Target Compounds 5a−5t.

129

Compounds 5a−5t were synthesized by the reaction of piperidine with corresponding

130

acids. The mixture of corresponding acid (0.40 mmol), 1-(3-dimethylaminopropyl)-3-

131

ethylcarbodiimide hydrochloride (EDCI) (0.45 mmol), and 1-hydroxybenzotriazole

132

(HOBt) (0.40 mmol) was stirred in dry CH2Cl2 (10 mL) in an ice-water bath for 1 h



133

under nitrogen atmosphere. Then compound 3b (0.38 mmol) was dissolved in 10 mL

134

CH2Cl2, and added to the reaction system, then Et3N (0.45 mmol) was added. The

135

reaction mixture was then stirred at room temperature overnight, after stopping the

136

reaction, the mixture washed with saturated sodium bicarbonate (20 mL) and brine (20

137

mL) respectively, dried over anhydrous sodium sulfate, and then concentrated under

138

reduced pressure. The residue was purified by column chromatography on silica gel

139

with a mixture of ethyl acetate /petroleum ether (60−90 °C fraction) (1:9−1:3, υ/υ) to

140

give compounds 5a−5t (59%−100%).

141

Analytical data for compound 5a: White solid; yield, 82%; m.p., 141−143 oC; 1H

142

NMR (400 MHz, DMSO-d6) δ 8.52 (s, 1H), 8.21 (s, 1H), 8.02 (d, J = 12.8 Hz, 2H),

143

7.96 – 7.81 (m, 2H), 4.60 (s, 1H), 3.83 (s, 1H), 3.43 (d, J = 47.0 Hz, 2H), 3.04 (d, J =

144

10.2 Hz, 1H), 2.17 (d, J = 42.0 Hz, 2H), 1.70 (s, 2H). 13C NMR (101 MHz, CDCl3) δ

145

173.08 (s), 160.18 (s), 153.88 (s), 147.81 (s), 142.45 (s), 140.41 (s), 137.79 (s),

146

131.37−130.20 (m), 128.35 (d, J = 39.3 Hz), 125.51 (s), 125.02 (s), 123.79 (s), 121.08

147

(s), 119.72 (s), 119.34 (s), 117.03 (s), 115.95 (s), 45.92 (s), 40.48 (s), 38.92 (s), 31.24

148

(s), 30.66 (s). HRMS (ESI) [M+H]+ calcd for C23H15Cl2F6N5OS2: 626.0077, found:

149

626.0071. Analytical data for compound 5f: White powder; yield, 72%; m.p., 109−111 oC;

150

H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1H), 7.36 (d, J = 7.1 Hz, 1H), 7.07 (dd, J =

151

1

152

13.9, 8.1 Hz, 3H), 4.46 (d, J = 13.1 Hz, 1H), 4.06 (d, J = 13.2 Hz, 1H), 3.80 (s, 2H),

153

3.37 (s, 1H), 3.19 (t, J = 12.5 Hz, 1H), 2.78 (t, J = 12.0 Hz, 1H), 2.06 (t, J = 11.0 Hz,

154

2H), 1.53 (d, J = 12.0 Hz, 2H). 13C NMR (101 MHz, CDCl3) δ 174.42 (s), 168.94 (s),


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164.22 (s), 161.77 (s), 154.96 (s), 148.83 (s), 143.44 (s), 137.32 (d, J = 7.9 Hz), 130.24

156

(d, J = 8.2 Hz), 124.35 (s), 116.86 (s), 115.69 (d, J = 21.7 Hz), 113.93 (d, J = 20.9 Hz),

157

45.75 (s), 41.58 (s), 40.67 (s), 40.01 (s), 32.28 (s), 31.85 (s). HRMS (ESI) [M+H]+ calcd

158

for C19H16Cl2FN3OS2:456.0174, found: 456.0171.

