Antifungal Activity of Griseofulvin Derivatives against Phytopathogenic

Agricultural and Environmental Chemistry

Subscriber access provided by BOSTON COLLEGE

Antifungal Activity of Griseofulvin Derivatives against Phytopathogenic Fungi In Vitro, In Vivo, and 3D-QSAR Analysis Yu-Bin Bai, Yu-Qi Gao, Xiao-Di Nie, Thi Mai Luong Tuong, Ding Li, and Jin-Ming Gao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00606 • Publication Date (Web): 14 May 2019 Downloaded from http://pubs.acs.org on May 15, 2019

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 24

Journal of Agricultural and Food Chemistry

1

Antifungal Activity of Griseofulvin Derivatives against Phytopathogenic Fungi In

2

Vitro, In Vivo, and 3D-QSAR Analysis

3 4

Yu-Bin Bai†‡∥, Yu-Qi Gao†∥, Xiao-Di Nie†, Thi-Mai-Luong Tuong†, Ding Li*†,

5

and Jin-Ming Gao*†

6 7 8 9 10 11

†Shaanxi

Key Laboratory of Natural Products & Chemical Biology, College of Chemistry & Pharmacy, Northwest A&F University, Yangling 712100, China ‡Shaanxi

Key Laboratory of Chemical Reaction Engineering, College of Chemistry and Chemical Engineering, Yan’an University, Yan’an 716000, China

12 13 14

*Corresponding author:

15

Fax/Tel: +86-29-87092335.

16

E-mail: [email protected] (J. M. Gao); [email protected] (D. Li)

17 18

1

ACS Paragon Plus Environment


19

Page 2 of 24

ABSTRACT

20

With environmental pollution, residual hazards accumulate, and severe drug resistance and

21

many other problems appear; some highly toxic drugs have been banned, and antifungal agents

22

are far from satisfactory. Natural products play an important role in the discovery and

23

development of new pesticides. The natural product griseofulvin (1) is known as an antifungal

24

agent in the treatment of dermatomycoses for decades. In this study, a series of new griseofulvin

25

derivatives were synthesized with good yields. Their structures were characterized by 1H and 13C

26

NMR and HR-MS (ESI). The antifungal activities of griseofulvin analogues were first evaluated

27

against ﬁve phytopathogenic fungi (Cytospora sp., Colletotrichum gloeosporioides, Botrytis

28

cinerea, Alternaria solani, and Fusarium solani) in vitro. Of significance is that most of them

29

showed excellent antifungal activities against C. gloeosporioides. The antifungal activities of

30

four best compounds (6a, 6c, 6e, and 6f) against Colletotrichum gloeosporioides were further

31

investigated in vivo by using infected apples. The results suggested that compounds 6c, 6e, and

32

6f (IC50 = 47.25±1.46 μg/mL, IC50 = 49.44±1.50 μg/mL, and IC50 = 53.63±1.74 μg/mL,

33

respectively) were better than thiophanate-methyl (IC50 = 69.66±6.07 μg/mL). Furthermore,

34

comparative molecular field analysis (CoMFA) was performed on the basis of the antifungal

35

activity results of all 22 of the compounds against C. gloeosporioides in vitro. The 3D coefficient

36

contour plots revealed that the suitable bulky and electronegative acyl substituted groups seem to

37

be more favorable for increasing activity at the 4 position of griseofulvin. The structure–activity

38

relationships were also discussed. Griseofulvin derivatives can be used for the development of

39

highly effective and safe agricultural fungicides.

40

_____________________________________________________________________________

41 42

KEY WORDS: Griseofulvin derivatives; antifungal activity; phytopathogenic fungi; structure-activity relationships

2


Page 3 of 24

44


INTRODUCTION

45

It is well known that plant pathogenic fungi can cause a tremendous loss of global

46

agricultural production. Despite that synthetic fungicides are effective and play an indispensable

47

role against pathogenic fungi, the available antifungal agents are far from satisfactory due to

48

several drawbacks, such as severe drug resistance, drug-related toxicity, and environmental

49

hazards[1-3] Therefore, novel antifungal agents are needed to effectively control the fungal

50

diseases of agricultural crops.

51

Griseofulvin (1, Scheme 1), a spirocyclic benzofuran-3-one natural antifungal product, was

52

initially isolated from Penicillium griseofulvum in 1939 by Oxford et al.[4]. It was the first oral

53

antifungal drug in the treatment of dermatomycoses, such as tinea capitis (ringworm of the scalp)

54

and tinea pedis (athlete’s foot), in animals and humans for decades.[5-8] Griseofulvin has gained a

55

lot of attention in research and academic circles[9-10]; more than 400 griseofulvin analogues have

56

been synthesized and used for drug screening.[10-12] In addition to the antifungal effect,

57

griseofulvin exhibits various other biological activities, including anticancer and antiviral

58

properties. Moreover, it is also useful against plant pathogenic fungi.[11-13] Zhao et al.[14] reported

59

that griseofulvin displayed clear inhibition of the growth of 8 different plant pathogenic fungi. In

60

our previously reported study, griseofulvin, as an isolated natural product, showed a significant

61

inhibitory activity against the pathogenic fungus Alternaria solani. [15]

62

In this work, a series of novel griseofulvin derivatives were designed and synthesized from

63

commercially available griseofulvin via efficient methods (Scheme 2). The antifungal activities

64

of newly synthesized compounds were investigated for some important agricultural fungi

65

diseases, including Cytospora sp., Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria

66

solani, and Fusarium solani in vitro. Active compounds with a high inhibitory effect against

67

Colletotrichum gloeosporioides were tested in vivo. The structure-antifungal activity

68

relationships of all of the griseofulvin analogues were also discussed based on 3D-QSAR

69

CoMFA analysis.

