Characterization of Diterpenes from Euphorbia prolifera and Their

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Characterization of Diterpenes from Euphorbia prolifera and Their Antifungal Activities against Phytopathogenic Fungi Jing Xu, Jing Kang, Xiangrong Cao, Xiaocong Sun, Shujing Yu, Xiao Zhang, Hongwei Sun, and Yuanqiang Guo J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02021 • Publication Date (Web): 11 Jun 2015 Downloaded from http://pubs.acs.org on June 19, 2015

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Journal of Agricultural and Food Chemistry

Characterization of Diterpenes from Euphorbia prolifera and Their Antifungal Activities against Phytopathogenic Fungi

Jing Xu,†,‡ Jing Kang,ǁ Xiangrong Cao,†,‡ Xiaocong Sun,†,‡ Shujing Yu,⊥ Xiao Zhang,⊥ Hongwei Sun,§ Yuanqiang Guo*,†,‡,ǁ

†

State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University,

Tianjin 300071, People’s Republic of China ‡

Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, People’s

Republic of China ǁ

College of Pharmacy, Harbin University of Commerce, Harbin 150076, People’s Republic of China

⊥

Research Institute of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, People’s

Republic of China §

Computational Centre for Molecular Science, College of Chemistry, Nankai University, Tianjin

300071, People’s Republic of China

*Corresponding author (Tel./fax: +86-22-23502595; E-mail: [email protected])

ACS Paragon Plus Environment


1

ABSTRACT: Euphorbia prolifera is a poisonous plant belonging to the Euphorbiaceae family. In

2

our survey on plant secondary metabolites to obtain bioactive substances for the development of new

3

antifungal agents of agriculture, the chemical constituents of the plant E. prolifera were investigated.

4

This procedure led to the isolation of six new and two known diterpenes. Their structures including

5

absolute configurations, were elucidated based on the extensive NMR spectroscopic data analyses,

6

and the time-dependent density functional theory ECD calculations. Biological screenings revealed

7

that these diterpenes possessed antifungal activities against three phytopathogenic fungi. The results

8

of our phytochemical investigation further revealed the chemical components of the poisonous plant

9

E. prolifera and biological screenings implied the extract or bioactive diterpenes from this plant may

10

be regarded as candidate agents of antifungal agrochemicals for crop protection products.

11

KEYWORDS:

12

agrochemicals

Euphorbia

prolifera,

diterpenes,

ECD

calculations, antifungal activities,

13 14 15 16 17 18 19 20 21 22 23 24 25 26 2


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INTRODUCTION

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Plant pathogenic fungi remain a continuous and huge threat to crops. Crops infected by fungal

29

pathogens can lead to significant yield reduction and dramatic economic losses in agriculture.1

30

Several important plant fungal pathogens, such as Gibberella zeae, Physalospora piricola, Alternaria

31

solani, Cercospora arachidicola, Cladosporium cucumerinum, and Phytophthora capsici, have

32

received much attention due to their perniciousness and universality. To protect crops from plant

33

pathogenic fungi, some synthetic antifungal agents have been used currently as an effective method.2

34

However, these available antifungal agents have several side effects, such as residual toxicity, severe

35

drug resistance, environmental pollution.3−6 Therefore, there is an urgent need to develop new

36

antifungal agents to control those crop diseases effectively. One strategy to develop new antifungal

37

agents is to search for biologically active substances or lead compounds from plant secondary

38

metabolites, which has been proven to be effective and practicable for new pesticides of agricultural

39

field7 and new drugs of medical field.8

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Euphorbia prolifera Buch-Ham., belonging to the Euphorbiaceae family, is a perennial herbaceous

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plant distributed mainly in southwest China.9 This plant is poisonous and its roots were documented

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as a poisonous Chinese medicine.10 Previous phytochemical investigations on E. prolifera led to the

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isolation and identification of terpenoids,11−23 especially diterpenoids, which displayed a broad

44

spectrum of biological effects, such as neuroprotective, cytotoxic, NO inhibitory, and

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anti-inflammatory activities, and modulation of multidrug resistance.13‒19 In the course of an ongoing

46

search for biologically active substances from plant secondary metabolites,24‒26 the poisonous plants

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documented in the literature evoked our attention, since their extracts or constituents may be

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potentially useful for the agricultural and medical industry due to their toxicity.27,28 In order to obtain

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new bioactive antifungal compounds for the development of new antifungal agents for agriculture,

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the chemical constituents of this poisonous plant E. prolifera were investigated. This investigation

51

resulted in the isolation of six new diterpenes. Their structures were elucidated based on the NMR

52

spectroscopic data analyses and the time-dependent density functional theory (TDDFT) ECD 3



53

calculations. The compounds were tested for antifungal activity against three phytopathogenic fungi.

54 55

MATERIALS AND METHODS

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General. Optical rotations were measured in CH2Cl2 using an Autopol IV automatic polarimeter

57

(Rudolph Research Analytical, Hackettstown, NJ). IR spectra were taken on a Bruker Tensor 27

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FT-IR spectrometer (Bruker Optics, Ettlingen, Germany) with KBr disks. ECD spectra were obtained

59

on a Chirascan spectrometer (Applied Photophysics Ltd., Leatherhead, UK). 1D and 2D NMR

60

spectra were recorded on a Bruker AV 400 instrument (Bruker Group, Fallanden, Switzerland, 400

61

MHz for 1H and 100 MHz for 13C) with TMS as an internal standard. ESIMS spectra were acquired

62

on a Thermo Finnigan LCQ-Advantage mass spectrometer (Finnigan Co., Ltd., San Jose, CA).

