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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])
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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.
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KEYWORDS:
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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
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pathogens can lead to significant yield reduction and dramatic economic losses in agriculture.1
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Several important plant fungal pathogens, such as Gibberella zeae, Physalospora piricola, Alternaria
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solani, Cercospora arachidicola, Cladosporium cucumerinum, and Phytophthora capsici, have
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received much attention due to their perniciousness and universality. To protect crops from plant
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pathogenic fungi, some synthetic antifungal agents have been used currently as an effective method.2
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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
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agents is to search for biologically active substances or lead compounds from plant secondary
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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
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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
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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
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resulted in the isolation of six new diterpenes. Their structures were elucidated based on the NMR
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spectroscopic data analyses and the time-dependent density functional theory (TDDFT) ECD 3
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calculations. The compounds were tested for antifungal activity against three phytopathogenic fungi.
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MATERIALS AND METHODS
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General. Optical rotations were measured in CH2Cl2 using an Autopol IV automatic polarimeter
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(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
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on a Chirascan spectrometer (Applied Photophysics Ltd., Leatherhead, UK). 1D and 2D NMR
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spectra were recorded on a Bruker AV 400 instrument (Bruker Group, Fallanden, Switzerland, 400
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MHz for 1H and 100 MHz for 13C) with TMS as an internal standard. ESIMS spectra were acquired
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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
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column used was a 20 × 250 mm i.d., 5 µm, YMC-pack ODS-AM (YMC Co., Ltd., Kyoto, Japan).
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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.
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G.), and a voucher specimen (No. 20100705) was deposited at the laboratory of the Research
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Department of Natural Medicine, College of Pharmacy, Nankai University, People’s Republic of
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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
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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
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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
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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
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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
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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
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following purification of subfractions F7-3 (73% MeOH in H2O) produced compounds 3 (tR = 35 min,
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13.1 mg), 6 (tR = 30 min, 13.8 mg), and 7 (tR = 26 min, 15.0 mg).
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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,
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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,
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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,
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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]+,
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C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz,
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calcd. for C39H47NNaO11, 728.3047.
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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,
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1638, 1592, 1456, 1367, 1269, 1220, 1110, and 1026 cm-1;
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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
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1
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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
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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,
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CDCl3) data (Tables 1 and 3); ESIMS m/z 668 [M + H]+; HR-ESIMS m/z 668.3062 [M + H]+, calcd.
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for C36H46NO11, 668.3071.
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Computational Methods. The ECD calculations were performed according to the reported method.29
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Antifungal Activity Assay. The antifungal activities of the isolated compounds were assayed
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against three pathogenic fungi (Physalospora piricola, Alternaria solani, and Gibberella zeae) using
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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
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corresponding positive controls. Compounds possessing good activities (inhibitory rate >50% at 50
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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
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six new (1−6) and two known (7 and 8) diterpenoids (Figure 1). The known compounds were
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identified by comparison of spectroscopic data with those reported in the literature as
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2α,5α,7β,15β-tetra-O-acetyl-3β-O-propinoyl-14α-O-benzoyl-14-deoxomyrsinol, 7,31 and 3β-O-
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propionyl-5α,10,15β-tri-O-acetyl-14β-O-nicotinoyl-7-oxo-10,18-dihydromyrsinol, 8.14
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Compound 1 was obtained as a white powder. Its molecular formula was assigned as C35H43NO13
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on the basis of HR-ESIMS. The 1H-NMR spectrum of 1 exhibited four oxygenated methine protons
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[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
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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
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Hz), 8.79 (1H, d, J = 4.9 Hz), and 9.22 (1H, br. s)], and a pair of oxygenated methylene protons [δH
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4.17, and 3.66 (each 1H, d, J = 9.8 Hz, H2-17)]. Additionally, six methyl signals were also exhibited
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in the 1H NMR spectrum. The
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From the 1H and 13C NMR spectra of 1, three acetyl groups were apparent from the methyl singlets
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(δH 2.24 s, 2.01 s, and 1.90 s) and the corresponding carbons (Table 1). The above mentioned four
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aromatic proton signals (Table 2), together with the typical carbon resonances, revealed the presence
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of one nicotinoyl group.14 In addition to these substituent groups, a propionyl moiety (Tables 1 and 2)
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were deduced and defined from the methyl and methylene proton signals and the observation of the
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corresponding carbon signals (Tables 1 and 2).13 Apart from the above 15 carbon signals for the
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substituent groups, there were additional 20 carbons displayed in the 13C NMR spectrum, including
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three methyls, three methylenes, nine methines, and five quaternary carbons based on the DEPT and
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HMQC spectra. These 20 typically skeletal carbons implied that compound 1 possessed a
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characteristic cyclomyrsinol-type diterpene skeleton according to the reported related diterpenes
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from the genus Euphorbia.32−36 This skeleton of cyclomyrsinol-type diterpene was further confirmed
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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).