70 71

MATERIALS AND METHODS

72

General Experimental Procedures. The melting points of the products were determined on an

73

M420 apparatus (Jinan hanon Instruments Co., Ltd., China) and are uncorrected. Nuclear 3



Page 4 of 24

74

magnetic resonance (NMR) spectra were recorded on a Bruker Vance III spectrometer (Unity

75

plus 500 MHz, Bruker Bios pin, Rheinstetten, Germany) and DD2 400-MR (Agilent NMR

76

Systems 400 MHz Spectrometer, America) with TMS as the internal standard. Thin-layer

77

chromatography (TLC) was performed on silica gel 60 F254 (Qingdao Marine Chemical Ltd.,

78

China), and column chromatography (CC) was performed on silica gel (200-300 mesh, Qingdao

79

Marine Chemical Ltd.). The high resolution electrospray ionization mass spectrometry

80

(HRESIMS) data were recorded on a Bruker maXis ESI-Q-TOF (Bruker Daltonics Inc.,

81

America).

82

Reagents. Chemicals and reagents are commercially available. Griseofulvin was purchased from

83

a commercial source (Shanghai Aladdin Bio-Chem Technology Co., Ltd.).

84

Synthetic Procedures.

85

General Synthetic Procedure for the Key Intermediates.

86

The intermediate 2 ((2S,6'R)-7-chloro-4-hydroxy-2',6-dimethoxy-6'-methyl-2-spiro[benzofuran-

87

2,1'-cyclohex[2]ene]-3,4'-dione) was synthesized from griseofulvin by a reported procedure.[8, 16]

88

Iodine pills (1.83 g, 7.2 mmol) were added to a solution of magnesium turnings (0.52 g, 21.6

89

mmol) in anhydrous Et2O (4 mL) and toluene (8 mL). This solution was refluxed at 80°C for

90

about 30 min. until the reaction mixture turned to be a colorless solution under argon. The

91

resulting solution was added to a solution of griseofulvin (1.41 g, 4 mmol) in dry toluene (10

92

mL) and heated to 80°C for 4 h. Then, H2O (20 mL) and Na2S2O3 were added and the reaction

93

mixture was poured into 5% hydrochloric acid (10 mL). The mixture was extracted with EtOAc

94

(3 × 20 mL), dried under MgSO4 and concentrated.[8,

95

chromatography (CH2Cl2: MeOH: AcOH 97:2:1) to yield the title compound as a white solid

96

(1.12 g, 81%). m.p. 130.7 – 133.2°C; [α]19.6 D = + 397.2 (c = 0.11 in CH3OH); 1H NMR (500

97

MHz, CDCl3) δ 6.18 (s, 1H, H-5), 5.56 (s, 1H, H-3), 3.97 (s, 3H, H-10), 3.65 (s, 3H, H-11), 2.95

98

(dd, J = 16.2, 13.2 Hz, 1H, H-5), 2.87 (dtd, J = 10.8, 6.4, 4.1 Hz, 1H, H-6), 2.48 (dd, J = 16.2,

99

4.1 Hz, 1H, H-5), 0.97 (d, J = 6.5 Hz, 3H, H-8); 13C NMR (125 MHz, CDCl3) δ 196.8 (C-4),

100

196.1 (C-3), 170.3 (C-2), 167.4 (C-7a), 165.7 (C-6), 156.1 (C-4), 105.1 (C-3), 104.1 (C-3a),

101

96.7 (C-7), 93.9 (C-5), 91.5 (C-2), 57.4 (C-10), 57.0 (C-11), 40.2 (C-5), 36.4 (C-6), 14.4 (C-8).

102

MS (ESI): m/z for C16H14ClO6: 337.05; found: 337.45 [M-H]-.

103

General synthetic procedure for intermediate 4 by the conventional method[8].

16]

The residue was purified by column

4


Page 5 of 24


104

Griseofulvin (3.52 g, 10 mmol) was dissolved in a mixture of ethanol (200 mL) and water (60

105

mL). Then, hydroxyl ammonium hydrochloride (2.26 g, 35 mmol) and sodium acetate trihydrate

106

(5.88 g, 43 mmol) were added to this solution. The mixture was refluxed for 6 h and then cooled

107

to room temperature, followed by the addition of cold water. The mixture was extracted with

108

Et2O (3 × 100 mL), dried (MgSO4), and concentrated[18-20]. The residue was purified by column

109

chromatography on silica gel, eluting with PE-actone (2:1) to yield the final compound as a light

110

yellow solid 3.42 g, yield 93%. m.p. 228.1 – 230.4°C; [α]19.4 D= +379.7 (c = 0.19 in CH3OH);

111

MS (ESI): m/z calcd. for C17H18ClNO6H: 368.09; found: 368.10 [M+H]+.

112

General synthetic procedure for compound 5 by the same method with intermediate 4.

113

(2S,6'R)-7-chloro-2',4,6-trimethoxy-4'-(methoxyimino)-6'-methyl-2-spiro[benzofuran-2,1'-cyclo

114

hex[2]ene]-3-one (5). Yield, 92%; white solid; m.p. 188.6 – 189.5°C; [α]19.6 D= +388.8 (c 0.15

115

in CH3OH). The product was a mixture of cis and trans isomers with a molar ratio about 5a: 5b =

116

0.8:1. 5a 1H NMR (500 MHz, CDCl3) δ 6.15 (s, 0.8H, H-5), 6.09 (s, 0.8H, H-3′), 4.00 (s, 3H,

117

H-10), 3.95 (s, 2.4H, H-9), 3.87 (s, 2.4H, -NOCH3), 3.57 (s, 2.4H, H-11), 2.99 – 2.93 (m, 0.8H,

118

H-5′), 2.54 (m, 0.8H, H-6′), 2.37 (dd, J = 15.1, 4.2 Hz, 0.8H, H-5′), 0.93 (d, J = 6.8, 2.4H, H-8).

