Design, Synthesis, Fungicidal Activity, and Unexpected Docking

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Design, Synthesis, Fungicidal Activity and Unexpected Docking Model of the First Chiral Boscalid Analogues Containing Oxazolines Shengkun Li, Dangdang Li, Taifeng Xiao, Shasha Zhang, Zehua Song, and Hongyu Ma J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b03464 • Publication Date (Web): 28 Oct 2016 Downloaded from http://pubs.acs.org on November 1, 2016

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Journal of Agricultural and Food Chemistry

1

Design, Synthesis, Fungicidal Activity and Unexpected Docking

2

Model of the First Chiral Boscalid Analogues Containing Oxazolines

3 4

Shengkun Li*, Dangdang Li, Taifeng Xiao, ShaSha Zhang, Zehua Song, Hongyu Ma,

5

Department of Pesticide Science, College of Plant Protection, Nanjing Agricultural

6

University, Weigang 1, Xuanwu District, Nanjing 210095, People’s Republic of

7

China.

8

Corresponding Author: Shengkun Li, Email [email protected]

9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 1

ACS Paragon Plus Environment


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30 31

Chirality

greatly

influences

a

pesticide's

biological

and

32

ABSTRACT:

33

pharmacological properties, and will contribute to unnecessary environment loading

34

and undesired ecological impact. No structure and activity relationship (SAR) of

35

enantiopure succinate dehydrogenase inhibitors (SDHIs) were documented during the

36

structure optimization of boscalids. Based on commercial SDHIs, oxazoline natural

37

products and versatile oxazoline ligands in organic synthesis, the first effort was

38

devoted to explore the chiral SDHIs and the preliminary mechanism thereof.

39

Fine-tuning furnished chiral nicotinamides 4ag as a more promising fungicidal

40

candidate against Rhizoctonia solani, Botrytis cinerea and Sclerotinia sclerotiorum,

41

with EC50 values of 0.58, 0.42 and 2.10 mg/L, respectively. In vivo bioassay and

42

molecular docking were investigated to explore the potential in practical application

43

and plausible novelty in action mechanism, respectively. The unexpected molecular

44

docking model showed the differently chiral effect on the binding site with the amino

45

acids residues. This chiral nicotinamides also featured easy synthesis and

46

cost-efficacy. It will provide a powerful complement to the commercial SDHI

47

fungicides with the introduction of chirality.

48

KEYWORDS: chiral pesticide, oxazoline, nicotinamide, spoxazomicin, fungicide

49 50 51 52 53 54 2


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55 56 57

INTRODUCTION

58

With an increasing trend for introduction of enantioenriched novel ones, around

59

one-third of all agrochemicals are chiral compounds[1]. Chirality greatly influences a

60

drug's biological and pharmacological properties[2]. Chiral centers are crucial for

61

biological processes, because the response of an organism to them often depends on

62

how these molecules fits the particular sites on biological receptors. The application

63

of chiral pesticides will contribute to unnecessary environment loading and undesired

64

toxic and ecological impact.

65

Recent analysis of past and present synthetic methodologies on medicinal

66

chemistry demonstrates the popularity and significance of amide formation, which is

67

also frequently used in agrochemical discovery. Amide fungicides are classic but

68

vibrant among different commercial available agrochemicals, the most famous class

69

are the succinate dehydrogenase inhibitors (SDHIs). Since the first SDHI carboxin

70

launched, 18 SDHIs have been developed as agricultural fungicides[3]. Homology

71

models and docking simulations were also developed to explain binding behaviors

72

and the peculiarities of the resistance profiles[4]. Schematic overview of structure

73

showed the SDHIs consist of other three parts (polar core + linker + substituent rest),

74

keeping the amide bond as constant. Significant progress were made on the

75

hydrophobic rest optimization, which was speculated to be embedded in the groove on

76

the target surface. With boscalid as a model, aryl ether and heterocycles, etc. are

77

typically hydrophobic units modification[5]. The latest commercial generation was 3



Page 4 of 26

78

developed by Syngenta, including isopyrazam, sedaxane and benzovindiflupyr

79

(Figure 1), we envisage the “dearomatization” may be a tendency for the hydrophobic

80

rest optimization, while no SAR of enantiopure SDHIs were discussed.

