Lathyrane diterpenes from Euphorbia prolifera and their inhibitory activities on LPS-induced NO production

Fitoterapia 83 (2012) 1205–1209

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Lathyrane diterpenes from Euphorbia prolifera and their inhibitory activities on LPS-induced NO production Jing Xu a, Da-qing Jin b, Haibin Song c, Yuanqiang Guo a, d,⁎, Yisha He a a College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China b School of Medicine, Nankai University, Tianjin 300071, China c State Key Laboratory of Elemento-Organic Chemistry, Nankai University, Tianjin 300071, China d State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, China

a r t i c l e

i n f o

Article history: Received 22 April 2012 Accepted in revised form 28 June 2012 Available online 7 July 2012 Keywords: Euphorbia prolifera Lathyrane diterpene X-ray crystallography Inhibitory activity on NO production

a b s t r a c t A new lathyrane diterpene (1), an unreported spectroscopic data lathyrane diterpenene (2), and two known analaogues (3 and 4) have been isolated from Euphorbia prolifera. Their structures were elucidated as (12E,2S,3S,4R,5R,6S,9S,11S,15R)-3-butyryloxy-5,15-diacetoxy-6,17-epoxylathyra12-en-14-one (1), (12E,2S,3S,4R,5R,6S,9S,11S,15R)-3-propionyloxy-5,15-diacetoxy-6,17- epoxylathyra12-en-14-one (2), (12E,2S,3S,4R,5R,6S,9S,11S,15R)-3-benzoyloxy-5,15-diacetoxy −6,17-epoxylathyra12-en-14-one (3), and 15-O-acetyl-17-hydroxyjolkinol (4) by spectroscopic methods (IR, ESIMS, HR-ESIMS, NMR, and X-ray crystallography). The inhibitory activities on LPS-induced NO production of these diterpenes were evaluated and compounds 1, 3 and 4 showed inhibitory effects. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Euphorbia prolifera Buch-Ham, belonging to the family Euphorbiaceae, is a perennial herbaceous plant distributed in southwest China [1]. Its roots have been used as a folk medicine for the treatment of inflammations and tumors [2]. Previous phytochemical investigations on E. prolifera led to the identification of diterpenes [3–7], especially myrsinane-type diterpenes [6,7], which are the main and characteristic constituents of E. prolifera. In our continuous search for bioactive natural products from medicinal plants [8–11], the chemical constituents of the roots of E. prolifera had been investigated. As a result, a new lathyrane diterpene (1), an unreported spectroscopic data lathyrane diterpenene (2), and two known analaogues (3 and 4) have been isolated from E. prolifera. Their structures were elucidated as (12E,2S,3S,4R,5R,6S,9S,11S,15R)-3-butyryloxy-

⁎ Corresponding author at: College of Pharmacy, State Key Laboratory of Medicinal Chemical Biology and Tianjin Key Laboratory of Molecular Drug Research, Nankai University, Tianjin 300071, China. Tel./fax: +86 22 23502595. E-mail address: [email protected] (Y. Guo). 0367-326X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.fitote.2012.06.014

5,15- diacetoxy-6,17-epoxylathyra-12-en-14-one (1), (12E,2S,3S,4R,5R,6S,9S,11S,15R)-3-propionyloxy- 5,15-diacetoxy-6,17-epoxylathyra-12-en-14-one (2), (12E,2S,3S,4R,5R,6S,9S,11S, 15R)-3- benzoyloxy-5,15-diacetoxy-6,17-epoxylathyra-12-en14-one (3), and 15-O-acetyl-17-hydroxyjolkinol (4) by spectroscopic methods (IR, ESIMS, HR-ESIMS, NMR, and Xray crystallography). The inhibitory activities on LPS-induced NO production of these diterpenes were also evaluated. This paper herein describes their isolation, structure elucidation and inhibitory effects on LPS-induced NO production in murine microglial BV-2 cells.