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HR-ESIMS were recorded by an IonSpec 7.0 T FTICR MS (IonSpec Co., Ltd., Lake Forest, CA).

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HPLC purifications were finished on a CXTH system, equipped with a UV3000 detector at 210 nm

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(Beijing Chuangxintongheng Instruments Co., Ltd., Beijing, People’s Republic of China). The HPLC

66

column used was a 20 × 250 mm i.d., 5 µm, YMC-pack ODS-AM (YMC Co., Ltd., Kyoto, Japan).

67

Silica gel (100-200 mesh) was used for column chromatography (Qingdao Haiyang Chemical Group

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Co., Ltd., Qingdao, People’s Republic of China). Chemical reagents for isolation were of analytical

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grade and purchased from Tianjin Chemical Reagent Co. (Tianjin, People’s Republic of China).

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Biological reagents were from Sigma Chemical Co.

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Plant Material. The roots of E. prolifera were collected in July 2010 from Kunming, Yunnan

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province, People’s Republic of China. The botanical identification was made by one of the authors (Y.

73

G.), and a voucher specimen (No. 20100705) was deposited at the laboratory of the Research

74

Department of Natural Medicine, College of Pharmacy, Nankai University, People’s Republic of

75

China.

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Extraction and Isolation. The air-dried roots of E. prolifera (3.8 kg) were extracted with

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methanol three times (3 × 20 L) under reflux. The organic solvent was evaporated to afford a crude

78

extract (900 g), which was suspended in H2O (0.9 L) and then partitioned with ethyl acetate 4


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(3 × 0.9 L) to give the EtOAc-soluble portion (150 g). This portion was fractionated by silica gel

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column chromatography (silica gel, 1.0 kg; column, 9 × 70 cm), using a gradient solvent system of

81

petroleum ether-acetone (100:0, 100:2, 100:4, 100:7, 100:11, 100:16, 100:23, 100:30, 100:40, and

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0:100, 21 L for each gradient elution), to afford nine fractions (F1−F9) according to the TLC analyses.

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Fraction F8 (eluted by petroleum ether-acetone, 100:30, and 100:40) was subjected to medium

84

pressure liquid chromatography (MPLC) over octadecylsilyl (ODS) eluting with a step gradient from

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60-90% MeOH in H2O to give four subfractions (F8-1‒F8-4). The subfraction F8-3 was further purified

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by preparative HPLC (YMC-pack ODS-AM, 250 × 20 mm i.d., 77% MeOH in H2O) to afford

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compound 1 (tR = 23 min, 7.2 mg), and the purification of F8-4 by the same HPLC system (82%

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MeOH in H2O) yielded compound 5 (tR = 27 min, 11.5 mg). Fraction F9 (eluted by petroleum

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ether-acetone, 100:40, and 0:100), using the above MPLC (60−90% MeOH in H2O), provided four

90

subfractions F9-1−F9-4. Compounds 2 (tR = 32 min, 13.6 mg) and 8 (tR = 21 min, 13.1 mg) were

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obtained from subfraction F9-2 by the same HPLC system (73% MeOH in H2O), and the following

92

purification of subfraction F9-4 (77% MeOH in H2O) afforded compound 4 (tR = 24 min, 13.2 mg).

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Using the same MPLC system as for the above fractions, F7 gave the subfractions F7-1−F7-4. The

94

following purification of subfractions F7-3 (73% MeOH in H2O) produced compounds 3 (tR = 35 min,

95

13.1 mg), 6 (tR = 30 min, 13.8 mg), and 7 (tR = 26 min, 15.0 mg).

96

Prolifepene A, 1: white powder; [α]15 D : +27.5 (c 0.08, CH2Cl2); CD (CH3CN): 216 (∆ε +4.14), 237

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(∆ε ‒1.44) nm, 311 (∆ε +9.42) nm; IR (KBr) νmax 3422, 2934, 2875, 1734, 1637, 1395, 1249, 1226,

98

1082, and 1023 cm-1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data (Tables 1

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and 2); ESIMS m/z 708 [M + Na]+; HR-ESIMS m/z 708.2632 [M + Na]+, calcd. for C35H43NNaO13,

100

708.2632.

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Prolifepene B, 2: white powder; [α]15 D : +16.9 (c 0.11, CH2Cl2); CD (CH3CN): 200 (∆ε +8.81), 215

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(∆ε +6.73), 234 (∆ε ‒6.69), 300 (∆ε +3.61) nm; IR (KBr) νmax 3435, 2962, 2933, 1728, 1654, 1591,

103

1373, 1282, 1230, 1109, and 1025 cm–1;

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CDCl3) data (Tables 1 and 2); ESIMS m/z 728 [M + Na]+; HR-ESIMS m/z 728.3041 [M + Na]+,

13

C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz,

5



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calcd. for C39H47NNaO11, 728.3047.

106

Prolifepene C, 3: white powder; [α]15 D : −43.8 (c 0.17, CH2Cl2); CD (CH3CN): 202 (∆ε +11.16), 216

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(∆ε ‒11.08), 244 (∆ε +1.48) nm; IR (KBr) νmax 2963, 2932, 2875, 1737, 1640, 1590, 1437, 1242,

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1150, and 1024 cm-1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data (Tables 1

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and 2); ESIMS m/z 668 [M + H]+; HR-ESIMS m/z 668.3067 [M + H]+, calcd. for C36H46NO11,

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668.3071. Prolifepene D, 4: White powder; [α]15 D : −12.0 (c 0.10, CH2Cl2); IR (KBr) νmax 2961, 2930, 1728,

111 112

1638, 1592, 1456, 1367, 1269, 1220, 1110, and 1026 cm-1;

13

C NMR (100 MHz, CDCl3) and 1H

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NMR (400 MHz, CDCl3) data (Tables 1 and 2); ESIMS m/z 787 [M + Na]+; HR-ESIMS m/z

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787.2841 [M + Na]+, calcd. for C43H44N2NaO11, 787.2843.