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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
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attributed to C-3, C-5, C-8, C-10, C-13, C-14, C-15, and C-17, respectively. The positions of the
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acyloxy groups were deduced from the HMBC spectrum. The HMBC correlation of the carbonyl
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signal at δC 173.2 with the proton signals at δH 5.53 (H-3), demonstrated that one acetoxy group was
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attached at C-3. Similarly, the long-range couplings of the carbonyl carbon signals at δC 173.5 and
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166.3 with the proton signals at δH 5.90 (H-5) and 5.35 (H-14) disclosed the presence of the
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propionyloxy group and the nicotinoyloxy group at C-5 and C-14, respectively. Because of no
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long-range correlations of the skeletal protons with the carbonyl carbons of the two remaining
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acetoxy groups, it was therefore concluded that the two acetoxy groups were attached to quaternary
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carbons of the cyclomyrsinol skeleton. Based on the chemical shifts of skeletally oxygenated carbons
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and similar cyclomyrsinol diterpenes in the literature,35,36 the residual acetoxy groups could only be
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located at C-10 and C-15, of which the C-10 acetoxy group was assigned by a NOESY experiment,
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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.
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The stereochemistry of 1 were elucidated as follows. The NOESY correlations of H-2/H-4,
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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
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arrangement for compound 1, the three rings A, B, and C (5/7/6) were trans-fused with each other,
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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
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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
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C-3 and C-10 and the C-8 hydroxy group were all β-oriented, and the C-5 propionyloxy group was in
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an α-position according to this conformation of 1 and the above NOESY correlations. The relative 8
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configuration of 1 was therefore designated as shown in Figure 3.
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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.
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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
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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
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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
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as
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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-
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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
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aromatic protons, and six methyl signals were observed (Table 2). The
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exhibited 39 carbon resonances. From the 1H and
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together with the corresponding carbon signals (Table 1) implied the occurrence of one acetyl group,
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and the aromatic proton signals and the corresponding carbon resonaces (Table 1) revealed the
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presence of one nicotinoyl and one benzoyl group.14,16 In addition, a butyryl group was also deduced
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and defined from the observation of the carbon signals (δC 170.1, 35.7, 17.7, and 13.5) and the
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corresponding proton signals (Table 2) based on those reported diterpenes with acyl groups in the
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literature.13 Apart from the above 19 resonances for the substituent groups, the remaining 20 skeletal
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carbons in the
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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
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In order to confirm the above deduction and position these substituent groups, the following
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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
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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
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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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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
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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
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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
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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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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
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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
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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
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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
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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.
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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
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Figure 2
24
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Figure 3
16
15
1 2
10
14
20
A 4
13
B
3
18
12
6
11
C
5
7 8 17
25
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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
Exptl. ECD of 3 Calcd ECD of 3
-50 -60 200
250
300
350
Wavelength (nm)
26
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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
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50 ▲
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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
Exptl. ECD of 1 Calcd ECD of 1
-40
Exptl. ECD of 2 Calcd ECD of 2
-20
-20 250
300
Wavelength (nm)
350
400
Exptl. ECD of 3 Calcd ECD of 3
-50
-25 200
-60
200
250
300
Wavelength (nm)
28
350
400
200
250
300
Wavelength (nm)
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Article
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
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 free 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 accessible to all readers and 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.
Journal of Agricultural and Food Chemistry 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.
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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])
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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
40
Euphorbia prolifera Buch-Ham., belonging to the Euphorbiaceae family, is a perennial herbaceous
41
plant distributed mainly in southwest China.9 This plant is poisonous and its roots were documented
42
as a poisonous Chinese medicine.10 Previous phytochemical investigations on E. prolifera led to the
43
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
45
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
47
documented in the literature evoked our attention, since their extracts or constituents may be
48
potentially useful for the agricultural and medical industry due to their toxicity.27,28 In order to obtain
49
new bioactive antifungal compounds for the development of new antifungal agents for agriculture,
50
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
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calculations. The compounds were tested for antifungal activity against three phytopathogenic fungi.