159

Analytical data for compound 5g: White solid; yield, 100%; m.p., >200 oC; 1H

160

NMR (400 MHz, CDCl3) δ 8.07 (s, 1H), 5.86 (s, 1H), 4.90 (d, J = 11.7 Hz, 2H), 4.60

161

(d, J = 13.4 Hz, 1H), 4.09 (d, J = 13.7 Hz, 1H), 3.40−3.19 (m, 2H), 2.89 (m, 1H), 2.23

162

(m, 7H), 1.89−1.64 (m, 3H).

163

154.97 (s), 148.84 (s), 148.13 (s), 143.46 (s), 140.73 (s), 116.89 (s), 105.81 (s), 50.89

164

(s), 44.84 (s), 41.92 (s), 40.00 (s), 32.31 (s), 31.84 (s), 13.48 (s), 11.17 (s). HRMS (ESI)

165

[M+H]+ calcd for C18H19Cl2N5OS2: 456.0486 found: 456.0485.

13

C NMR (101 MHz, CDCl3) δ 174.37 (s), 165.25 (s),

Analytical data for compound 5h: White powder; yield, 70%; m.p., 127−129 oC;

166

H NMR (400 MHz, CDCl3) δ 8.37 – 8.01 (m, 1H), 4.82 (d, J = 51.1 Hz, 1H), 4.08 (d,

167

1

168

J = 52.0 Hz, 1H), 3.59 – 3.18 (m, 2H), 3.11 – 2.75 (m, 1H), 2.59 – 2.16 (m, 4H), 1.90

169

(d, J = 49.0 Hz, 2H), 1.20 (t, J = 28.1 Hz, 1H), 0.87 – 0.54 (m, 2H), 0.48 – 0.11 (m,

170

2H).

171

143.40 (s), 116.86 (s), 45.32 (s), 41.21 (s), 40.29 (s), 38.62 (s), 32.71 (s), 32.02 (s), 7.34

172

(s), 4.55 (s). HRMS (ESI) [M+H]+ calcd for C16H17Cl2N3OS2: 402.0268, found:

173

402.0264.

13

C NMR (101 MHz, CDCl3) δ 174.74 (s), 171.09 (s), 155.02 (s), 148.78 (s),

Analytical data for compound 5i: White powder; yield, 85%; m.p., 166−168 oC;

174

H NMR (400 MHz, DMSO-d6) δ 8.47 (d, J = 6.8 Hz, 1H), 7.50 (s, 1H), 7.29 (s, 3H),

175

1

176

4.54 (s, 1H), 3.63 (s, 1H), 3.42 (s, 1H), 3.23 (s, 1H), 2.99 (s, 1H), 2.11 (d, J = 52.2 Hz,



13

C NMR (101 MHz, CDCl3) δ 174.39 (s), 169.00 (s), 163.82 (s),

177

2H), 1.72 (s, 2H).

178

161.36 (s), 154.95 (s), 148.85 (s), 143.48 (s), 137.86 (s), 130.38 (s), 122.59 (s), 116.93

179

(s), 114.16 (s), 47.15 (s), 41.84 (s), 40.22 (s), 32.54 (s), 32.03 (s). HRMS (ESI) [M+H]+

180

calcd for C18H14Cl2FN3OS2: 442.0017, found: 442.0008. Analytical data for compound 5q: White crystal; yield, 65%; m.p., 155−157 oC;

181

H NMR (400 MHz, DMSO-d6) δ 8.49 (s, 1H), 4.41 (d, J = 30.6 Hz, 2H), 3.42 (d, J =

182

1

183

9.5 Hz, 1H), 3.29 (d, J = 10.4 Hz, 1H), 2.79 (d, J = 10.2 Hz, 1H), 2.27 – 1.98 (m, 3H),

184

1.62 (d, J = 57.9 Hz, 2H), 0.74 (d, J = 5.5 Hz, 4H).

185

174.85 (s), 171.92 (s), 155.04 (s), 148.76 (s), 143.37 (s), 116.86 (s), 45.18 (s), 41.89 (s),

186

40.46 (s), 32.40 (d, J = 77.0 Hz), 11.05 (s), 7.40 (s). HRMS (ESI) [M+H]+ calcd for

187

C15H15Cl2N3OS2: 388.0112, found: 388.0105.