2. Experimental 2.1. General Melting points were determined with an XT-4 microscopic thermometer. The optical rotations were measured in CH2Cl2 using a Rudolph Autopol IV automatic polarimeter. The IR spectra were recorded on a Bio-Rad FTS 6000 Fourier transform infrared (FTIR) spectrometer with KBr discs (DeFelsko Co. Ltd.,

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America). The ESIMS spectra were obtained on an LCQAdvantage mass spectrometer (Finnigan Co. Ltd., America). HR-ESIMS spectra were recorded by an IonSpec 7.0 T FTICR MS (IonSpec Co. Ltd., America). 1D and 2D NMR data were recorded on a Bruker AV 400 instrument (400 MHz for 1H and 100 MHz for 13C) with TMS as an internal standard. HPLC separations were performed on a CXTH system, equipped with a UV3000 detector at 210 nm (Beijing Chuangxintongheng Instruments Co. Ltd., China), and a YMC-pack ODS-AM (250× 20 mm) column (YMC Co. Ltd., Japan). X-ray crystallographic analysis was carried out on a Rigaku Saturn 944 CCD diffractometer equipped with multilayer-monochromator and Cu Kα-radiation (λ = 1.54187 Å) (Rigaku Co. Ltd., Japan). The structure was solved by direct methods (SHELXL-97), expanded using Fourier techniques, and refined with full-matrix least-squares on F 2 (SHELXL-97). Silica gel was used for column chromatography (200–300 mesh, Qingdao Marine Chemical Group Co. Ltd., China). Chemical reagents for isolation were of analytical grade and purchased from Tianjin Yuanli Co. Ltd., China. Biological reagents were from Sigma Company. The murine microglial BV-2 cell line was from Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences (China). 2.2. Plant material The roots of E. prolifera were collected from Kunming, Yunnan Province, China, in July 2010. The botanical identification was made by Dr. Yuanqiang Guo (College of Pharmacy, Nankai University, China), and a voucher specimen (No. 20100705) was deposited at the laboratory of the Research Department of Natural Medicine, College of Pharmacy, Nankai University, China. 2.3. Extraction and isolation The air-dried roots of E. prolifera (3.8 kg) were powdered and extracted with MeOH (3 × 20 L) under reflux. The solvent was evaporated to obtain a crude extract (900 g). The extract was suspended in H2O (0.9 L) and partitioned with EtOAc (3 × 0.9 L). The EtOAc soluble part (150.0 g) was subjected to a silica gel column chromatography using a gradient solvent system from 1 to 40% acetone in petroleum ether to give eight fractions (F1−F8) based on TLC analyses. The following acetone was used as the eluent to afford the last fraction (F9). F2 (6.7 g) was separated by MPLC over ODS eluting with a step gradient from 65% to 90% MeOH in H2O to give three subfractions (F2-1−F2-3). F2-3 was purified by preparative HPLC (YMC-pack ODS-AM, 20 × 250 mm, 84% MeOH in H2O) to afford compounds 1 (tR = 35 min, 11.9 mg), 2 (tR = 31 min, 12.6 mg), and 3 (tR = 42 min, 15.1 mg). Fraction F9 (13.2 g) was separated by MPLC over ODS eluting with a step gradient from 55 to 85% MeOH in H2O to obtain four subfractions (F9-1−F9-4). Subfraction F9-1 was further purified by preparative HPLC (YMC-pack ODS-AM, 20 × 250 mm, 60% MeOH in H2O) to afford compound 4 (tR = 30 min, 12.4 mg).

ð12E; 2S; 3S; 4R; 5R; 6S; 9S; 11S; 15RÞ  3  Butyryloxy  5; 15  diacetoxy  6; 17  epoxylathyra  12  en  14  one ð1Þ

White powder; [α]D20 + 72.6 (c 0.31, CH2Cl2); IR (KBr) νmax 2930, 1742, 1650, 1372, and 1234 cm −1; 1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data see Table 1; ESIMS m/z 527 [M + Na] +; HR-ESIMS m/z 527.2618 [M + Na] + (calcd. for C28H40O8Na, 527.2621). ð12E; 2S; 3S; 4R; 5R; 6S; 9S; 11S; 15RÞ  3  Propionyloxy5; 15  diacetoxy  6; 17  epoxylathyra  12  en  14  one ð2Þ