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Prolifepene E, 5: white powder; [α]15 D : −10.7 (c 0.60, CH2Cl2); IR (KBr) νmax 2966, 2936, 1739,

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1641, 1591, 1458, 1371, 1269, 1244, 1151, 1083, and 1025 cm-1; 13C NMR (100 MHz, CDCl3) and

117

1

118

676.2734 [M + Na]+, calcd. for C35H43NNaO11, 676.2734.

H NMR (400 MHz, CDCl3) data (Tables 1 and 3); ESIMS m/z 676 [M + Na]+; HR-ESIMS m/z

119

Prolifepene F, 6: white powder; [α]15 D : −11.7 (c 0.12, CH2Cl2); IR (KBr) νmax 2967, 2936, 1741,

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1643, 1513, 1455, 1370, 1275, and 1224 cm-1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz,

121

CDCl3) data (Tables 1 and 3); ESIMS m/z 668 [M + H]+; HR-ESIMS m/z 668.3062 [M + H]+, calcd.

122

for C36H46NO11, 668.3071.

123 124

Computational Methods. The ECD calculations were performed according to the reported method.29

125

Antifungal Activity Assay. The antifungal activities of the isolated compounds were assayed

126

against three pathogenic fungi (Physalospora piricola, Alternaria solani, and Gibberella zeae) using

127

a mycelium growth inhibition method, whose details have been previously reported.30 The

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commercially available agricultural fungicides chlorothalonil and carbendazol were used as the

129

corresponding positive controls. Compounds possessing good activities (inhibitory rate >50% at 50

130

µg/mL) were further evaluated using the above-mentioned method with different concentrations. 6


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RESULTS AND DISCUSSION

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The ethyl acetate-soluble part of the methanol extract of the aerial parts of E. prolifera was

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fractionated by silica gel column chromatography and MPLC and further purified by HPLC to afford

134

six new (1−6) and two known (7 and 8) diterpenoids (Figure 1). The known compounds were

135

identified by comparison of spectroscopic data with those reported in the literature as

136

2α,5α,7β,15β-tetra-O-acetyl-3β-O-propinoyl-14α-O-benzoyl-14-deoxomyrsinol, 7,31 and 3β-O-

137

propionyl-5α,10,15β-tri-O-acetyl-14β-O-nicotinoyl-7-oxo-10,18-dihydromyrsinol, 8.14

138

Compound 1 was obtained as a white powder. Its molecular formula was assigned as C35H43NO13

139

on the basis of HR-ESIMS. The 1H-NMR spectrum of 1 exhibited four oxygenated methine protons

140

[δH 5.53 (1H, t, J = 3.8 Hz, H-3), 5.90 (1H, d, J = 11.1 Hz, H-5), 3.91 (1H, d, J = 5.5 Hz, H-8), and

141

5.35 (1H, br. s, H-14)], four aromatic protons [δH 8.36 (1H, d, J = 7.9 Hz), 7.40 (1H, dd, J = 7.9, 4.9

142

Hz), 8.79 (1H, d, J = 4.9 Hz), and 9.22 (1H, br. s)], and a pair of oxygenated methylene protons [δH

143

4.17, and 3.66 (each 1H, d, J = 9.8 Hz, H2-17)]. Additionally, six methyl signals were also exhibited

144

in the 1H NMR spectrum. The

145

From the 1H and 13C NMR spectra of 1, three acetyl groups were apparent from the methyl singlets

146

(δH 2.24 s, 2.01 s, and 1.90 s) and the corresponding carbons (Table 1). The above mentioned four

147

aromatic proton signals (Table 2), together with the typical carbon resonances, revealed the presence

148

of one nicotinoyl group.14 In addition to these substituent groups, a propionyl moiety (Tables 1 and 2)

149

were deduced and defined from the methyl and methylene proton signals and the observation of the

150

corresponding carbon signals (Tables 1 and 2).13 Apart from the above 15 carbon signals for the

151

substituent groups, there were additional 20 carbons displayed in the 13C NMR spectrum, including

152

three methyls, three methylenes, nine methines, and five quaternary carbons based on the DEPT and

153

HMQC spectra. These 20 typically skeletal carbons implied that compound 1 possessed a

154

characteristic cyclomyrsinol-type diterpene skeleton according to the reported related diterpenes

155

from the genus Euphorbia.32−36 This skeleton of cyclomyrsinol-type diterpene was further confirmed

156

by HMBC and 1H-1H COSY experiments, in which the carbonyl signal at δC 211.2 was assigned to

13

C NMR spectrum of 1 showed 35 carbon resonances (Table 1).