13

C NMR (101 MHz, CDCl3) δ

Analytical data for compound 5t: White powder; yield, 100%; m.p., 168−170 oC;

188

H NMR (400 MHz, DMSO-d6) δ 8.46 (s, 1H), 8.08 (s, 1H), 7.68 (s, 1H), 4.32 (s, 2H),

189

1

190

3.86 (s, 3H), 3.41 (d, J = 8.3 Hz, 1H), 3.08 (s, 2H), 2.11 (s, 2H), 1.66 (s, 2H). 13C NMR

191

(101 MHz, CDCl3) δ 173.56 (s), 162.66 (s), 153.96 (s), 147.80 (s), 142.41 (s), 137.73

192

(s), 131.34 (s), 116.15 (s), 115.88 (s), 39.32 (s), 38.16 (s), 31.35 (s), 28.67 (s). HRMS

193

(ESI) [M+H]+ calcd for C16H15Cl2N5OS2: 428.0173, found: 428.0167.

194

In Vitro Fungicidal Activity Test

195

Fungicidal activities of the compounds 3−5 against Alternaria solani (A. s),

196

Botrytis cinerea (B. c), Cercospora arachidicola (C. a), Gibberella zeae (G. z),

197

Physalospora piricola (P. p), Pellicularia sasakii (P. s), Rhizoctonia cerealis (R. c) and

198

Sclerotinia sclerotiorum (S. s) were tested in vitro at a concentration of 50 mg/L using


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199

the mycelium growth inhibition method.23 The target fungus was plated on a potato

200

dextrose agar (PDA) culture plate and precultured at 25±1°C for 5 days. And then the

201

fungi cake with the same diameter of 4 mm were removed for inoculation on the center

202

of culture plate with medicine and water by inoculation needle under sterilized

203

condition. Each experiment was repeated for three times. The diameter of fungi spread

204

was measured after 5 days. The average growth diameter was obtained by calculating

205

the average of three replicates. Growth inhibition was then calculated with a formula

206

as: Efficacy (%) = [(CK0 - PT1)/(CK0 - 4)] × 100%; CK0 means the average growth

207

diameter of fungi cake cultured on a plate with water; PT1 means the average growth

208

diameter of fungi cake cultured on a plate with fungicide. And based on the results of

209

in vitro antifungal activities, those compounds with inhibition above 70% in vitro at a

210

concentration of 50 mg/L were further measured for their median effective

211

concentration (EC50).23

212

In Vivo Fungicidal Activity Test

213

The protective activities of target compounds 3−5 against Pseudoperonospora

214

cubensis (Berk. et Curt.) Rostov (P. cubensis), Phytophthora infestans (P. infestans),

215

Sphaerotheca fuliginea (S. fuliginea) and Puccinia sorghi Schwat (P. sorghi Schwat)

216

causing disease in cucumber, tomato, potato and corn respectively were determined

217

according to the ref. 26 in Shenyang Research Institute of Chemical Industry (Shenyang,

218

China) at a concentration of 100 mg/L by pot bioassay. Those compounds with

219

inhibition over 90% were further tested at lower concentrations.

220

Field Efficacy Trials of Compound 5l



221

According to the good inhibitory activity against P. cubensis in vivo, efficacy of

222

compound 5l was further evaluated by cucumber field experiment for fungicidal

223

activity validation. Compound 5l was prepared as 5.32 % emulsifiable concentrate, and

224

diluted to 0.5 g ai/667m2, 1 g ai/667m2 and 2 g ai/667m2 with water for application. In

225

the early onset of P. cubensis, the cucumbers were uniformly sprayed with the

226

compound 5l. Equal amount of water was used as the blank and oxathiapiprolin (10 %,

227

flowable agent) was used as positive control. The disease indexes (DI) and control

228

efficacies were calculated according to the disease level of the investigated leaves. DI

