Bioactive Dihydronaphthoquinone Derivatives from Fusarium solani

Aug 28, 2014 - New dihydronaphthoquinone derivatives, karuquinone A (1), karuquinone B (2), and karuquinone C (3), were isolated from a fungal culture...
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Bioactive Dihydronaphthoquinone Derivatives from Fusarium solani Kenji Takemoto,† Shinji Kamisuki,*,† Pei Thing Chia,† Isoko Kuriyama,‡ Yoshiyuki Mizushina,‡,§ and Fumio Sugawara† †

Department of Applied Biological Science, Tokyo University of Science, Noda, Chiba 278-8510, Japan Laboratory of Food & Nutritional Sciences, Faculty of Nutrition, Kobe Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan § Cooperative Research Center of Life Sciences, Kobe Gakuin University, Nishi-ku, Kobe, Hyogo 651-2180, Japan ‡

S Supporting Information *

ABSTRACT: New dihydronaphthoquinone derivatives, karuquinone A (1), karuquinone B (2), and karuquinone C (3), were isolated from a fungal culture broth of Fusarium solani. The structures were determined by interpretation of spectroscopic data (1D/2D NMR, MS, and IR). Three known compounds, javanicin (4), 2,3-dihydro-5-hydroxy-8methoxy-2,4-dimethylnaphtho[1,2-b]furan-6,9-dione (5), and 5-hydroxydihydrofusarubin C (6), were also isolated. The six isolated compounds were tested for cytotoxicity against three human cancer cell lines and a human umbilical vein endothelial cell (HUVEC) line. Of these, karuquinone A exhibited the strongest cytotoxic activity. Karuquinone B did not affect the proliferation of the cancer cell lines but did inhibit the proliferation of HUVEC. Additionally, we demonstrated that karuquinone A induces apoptosis in cancer cells through the generation of reactive oxygen species (ROS).

karuquinone B (2), and karuquinone C (3). Three known naphthoquinones, javanicin (4),14 2,3-dihydro-5-hydroxy-8methoxy-2,4-dimethylnaphtho[1,2-b]furan-6,9-dione (5),15 and 5-hydroxydihydrofusarubin C (6),16 are also described. Isolated metabolites were evaluated for their cytotoxicity against four different cell lines. We also examined whether the dihydronaphthoquinone derivatives induce apoptosis by producing ROS.

Quinones and naphthoquinones are ubiquitous in nature and have a wide range of biological activities, including cytotoxicity against cancer cell lines as well as anti-inflammatory and antibacterial activities.1 The best studied naphthoquinone is vitamin K, which is known to display anticancer effects.2 Menadione (vitamin K3), a synthetic derivative of vitamin K, exhibits cytotoxicity against various human cancer cell lines.3,4 The effectiveness of menadione against cancerous cells is believed to be due to the induction of oxidative stress via redox cycling of the quinone to produce reactive oxygen species (ROS).2 Several naphthoquinones are known to generate ROS, thereby targeting various cellular compounds that regulate different signaling pathways. The naphthoquinone plumbagin, which is found in several flowering plants of the Droseraceae or Plumbaginaceae family, has been extensively investigated.5,6 Plumbagin displays antitumor activity both in vitro and in vivo by targeting cellular components through the generation of ROS.7 It has been reported that cancer cells possess higher levels of ROS than normal cells, and selective death of cancer cells by targeting the stress response to ROS has become a novel approach for cancer chemotherapy.8,9 In order to construct a natural products library, we previously focused on isolating new and known compounds derived from fungi isolated from plants and soils. Using our library to screen for bioactive compounds, we have identified DNA polymerase inhibitors,10 cytotoxins,11 a neuroprotective compound,12 and antihepatitis C virus agents.13 In this study, we report on the isolation, structural elucidation, and biological activities of new dihydronaphthoquinone derivatives, karuquinone A (1), © XXXX American Chemical Society and American Society of Pharmacognosy

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RESULTS AND DISCUSSION Repeated separation of a culture extract from Fusarium solani using silica gel yielded compounds 1−6. The molecular formula of C14H14O5 for compound 1 was determined by HRESIMS. The IR spectrum showed absorption bands at 1718 and 1637 cm−1 corresponding to carbonyl groups. 1H NMR signals at δ 12.53 (1H, s) and δ 12.50 (1H, s) indicated the presence of two hydrogen-bonded hydroxy groups, as shown in Table 1. The 13 C NMR spectrum revealed all 14 carbon atoms, comprising three carbonyl carbons, six aromatic carbons, three methylene carbons, and two methyl carbons. It was noteworthy that each chemical shift value of C-3, C-4, C-4a, C-5, and C-6 was almost the same as those of C-2, C-1, C-8a, C-8, and C-7, respectively. A singlet signal at δ 3.04, implying four protons in the 1H NMR spectrum, was assigned to four methylene protons on the basis of HMQC correlations from these protons to two methylene carbons, C-2 (δ 36.1) and C-3 (δ 36.2). Moreover, the δ 3.04 Received: February 24, 2014