7



157

C-7, and the oxygenated carbon signals at δC 77.3, 71.7, 72.1, 78.2, 89.6, 84.1, 90.2, and 66.9 were

158

attributed to C-3, C-5, C-8, C-10, C-13, C-14, C-15, and C-17, respectively. The positions of the

159

acyloxy groups were deduced from the HMBC spectrum. The HMBC correlation of the carbonyl

160

signal at δC 173.2 with the proton signals at δH 5.53 (H-3), demonstrated that one acetoxy group was

161

attached at C-3. Similarly, the long-range couplings of the carbonyl carbon signals at δC 173.5 and

162

166.3 with the proton signals at δH 5.90 (H-5) and 5.35 (H-14) disclosed the presence of the

163

propionyloxy group and the nicotinoyloxy group at C-5 and C-14, respectively. Because of no

164

long-range correlations of the skeletal protons with the carbonyl carbons of the two remaining

165

acetoxy groups, it was therefore concluded that the two acetoxy groups were attached to quaternary

166

carbons of the cyclomyrsinol skeleton. Based on the chemical shifts of skeletally oxygenated carbons

167

and similar cyclomyrsinol diterpenes in the literature,35,36 the residual acetoxy groups could only be

168

located at C-10 and C-15, of which the C-10 acetoxy group was assigned by a NOESY experiment,

169

indicating the NOESY correlations of H3-19 (δH 1.59) to the methyl protons (δH 1.90) of the C-10

170

acetoxy group. Consequently, the other remaining acetoxy was assigned to C-15. By further

171

analyzing the HMQC, HMBC, and 1H-1H COSY spectra (Figure 2), all the proton and carbon signals

172

were assigned unambiguously. Thus, the planar structure of 1 was established.

173

The stereochemistry of 1 were elucidated as follows. The NOESY correlations of H-2/H-4,

174

H-4/H-14, H-4/H2-17, H-5/H-12, H-12/H3-20, H-8/H-9, H3-19/H-11, and H3-19/H-9, but not for

175

H-3/H-5, together with a Chem3D modeling revealed a conformation as shown in Figure 2. In this

176

arrangement for compound 1, the three rings A, B, and C (5/7/6) were trans-fused with each other,

177

the H-12 proton and the C-15 acetoxy group were found to be in a β-position, and the H-4 and C-17

178

were in an α-position. Another four-membered ring in the skeleton was cis-fused with the ring C, and

179

the H-9 and H-11 proton were assigned to be α-oriented correspondingly. In turn, the three methyl

180

groups Me-16, Me-19, and Me-20 were determined as β-, α-, and β-oriented, the acetoxy groups at

181

C-3 and C-10 and the C-8 hydroxy group were all β-oriented, and the C-5 propionyloxy group was in

182

an α-position according to this conformation of 1 and the above NOESY correlations. The relative 8


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183


configuration of 1 was therefore designated as shown in Figure 3.

184

The absolute conﬁguration of 1 was established by the TDDFT ECD calculations37−39 and

185

substantiated by the octant rule. Starting from the conformation of 1 deduced from the NOESY

186

correlations and Chem3D modeling, conformational searches with the MMFF94 force field by MOE

187

software40 and geometry optimizations by the Gaussian 09 package41 were performed. Then, the

188

ECD spectra were calculated at the CAM-B3LYP/SVP level with the CPCM model in acetonitrile.

189

The obtained ECD spectrum of 1 (Figure 4) matched the experimental results closely, which

190

suggested an absolute configuration of 2S,3S,4R,5R,6S,8R,9R,10S,11S,12R,13R,14S, and 15R for

191

compound 1. The experimental ECD spectrum of 1 displayed a positive Cotton effect at 311 nm (∆ε

192

+9.42), corresponding to the n−π* transition of the cyclohexanone chromophore, which confirmed

193

the absolute configuration of 2S,3S,4R,5R,6S,8R,9R,10S,11S,12R,13R,14S, and 15R unequivocally

194

according to the octant rule.42 On the basis of the above evidence, the structure of 1 was established

195

as

196

O-nicotinoylcyclomyrsinol, which has been named prolifepene A.

(2S,3S,4R,5R,6S,8R,9R,10S,11S,12R,13R,14S,15R)-3β,10β,15-tri-O-acetyl-5α-O-propionyl-14β-

197

Compound 2 had a molecular formula of C39H47NO11 based on the HR-ESIMS. From the 1H NMR

198

spectrum of 2, three oxygenated methine protons, a set of oxygenated methylene protons, nine

199

aromatic protons, and six methyl signals were observed (Table 2). The

200

exhibited 39 carbon resonances. From the 1H and

201

together with the corresponding carbon signals (Table 1) implied the occurrence of one acetyl group,

202

and the aromatic proton signals and the corresponding carbon resonaces (Table 1) revealed the

203

presence of one nicotinoyl and one benzoyl group.14,16 In addition, a butyryl group was also deduced

204

and defined from the observation of the carbon signals (δC 170.1, 35.7, 17.7, and 13.5) and the

205

corresponding proton signals (Table 2) based on those reported diterpenes with acyl groups in the

206

literature.13 Apart from the above 19 resonances for the substituent groups, the remaining 20 skeletal

207

carbons in the

208

skeleton.35,36

13

13

13

C NMR spectrum of 2

C NMR spectra, the methyl singlet (δH 1.88 s),

C NMR spectrum constituted a characteristic premyrsinol-type diterpene

9



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209

In order to confirm the above deduction and position these substituent groups, the following

210

HMQC, HMBC, and 1H-1H COSY experiments were performed. By interpretation of the 2D NMR

211

spectra, the presence of the above deduced acyl groups were substantiated and the characteristic

212

premyrsinol-type diterpene skeleton was elucidated unequivocally, where the oxygenated carbons at

213

δC 78.2, 69.2, 70.3, 79.6, 84.3, and 62.3 were attributed to C-3, C-5, C-7, C-13, C-15, and C-17,

214

respectively. The locations of these acyloxy groups were determined by HMBC correlations. The

215

long-range couplings of the protons H-3 (δH 5.61), H-5 (δH 6.07), H-7 (δH 5.06), and H2-17 (δH 5.33

216

and 4.57) with the carbonyl carbons at δC 170.1, 165.0, 171.8, and 165.0, demonstrated that the