229

was caculated by formula as DI=Σ(A×B)×100/(C×9), and the control efficacy (CE) was

230

evaluated by formula as CE(%)=[1－CK0×pt1/(CK1×pt0)]×100, among of them, A was

231

the number of diseased leaves, B was the corresponding disease level, C was the total

232

number of leaves investigated, CK0 indicated the disease index of the blank before the

233

pesticide application, CK1 indicated the disease index of the blank after the pesticide

234

application, pt0 was the disease index of the pesticide treatment group before compound

235

application, pt1 indicated the disease index of the pesticide treatment group after

236

compound application. The statistical analysis of control effects was established via

237

reverse arcsine transformation and variance analysis. Moreover, the significant

238

difference between each treatment was compared by LRS method.27

239

The Modeling and Docking Analysis Between Compounds and PiORP1

240

Modeling the structure of P. infestans oxysterol-binding protein (PiORP1, Protein

241

ID: XP_002902250.1) was conducted by YASARA program28 with default parameters.

242

The ligand binding center of PiORP1 was identified by Autodock Tools.29,30 The


Page 12 of 42

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243

structures of ligands were optimized with the ligand minimization protocol. The

244

molecular docking analysis was performed by the Autodock Tools with default values.

245

The optimal structure in complex was selected according the visual inspection and

246

docking score. The structures of complexes were showed by Pymol.31

247

Fluorescence Quenching Analysis of Oxysterol-binding Protein

248

Full-length yeast oxysterol-binding protein (Osh4p) was cloned into pGEX-4T-3

249

vector to code for glutathione S-transferase (GST)-fused constructs, which was gifted

250

by Prof. Guillaume Drin (CNRS, France), the methods of protein induction and

251

purification for Osh4p were conducted as document description.32 Fluorescence

252

quenching analysis was preformed using F-4500 fluorescence spectrophotometer

253

(Hitachi, Tokyo, Japan). Fluorescence emission spectra were recorded at a wavelength

254

of 290-520 nm, and an excitation wavelength of 275 nm at 4 oC, with emission band

255

width setting at 10 nm and medium sensitive. Oxathiapiprolin and 5i with 0 to 9 mg/L

256

was added to Osh4p for fluorescence determination. The highest concentration of N,N-

257

Dimethylformamide (DMF) in Osh4p solution was used as blank, the highest

258

concentration of oxathiapiprolin or 5i in PBS were also determined separately.

259

RNA Sequencing and Data Analysis

260

S. sclerotiorum, which grew in PDA at 25 oC for 1 week, was treated by compound

261

5i (0.1mg/L) for 36 h and the same amount of DMF was used as control. Total RNAs

262

of samples were extracted using TRIzol Reagent (Invitrogen, USA). cDNA Libraries

263

were constructed using a Truseq stranded mRNA kit (Illumina, San Diego, USA).

264

Sequencing was conducted on an Illumina MiSeq sequencing system using the HiSeq



Page 14 of 42

265

4000 SBS Kit (Illumina, San Diego, USA) following the manufacturer’s instructions.

266

S.

267

(http://www.broadinstitute.org/annotation/genome/sclerotinia_sclerotiorum/MultiHo

268

me.html) were used as a reference for mapping the short reads. The count data were

269

normalized to generate effective library sizes using the scaling method Trimmed Means

270

of Means values (TMM). Statistical analysis was performed using a generalized linear

271

model linked to the negative binomial distribution performed using the EdgeR package.

272

The different expression genes were identified with false discovery rate (FDR)≤0.05

273

and |log2[fold change]|＞1. The Gene ontology (GO) terms and kyoto encyclopedia of

274

genes and genomes (KEGG) pathway for each gene were extracted.