81

Rethinking the aforementioned the importance of chirality on biological processes，

82

we envision that this “invaginated hydrophobic groove” on the target surface may be

83

sensitive to chirality. Herein, we report the first effort to explore the novel SDHIs

84

with chiral hydrophobic rest and the preliminary mechanism will also be discussed.

85

MOLECULAR DESIGN

86

A literature survey confirmed the popularity of chiral oxazoline in organic

87

synthesis[6], natural products and pesticide chemistry (Figure 2). Since the

88

development of commercially available oxazoline insecticide etoxazole

89

etoxazole annalogues with oxazoline unit were patented with acaricidal/insecticidal

90

activities[8]. DuPont Company explored the structure activity relationships and

91

evaluate the potential of 2,4-diaryloxazolines as insecticides and acaricides

92

Syngenta group showed novel oxazolines sub-group with chiral Indanyl core as

93

insecticides[10]. Recently, Wang’s group documented many elegant works on this kind

94

of bioactive agrochemicals as acaricidal/insecticidal ingredients[11]( Figure 2). In 2011,

95

the Spoxazomicins A-C were isolated by Kazuro Shiomi

96

antitrypanosomal activity without cytotoxicity or obvious antibacterial and antifungal

97

activities; in the same year, the new oxazoline compound, nocazoline A was isolated

98

by Zhu[13]. Three years later, the enantiomer of nocazoline A, Yanglingmycin, was

99

isolated by Wu, interestingly, it exhibited good and broad antibacterial activities.

100

[7]

, many

[9]

, the

[12]

, those alkaloids showed

The chiral difference in biological effect and the importance of oxazolines as 4


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101

“privileged ligands” in organic synthesis and medicinal chemistry intrigued us deeply.

102

We envision that this simple but amusing scaffold can also act as good “ligands” for

103

biological “acceptor” to facilitate novel pesticide discovery. A novel class of

104

nicotinamides containing chiral oxazoline was designed and synthesized, which

105

featured “easy synthesis, C-ring dearomatization and chirality formation” (Figure 3).

106

MATERIALS AND METHODS

107

Instruments and Chemicals. All solvents and reagents were purchased from

108

commercial sources (Energy, Meryer or Aladdin Chemicals etc.), they were

109

analytically pure and used as received. Anhydrous solvents were dried and distilled by

110

standard techniques before use; chlorobenzene was dried over 4A MS for 72hrs

111

before use, ZnCl2 was flame-dried under vacuum just before use. Silica gel GF254 and

112

column chromatography silica gel for isolation (200~300 mesh) were both purchased

113

from Qingdao Broadchem Industrial Co., Ltd.

114

thin-layer chromatography (TLC) on silica gel GF254 with ultraviolet (UV254nm)

115

detection or visualized with phosphomolybdic acid. Yields of all the tiltle compounds

116

were not optimized. Melting points (M.P.) were recorded on Shenguang WRS-1B

117

melting point apparatus and are uncorrected. 1HNMR and

118

carried out utilizing a Bruker AV400 spectrometer with CDCl3 as solvent and

119

tetramethylsilane as the internal standard and the chemical shifts (δ) were recorded in

120

parts per million (ppm). Data for 1H-NMR are reported as follows: chemical shift (δ:

121

ppm), multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet),

122

coupling constant (Hz), integration and assignment (italic H). Data for 13C NMR are

123

reported in terms of chemical shift (δ: ppm). (C) stands for quaternary carbon, (CH)

Reaction progress was monitored by

5


13

C NMR spectra were


124

stands for tertiary carbon, (CH2) stands for secondary carbon, (CH3) stands for

125

primary carbon. Elemental analyses were performed on a CHN-O-Rapid instrument.

126

Mass spectrometry (MS) data were obtained with Waters Xevo TQ-S Micro

127

Spectrometer. QSAR Analyses and Molecular Docking were performed with Tripos

128

SYBYL X 2.0 program.