217

butyryloxy group was located at C-3, the benzoyloxy group was at C-5, the acetoxy group was at C-7,

218

and the nicotinoyloxy group was at C-17, respectively. There were no additional acyloxy groups in

219

compound 2, indicating that the substituent groups at C-13 and C-15 could only be hydroxy groups,

220

which was verified by the HR-ESIMS data. Further analyses of the HMQC, HMBC, and 1H-1H

221

COSY data led to the assignments of all the proton and carbon signals. The planar structure with a

222

premyrsinol-type diterpene skeleton for 2 was therefore elucidated. The relative configuration was

223

elucidated on the basis of NOESY spectrum, which disclosed the correlations of H-2/H-4, H-3/H-4,

224

H-4/H3-20, H-5/H-12, H-7/H2-17, H-9/H-11, H2-17/H3-20, and H-11/H3-20. These NOESY

225

correlations and the Chem3D modeling revealed that the three rings (5/7/6) forming the

226

premyrsinol-type skeleton were trans-fused as those in 1, the C-15 hydroxy group and H-12 proton

227

were in a β-position, and C-17 and H-4 were in an α-position. Consequently, the C-3 butyryloxy, C-7

228

acetoxy, and C-13 hydroxy group were assigned to be β-oriented, and the C-5 benzoyloxy group and

229

H-9 and H-11 proton were determined as α-oriented according to the NOESY correlations. The

230

relative configuration of compound 2 was therefore established. Based on the relative configuration

231

of 2 deduced from the NOESY spectrum, the same procedures, conformational searches, geometry

232

optimizations, and calculated ECD spectra as in the case of 1 were performed to determine the

233

absolute configuration of 2. As shown in Figure 5, the calculated ECD spectrum for

234

(2S,3S,4R,5R,6R,7R,9S,11S,12R,13S,15R)-2 was in good agreement with the experimental one. So, 10


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235

the absolute configuration of 2 was assigned as 2S,3S,4R,5R,6R,7R,9S,11S,12R,13S, and 15R. Thus,

236

compound 2 was characterized as (2S,3S,4R,5R,6R,7R,9S,11S,12R,13S,15R)-3β-O-butyryl-5α-

237

O-benzoyl-7β-O-acetyl-17-O-nicotinoylpremyrsinol, and was given a trivial name prolifepene B.

238

Compound 3 was obtained as a white powder. Its HR-ESIMS corresponded to the molecular

239

formula C36H45NO11. From the 1H and

13

240

nicotinoyl group were evident based those acyl groups in compounds 1 and 2. Apart from these

241

carbon resonances for the acyl groups, there were 20 residual ones present in the 13C NMR spectrum.

242

The 20 skeletal carbons were sorted into three methyls, three methylenes, ten methines, and four

243

quaternary carbons with the aid of DEPT and HMQC spectra. According to these spectroscopic

244

features, especially the terminal double bond (C-10 and C-18), and the diterpenoids isolated from the

245

genus Euphorbia,13,14 it could be deduced that compound 3 had a 14-deoxomyrsinol-type skeleton,

246

which was confirmed by the following HMBC and 1H-1H COSY experiments. Correspondingly, the

247

skeletal olefinic and oxygenated carbon signals at δC 123.1, 133.1, 147.2, 112.8, 76.5, 69.0, 63.6,

248

89.1, 82.0, 89.6, and 69.2 were assigned to C-8, C-9, C-10, C-18, C-3, C-5, C-7, C-13, C-14, C-15,

249

and C-17, respectively. The locations of acyloxy groups were deduced from the HMBC spectrum,

250

exhibiting the correlations of the protons H-3 (δH 5.31), H-5 (δH 5.95), H-7 (δH 4.86), and H-14 (δH

251

5.25) with the corresponding carbonyl carbons at δC 173.4, 169.2, 170.4, and 164.4. These HMBC

252

correlations demonstrated the two acetoxy, one butyryloxy, and one nicotinoyl group to be attached

253

at C-5, C-7, C-3, and C-14, respectively. The residual one acetxoy groups should be attached to

254

quaternary carbons of the myrsinol skeleton due to the lack of long-range correlations of skeletal

255

protons to the carbonyl carbon of the acyloxy moiety. Based on the chemical shifts of skeletally

256

oxygenated carbons, this residual acetoxy group could only be located at C-15.13,16 By further

257

analyzing the 2D NMR spectra, the planar structure of 3 was elucidated.

C NMR spectra, three acetyl, one butyryl, and one

258

The relative configuration of compound 3 was established by analyses of the NOESY spectrum,

259

which showed the correlations of H-2/H-4, H-3/H-4, H-4/H-14, H-4/H2-17, H-5/H-12, H-7/H-11,

260

and H-12/H3-20. According to these NOESY correlations, the relative configuration of 3 was 11



261

depicted as in Figure 1, where the three rings (5/7/6) were the same trans-fused with each other as

262

those in compounds 1 and 2, H-4 and H-11 protons and C-17 were found to be in an α-position, the

263

C-3 butyryloxy, the C-7 and C-15 acetoxy groups, and the C-14 nicotinoyloxy were all in a

264

β-position, and the C-5 acetoxy were assigned as α-oriented. After defining the relative configuration

265

of 3, the absolute configuration was assigned as 2S,3S,4R,5R,6R,7R,11S,12R,13R,14S, and 15R, by

266

comparison of its experimental and calculated ECD spectra (Figure 6) using TDDFT method. On the

267

basis of the above evidence, the structure of compound 3 (prolifepene C) was established as