119

13C

120

151.1 (C-4), 105.8 (C-3a), 97.3 (C-7), 93.3 (C-3′), 91.6 (C-2), 89.3 (C-5), 61.5 (-NOCH3), 57.0

121

(C-10), 56.4 (C-9), 56.2 (C-11), 36.6 (C-5′), 26.2 (C-6′), 14.4 (C-8). 5b 1H NMR (500 MHz,

122

CDCl3) δ 6.09 (s, 1H, H-5), 5.59 (s, 1H, H-3′), 4.00 (s, 3H, H-10), 3.95 (s, 3H, H-9), 3.88 (s, 3H,

123

-NOCH3), 3.53 (s, 3H, H-11), 3.01 (dd, J = 16.8, 4.9 Hz, 1H, H-5′), 2.68 – 2.60 (m, 1H, H-6′),

124

2.54 (m, 1H, H-5′), 0.91 (d, J = 6.7 Hz, 3H, H-8). 13C NMR (125 MHz, CDCl3) δ 194.3 (C-3),

125

169.6 (C-2′), 164.4 (C-7a), 158.5 (C-4′), 157.6 (C-6), 154.4 (C-4), 105.8 (C-3a), 99.3 (C-3′), 97.3

126

(C-7), 91.5 (C-2), 89.3 (C-5), 61.8 (-NOCH3), 57.0 (C-10), 56.4 (C-9), 56.0 (C-11), 35.5 (C-5′),

127

31.2 (C-6′), 14.5 (C-8). HR-MS (ESI): m/z calcd. for C18H20ClNO6Na: 404.0877; found:

128

404.0873 [M+Na]+.

129

The general procedure of synthesizing compounds 3a-3g was a reported method[17], and

130

general synthetic procedures of compounds 6a-6k, 7 were based on a conventional method;

131

please see these details in supporting information.

NMR (125 MHz, CDCl3) δ 194.2 (C-3), 169.6 (C-2′), 164.4 (C-7a), 161.1 (C-4′), 157.6 (C-6),

132 133

Antifungal Activity Assay in Vitro 5



Page 6 of 24

134

The antifungal activities of griseofulvin and its derivatives were evaluated against five plant

135

phytopathogenic fungi: Cytospora sp, Colletotrichum gloeosporioides, Botrytis cinerea,

136

Alternaria solani, and Fusarium solani by the mycelial growth inhibitory rate method according

137

to previously reported approaches.[2] PDA medium was prepared and sterilized. The compounds

138

were dissolved in acetone before mixing with molten agar at 40°C; the concentration of the

139

compounds in the medium was ﬁxed at 100 μg/mL. The mixture was then poured into sterilized

140

Petri dishes. After cooling, a mycelial disk of approximately 4 mm diameter from the culture

141

medium of test fungi was picked up with a sterilized inoculation needle and inoculated in the

142

center of the fresh PDA Petri dishes. The Petri dishes were incubated at 28°C for 3–4 d. Acetone

143

was used as the negative control, while the hymexazol (Hy) and thiophanate-methyl (Tpm), both

144

commercially available agricultural fungicides for direct application, were used as the positive

145

controls. Each sample was measured in triplicate, and each colony diameter of all triplicates was

146

measured 4 times by the cross bracketing method. After the mycelia grew completely, the

147

diameters of the mycelia were measured and the inhibition rate was calculated according to the

148

formula:

149

Inhibition rate (%) = (C－T)/ (C－4 mm) × 100%,

150

where C represents the diameter of fungal growth on untreated PDA, and T represents the

151

diameter of fungal growth on treated PDA.

152

The compounds with high activity against the target fungi were prepared in 7 fresh PDA Petri

153

dishes with different concentrations (100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 μg/mL), and their

154

precise antifungal effects (IC50 value) were further investigated.

155

Antifungal Activity Assay in Vivo

156

According to the results of in vitro antifungal activity, the 4 best activity compounds 6a, 6c,

157

6e, and 6f against Colletotrichum gloeosporioides were further tested in vivo. The same kind of

158

ripened apples with similar shapes were picked in the same orchard for testing. The apples were

159

soaked in 1% sodium hypochlorite for 2 minutes to disinfect them before they were well rinsed

160

under running water for 5 minutes. Then, a hole (3 mm × 3 mm) was punched on the apple

161

surface. After the drying of holes, a dose of 20 μL solution (each compound was dissolved in

162

acetone and five concentration gradients 100, 50, 25, 12.5, and 6.25 ug/mL were set) was added

163

to separate samples. As the acetone solution of the compound dried, a 20-μL spore suspension 6


Page 7 of 24


164

solution (CFU = 5 × 104 /mL) was added to each specimen. Upon drying, all of the apples

165

were placed in green room at 28°C, and moisture was retained for 7–8 d. A spore suspension

166

solution served as the blank while sterile water was a negative control. The commercially

167

available agricultural fungicide thiophanate-methyl (Tpm) was used as a positive control. Each

168

sample was measured in triplicate. The same method was followed for the preparation of

169

solutions of different concentrations, and the results were analyzed in vitro.

170

3D-QSAR Analysis

171

Data Sets for 3D-QSAR Analysis. All of the molecular modeling and calculations were

172

performed using SYBYL-X1.3 software (Tripos, Inc.) in a CCNUGrid-based computational

173

environment[21]. The results of biological activities of all 22 griseofulvin analogues were used to

174

derive the CoMFA analyses model listed in Table 4. The structure of griseofulvin was used as a

175

template to construct the other molecular structures by using the “SKETCH” option function in

176

SYBYL. The Gasteiger-Hückel charge was used to calculate the partial atomic charges. Each

177

structure was fully geometrically-optimized using a conjugate gradient procedure based on the

178

TRIPOS force field with the Powell conjugate gradient minimization algorithm and a

179

convergence criterion of 0.05 kcal/mol·Å. These compounds share a common skeleton; the

180

cyclization atoms marked with an asterisk (Scheme 1) were used as the reference atoms, and GF

181

was chosen as template molecule to fit the remaining compounds by using the Database Align

182

function in SYBYL.