White powder; [α]D20 + 29.3 (c 0.21, CH2Cl2); IR (KBr) νmax 2924, 1736, 1624, 1374, and 1232 cm –1; 1H (400 MHz, CDCl3) and 13C (100 MHz, CDCl3) NMR data see Table 1; ESIMS m/z 513 [M + Na] +; HR-ESIMS m/z 513.2457 [M + Na] + (calcd. for C27H38O8Na, 513.2464). X  raycrystal data of ð12E; 2S; 3S; 4R; 5R; 6S; 9S; 11S; 15RÞ  3  benzoyloxy  5; 15  diacetoxy  6; 17  epoxylathyra  12  en  14  one ð3Þ

C31H38O8, Mr = 538.61, monoclinic, space group P12(1)1, a = 10.2190(10) Å, b = 17.8340(18) Å, c = 16.1690(16) Å, V = 2872.9(5) Å 3, Z = 4, Dcalc = 1.245 g/cm 3, crystal dimensions 0.16 × 0.12 × 0.10 mm were used for measurements. The total number of reflections measured was 31,117, of which 10,866 were unique and 10,213 were observed, I > 2σ(I). Final indices: R1 = 0.0385, wR2 = 0.0899 for observed reflections, and R1 = 0.0420, wR2 = 0.0947 for all reflections. 2.4. Bioassay for NO production Murine microglial BV-2 cells were cultured at 37 °C in DMEM supplemented with 10% (v/v) inactivated fetal bovine serum and 100 U/mL penicillin/streptomycin under a watersaturated atmosphere of 95% air and 5% CO2. The cells were seeded in 96-well culture plates (5 × 10 4 cells/well) and allowed to adhere for 24 h at 37 °C. The cells were incubated for 20 h with or without 0.4 μg/mL of LPS (Sigma Chemical Co., St. Louis, MO, U.S.A.) in the absence or presence of the test compounds. SMT was used as a positive control. As a parameter of NO synthesis, the nitrite concentration was measured by the Griess reaction using the supernatant of the BV-2 cells. Briefly, 50 μL of the cell culture supernatant were reacted with 50 μL of Griess reagent [1:1 mixture of 0.1% N-(1-naphtyl)ethylenediamine in H2O and 1% sulfanilamide in 5% phosphoric acid] in a 96 well plate and the absorbance was read with a microplate reader (Thermo Fisher Scientific Inc. America) at 550 nm. The experiment was performed three times, and the IC50 values for the inhibition of NO production were determined on the basis of linear or nonlinear regression analysis of the concentration–response data curves. 3. Results and discussion Compound 1 was obtained as a white powder. Its molecular formula was determined to be C28H40O8 by HR-ESIMS (m/z 527.2618 [M+ Na]+, calcd. for C28H40O8Na, 527.2621), which was compatible with the NMR data. The 1H and 13C NMR spectra of 1 revealed the occurrence of two acetyl groups [δH 2.09 s, and 2.05 s; δC 170.5, 169.5, 21.7, and 20.9], and one

J. Xu et al. / Fitoterapia 83 (2012) 1205–1209

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Table 1 1 H and 13C NMR data for compounds 1−3 (in CDCl3, δ in ppm, J in Hz). position

1

2

δC

δH

1

47.9

2 3 4 5 6 7

37.5 80.0 50.0 65.1 58.8 33.5

3.39 1.48 2.07 5.48 1.87 6.18

dd (14.2, 8.4) dd (14.2, 12.0) m br. s dd (9.3, 3.1) d (9.3)

8

19.9

9 10 11 12 13 14 15 16 17

34.7 25.5 28.9 143.6 135.9 196.8 91.7 13.8 55.3

0.93 2.08 2.06 1.71 1.06

m m m m m

18 19 20 3-OR

28.8 16.6 12.3 172.6 36.2 18.1 13.9

5-OAc 15-OAc

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

170.5 20.9 169.5 21.7

1.46 dd (11.4, 8.4) 6.57 d (11.4)

0.84 2.47 2.30 1.19 1.19 1.84

d (6.7) d (3.2) d (3.2) s s s

2.25 t (7.6) 1.63 m 0.95 t (7.4)

2.05 s 2.09 s

3

δC

δH

47.9

3.38 1.48 2.07 5.47 1.86 6.17

dd (14.2, 8.4) dd (14.2, 11.0) m br. s dd (9.3, 3.1) d (9.3)