A

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Table 1. 1H (400 MHz) and 13C (100 MHz) NMR Spectroscopic Data for Compounds 1−3 in CDCl3 1 position 1 1-OH 2 3

δC, type

2 δH (J in Hz)

201.5, C

3

δC, type

δH (J in Hz)

68.4, CH

5.20, m 2.84 brd (6.7) 2.44, dddd (12.7,4.8, 4.8, 4.8) 2.14, m 2.78, ddd (17.2, 4.8, 4.8) 2.60, ddd (17.2, 13.1, 4.8)

36.1, CH2

3.04, s

31.9, CH2

36.2, CH2

3.04, s

35.6, CH2

δC, type

δH (J in Hz)

157.7, C 106.7, CH

12.09, s 6.70, s

156.5, C

4 4-OH 4a 5 5-OH 6 7 8 8-OH 8a 9

201.3, C

202.6, C

146.2, C

111.9, C 153.4, C

112.4, C 154.5, C

113.8, C 202.7, C

112.5, C 41.5, CH2

3.94, s

123.7, C 40.9, CH2

10 11 12 13

204.3, C 30.0, CH3 12.7, CH3

2.29, s 2.24, s

206.3, C 29.5, CH3 13.1, CH3

12.57, s

12.53, s 133.7, C 138.8, C 153.9, C

12.49, s 123.2, C 137.0, C 145.9, C

12.50, s

48.6, CH 44.1, CH 201.3, C

3.25, dt (10.9, 4.8) 3.13, dq (10.9, 6.7)

8.23, s 3.87, d (16.9) 3.82, d (16.9) 2.24, s 2.18, s

107.1, C 40.8, CH2 205.7, 30.3, 13.7, 56.6,

C CH3 CH3 CH3

2.98, dd (17.9, 4.8) 2.92, dd (17.9, 4.8) 2.29, s 1.33, d (6.7) 3.96, s

connected at C-6 of the naphthoquinone moiety on the basis of HMBC correlations from H-9 (δ 3.94) to C-5 (δ 153.4), C-6 (δ 133.7), and C-7 (δ 138.8). A methyl group was found to be located at C-7 of the naphthoquinone by the HMBC correlations from H-12 (δ 2.24) to C-6 (δ 133.7), C-7 (δ 138.8), and C-8 (δ 153.9). Thus, the structure of compound 1 was determined (Figure 1) and named karuquinone A. The molecular formula of C14H16O5 for compound 2 was determined by HRESIMS and found to differ from compound 1 by two hydrogen atoms. A broad absorption band at 3267 cm−1 attributable to a hydroxy group was observed in the IR spectrum. The 1H and 13C NMR spectra of compound 2 were similar to those of 1, except for the presence of an oxymethine proton at H-1 (δ 5.20) in 2 and the absence of the carbonyl carbon at C-1 in 1 (Table 1). These data indicated that the carbonyl group in 1 was replaced by a hydroxy group in 2, in agreement with the HRESIMS (which identified two hydrogen atoms in the molecular formula). 1H−1H COSY correlations between H-1 and H-2 and between H-2 and H-3 supported the location of the hydroxy group at C-1. Thus, the structure of 2 (karuquinone B) was elucidated (Figure 1) and was further confirmed by DEPT, HMQC, and HMBC experiments (Figure 2). Due to the low reactivity of hydroxy groups of karuquinone B and shortage of material, the absolute configuration still remains to be determined. The molecular formula of C15H16O6 for compound 3 was determined by HRESIMS and found to differ from javanicin (4), a known cytotoxic naphthoquinone, by two hydrogen atoms. As shown in Table 1, the 1H and 13C NMR spectra suggested that the structure of compound 2 was similar to that of javanicin except for the presence of two methine protons at H-6 (δ 3.25) and H-7 (δ 3.13) in 3 (Table 1). 13C NMR signals of two aromatic carbons, C-6 and C-7, in compound 4 were replaced in compound 3 by those of methine carbons (δ 48.6, C-6 and δ 44.1, C-7), which were assigned on the basis of HMBC correlations from H-12 to C-6, C-7, and C-8, and

signal correlated with C-1 (δ 201.5), C-4 (δ 201.3), C-4a (δ 111.9), and C-8a (δ 112.5) in the HMBC spectrum (Figure 2).