183

CoMFA Descriptors. The CoMFA modeling was done by following a previously described

184

procedure[22, 23]. The CoMFA steric and electrostatic interaction fields were calculated on a 3D

185

cubic lattice with a regularly spaced grid of 2.0 Å. The grid pattern was generated automatically

186

by the SYBYL/CoMFA routine, and an sp3 carbon atom probe with a van der Waals radius of

187

1.52 Å and a charge of +1.0 was used to calculate the steric (Lennard-Jones 6–12 potential) field

188

energies and electrostatic (Coulombic potential) fields with a distance-dependent dielectric at

189

each lattice point. Values of the steric and electrostatic fields were truncated at 30.0 kcal/mol.

190

The CoMFA steric and electrostatic fields generated were scaled by the CoMFA-STD method in

191

SYBYL.

192

A partial least-squares (PLS) approach was used to derive the 3D-QSAR, in which the

193

CoMFA descriptors were used as independent variables, and the IC50 values were used as 7



Page 8 of 24

194

dependent variables. The cross-validation was carried out with the leave-one-out (LOO) option

195

and the SAMPLS program, rather than column filtering, to obtain the optimal number of

196

components to be used in the final analysis. After the determination of the optimal number of

197

components, a non-cross-validated analysis was performed without column filtering. The

198

modeling capability (goodness of fit) was judged by the correlation coefficient square r2, and the

199

prediction capability (goodness of prediction) was implied by the cross-validated r2 (q2).

200 201

RESULTS AND DISCUSSION

202

Chemistry

203

The synthesis procedures of the compounds are outlined in Scheme 2. Intermediate 2 was

204

obtained by MgI2, which can selectively remove the methyl of griseofulvin at position 4 with a

205

high yield (81%). The corresponding compounds 3a~3g were synthesized with various acyl

206

chlorides and sodium hydride in dry acetone with yields of 71% ~ 81%. The intermediate 4 and

207

derivative 5 were synthesized by oximation with the addition of hydroxylamine hydrochloride

208

and methoxyamine hydrochloride with high yields of 93% and 92%, respectively. Furthermore,

209

the analogues 6a~6k were obtained by esterification through the use of triethylamine and

210

different acetyl chlorides in dry acetone with good yields (74%~95%). Compound 7 was

211

obtained (with a good yield, 90%) when intermediate 4 and metallic sodium were refluxed in

212

ethyl alcohol. It should be noticed that the methods to synthesize these series of compounds are

213

simple and effective.

214

All of these compounds, 2 intermediates, and the newly prepared 19 derivatives were

215

confirmed by 1H NMR,

13C

216

analogues, 17 compounds (3b-3g, 5, 6a-6c, 6e-6k, 7) are new based on searching in SciFinder

217

web. The antifungal activities of all of the synthesized analogues, including known compounds,

218

are also reported here for the first time.

NMR, and MS, as well as HRESIMS spectral data. Out of the 22

219 220

Evaluation of Inhibitory Efficacy In Vitro

221

The preliminary inhibition rates of all of the title compounds,[24] including griseofulvin and

222

intermediates 2 and 4, and two commercial agricultural fungicides, against five plant-pathogenic

223

fungi (Cytospora sp., Colletotrichum gloeosporioides, Botrytis cinerea, Alternaria solani, and 8


Page 9 of 24


224

Fusarium solani) are shown in supporting information. The results suggested that some

225

synthesized compounds have significant activities with a 65% higher inhibition rate against five

226

plant pathogenic fungi at a concentration of 100 μg/mL. Furthermore, in order to evaluate their

227

antifungal effects precisely, their IC50 (the half-maximal inhibitory concentration) values were

228

determined by the method presented in the antifungal activity assay part, and the data are listed

229

in Table 1. According to the data, griseofulvin showed more remarkable inhibitory activities

230

against Colletotrichum gloeosporioides (IC50 = 11.06±0.42 μg/mL) and Alternaria solani (IC50 =

231

2.68±0.33 μg/mL) than the positive control hymexazol (IC50  100 μg/mL for Colletotrichum

232

gloeosporioides, IC50 = 44.62±0.59 μg/mL for Alternaria solani), but slightly less activity against

233

B. cinerea than hymexazol. While compared to thiophanate-methyl, griseofulvin was similar or a

234

bit more potent against Colletotrichum gloeosporioides, Botrytis cinereal, and Alternaria solani,

235

but less against Cytospora sp. and Fusarium solani. The intermediate 2 reduced the antifungal

236

activity of griseofulvin and only had moderate activity with respect to Colletotrichum

237

gloeosporioides (IC50 = 33.07±0.83 μg/mL). The intermediate 4 was barely able to maintain the

238

antifungal activity against Colletotrichum gloeosporioides (IC50 = 24.38±0.59 μg/mL) and

239

Alternaria solani (IC50 = 19.50±0.99 μg/mL). The overall results revealed that the modifications

240

at position 4 of the griseofulvin molecule decreased the antifungal activity against all five plant

241

pathogenic fungi species analyzed, while the modifications at the 4 position would maintain or

242

improve the antifungal potency of the parent compound up to 10-fold.

243

For instance, some compounds in series 3 (compounds 3a~3g) including intermediate 2, were

244

exceptionally less active than griseofulvin and most of them were completely inactive.

245

Fortunately, some maintained specific activity against Colletotrichum gloeosporioides, and

246

compound 3b had the highest activities (IC50 = 35.56±1.3 μg/mL). Moreover, among these

247

compounds, it was observed that the activities of aliphatic acyl analogues (3a, IC50 = 46.74±0.49

248

μg/mL) were superior to those of the aromatic acyl analogues (3f, IC50 = 68.52±0.11 μg/mL), and

249

acyl analogues were more significant in inhibitory efficacy than sulfonyl analogues.