268

(2S,3S,4R,5R,6R,7R,11S,12R,13R,14S,15R)-3β-O-butyryl-5α,7β,15β-tri-O-acetyl-14β-O-nicotinoyl-

269

14-deoxomyrsinol. The 1H and 13C NMR spectra of compounds 4−6 were found to be similar to each other. Analyses

270

13

C and 1H NMR spectra of the three compounds, indicated that compounds 4−6 all possess

271

of the

272

the same 14-deoxomyrsinol-type diterpene scaffold as compound 3.35,36 The main differences

273

between compounds 4−6 were found to be in the different substituent groups that occurred in each

274

case. For compound 4, five acyl groups (two acetyl, two nicotinoyl, and one benzoyl group) were

275

deduced and determined according to its

276

NOESY experiments as for compounds 1‒3, the positions of these substituent groups and the relative

277

configuration of compound 4 were determined, where one acetoxy group was located at C-5 with an

278

α-orientation, the other acetoxy group was at C-15 with a β-orientation, the benzoyloxy group was at

279

C-7 with a β-orientation, and the two nicotinoyloxy groups were at C-3 and C-14 both in an

280

β-position. For compound 5, besides the same three acetoxy groups and one nicotinoyloxy group as

281

present in compound 3, a propionyloxy moiety was deduced and defined on the basis of its 13C and

282

1

283

6 were found to have the same acyl groups (three acetyl, one nicotinoyl, and one butyryl group) as

284

compound 3. From the evidence of the HMBC spectrum, the butyryloxy group was found to be

285

located at C-3, the nicotinoyloxy group was at C-7, and the three acetoxy groups were at C-5, C-14,

286

and C-15, respectively. The same relative configuration for all of compounds 3‒6 was validated by

13

C and 1H NMR spectra. Using the same HMBC and

H NMR data, which was located at C-3, replacing the butyryloxy group in compound 3. Compound

12


Page 12 of 28

Page 13 of 28


287

the careful comparison of their NOESY spectra. Based on the above spectroscopic evidence,

288

biosynthetic considerations, and the absolute configuration of 3 elucidated by the TDDFT ECD

289

method,

290

(2S,3S,4R,5R,6R,7R,11S,12R,13R,14S,15R)-3β,14β-di-O-nicotinoyl-5α,15β-di-O-acetyl-7β-O-

291

benzoyl-14-deoxomyrsinol, (2S,3S,4R,5R,6R,7R,11S,12R,13R,14S,15R)-3β-O-propinoyl-5α,7β,15β-

292

tri-O-acetyl-14β-O-nicotinoyl-14-deoxomyrsinol, and (2S,3S,4R,5R,6R,7R,11S,12R,13R,14S,15R)-

293

3β-O-butyryl-5α,14α,15β-tri-O-acetyl-7β-O-nicotinoyl-14-deoxomyrsinol, which have been named

294

as prolifepenes D, E, and F, respectively.

the

structures

of

compounds

4−6

were

therefore

elucidated

as

295

E. prolifera is a poisonous plant and its chemical constituents may be useful for the agricultural

296

and medical industry.27,28 In order to explore the potential biological effects of these diterpenes to

297

serve agriculture, these isolated compounds from E. prolifera were preliminarily investigated for

298

their antifungal activities against three pathogenic fungi (G. zeae, P. piricola, A. solani), which are

299

common to infect crops, leading to severe crop yield reduction and dramatic economic losses in

300

agriculture.1 Carbendazol was used as a positive control and had an EC50 value of

301

against P. piricola, and the other positive control chlorothalonil gave the corresponding EC50 values

302

of 27.34 and 10.10 µg/mL against A. solani and G. zeae, respectively. At the concentration 50 µg/mL,

303

all the evaluated compounds exhibited antifungal activities. Their preliminary antifungal activities

304

are shown in Figure 7 (compound 1 was not assayed for the antifungal effects because of inadequate

305

amounts). For the pathogenic fungi G. zeae, all the compounds showed weak or moderate antifungal

306

effects at the concentration of 50 µg/mL. For the fungus A. solani, compound 6 showed a strong

307

activity with an inhibitory rate of 53% and an EC50 value of 29.60 µg/mL. For the fungus P. piricola,

308

compound 3 had a stronger activity than other compounds with an inhibitory rate of 64% and an

309

EC50 value of 28.26 µg/mL.

0.43 µg/mL

310

In conclusion, six new and two known diterpenes were obtained from the aerial parts of the plant

311

E. prolifera. Their structures including the absolute configurations were elucidated by the NMR data

312

analyses, the TDDFT ECD calculations, and the octant rule. The subsequent biological screenings 13



Page 14 of 28

313

indicated that these isolated compounds had antifungal activities against three phytopathogenic fungi.

314

The results of our phytochemical investigation further revealed the chemical components of E.

315

prolifera as a poisonous and medicinal plant and the biological screenings suggested that E. prolifera

316

may be potentially useful to protect crops against phytopathogenic fungi and the bioactive

317

compounds may probably be considered as candidate agents of antifungal agrochemicals for crop

318

protection products.

319 320

ASSOCIATED CONTENT

321

Supporting Information

322

The 1D and 2D NMR and HR-ESIMS spectra of compounds 1−6. This material is available free of

323

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

324

AUTHOR INFORMATION

325

Corresponding Author

326

*Tel/fax: +86-22-23502595. E-mail: [email protected].

327

Funding

328

This work was supported by the National Natural Science Foundation of China (Nos. 21372125 and

329

81102331).

330

Notes

331

The authors declare no competing financial interest.