54 55
MATERIALS AND METHODS
56
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
58
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).
63
HR-ESIMS were recorded by an IonSpec 7.0 T FTICR MS (IonSpec Co., Ltd., Lake Forest, CA).
64
HPLC purifications were finished on a CXTH system, equipped with a UV3000 detector at 210 nm
65
(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
68
Co., Ltd., Qingdao, People’s Republic of China). Chemical reagents for isolation were of analytical
69
grade and purchased from Tianjin Chemical Reagent Co. (Tianjin, People’s Republic of China).
70
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
72
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.
76
Extraction and Isolation. The air-dried roots of E. prolifera (3.8 kg) were extracted with
77
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
80
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
82
0:100, 21 L for each gradient elution), to afford nine fractions (F1−F9) according to the TLC analyses.
83
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
85
60-90% MeOH in H2O to give four subfractions (F8-1‒F8-4). The subfraction F8-3 was further purified
86
by preparative HPLC (YMC-pack ODS-AM, 250 × 20 mm i.d., 77% MeOH in H2O) to afford
87
compound 1 (tR = 23 min, 7.2 mg), and the purification of F8-4 by the same HPLC system (82%
88
MeOH in H2O) yielded compound 5 (tR = 27 min, 11.5 mg). Fraction F9 (eluted by petroleum
89
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
91
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).
93
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
97
(∆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
99
and 2); ESIMS m/z 708 [M + Na]+; HR-ESIMS m/z 708.2632 [M + Na]+, calcd. for C35H43NNaO13,
100
708.2632.
101
Prolifepene B, 2: white powder; [α]15 D : +16.9 (c 0.11, CH2Cl2); CD (CH3CN): 200 (∆ε +8.81), 215
102
(∆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;
104
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,
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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
107
(∆ε ‒11.08), 244 (∆ε +1.48) nm; IR (KBr) νmax 2963, 2932, 2875, 1737, 1640, 1590, 1437, 1242,
108
1150, and 1024 cm-1; 13C NMR (100 MHz, CDCl3) and 1H NMR (400 MHz, CDCl3) data (Tables 1
109
and 2); ESIMS m/z 668 [M + H]+; HR-ESIMS m/z 668.3067 [M + H]+, calcd. for C36H46NO11,
110
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
113
NMR (400 MHz, CDCl3) data (Tables 1 and 2); ESIMS m/z 787 [M + Na]+; HR-ESIMS m/z
114
787.2841 [M + Na]+, calcd. for C43H44N2NaO11, 787.2843.
115
Prolifepene E, 5: white powder; [α]15 D : −10.7 (c 0.60, CH2Cl2); IR (KBr) νmax 2966, 2936, 1739,
116
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,
120
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
128
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
132
The ethyl acetate-soluble part of the methanol extract of the aerial parts of E. prolifera was
133
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
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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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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
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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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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
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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
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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
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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
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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
ACS Paragon Plus Environment
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
Journal of Agricultural and Food Chemistry
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
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Journal of Agricultural and Food Chemistry
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
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Journal of Agricultural and Food Chemistry
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.
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Page 23 of 28
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
Nic
Journal of Agricultural and Food Chemistry
Figure 2
24
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Page 25 of 28
Journal of Agricultural and Food Chemistry
Figure 3
16
15
1 2
10
14
20
A 4
13
B
3
18
12
6
11
C
5
7 8 17
25
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9
19
Journal of Agricultural and Food Chemistry
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
Exptl. ECD of 3 Calcd ECD of 3
-50 -60 200
250
300
350
Wavelength (nm)
26
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400
Page 27 of 28
Journal of Agricultural and Food Chemistry
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
ACS Paragon Plus Environment
50 ▼
50 ▲
µg/mL
Journal of Agricultural and Food Chemistry
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
Exptl. ECD of 1 Calcd ECD of 1
-40
Exptl. ECD of 2 Calcd ECD of 2
-20
-20 250
300
Wavelength (nm)
350
400
Exptl. ECD of 3 Calcd ECD of 3
-50
-25 200
-60
200
250
300
Wavelength (nm)
28
350
400
200
250
300
Wavelength (nm)
ACS Paragon Plus Environment
350
400