250

From the results, it is obvious that compounds in series 6 (compounds 6a~6j) had meaningful

251

activities against Colletotrichum gloeosporioides, Botrytis cinereal, and Alternaria solani. The

252

most aliphatic acyl derivatives, obtained by modifications at the 4 position, improved antifungal

253

activities, as compared to griseofulvin, against Colletotrichum gloeosporioides and Botrytis 9



Page 10 of 24

254

cinerea. In particular, compound 6c displayed the most promising inhibitory activities against

255

four out of five targeted fungi. Furthermore, the antifungal activities of aliphatic acyl analogues

256

were better than those of aromatic acyl analogues. Compared with the positive control, six

257

analogues (6a~6e and 6i) in this series had more influential activities against Colletotrichum

258

gloeosporioides and Alternaria solani, specifically Colletotrichum gloeosporioides. Although

259

compounds 6a~6c, 6e and 6f were less active than hymexazol against Botrytis cinerea, they still

260

showed similar or even higher inhibition potency than thiophanate-methyl. As presented in Table

261

1, compound 6c had the most extensive and remarkable activity against almost all of the target

262

fungal species. In general, the results revealed that the griseofulvin analogues obtained by

263

modifications at position 4′ are more significant for antifungal activity and might be worthy of

264

further development as potential agricultural fungicides.

265 266

Evaluation of Inhibitory Efficacy In Vivo.

267

The 4 compounds (6a, 6c, 6e, and 6f) with the highest activities against Colletotrichum

268

gloeosporioides in vitro were further investigated in vivo. The results of in vivo inhibition

269

activities are summarized in Table 2 and showed that the antifungal activities in vivo were less

270

pronounced than those in vitro. All of the above 4 compounds not only presented significant

271

inhibition against C. gloeosporioides but they also protected the fruit from the infection of the

272

fungal pathogen. The results for 7 days inhibitory efficacy of compounds are presented in Table

273

3. It was found that compounds 6c (IC50 = 47.25±1.46 μg/mL), 6e (IC50 = 49.44±1.50 μg/mL)

274

and 6f (IC50 = 53.63±1.74 μg/mL) possessed better restraint of fungal strains than the positive

275

control Thiophanate-methyl (IC50 = 69.66±6.07 μg/mL). The present results also demonstrated

276

that compound 6e has the highest in vitro and in vivo antifungal activities against Colletotrichum

277

gloeosporioides, while compound 6c showed very good in vitro and in vivo antifungal activities

278

against Colletotrichum gloeosporioides and the highest in vitro antifungal activities against other

279

fungal species. These findings strongly suggest that griseofulvin derivatives can be used for

280

development as potential fungicides.

281 282 283

3D-QSAR CoMFA Analysis

Comparative Molecular Field Analysis (CoMFA) mapped the interaction fields that 10


Page 11 of 24


284

surrounded the structures according to their impact on given activities.[25] To derive a reliable

285

CoMFA analysis model, all 22 of the griseofulvin analogues with distinct in vivo activity against

286

Colletotrichum gloeosporioides were studied by cross validation with the LOO option, and the

287

SAMPLS program was used to determine the optimal number of components in CoMFA

288

3D-QSAR analyses. Then, a non-cross-validated analysis was performed without column

289

filtering. The model had three components q2 (cross-validated r2) = 0.648, r2 (non-crossvalidated

290

r2) = 0.929, and SEE (standard error of estimate) = 0.151, defined in SYBYL. The observed and

291

calculated activity values are listed in Table 4. The models exhibited a good predictability for

292

these compounds. The steric and electrostatic contribution contour maps of CoMFA are plotted

293

in Figure 1 and 2, respectively. The 3D coefficient contour plots showed the field effect on the

294

target property; they were helpful to identify important regions that changed in the steric and

295

electrostatic fields. These can help to calculate the possible interaction sites. Compound 6c was

296

illustrated to explain the field contributions of different properties obtained from the CoMFA

297

analyses.

298

Figure 1 shows that there are two steric unfavorable yellow contours: one near the 4 position

299

and the other located beside the propionyl group of 6c. The first steric yellow contours showed

300

that an increase in steric bulk would decrease the activity, which revealed that the more bulky

301

group modifications at 4 position of griseofulvin would result in a decreased antifungal activity.

302

The second steric yellow contours together with the green polyhedral were the most important

303

characteristic of Figure 1, which indicates that too small or bulky substituents were not

304

inappropriate in the 4′ position of griseofulvin. The potency difference between 4 and 6c (6c and

305

6g) could be explained by changes from the -H to -COCH2CH3 (-COCH2CH3 to -COC6H4-CH3)

306

group.

307

The blue contour near the 4 position of 6c insinuated that increasing electron density was

308

unfavorable in this region, as depicted in Figure 2. Besides the blue contours, a predominant

309

feature of the electrostatic plot was the presence of the red contour proximal to the acyl carbonyl

310

oxygen of 6c. It could be reasonably presumed that there was a significant electrostatic

311

interaction between the acyl carbonyl oxygen and the possible receptor.

312

According to the CoMFA/PLS analysis, the steric and electrostatic field properties

313

contributed in a 52/48 ratio to the total variance, which specified that both the steric field and 11



Page 12 of 24

314

electrostatic descriptors have the same importance in explaining the dependent variable of the

315

CoMFA model. The 3D coefficient contour plots revealed that the suitable bulky and

316

electronegative acyl substituted groups seemed to be more favorable for increasing activity at the

317

4 position of griseofulvin. Although the CoMFA model was constructed using only the

318

antifungal activities against Colletotrichum gloeosporioides, it seemed to be helpful to

319

understand structure–activity relationships of these griseofulvin analogues against three other

320

target fungi. Most importantly, these results offered important new insights into designing highly

321

active compounds prior to future synthesis.

322 323

Conclusion

324

A series of griseofulvin derivatives were synthesized by effective approaches, with good yields,

325

and 17 of the 22 were novel. Antifungal activities results indicated that griseofulvin and the

326

majority derivatives have the strongest activities against different phytopathogenic fungi in vitro.