332

REFERENCES

333 334

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18


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Page 19 of 28


Table 1. 13C NMR Data (δC) of Compounds 1–6 position 1 2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 3-OR

5-OR

7/10-OAc 14/17-OR

15-OAc

1 2 3 4 1 2/6 3/5 4 7 1 2 1 2 3 4 5 6 1 2

42.0 36.3 77.3 51.1 71.7 62.1 211.2 72.1 31.0 78.2 42.0 42.2 89.6 84.1 90.2 13.9 66.9 33.5 24.8 21.8 173.2 21.2

173.5 27.5 9.1

169.1 22.0 126.2 137.5 123.1 153.5 151.2 166.3 168.3 23.0

46.4 35.7 78.2 50.9 69.2 47.7 70.3 23.0 20.3 19.3 22.7 41.9 79.6 210.0 84.3 15.4 62.3 15.4 28.5 22.1 170.1 35.7 17.7 13.5 129.1 129.5 128.1 133.0 165.0 171.8 21.3 125.5 136.7 123.3 153.5 150.8 165.0

position

3

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 1 2 3 4 5 6 1 2 1 2 3 4 5 6 7 1 2 3 4 5 6 1 2

3-OR

5-OR 7-OR

14-OR

15-OAc

19

43.7 36.9 76.5 51.6 69.0 53.9 63.6 123.1 133.1 147.2 42.2 40.5 89.1 82.0 89.6 14.1 69.2 112.8 19.9 24.6 173.4 36.4 18.1 13.8

169.2 20.8 170.4 21.0

125.8 137.5 123.4 153.4 150.9 164.4 168.2 22.3


4 44.2 37.2 78.1 51.9 69.1 54.4 64.5 123.0 133.4 146.9 42.0 40.7 89.3 82.2 89.9 14.2 69.2 113.1 20.4 24.7 125.7 137.5 123.4 153.6 150.9 164.5 168.8 20.6 130.4 129.3 128.0 132.6 128.0 129.3 165.7 126.8 136.9 123.0 152.9 150.3 165.4 168.2 22.6

5

6

43.8 36.8 76.5 51.6 69.0 53.9 63.6 123.0 133.2 147.2 42.2 40.5 89.1 82.0 89.6 14.0 69.2 112.8 19.9 24.6 174.1 27.8 8.9

43.6 36.6 76.5 51.7 69.2 54.9 65.2 123.3 134.0 147.0 41.1 40.8 89.0 81.1 90.1 14.1 69.1 112.2 21.3 24.5 173.3 36.3 17.9 13.7

169.2 20.8 170.4 21.0

169.0 20.7 126.6 137.1 123.0 153.0 150.7 164.5

125.7 137.5 123.3 153.5 150.9 164.4 168.2 22.3

170.2 20.9

168.5 22.6


Page 20 of 28

Table 2. 1H NMR Data (δH) of Compounds 1–6a position

1

2

position

3

4 b

5

6

1α

2.94 dd (15.5,10.3)

2.75 dd (14.7,10.9)

1α

2.78 dd (15.8,10.9)

2.86

2.76 dd (15.8,11.0)

2.83 dd (15.9,10.8)

β

2.46b

1.80 dd (14.7,8.0)

β

2.69 dd (15.8,9.1)

2.83b

2.65 dd (15.8,9.0)

2.54 dd (15.9,9.3)

2

2.28 m

2.46 m

2

2.13 m

2.28 m

2.18 m

2.06 m

3

5.53 t (3.8)

5.61 t (4.5)

3

5.31 t (3.2)

5.54 t (3.5)

5.31 t (3.5)

5.27 t (3.3)

4

2.99 dd (11.1,3.8)

3.01 dd (10.6,4.5)

4

3.18 dd (11.1,3.2)

3.42 dd (11.2,3.5)

3.18 dd (11.1,3.5)

3.10 dd (11.1,3.3)

5

5.90 d (11.1)

6.07 d (10.6)

5

5.95 d (11.1)

6.23 d (11.2)

5.96 d (11.1)

5.96 d (11.1)

5.06 dd (5.8,3.2)

7

4.86 d (6.1)

5.07 d (6.4)

4.85 d (6.1)

5.07 d (6.5)

3.91 d (5.5)

1.61 m

8

6.10 dd (9.7,6.1)

6.26 dd (9.8,6.4)

6.10 dd (9.8,6.1)

6.28 dd (9.7,6.5)

0.90 m

9

5.80 dd (9.7,4.7)

5.85 dd (9.8,5.4)

5.80 dd (9.8,5.0)

5.94 dd (9.7,5.5)

7 8

9

2.52 m

11

3.29 m

3.34 m

3.24 m

3.15 m

11

2.36 m

0.95 m

12

3.28 br.s

3.50 d (3.8)

3.28 m

3.32 m

12

4.53 d (12.2)

2.35 d (7.6)

14

5.25 br.s

5.38 br.s

5.24 s

5.03 br.s

14

5.35 br.s

16

0.83 d (6.6)

0.89 d (6.7)

0.83 d (6.7)

0.81 d (6.7)

16

0.88 d (6.6)

0.97 d (6.8)

17

4.06 d (8.7)

4.19 d (8.8)

4.06 d (8.8)

4.06 d (8.8)

17

4.17 d (9.8)

5.33 d (12.1)

3.53 d (8.7)

3.65 d (8.8)

3.53 d (8.8)

3.60 d (8.8)

3.66 d (9.8)

4.57 d (12.1)

4.99 s

4.93 s

4.99 s

4.76 s

α 2.47b

1.03 s

4.86 s

4.83 s

4.85 s

4.64 s

1.88 s

1.88 s

1.88 s

1.87 s

18

19

β 3.04 dd (12.2,9.0) 19

1.59 s

1.11 s

20

1.22 s

1.61 s

2.01 s

2.12 m

3-OR

2

1.31 s

1.37 s

1.31 s

1.29 s

2

2.27 m

8.28 dt (7.9,1.4)