129

General Procedure for the Synthesis of Compound 2. A three-neck round-bottom

130

schlenk flask fitted with a magnetic stir bar, a reflux condenser and an addition funnel,

131

was charged with sodium borohydride (0.95g, 25mmol) and 50mL dry

132

tetrahydrofuran (THF) under N2 atmosphere; then 1.65g of L-Phenylalanine(10mmol)

133

was added in one portion and cooled to 0 °C with an ice bath. A solution of iodine

134

（2.54g, 10mmol）in dry THF (25 mL) was added slowly and dropwise with an

135

addition funnel under vigorous stirring. After completion of the iodine addition, the

136

whole system was put into a preheated oil bath (80 °C), the progress of the reaction

137

was monitored by TLC until the reaction was complete (~12h). The flask was then

138

cooled to room temperature, and cold water was added cautiously to quench the

139

reaction. The solvent was removed under vacuum, 20mL of 20% aqueous KOH was

140

added to the white paste and the solution was stirred for 1 h and extracted by

141

dichloromethane (DCM, 30mL X 4), The organic extracts were combined and dried

142

over sodium sulfate and concentrated in vacuum to afford a white semisolid 1.298g

143

(yield 86%) and was used for next step without further purification.

144

General Procedure for the synthesis of Compound 3. Method 1[14]; an oven dried

145

two-necked schlenk flask was purged with nitrogen and charged with freshly flame 6


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146

dried ZnCl2 (405 mg, 3 mmol), anhydrous chlorobenzene (6 mL), 2-aminobenzonitrile

147

(118 mg, 1 mmol) and (R)-2-amino-2-phenylethanol (1.2 mmol) respectively. The

148

mixture was stirred at reflux and the reaction progress was monitored by TLC until

149

the reaction was complete (24 h~48h). The solvent was then removed under vacuum

150

and the residue was stirred with 30% NaOH (10mL) for 0.5 h. The product was

151

extracted with dichloromethane(15mL X 3) and purified by flash column

152

chromatography on 200~300 mesh silica gel (hexane/EtOAc, 2:1) to give the

153

(R)-2-(4-phenyl-4,5-dihydrooxazol-2-yl)aniline as white solid (Yield 60.1%).

154

Method 2: A tiny modification from the literature report procedure[15]. To an oven

155

dried tube under nitrogen atomosphere, was added 2-aminobenzonitrile (118 mg, 1

156

mmol), (R)-2-aminopropan-1-ol, and freshly flame dried ZnCl2 (13mg, 10%mmol).

157

The mixture was sealed with teflon tape and stirred at 150℃，the reaction progress

158

was monitored by TLC until the consumption of aminobenzonitrile （6h~8h）. The

159

reaction mixture was quenched and suspended with ethyl acetate（50mL），30% NaOH

160

(5 mL) was added and the organic phase was washed with H2O (10mL X 2) and

161

saturated aqueous NaCl（10mL）respectively，then dried over anhydrous sodium

162

sulfate, filtered, and concentrated by evaporation under vacuum to give the crude

163

products，which was subject to flash chromatography purification on silica gel

164

(hexane/EtOAc, v/v = 2:1)to give (R)-2-(4-methyl-4,5-dihydrooxazol-2-yl)aniline as a

165

light yellow oil(68.4%).

166 167

The other 2-(4, 5-dihydrooxazol-2-yl)anilines were synthesized accordingly, with yields ranging from 55.4% to 72.8%. 7



168

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General Procedure for the synthesis of Compound 4.

169

With Acyl Chloride: To a dried schlenk flask charged with the aforementioned

170

synthesized 2-(4,5-dihydrooxazol-2-yl)aniline 3 (1mmol)，were successively added

171

anhydrous DCM (10mL) and distilled NEt3 under N2 atomosphere. The solution of

172

nicotinoyl chloride in anhydrous DCM was added slowly under ice-bath. The mixture

173

was allowed to warm gradually to ambient temperature and stirred for 24 h. Saturated

174

aqueous NH4Cl（15mL）was added to quench the reaction, separated and extracted

175

with dichloromethane (15mL X 3). Then the combined organic phase was washed

176

with water (10mL X 2) and saturated aqueous NaCl (10mL), dried over anhydrous

177

sodium sulfate, and concentrated to give the crude products, which was subject to

178

flash chromatography purification on silica gel (hexane/EtOAc, v/v =2:1) to give the

179

desired product.

180

Compounds 4ba−4bd were synthesized according to this procedure.