327

In particular, compounds (6a~6f, 6i) were found to have significant antifungal potential and were

328

superior to commercial fungicides hymexazol and thiophanate-methyl. Inhibitory efficacy in vivo

329

further demonstrated that the griseofulvin derivatives can be used as potential fungicides. The

330

SAR of the 4 position derivatives against all five fungi had the same trend, with the majority

331

being less active than griseofulvin. The modification of the 4′ position can improve the

332

antifungal activity against Colletotrichum gloeosporioides and Botrytis cinerea, even up to

333

10-fold more than the inhibition potency of the parent compound. To further explore the

334

structure–activity relationship, comparative molecular field analysis (CoMFA) was performed on

335

the basis of all 22 compounds with their inhibition of C. gloeosporioides. The 3D coefficient

336

contour plots revealed that the suitable bulky and electronegative acyl substituted groups at the 4

337

position of griseofulvin seem to be more favorable for increasing activity. In conclusion,

338

griseofulvin derivatives can be used for the development of potential agricultural fungicides.

339 340

■ AUTHOR INFORMATION

341

To whom Correspondence should be addressed.

342

Tel: +86-29-87092335.

343

E-mail: [email protected] (J.M. Gao); [email protected] (D. Li) 12


Page 13 of 24


344

ORCID

345

Jin-Ming Gao: 0000-0003-4801-6514

346

Ding Li: 0000-0003-3130-6099

347 348

Author Contributions Yu-Bin Bai and Yu-Qi Gao contributed equally to this work.

∥

349 350 351

Funding

352

This work was supported by the Natural Science Foundation of China (21502152), Natural

353

Science Foundation of Shaanxi Province (2014JQ2075), China Postdoctoral Science Foundation

354

(2014M562452), the Program of Unified Planning Innovation Engineering of Science &

355

Technology in Shaanxi Province (No. 2015KTCQ02-14), and Doctoral Scientific Research

356

Start-Up Fund of NWSUAF (1012013BSJJ037).

357

Notes The authors declare that they do not have any competing financial interests.

358 359

■ ACKNOWLEDGMENTS

360

The authors would like to express their thanks to Prof. Jian Wan at Central China Normal

361

University for providing computing resources.

362 363

■ REFERENCES

364

1.

365

Ye, Y.-H., Design, synthesis, antifungal, and antioxidant activities of (E)-6-((2-Phenyl-

366

hydrazono)methyl)quinoxaline derivatives. J. Agric. Food Chem. 2014, 62 (40), 9637-9643.

367

2.

368

2-chloromethyl-1H-benzimidazole derivatives against phytopathogenic fungi in vitro. J. Agric.

369

Food Chem. 2013, 61 (11), 2789-2795.

370

3.

371

Synthesis of 4′-thiosemicarbazonegriseofulvin and its effects on the control of enzymatic

372

browning and postharvest disease of fruits. J. Agric. Food Chem. 2012, 60 (43), 10784-10788.

373

4.

374

griseofulvin, C17H17O6Cl, a metabolic product of penicillium griseofulvum dierckx. Biochem. J.

375

1939, 33 (2), 240-248.

Zhang, M.; Dai, Z.-C.; Qian, S.-S.; Liu, J.-Y.; Xiao, Y.; Lu, A.-M.; Zhu, H.-L.; Wang, J.-X.;

Bai, Y.-B.; Zhang, A.-L.; Tang, J.-J.; Gao, J.-M., Synthesis and antifungal activity of

Pan, Z.-Z.; Zhu, Y.-J.; Yu, X.-J.; Lin, Q.-F.; Xiao, R.-F.; Tang, J.-Y.; Chen, Q.-X.; Liu, B.,

Oxford, A. E.; Raistrick, H.; Simonart, P., Studies in the biochemistry of micro-organisms:

13



Page 14 of 24

376

5.

Gentles, J. C., Experimental ringworm in guinea pigs: oral treatment with griseofulvin.

377

Nature 1958, 182 (4633), 476-477.

378

6.

379

biosynthesis: a spirocycle-forming P450 in the concise pathway to the antifungal drug

380

griseofulvin. ACS Chem. Biol. 2013, 8 (10), 2322-2330.

381

7.

382

Am. Chem. Soc. 1991, 113 (22), 8561-8562.

383

8.

384

single crystal X-ray analysis of two griseofulvin metabolites. Tetrahedron Lett. 2010, 51 (45),

385

5881-5882.

386

9.

387

griseofulvin. Chem. Rev. 2014, 114 (24), 12088-12107.

388

10. Rønnest, M. H.; Rebacz, B.; Markworth, L.; Terp, A. H.; Larsen, T. O.; Krämer, A.;

389

Clausen, M. H., Synthesis and structure−activity relationship of griseofulvin analogues as

390

inhibitors of centrosomal clustering in cancer cells. J. Med. Chem. 2009, 52 (10), 3342-3347.

391

11. Rønnest, M. H.; Raab, M. S.; Anderhub, S.; Boesen, S.; Krämer, A.; Larsen, T. O.; Clausen,

392

M. H., Disparate SAR data of griseofulvin analogues for the dermatophytes trichophyton

393

mentagrophytes, T. rubrum, and MDA-MB-231 cancer cells. J. Med. Chem. 2012, 55 (2),

394

652-660.

395

12. Dong, N.; Li, X.; Wang, F.; Cheng, J.-P., Asymmetric michael-aldol tandem reaction of

396

2-substituted benzofuran-3-ones and enones: a facile synthesis of griseofulvin analogues. Org.

397

Lett. 2013, 15 (18), 4896-4899.

398

13. Friedrich, M.; Meichle, W.; Bernhard, H.; Rihs, G.; Otto, H.-H., Sulfogriseofulvin

399

derivatives. synthesis by [4+2]cycloaddition, structure, properties, crystal structure analysis, and

400

antifungal activity of spiro[1,3-benzoxathiole-2,1′-cyclohex-2′-en]-4′-one-3,3-dioxides. Arch.