2.31 q (7.5)

2.12 m

3

1.62 m

7.43 dd (7.9,4.8)

1.12 t (7.5)

1.44 m

0.94 t (7.4)

1.43 m

4

4

0.77 d (7.4)

5

8.80 dd (4.8,1.4)

0.78 t (7.4)

9.11 d (1.4)

5-OAc

2

1.99 s

1.96 s

1.99 s

1.94 s

7/10-OR

2

1.99 s

7.89 d (7.8)

1.99 s

8.25 d (7.9)

2/6

2.29 m

7.67 d (7.8)

3/5

1.12 t (7.6)

7.08 t (7.8)

3

7.31 t (7.8)

7.35 dd (7.9,4.9)

7.30 t (7.8)

4

7.46 t (7.8)

8.74 d (4.9)

1.88 s

5

7.31 t (7.8)

9.14 s

4 7/10-OAc

20 3-OR

3

5 5-OR

18

2

1.90 s

20


Page 21 of 28


Table 2. (Continued) position 14/17-OR

15-OAc

1

2

position

3

2

8.36 d (7.9)

7.95 d (7.9)

7/10- OR

6

3

7.40 dd (7.9,4.9)

7.18 dd (7.6,4.9)

14/17-OR

2

4

8.79 d (4.9)

8.68 d (4.9)

3

5

9.22 br.s

9.08 br.s

4

2

2.24 s

5 2

15-OAc

4

5

6

7.89 d (7.8) 8.25 d (8.0)

8.06 dt (7.8,1.4)

8.25 dt (7.9,1.4)

7.41 dd (8.0,4.3)

7.18 dd (7.8,4.8)

7.41 dd (7.9,4.8)

8.79 d (4.3)

8.67 dd (4.8,1.4)

8.78 dd (4.8,1.4)

9.06 s

9.07 d (1.4)

9.07 d (1.4)

2.08 s

2.33 s

2.08 s

a

2.06 s

2.21 s

Assignments of 1H NMR data are based on 1H-1H COSY, HMQC, and HMBC experiments. bSignals were in overlapped regions of the spectra, and the multiplicities could not be discerned.

21



Figure Captions Figure 1. Structures of compounds 1− −8 from E. prolifera. Figure 2. 1H-1H COSY and selected HMBC correlations of compound 1. Figure 3. Conformation and key NOESY correlations of compound 1. Figure 4. Calculated and experimental ECD spectra for compounds 1 (A), 2 (B), and 3 (C) in acetonitrile. Figure 5. Antifungal effects of compounds 2‒8 against three pathogenic fungi. Three common pathogenic fungi (P. piricola, A. solani, and G. zeae) were selected to evaluate the antifungal activities of compounds 2‒8. The biological data presented are the mean scores for each treatment across replicates. The symbols ▼ and▲ indicated the positive controls chlorothalonil and carbendazol, respectively.


Page 22 of 28

Page 23 of 28


Figure 1 NicO AcO15 14 13 1

O

2 16 3

17

4

20

19 10

HH 12

8

OH HH

H OH

BzO AcO O

NicO AcO

H

H

OAc

H

O

9

R1O H AcO

3 4 5 6

R1 Bu Nic Prop Bu

1

Ac

2

1

O

R3 Nic Nic Nic Ac

3

Prop 2

O 2

2

R2 Ac Bz Ac Nic

8

O

O

R 2O

O

H PropO AcO

AcO 7

18 11

12

AcO PropO H AcO

10

H

18

2

1

19

O

10

9

H BuO BzO NicO AcO

R3O AcO

19

12 11

18

6

O

O

OAc

11 9

5 AcO H PropO

HO

1

3

3

1

Bu

3

5

N

4

6

Bz

23

2 1

4

7

4

O

6

5


Nic


Figure 2

24


Page 24 of 28

Page 25 of 28


Figure 3

16

15

1 2

10

14

20

A 4

13

B

3

18

12

6

11

C

5

7 8 17

25


9

19


Page 26 of 28

Figure 4 20

A

CD[mdeg]

10

0

-10

Exptl. ECD of 1 Calcd ECD of 1 -20 200

25

250

300

350

400

Wavelength (nm)

B

20 15

CD[mdeg]

10 5 0 -5 -10 -15

Exptl. ECD of 2 Calcd ECD of 2

-20 -25 200 60

C

250

300

350

400

Wavelength (nm)

50 40 30

CD[mdeg]

20 10 0 -10 -20 -30 -40


-50 -60 200

250

300

350

Wavelength (nm)

26


400

Page 27 of 28


Figure 5 Antifungal activity (Inhibitory Rate)

105 Physalospora piricola

Alternaria solani

Gibberella zeae

70

35

0 Conc. Comp.

50 2

50 3

50 4

50 5

50 6

27

50 7

50 8


50 ▼

50 ▲

µg/mL


Page 28 of 28

Table of Contents Graphic NicO AcO O

H

H BuO AcO 20

25

60

20

50

15

40

10

AcO 3

30

10 5

CD[mdeg]

CD[mdeg]

CD[mdeg]

20

0

0 -5

10 0 -10 -20

-10 -10

-30

-15


-40


-20

-20 250

300

Wavelength (nm)

350

400


-50

-25 200

-60

200

250

300

Wavelength (nm)

28

350

400

200

250

300

Wavelength (nm)


350

400

Characterization of Diterpenes from Euphorbia prolifera and Their

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