275

Results and Discussion

276

Chemistry

sclerotiorum

transcripts

available

in

the

database

277

The target compounds 5a−5t were synthesized by a condensation reaction from

278

piperidine and different acids in the presence of Et3N in dry CH2Cl2 using EDCI and

279

HOBt as condensation reagent and had favorable yields. The crystal data and structure

280

refinement of compound 5p, which was cultured from the mixture of CH2Cl2 and ethyl

281

acetate, were shown in Figure 4. The key intermediate 3a was obtained by condensation

282

of an α-halocarboxylates (2-bromo-1-(3,4-dichloroisothiazol-5-yl) ethan-1-one) and

283

thioamides (tert-butyl 4-carbamothioylpiperidine-1-carboxylate),33 and the succeeding

284

reaction of the key intermediate 3a with hydrogen halide formed compound 3b. α-

285

Halocarboxylates 1 was synthesized by the reaction of 1-(3,4-dichloroisothiazol-5-

286

yl)ethan-1-one with pyridinium tribromide in the presence of hydrogen bromide–acetic


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287

acid.23 Thioamide 2 was synthesized via the reaction between the -NH-Boc protected

288

hexahydroisonicotinamide and Lawesson's Reagent.

289

In Vitro Antifungal Activity

290

Table 2 showed the in vitro activity of compounds 3−5 against A. solani, B. cinerea,

291

C. arachidicola, G. azeae, P. piricola, P. sasakii, R. cerealis and S. sclerotiorum at 50

292

mg/L. The commercial fungicides oxathiapiprolin, isotianil and azoxystrobin were used

293

as positive controls. All the compounds exhibited various degrees of inhibitory

294

activities against fungi tested. Most compounds exhibited good activity against S.

295

sclerotiorum at 50 mg/L in vitro. Particularly, compounds 3b, 4, 5i and 5k showed

296

outstanding activity (100%), which was higher than those of positive controls

297

oxathiapiprolin (90%) and isotianil (45%).

298

Based on the results of antifungal activities in vitro, the compounds with inhibition

299

over 70% were further determined for their EC50 (Table 3). Compound 3b, with EC50

300

of 5.99−11.44 mg/L against B. cinerea, S. sclerotiorum and R. cerealis, exhibited

301

broad-spectrum of fungicidal activity. In particular, compound 5i strongly inhibited the

302

growth of B. cinerea, C. arachidicola and S. sclerotiorum with EC50 of 14.54 mg/L,

303

5.57 mg/L and 0.30 mg/L respectively, it was much better than those of positive control

304

oxathiapiprolin with corresponding EC50 of 35.91 mg/L (2.45 times), 79.20 mg/L

305

(14.22 times) and 5.98 mg/L (19.90 times) against B. cinerea, C. arachidicola and S.

306

sclerotiorum respectively, and at the same level as that of the positive control

307

azoxystrobin with the EC50 of 15.11 mg/L against B. cinerea,and more active (13.47

308

times) than the positive control azoxystrobin with the EC50 of 4.04 mg/L against S.



309

sclerotiorum. The activity of compound 5i was more than 10 times than these of both

310

positive controls oxathiapiprolin and azoxystrobin against S. sclerotiorum.

311

In Vivo Antifungal Activity

312

The protective activities of pot bioassay for the compounds 3−5 against P. cubensis,

313

P. infestans, S. fuliginea and P. sorghi Schw were detected in Shenyang Research

314

Institute of Chemical Industry (Shenyang, China), the results were listed in Table 4 and

315

Table 5. The commercial fungicides oxathiapiprolin and isotianil were used as the lead

316

compounds and positive controls, the commercial fungicides ethirimol and

317

azoxystrobin were also used as positive controls for S. fuliginea and P. sorghi. Most

318

target compounds, commercial fungicides oxathiapiprolin and isotianil had no

319

inhibitory activity against S. fuliginea and P. sorghi, only compounds 3a, 5q and 5r

320

showed 20%, 20% and 30% in vivo inhibition against S. fuliginea at a concentration of

321

100 mg/L respectively. However, most of the compounds exhibited excellent inhibitory

322

activity in vivo against P. cubensis and P. infestans at 100 mg/L. Among them,

323

compounds 5c, 5h, 5l and 5r exhibited better activities (>80% inhibition at 1 mg/L)

324

than isotianil (70% inhibition at 1 mg/L) against P. cubensis. And 5l also exhibit great

325

activity against P. infestans at 1 mg/L (80% inhibition) in vivo.