401

Pharm. (Weinheim, Ger.) 1996, 329 (7), 361-370.

402

14. Zhao, J. H.; Zhang, Y. L.; Wang, L. W.; Wang, J. Y.; Zhang, C. L., Bioactive secondary

403

metabolites from nigrospora sp. LLGLM003, an endophytic fungus of the medicinal plant

404

moringa oleifera Lam. World J. Microbiol. Biotechnol. 2012, 28 (5), 2107-2112.

405

15. Tang, H.-Y.; Zhang, Q.; Li, H.; Gao, J.-M., Antimicrobial and allelopathic metabolites

406

produced by penicillium brasilianum. Nat. Prod. Res. 2015, 29 (4), 345-348.

407

16. Bao, K.; Fan, A.; Dai, Y.; Zhang, L.; Zhang, W.; Cheng, M.; Yao, X., Selective

408

demethylation and debenzylation of aryl ethers by magnesium iodide under solvent-free

409

conditions and its application to the total synthesis of natural products. Org. Biomol. Chem.

410

2009, 7 (24), 5084-5090.

411

17. Wiles, C.; Watts, P.; Haswell, S. J.; Pombo-Villar, E., Solution phase synthesis of esters

Cacho, R. A.; Chooi, Y.-H.; Zhou, H.; Tang, Y., Complexity generation in fungal polyketide

Pirrung, M. C.; Brown, W. L.; Rege, S.; Laughton, P., Total synthesis of (+)-griseofulvin. J. Rønnest, M. H.; Harris, P.; Gotfredsen, C. H.; Larsen, T. O.; Clausen, M. H., Synthesis and

Petersen, A. B.; Rønnest, M. H.; Larsen, T. O.; Clausen, M. H., The chemistry of

14


Page 15 of 24


412

within a micro reactor. Tetrahedron 2003, 59 (51), 10173-10179.

413

18. Montgomery, R. S.; Dougherty, G., The interconversion and Beckmann rearrangement of

414

some α, β-unsaturated cyclic oximes J. Org. Chem. 1952, 17 (6), 823-826.

415

19. Dijkstra, D.; Rodenhuis, N.; Vermeulen, E. S.; Pugsley, T. A.; Wise, L. D.; Wikström, H.

416

V., Further characterization of structural requirements for ligands at the dopamine D2 and D3

417

receptor: exploring the thiophene moiety. J. Med. Chem. 2002, 45 (14), 3022-3031.

418

20. De Sousa, D.; Schefer, R.; Brocksom, U.; Brocksom, T., Synthesis and antidepressant

419

evaluation of three para-benzoquinone mono-oximes and their oxy derivatives. Molecules 2006,

420

11 (2), 148.

421

21. Li, D.; Gui, J.; Li, Y.; Feng, L.; Han, X.; Sun, Y.; Sun, T.; Chen, Z.; Cao, Y.; Zhang, Y.,

422

Structure-based design and screen of novel inhibitors for class II 3-hydroxy-3-methylglutaryl

423

coenzyme A reductase from streptococcus pneumoniae. J. Chem. Inf. Model. 2012, 52 (7),

424

1833-1841.

425

22. Liu, Y.-X.; Wei, D.-G.; Zhu, Y.-R.; Liu, S.-H.; Zhang, Y.-L.; Zhao, Q.-Q.; Cai, B.-L.; Li,

426

Y.-H.; Song, H.-B.; Liu, Y.; Wang, Y.; Huang, R.-Q.; Wang, Q.-M., Synthesis, herbicidal

427

activities, and 3D-QSAR of 2-cyanoacrylates containing aromatic methylamine moieties. J.

428

Agric. Food Chem. 2008, 56 (1), 204-212.

429

23. Zhang, Q.; Yang, J.; Liang, K.; Feng, L.; Li, S.; Wan, J.; Xu, X.; Yang, G.; Liu, D.; Yang,

430

S., Binding interaction analysis of the active site and its inhibitors for neuraminidase (N1

431

subtype) of human influenza virus by the integration of molecular docking, FMO calculation and

432

3D-QSAR CoMFA modeling. J. Chem. Inf. Model. 2008, 48 (9), 1802-1812.

433

24. Xiao, J.; Zhang, Q.; Gao, Y. Q.; Tang, J.-J.; Zhang, A.-L.; Gao, J.-M. Secondary metabolites

434

from the endophytic Botryosphaeria dothidea of Melia azedarach and their antifungal,

435

antibacterial, antioxidant, and cytotoxic activities. J. Agric. Food Chem. 2014, 62, 3584−3590.

436

25. Cramer, R. D.; Patterson, D. E.; Bunce, J. D., Comparative molecular field analysis

437

(CoMFA). 1. effect of shape on binding of steroids to carrier proteins. J. Am. Chem. Soc. 1988,

438

110 (18), 5959-5967.

15



11

9

OMe O OMe *

4 5

*

*

10

MeO

6

*

*

7

2'

3a * *

*

O

7a

Cl 440 441

*

*3 *

2 * 6'

3' * 4'

* 5'

*

8

1 Scheme 1. Structure of griseofulvin

442

16


O

Page 16 of 24

Page 17 of 24


Scheme 2. Synthetic Route of Griseofulvin Analoguesa

443

OH

a

b O

O

OMe

O

O

Cl

1

NOH

c1

Cl OMe

c2

O

NOR2 Cl

d

6a~6k

OEt

OMe

O

444 445 446 447 448 449 450 451 452 453 454 455

Cl

5

a

OMe NOH

NOMe O

MeO

OMe

O

MeO

4

O

e

O

MeO

3a~3g

OMe

OMe

O

MeO

Cl

2

Cl

OMe

O O

MeO

O

MeO OMe

OMe

O

OR1

OMe

O

O

EtO Cl

7

Reagents and conditions: (a) MgI2, Et2O/tolune; (b) NaH, THF; (c1) NH2OH·HCl, AcONa·3H2O /EtOH; (c2) MeONHOH·HCl, AcONa·3H2O /EtOH; (d) Na, EtOH reflux; (e) triethylamine, acetone. R1= 3a: -COCH3 3b: -COCH2CH3 3c: -COCH2CH2CH3 3d: -COCH2CH2CH2CH3 R2= 6a: -COCH3 6b: -COCH2Cl 6c: -COCH2CH3 6d: -COCHCH2 6e: -COCH2CH2CH3 6f: -COCH2CH2CH2CH3