181

With Pyridyl acid: To a dried schlenk flask charged with the aforementioned

182

synthesized 2-(4,5-dihydrooxazol-2-yl)aniline 3 (1mmol) and the pyridyl acid (1.05

183

mmol), were added anhydrous DCM (8 mL) and DiPEA (1.5mmol) and the mixture

184

was

185

ethylcarbodiimide

186

4-dimethylaminopyridine (DMAP, 0.012 g, 0.1 mmol), and then the mixture was

187

stirred overnight at room temperature until the full consumption of compound 3

188

detected by TLC. The mixture was quenched by the addition of a saturated aqueous

189

solution of NH4Cl (20 mL) and separated. The water phase was extracted with

vigorously

stirred

for

hydrochloride

dissolution, (EDCI-HCl)

N-(3-Dimethylaminopropyl)(0.211

8


g,

1.1

mmol)

N′and

Page 9 of 26


190

dichloromethane (15mL X 3), and the combined organic phase was washed with water

191

(10mL X 2) and saturated aqueous NaCl (10mL) successively, dried over anhydrous

192

sodium sulfate, concentrated under vacuum and purified by chromatography on silica

193

gel (hexane/EtOAc, v/v = 4:1 ~ 2:1) to give the desired product. Unless otherwise mentioned, the other oxazoline nicotinamides were synthesized

194 195

according to this procedure.

196

Take compound 4be as an example for physicochemical detection and structure

197

elucidation: (R)-N-(2-(4-methyl-4,5-dihydrooxazol-2-yl)phenyl)nicotinamide (4be),

198

white solid, m.p. 88.1~88.4℃, purified on silica chromatography (Hexane/ Ethyl

199

acetate= 4:1), Yield 79%. 1H-NMR (CDCl3, 400 MHz) δ: 1.47(d, J = 5.08Hz, 3H,

200

CH3), 3.97(dd, J1 = 5.92Hz, J2 = 5.76 Hz, 1H, 1H in OCH2), 4.52~4.61(m, 2H, 1H in

201

OCH2 and 1H in CHN-CH3), 7.17 (ddd, 1H , J1 = 6.32Hz, J2= 5.88Hz, J3 = 0.84 Hz,

202

1H, aromatic H in phenyl ring) 7.53~7.57(m, 2H, aromatic H in phenyl ring and

203

Pyridyl ring ), 7.92(dd, J1 = 6.32Hz, J2 = 1.24 Hz, 1H, aromatic H in phenyl ring),

204

8.53(m, 1H, aromatic H in Pyridyl ring), 8.79(dd, J1 = 3.88Hz, J2 = 1.08 Hz, 1H,

205

aromatic H in phenyl ring), 8.90(dd, J1 = 6.20Hz, J2 = 0.56 Hz, 1H, aromatic H in

206

Pyridyl ring), 9.37(d, 1H, J = 1.40 Hz, aromatic H in Pyridyl ring), 13.41(s, 1H, NH).

207

13

208

113.8(C), 119.9(CH), 123.1(CH), 124.1(CH), 129.4(CH), 131.7(C), 132.8(CH),

209

137.2(CH), 139.7, 147.5(CH), 150.6(CH), 163.2(C), 163.9(C). Elemental anal. calcd

210

for C16H15N3O2: C, 68.31; H, 5.37; N, 14.94; Found: C, 68.42; H, 5.41; N, 14.96.

211

ESI-MS, Calcd for [M+H] 282.12, found 282.19.

C-NMR and DEPT135 (CDCl3, 100 MHz) δ: 21.6(CH3), 61.9(CH), 72.9(OCH2),

9



212

Biological Assay. The fungi were provided by the Department of Pesticide, College

213

of Plant Protection, Nanjing Agricultural University (Nanjing, China). The fungicidal

214

activity of the target compounds was tested in vitro against the three plant pathogenic

215

fungi using the mycelium growth rate test. All the tested compounds were dissolved

216

in DMSO at a concentration of 10 mg·mL−1. The media containing compounds at a

217

concentration of 50 µg·mL−1 were then poured into Petri dishes for initial screening.

218

In the precision antifungal test, the 10 mg·mL−1 solution was diluted to 25, 12.5, 6.25,

219

3.125, 1.56 µg·mL−1 and the above experiments were repeated three times, the

220

inhibition rates were calculated separately.

221

The in vivo fungicidal activity of the target compounds was carried out on tomato.