0.93 2.08 2.06 1.67 1.05

m m m m m

37.5 80.0 49.9 65.1 58.8 33.4 19.9 34.7 25.5 28.9 143.6 135.8 196.8 91.7 13.9 55.3 28.8 16.6 12.2 173.3 27.6 8.9

170.4 20.9 169.5 21.7

1.45 dd (11.0, 8.4) 6.56 d (11.4)

0.83 2.45 2.28 1.17 1.17 1.82

d (6.6) d (3.3) d (3.3) s s s

2.28 q (7.6) 1.10 t (7.6)

2.03 s 2.07 s

δC

δH

48.0

3.47 1.61 2.25 5.72 1.98 6.34

dd (14.2, 8.2) dd (14.2, 12.5) m br. s dd (8.9, 3.3) d (8.9)

0.92 2.06 2.03 1.73 1.09

m m m m m

38.1 80.8 49.8 64.9 58.9 33.3 20.0 34.7 25.6 29.0 143.7 135. 9 196.6 91.9 14.0 55.1 28.8 16.7 12.3 129.7 129.5 128.2 133.1 165.7 170.2 20.5 169.4 21.7

1.51 dd (11.1, 8.2) 6.66 d (11.4)

0.90 2.50 2.34 1.21 1.23 1.88

d (6.7) d (3.3) d (3.3) s s s

8.00 d (7.8) 7.44 t (7.8) 7.58 t (7.8)

1.81 s 2.23 s

The assignments of proton and carbon signals are based on DEPT, 1H–1H COSY, HMQC, and HMBC experiments.

butyryl group (δH 2.25 t, 1.63 m, and 0.95 t; δC 172.6, 36.2, 18.1, and 13.9) (Table 1). Additionally, three methyls (δH 0.84 d, and 1.19 × 2 s), one methyl group bearing a double bond (δH 1.84 s), and one olefinic proton (δH 6.57 d) were also revealed by the 1H NMR spectrum. The 13C NMR spectrum showed 28 carbon resonances including the above eight carbons for the substituents (two acetoxy and one butyryloxy groups). The remaining 20 carbons in the 13C NMR spectrum, including four methyls, four methylenes, six methines, and six quaternary carbons, constitute a characteristic 6,17-epoxylathyrol diterpene skeleton for 1 based on the reported 6,17-epoxylathyrol diterpenes from the genus Euphorbia [12–15]. In order to confirm the 6,17epoxylathyrol skeleton and determine the positions of the acyloxy groups, the following HMQC and HMBC experiments were performed. By the interpretation of 1D and 2D NMR spectra, the 6,17-epoxylathyrol skeleton was defined (Fig. 1), where the double bond was attributed to C-12 and C-13. The HMBC correlations of the carbonyl signal at δC 172.6 (butyryl CO) with the proton signals at δH 5.48 (H-3), 2.25, and 1.63 indicated the presence of the butyryloxy group at C-3. Similarly, the long-range coupling of the carbonyl carbon signal at δC 170.5 with the proton signal at δH 6.18 (H-5) demonstrated the presence of acetyl group at C-5. Based on the 13C NMR spectroscopic data and similar 6,17-epoxylathyrol diterpenes in the literature [12,13], the

residuary acetyl group could only be located at C-15. By further analyzing the HMQC, HMBC, and 1H- 1H COSY spectra (Fig. 2), all the proton and carbon signals were assigned unambiguously. Thus, the planar structure of 1 was established. The configuration of 1 was elucidated as follows. For the reported natural lathyrane diterpenes, the five membered ring and the macro-ring forming the skeleton are trans-fused, H-4 is α-oriented based on biosynthesis considerations, and H3-16, and the C-15 acyloxy group are β-oriented [12−15]. NOESY correlations observed for H-3/H-4, H-2/H-4, H-9/ H-11, H-9/H3-18, H-11/H3-18, but not for H-3/H-5 and H-12/ H3-20, suggested the presence of an E-configuration for the double bond, and revealed that H-3 and the C-5 acetoxy group were α-oriented, and H-9 and H-11 were on the same side of the three-membered ring. These assignments were consistent with the configuration of reported lathyrane diterpenes [12–15]. Further by comparing the chemical shifts of the skeletal carbons of 1 with those of compound 3 possessing the same parent skeleton, whose absolute configuration was determined by X-ray crystallography, the absolute configuration of 1 was elucidated. Therefore, compound 1 was determined as (12E,2S,3S,4R,5R,6S,9S,11S,15R)-3- butyryloxy-5,15-diacetoxy6,17-epoxylathyra-12-en-14-one. The molecular formula of compound 2 was determined as C27H38O8 on the basis of HR-ESIMS (m/z 513.2457 [M + Na] +,

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Fig. 1. Structures of compounds 1−4 from E. prolifera.