326

The results of in vitro (Table 3) and in vivo (Table 4 and Table 5) indicate that the

327

methylene between N-formylpiperidine and D ring can’t increase the activity, and the

328

D ring doesn’t necessarily need to be a pyrazole ring. However, when the D ring is an

329

ordinary substituted benzene ring, the compounds have better fungicidal activity.

330

Further, if the substituent on the benzene ring contains an F atom capable of forming


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331

an H bond, the activity of the compounds can be further increased.

332

Analysis of Field Efficacy Trials

333

A large number of cucumber leaves had black mold layer in the blank, and their

334

DI values increased from 1.72 to 19.81. While the conditions of cucumber leaves treated

335

with the different compounds were well controlled, and the DI values were between

336

3.86 and 6.47. Efficacies of compound 5l against P. cubensis in the cucumber field were

337

shown in Table 6. The efficacy of high dosage (2 g ai/667m2) of 5l application showed

338

no significant difference as compared with positive control oxathiapiprolin with

339

medium (1 g ai/667m2) and low (0.5 g ai/667m2) dosage application, which was lower

340

than that of the high dosage (2 g ai/667m2) application of oxathiapiprolin. There were

341

no obvious efficacy differences between the high and middle dosages application of 5l,

342

and both of them were evidently higher than the corresponding efficacy of the low dose

343

application. To control P. cubensis in field, compound 5l was recommended at the

344

dosage of 1 g ai/667m2 to 2 g ai/667m2 once a week for 3 times.

345

Docking Analysis

346

Docking analysis was employed to elucidate the binding modes and affinities between

347

compounds and protein. The results (Figure 5A, 5B, 5D) showed that 5i and 5l could

348

bind with the active center of PiORP1, the thiazole ring in 5i, 5l and oxathiapiprolin all

349

had a hydrogen bond with PiORP1 on Asn835. Since the crystal structure of OSBP

350

from oomycetes has not been elucidated yet, we have constructed the structure of

351

PiORP1 by homology modeling base on 5H2D.34 The substrate of 5H2D was ergosterol,

352

so the binding mode of ergosterol to OSBP was docked, although ergosterol was not



353

present in oomycetes. As shown in Figure 5E, ergosterol could bind to OSBP with

354

hydrophobic interaction. As were known, no hydrogen bond on Asn835 was a key

355

binding amino acid34,35 in the transport of steroids by oxidized sterol-binding proteins

356

and hydrogen bonding force was stronger than hydrophobic force. As shown in Figure

357

5C and 4F, 5i and 5l also had a same combination mode with oxathiapiprolin except

358

for 5-methyl-3-(trifluoromethyl)-1H-pyrazol-1-yl. In summary, we indicated that the

359

target compounds 5i and 5l had suitable binding positions in its potent target PiORP1,

360

therefore, 5i and 5l might have a same mechanism with oxathiapiprolin. 5-Methyl-3-

361

(trifluoromethyl)-1H-pyrazol-1-yl was also an important group of oxathiapiprolin,

362

which led the higher activity of oxathiapiprolin. Compounds 5i and 5l contained no F

363

atoms in their structures as compared with oxathiapiprolin, both methyl and

364

trifluoromethyl can be introduced into 5i and 5l on the same ring for future comparative

365

study.

366

RNA Sequencing Analysis

367

Because of the good fungicidal activity in vitro and in vivo, compound 5i showed

368

very different novel chemical structure and the potent similar target at OSBP as

369

compared with the positive control oxathiapiprolin, to validate this novel mode of

370

action of the target compounds, RNA sequencing was conducted to identify the

371

differential expression genes (DGEs) affected by compound 5i. The results indicated

372

that, totally 3080 DGEs with fold changes >1.50 or

Discovery of Novel Piperidinyl-thiazole Derivatives as Broad-spectrum

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