222

Appropriate amounts of all the test samples, including synthesized nicotinamides and

223

positive control boscalid, in 0.2 mL of dimethyl sulfoxide (DMSO) were suspended in

224

20mL distilled water with triton (0.1%) at a concentration of 100 mg/L. Each

225

suspension was sprayed onto the fruits of tomato (Lycopersicum esculentum), which

226

is washed and treated with water and 75% aqueous ethyl alcohol in advance. After

227

evaporation under ambient environment (~28℃), the epidermis (Ø 4.5 cm) on fruits

228

was punctured with inoculating needle, then each pathogen was inoculated. 1%

229

aqueous DMSO containing 0.1% triton was set up as blank control. All the treated

230

fruits were then placed into illumination incubator (25℃, 100% relative humidity) for

231

4 days. This experiments were repeated for three times.

232

The statistical analyses were performed by SPSS software (SPSS Statistic19.0).

10


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233

QSAR Analyses and Molecular Docking. The 3D-Quantitative Structure-Activity

234

Relationship (3D-QSAR), and Molecular Docking were performed with the SYBYL

235

X 2.0 program, using Topomer CoMFA and Surflex-Dock respectively. Besides the

236

core of the molecule, we split the functional groups of synthesized compounds into

237

two R-groups that refer to the amine moiety and carboxylic acid moiety groups. The

238

synthesized oxazoline amides were used to create a data set in which the inhibition

239

rate and EC50 of all compounds was determined against Botrytis cinerea.

240

Three-dimensional structures of the target compounds were built by the ChemBio 3D

241

Ultra software version 12.0. The Structures were imported to Tripos SYBYL X 2.0

242

and optimized for lowest energy geometry. Crystal structure of succinate

243

dehydrogenase (SDH) was downloaded from RCSB Protein Data Bank (PDB code:

244

2FBW). The dabigatran was docked in the corresponding binding site by an empirical

245

scoring function in Surflex-Dock. Before the docking process, the ligand (P/CBE 202)

246

was extracted and all water molecules were removed from the crystal structure.

247 248

RESULTS AND DISCUSSION

249

Synthesis. The chiral amino alcohols were synthesized by the reduction of the

250

corresponding chiral amino acids with previously reported systems of NaBH4-H2SO4

251

or NaBH4-I2 in good to excellent yields[16]. In most case, the NaBH4-I2 was used to

252

provide chiral amino alcohols. The chiral oxazoline units were established by

253

condensation of commercially available aromatic nitrile and different amino alcohols

254

based on the ZnCl2 catalyzed Witte-Seeliger reaction[14], It can also be synthesized

255

under microwave irradiation with higher temperature[15]. For the sake of easy workup, 11



256

we carried out this transformation with the modified Witte-Seeliger reaction in a

257

sealed tube at 150℃ catalyzed by freshly flame dried ZnCl2 under solvent free

258

conditions.

259

either by direct condensation of acyl chloride with amine or DMAP catalyzed steglich

260

type reaction from organic acids activated and dehydrated by EDCI (Scheme 1).

The amide bond formation was realized in good to excellent yields

261

Antifungal Activity and SAR Discussion. We began our investigation with the

262

simple and easily synthesized nicotinamide containing the unsubstituted oxazoline

263

unit (compound 4ba), the initial attempt for the bioactivity screening was not so

264

successful because of its poor physicochemical properties. This was readily solved by

265

the introduction of substituents on either the pyridinyl ring (4aa, 4ca and 4da) or the

266

oxazoline subuinit (4bb~4be). For the preliminary assessment of steric effect of the

267

substituents on the bioactivities, the smallest CH3 and bulky aromatic Phenyl ring

268

were designed and decorated on the 4-position of oxazoline, methyl effect showed

269

more advantages over the phenyl counterpart. The Hydroxyl group on the pyridinyl

270

ring is detrimental for the antifungicidal activity, as can be seen from the table 1, the

271

introduction of OH will lead sharp decrease in bioactivity, no matter what the

272

substituent and the configuration of the oxazoline ring is, especially for the

273

synthesized amides against the Botrytis cinerea and Sclerotinia sclerotiorum” (table1,

274

4ca~4ce). The importance of nicotinamide subunit on the bioactivity was confirmed

275

by the synthesis and bioassay of the corresponding nicotinic acid ester and benzoate

276

(Figure 4).