Fig. 2. Selected HMBC and 1H–1H COSY correlations of compound 1.

calcd. for C27H38O8Na, 513.2464). The 1H and 13C NMR spectra revealed the presence of one propionyloxy and two acetoxy groups, and a 6,17-epoxylathyrol skeleton consisting of 20 carbons. Close similarities of the chemical shifts from C-1 to C-20 in 2 with those in 1 (Table 1) suggested that compounds 1 and 2 had the same parent skeleton [12,13]. The following HMQC and HMBC experiments were performed, which demonstrated the presence of the 6,17-epoxylathyrol skeleton, two acetoxy groups, and one propionyloxy group. The locations of the acyloxy groups in 2 were determined as in the

case of 1. In the HMBC spectrum, the long-range correlations of H-3 (δH 5.47) to the carbonyl carbon (δC 173.3) of the propionyloxy, H-5 (δH 6.17) to the carbonyl carbon (δC 170.4) of one acetoxy, revealed that the propionyloxy and one acetoxy group were attached at C-3, and C-5, respectively. Consequently, the other acetoxy group in 2 could be assigned only to C-15. The same relative configuration was inferred for compounds 2 and 1 on the basis of comparison of the NOESY spectra of 1 and 2. Considering the biogenetic grounds and the similar chemical shifts for skeletal carbons of compounds 1−3, compound 2 had the same absolute configuration as 1 and 3 and was elucidated therefore as (12E,2S,3S,4R,5R,6S,9S,11S,15R)-3-propionyloxy-5, 15-diacetoxy-6,17-epoxylathyra-12-en-14-one. This compound was included in the patent published in 2011 [16], but there was only the structure, and no spectroscopic data were presented. Compound 3, colorless small quadrate crystals, was reported by us as a naturally occurring lathyrane diterpene for the first time. Appendino Giovanni et al. obtained this compound by structure modification of the compound Euphorbia Factor L3 [15]. The structure of 3 was elucidation by comparison of experimental and literature spectroscopic data [15]. In order to confirm the absolute configuration of compound 3 and the similar

Fig. 3. Thermal ellipsoid representation of 3.

J. Xu et al. / Fitoterapia 83 (2012) 1205–1209

In conclusion, four lathyrane diterpenes, including a new diterpene (1), an unreported spectroscopic data diterpenene (2), and two known analaogues (3 and 4) have been isolated from E. prolifera. Their structures were elucidated based on the analyses of IR, MS, and NMR spectra, and X-ray crystallography data and the inhibitory effects on NO production were reported for the first time. The presence of lathyrane diterpenes in E. prolifera demonstrated that numerous myrsinol diterpenes in the same plant are derived from lathyrane diterpenes [20].

120

NO inhibition (%)

1209

80

40

Acknowledgments The project was supported by the Natural Science Foundation of China (No. 81102331).

0 Conc.

Comp.

30

100

30 1

10

100

30 3

10 100

30

10

M

4

Fig. 4. Inhibitory effects of compounds 1, 3, and 4 on LPS-induced NO production. BV-2 cells were treated with LPS alone or together with each compound at the concentrations indicated. After 20 h incubation, the supernatants were tested by Griess assay and the NO inhibitory rates were calculated. The experiment was performed three times, and the data are expressed as mean ± SD values. The inhibitory rate on NO production was calculated as follows: Inhibitory rate (%) = (1 − (LPS/sample − untreated)/ (LPS − untreated)) × 100. ♦ Indicated positive control, SMT.