3e: -COC6H4-NO2 (p) 3f: -COC6H4-F (p) 3g: -COC6H3-F2 (3, 4)

6g: -COC6H4-CH3 (p) 6h: -SO2C6H4-CH3 (p) 6i: -COC6H4-F (p) 6j: -COC6H4-Cl (p) 6k: -COC6H4-Br (p)

456 457

17



Page 18 of 24

458 459 460 461

Figure 1. Steric contour maps from the CoMFA model. Compound 6c is shown inside the field. Green contours (70% contribution) encompass regions where the steric interaction is favored, whereas in yellow contoured areas (30% contribution), the steric interaction is disfavored.

462

18


Page 19 of 24


463

464 465 466 467

Figure 2. Electrostatic contour maps from the CoMFA model. Compound 6c is shown inside the field. Blue contours (70% contribution) encompass regions where negative charge will decrease inhibitory activity, whereas in red contoured areas (30% contribution), negative charges are favorable.

468

19



Table 1. Antifungal activity of compounds in vitro

469

IC50±SD/ (μg /mL)

Compd

470 471 472

Page 20 of 24

C.s.

C. g.

B.c.

A.s.

F.s.

1

>50

11.06±0.42

>50

2.68±0.33

>50

2

>50

33.07±0.83

>50

>50

>50

3a

>100

46.74±0.49

>100

>100

>100

3b

>100

35.56±1.3

>100

>100

>100

3c

>100

51.65±2.49

>100

>100

>100

3d

>100

48.58±0.14

>100

>100

>100

3e

>100

44.07±2.28

>100

>100

>100

3f

>100

68.52±0.11

>100

>100

>100

3g

>100

52.51±0.22

>100

>100

>100

4

>50

24.38±0.59

19.50±0.99

>50

5

108.49±5.84

126.33±3.48

14.19±1.65

>100

29.94±0.51

6a

>50

3.03±0.11

22.28±0.32

19.43±0.43

>50

6b

>50

5.53±0.48

26.57±0.66

9.21±0.16

>50

6c

>50

3.38±0.30

19.78±0.27

6.24±0.21

24.94±2.50

6d

>50

5.11±0.32

>50

21.90±0.82

>50

6e

>50

1.80±0.18

33.35±2.22

>100

>50

6f

>50

4.18±0.30

36.61±2.07

26.49±0.92

>50

6g

>50

11.13±1.64

>50

41.95±2.12

>100

6h

>100

50.21±2.34

>100

60.22±3.79

>100

6i

>50

9.67±0.50

>50

16.87±0.35

>50

6j

>100

16.26±0.22

>50

53.68±7.47

>100

6k

>100

13.58±0.10

>100

>50

>100

Hy

>100

>100

7.11±0.16

44.62±0.59

88.63±2.45

Tpm

62.22±1.47

100

>100

48.00±3.46

56.62±1.97

Note: C.s., Cytospora sp.; C.g., Colletotrichum gloeosporioides; B.c., Botrytis cinerea; A.s., Alternaria solani; F.s.; Fusarium solani; Hy, Hymexazol; Tpm, Thiophanate-methyl, (a commercial available agricultural fungicide for direct application bought from a pesticide shop, effective activity constituent is 75%).

20


Page 21 of 24

473


Table 2. The half-maximal inhibitory concentration values of compounds in vivo Compds against C. g. IC50±SD/ (μg /mL)

6a

79.14±7.07

6c

6e

47.25±1.46

49.44±1.50

21


6f

53.63±1.74

Tpm

69.66±6.07


Page 22 of 24

Table 3. Inhibitory efficacy of compounds in vivo

475

Concentration (μg/mL)

Pictures of 3 days inhibitory efficacy of compound 6f 1 6f

Pictures of 7 days inhibitory efficacy of compounds 6a

6c

6e

6f

100

50

25

12.5

6.25

Sterile water (negative control) Spore suspension (blank control) 476

1: 3 days inhibitory efficacy of compounds 6a, 6c and 6e is identical or similar to that of compound 6f.

22


Tpm

Page 23 of 24

478


Table 4. Data Sets for 3D-QSAR Analysis. pIC50 Compd. Actual

Calculated

Residual

1

4.5037

4.2012

0.3025

2

4.0104

4.1339

-0.1235

4

4.1786

4.023

0.1556

5

3.4803

3.7861

-0.3058

3a

3.911

4.0729

-0.1619

3b

4.0454

4.0391

0.0063

3c

3.8985

3.9729

-0.0745

3d

3.9397

3.9628

-0.0231

3e

4.0441

3.8685

0.1756

3f

3.8277

3.9137

-0.086

3g

3.9599

3.9201

0.0398

6a

5.1311

5.0414

0.0898

6b

4.9049

4.9406

-0.0357

6c

5.0983

5.1763

-0.078

6d

4.9167

5.0384

-0.1217

6e

5.3861

5.2261

0.16

6f

5.0339

5.2146

-0.1807

6g

4.6401

4.577

0.063

6h

4.0169

3.9314

0.0855

6i

4.7047

4.6458

0.0589

6j

4.4933

4.5314

-0.038

6k

4.6081

4.5163

0.0918

479 480 481 482 483 23



484

TOC

485

486

24


Page 24 of 24

Antifungal Activity of Griseofulvin Derivatives against Phytopathogenic

Recommend Documents