277

The electron donating groups (EDGs) are more beneficial for the antifungnal 12


Page 12 of 26

Page 13 of 26


278

bioactivity of nicotinamide with unmodified oxazoline ring (MeO > Cl, 4aa vs 4da),

279

while the situation changed inversely when the oxazoline part was substituted and the

280

chirality was introduced (Cl > MeO, 4ab~4ae and 4db~4de). Replacement of

281

nicotinyl group by isonicotinyl gave comparable activities in most cases, while when

282

the 4-position of oxazoline units was decorated by phenyl ring with R-configuration,

283

the inhibition against Rhizoctonia solani and Sclerotinia sclerotiorum dropped sharply

284

with the increment of EC50 from 5.34 and 9.75 to 17.57 and 18.02 mg/L, respectively

285

(compound 4be vs 4ee). Fine-tuning of the substituent on pyridyl ring protrude

286

2-Cl-Pyridinyl amide as good candidates for further optimization. The effect of the

287

substitution and the chiral properties or steric configuration in the 4-position of

288

oxazoline on the fungicidal activity is prominent. For most case, the R-configuration

289

is preferred for both nicotamides and isonicotamides, with similar if not superior

290

inhibitory effect.

291

With this interesting and encouraging results, we envisage that fine tuning of the

292

size and configuration of substituents may be beneficial for the discovery of more

293

promising fungicidal agents. Keeping 2-Cl-nicotamide as constant, compounds

294

4af~4am were synthesized and screened (Table 2). The antifungal activity was

295

enhanced when the bulkier aliphatic groups were introduced, including Et, iPr and

296

Butyl, with the R configuration as promising enantiomers, and it followed Ph < Me
12.5

Botry tis cinerea

3.17

>200

>12.5

Scl er otini a scler oti orum

11.69

>200

>12.5

O

Figure 4. Importance of nicotinamide unit on bioactivity

Figure 5. In vivo activity of 4af and 4ag

Figure 6. Docking model of 4af, 4ag and boscalid

22


Page 23 of 26


Scheme

Scheme 1. Synthesis of Chiral Nicotamides

23



Page 24 of 26

Tables Table 1. Preliminary Synthesis and Antifungal activity of Chiral Nicotinamides Bioactivity (EC50, mg/L)