compounds 1 and 2, a single-crystal X-ray crystallographic analysis using anomalous scattering of Cu Kα radiation was carried out [17]. A thermal ellipsoid representation, with the atom numbering indicated, was shown in Fig. 3. Thus, compound 3 was characterized as (12E,2S,3S,4R,5R,6S,9S,11S,15R)-3benzoyloxy-5,15-diacetoxy- 6,17-epoxylathyra-12-en-14-one (3). Based on the spectroscopic analyses and the comparison with the literature, the known compound 4 was identified as 15-O-acetyl-17-hydroxyjolkinol [18]. In order to explore the undiscovered and potential pharmacologically activities of these lathyrane diterpenes isolated from E. prolifera, compounds 1−4 were evaluated for their inhibitory activities on LPS-induced NO production in murine microglial BV-2 cells by the Griess reagent [19]. 2-Methyl-2-thiopseudourea, sulfate (SMT) was used as a positive control (IC50 4.70 μM). The inhibitory effects of these myrsinol diterpenes were shown in Fig. 4. Compounds 1 and 4 inhibited LPS-induced NO production in BV-2 cells dose-dependently with IC50 values of 14.56 and 82.56 μM, respectively. Compound 3 showed weak inhibitory effects (IC50 113.5 μM), and compound 2 was inactive. MTT assay indicated that all the compounds had no significant cytotoxicity to the BV-2 cells at their effective concentration for the inhibition of NO production (data not shown).

Appendix A. Supplementary data The cif file of the X-ray data for compound 3 can be found, in the online version, at http://dx.doi.org/10.1016/j.fitote. 2012.06.014. References [1] Editorial Committee of Flora of China. Chinese Academy of Sciences, Flora of China, vol. 44(3). Beijing: Science Press; 1997. p. 118. [2] Wu DG, Sorg B, Hecker E. Phytother Res 1994;8:95. [3] Zhang J, Yang CJ, Wu DG. Acta Bot Yunnan 1995;17:111. [4] Wu D, Sorg B, Hecker E. J Nat Prod 1995;58:408. [5] Zhang WJ, Chen DF, Hou AJ. Chin J Chem 2004;22:103. [6] Li J, Xu L, Wang FP. Helv Chim Acta 2010;93:746. [7] Xu J, Guo Y, Xie C, Li Y, Gao J, Zhang T, et al. J Nat Prod 2011;74:2224. [8] Xu J, Jin D, Shi D, Ma Y, Yang B, Zhao P, et al. Fitoterapia 2011;82:508. [9] Guo P, Li Y, Xu J, Guo Y, Jin D, Gao J, et al. Fitoterapia 2011;82:1123. [10] Guo P, Li Y, Xu J, Guo Y, Liu C, Ma Y, et al. J Nat Prod 2011;74:1575. [11] Xu J, Yang B, Guo Y, Jin DQ, Guo P, Liu C, et al. Fitoterapia 2011;82:849. [12] Jiao W, Dong W, Li Z, Deng M, Lu R. Bioorg Med Chem 2009;17:4786. [13] Ayatollahi AM, Ghanadian M, Afsharypuor S, Choudhary MI, Kobarfard F, Rahmati M. Fitoterapia 2010;81:891. [14] Appendino G, Belloro E, Tron GC, Jakupovic J, Ballero M. J Nat Prod 1999;62:1399. [15] Appendino G, Cravotto G, Jarevang T, Sterner O. Eur J Org Chem 2000;2000:2933. [16] Kato M, Ogami N, Ita M, Taguchi N. Age-related or noise-induced hearing loss and tinnitus prophylactic/therapeutic agents containing endothelin B receptor expression promoters, compositions containing the agents, production of transient hearing loss mouse model, use thereof, drug screening using the mouse, and detection or prediction of the hearing disorders. Jpn Kokai Tokkyo Koho 2011:56. [17] Crystallographic data for compound 3 have been deposited in the Cambridge Crystallographic Data Centre (CCDC 876213). Copies of the data can be obtained, free of charge, on application to the director, CCDC, 12 Union Road, Cambridge CB2 1EZ, UK (fax: +44-(0) 1223-336033 or e-mail: [email protected]). [18] Adolf W, KoehlerI Hecker E. Phytochemistry 1984;23:1461. [19] Barger SW, Harmon ADE. Nature 1997;388:878. [20] Shi QW, Su XH, Kiyota H. Chem Rev 2008;108:4295.