Structure

Properties

Compd

S.P. =

R=

R. S.

B. C.

S. S.

ClogP

tPSA

4aa

2-Cl-nicotinyl

H

56.51±0.69 l

151.17±0.57 l

99.73± 0.53 m

2.160

63.05

4ab

2-Cl-nicotinyl

S-CH3

11.18±0.24 e

16.15±0.28 de

25.75±0.51 h

2.679

63.05

4ac

2-Cl-nicotinyl

S-Ph

29.56±1.44 i

20.24± 1.86 f

30.28±1.67 j

3.718

63.05

4ad

2-Cl-nicotinyl

R-CH3

4.99±0.13 b

3.14±0.10 a

11.29±0.31 c

2.679

63.05

4ae

2-Cl-nicotinyl

R-Ph

27.10±0.67 h

13.99±0.15 cde

21.33±0.42 g

3.718

63.05

4ba

nicotinyl

H

-

-

-

2.198

63.05

4bb

nicotinyl

S-CH3

8.31±0.33 cd

23.91±0.13 g

19.73±0.15 f

2.717

63.05

4bc

nicotinyl

S-Ph

21.61±1.16 g

21.67±1.11 fg

25.58±0.46 h

3.756

63.05

4bd

nicotinyl

R-CH3

8.21±0.07 cd

11.78±0.29 bc

20.92±0.45 fg

2.717

63.05

4be

nicotinyl

R-Ph

5.34±0.13 b

10.60±0.42 b

9.75±0.08 b

3.756

63.05

4ca

2-OH-nicotinyl

H

114.44±3.71 p

>200

>200

3.486

83.23

4cb

2-OH-nicotinyl

S-CH3

94.26±0.72 n

>200

>200

4.005

83.23

4cc

2-OH-nicotinyl

S-Ph

44.31±0.62 k

>200

>200

5.044

83.23

4cd

2-OH-nicotinyl

R-CH3

99.96±2.11 o

138.79±1.81 k

>200

4.005

83.23

4ce

2-OH-nicotinyl

R-Ph

86.96±0.45 m

174.31±8.99 m

>200

5.044

83.23

4da

2-OCH3-nicotinyl

H

9.41±0.04 d

23.57±0.43 g

28.62±0.16 i

2.943

72.28

4db

2-OCH3-nicotinyl

S-CH3

16.34±0.13 f

17.01±0.43 e

31.35±0.11 j

3.453

72.28

4dc

2-OCH3-nicotinyl

S-Ph

134.66±0.52 r

57.02±0.57 j

147.88±2.01 n

4.492

72.28

4dd

2-OCH3-nicotinyl

R-CH3

12.77±0.12 e

13.65±0.20 bcd

54.53±0.32 l

3.453

72.28

4de

2-OCH3-nicotinyl

R-Ph

127.99±0.45 q

14.37±0.21 cde

40.33± 0.46 k

4.492

72.28

4ea

isonicotinyl

H

9.57±0.32 d

28.18±0.20 h

28.44±0.43 i

2.198

63.05

4eb

isonicotinyl

S-CH3

7.34± 0.15 c

12.31±0.12 bc

25.57±0.43 h

2.717

63.05

4ec

isonicotinyl

S-Ph

37.67±0.43 j

34.02±0.28 i

18.13±0.15 e

3.756

63.05

4ed

isonicotinyl

R-CH3

5.71±0.14 b

11.45±0.18 bc

15.09±0.24 d

2.717

63.05

4ee

isonicotinyl

R-Ph

17.57±0.33 f

16.32±0.34 de

18.02±0.48 e

3.756

63.05

1.59±0.11 a

1.66±0.12 a

0.34±0.05 a

3.437

41.46

Boscalid

Note: R. S.: Rhizoctonia solani; B. C.: Botrytis cinerea; S. S.: Sclerotinia sclerotiorum. The EC50 values are presented as mean ± standard deviation of triplicate experiments. Different small letters in the same column showed significant difference at P < 0.05 level, through Duncan’s multiple range test in SPSS statistics 19.0. The alphabetical order is consistent with the high to low order of the antifungal activity.

24


Page 25 of 26


Table 2. Further Optimization and Discovery of More Potent Chiral Nicotinamides Structure

Bioactivity (EC50,mg/L)

Properties

Compd

R=

R.S.

B.C.

S.S.

ClogP

tPSA

4ab

S-CH3

11.18±0.24 k

16.14±0.27 h

25.75±0.51f

2.679

63.05

4ad

R- CH3

4.99±0.13 e

3.14±0.10 f

11.29±0.31 e

2.679

63.05

4af

S-Et

3.58±0.11 d

2.30±0.12 e

4.58±0.12 c

3.208

63.05

4ag

R-Et

0.58±0.09 a

0.42±0.04 a

2.10±0.03 b

3.208

63.05

4ah

S-iPr

7.36±0.11 h

3.29±0.11 f

4.55±0.07 c

3.607

63.05

4ai

R-iPr

2.95±0.21 c

0.51±0.03 a

2.25±0.04 b

3.607

63.05

4aj

S-iBu

9.89±0.20 j

3.32±0.03 f

4.87±0.08 c

4.136

63.05

4ak

R-iBu

8.17±0.08 i

1.15±0.08 c

0.63±0.05 a

4.136

63.05

4al

S-s-Bu

6.16±0.09 f

0.70±0.02 b

2.11±0.03 b

4.136

63.05

4am

S-t-Bu

6.63±0.05 g

3.83±0.15 g

6.36±0.12 d

4.006

63.05

Boscalid

1.59±0.11 b

1.66±0.12 d

0.34±0.05 a

3.437

41.46

Note: R. S.: Rhizoctonia solani; B. C.: Botrytis cinerea; S. S.: Sclerotinia sclerotiorum. The EC50 values are presented as mean ± standard deviation of triplicate experiments. Different small letters in the same column showed significant difference at P < 0.05 level, through Duncan’s multiple range test in SPSS statistics 19.0. The alphabetical order is consistent with the high to low order of the antifungal activity.

Graphic for table of contents 25



26


Page 26 of 26

Design, Synthesis, Fungicidal Activity, and Unexpected Docking

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