Figure 1. Structures of compounds 1−6.

Figure 2. Key HMBC and 1H−1H COSY correlations of compounds 1 and 2.

These data revealed the presence of a symmetrical 5,8dihydroxy-2,3-dihydro-1,4-naphthoquinone moiety. HMBC correlations from H-11 (δ 2.29) to C-9 (δ 41.5) and C-10 (δ 204.3) suggested the presence of an acetonyl group, which was B

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H−1H COSY correlations, which suggested the reduction of C-6 and C-7 in 4 (Figure 3). The structure of compound 3 was

compound 3, showed similar cytotoxicity, with IC50 values of 7.7−15.4 μM. Compound 5 was found to be inactive against all cell lines tested, and 6 showed only a weak level of cytotoxicity against HuH-7 and HUVEC. Several naphthoquinones (e.g., menadione) are known to induce cytotoxicity through oxidative stress by inducing the production of ROS. With this in mind, we investigated whether compound 1 might also induce the production of ROS in HeLa cells. Cells were incubated in the presence of compound 1 (0− 10 μM) for 1 h before evaluating ROS levels using a CMH2DCFDA probe. This CM-H2DCFDA probe diffuses through the cell membrane and is hydrolyzed by intracellular esterases to DCFH. In the presence of ROS, DCF is oxidized to fluorescent DCF, and its level directly corresponds to the level of ROS. Our results show that 1 induces ROS generation in HeLa cells at a lower concentration (6 μM) than that of menadione (Figure 4). 1 (10 μM) seemed to induce cell death

Figure 3. Key HMBC, 1H−1H COSY, and NOESY correlations of compound 3.

in agreement with the molecular formula determined by HRESIMS and further confirmed by DEPT, HMQC, and HMBC experiments. The relative configuration of compound 3 was determined by 1H−1H coupling constants and NOESY correlations (Figure 3). The anti relationship of H-6/H-7 was deduced from the coupling constants (J6,7 = 10.9 Hz), which was supported by NOESY correlations between H-6 and H-12 and between H-7 and H-9 (Figure 3). Thus, the structure of compound 3 was determined (Figure 1) and named karuquinone C. Since optical rotation of compound 3 was essentially zero and this compound was easily oxidized to compound 4, we could not determine the optical activity of compound 3 at this time. Three known compounds, 4, 5, and 6, were identified as javanicin, 2,3-dihydro-5-hydroxy-8-methoxy-2,4dimethylnaphtho[1,2-b]furan-6,9-dione, and 5-hydroxydihydrofusarubin C by comparison of their 1H and 13C NMR and MS with data previously reported in the literature.16−18 The absolute configurations for 5 and 6 have not been determined. Compounds 1−6 were evaluated for their in vitro cytotoxicity against three human cancer cell lines (i.e., cervix (HeLa), hepatocarcinoma (HuH-7), and colon carcinoma (HCT116) cell lines) as well as a human umbilical vein endothelial cell (HUVEC) line. The IC50 values of the compounds are shown in Table 2. Of the six compounds, 1 exhibited the strongest cytotoxic effect against all the cell lines tested, with IC50 values of 1.1−4.0 μM. Compound 2, a reduced form of 1, had no detectable effect on the proliferation of any of the three cancer cell lines, suggesting the dihydroquinone moiety of 1 is likely to be important for cytotoxicity. Interestingly, compound 2 selectively suppressed the proliferation of HUVECs with an IC50 value of 5.8 μM, implying that this compound may have the potential to affect angiogenesis. Javanicin (4) displayed cytotoxicity against all of the cell lines, and its reductive form,

Figure 4. Generation of ROS in HeLa cells treated with compound 1. HeLa cells were treated with 1 for 3 h, and ROS levels were detected with a microplate reader using CM-H2DCFDA probes (n = 3) [p < 0.05 (*) indicates differences between control and 1 or menadionetreated cells].

during 1 h treatment, and thus the level of ROS generation was decreased. To identify a correlation between ROS production and cell death, HeLa cells were treated with compound 1 in the concentration range 0−30 μM for 48 h in the presence or absence of NAC (N-acetylcysteine), a ROS scavenger.19 Cell viability was then evaluated. As shown in Figure 5, NAC significantly decreased the cytotoxicity level of compound 1. Cytotoxicity of tamoxifen, a well-known anticancer drug, was not impaired by NAC (data not shown). These results

Table 2. IC50 of Compounds on the Proliferation of Three Human Cancer Cell Lines and a Human Umbilical Vein Endothelial Cell Line IC50 value (μM)a compound 1 2 3 4 5 6 doxorubicin

HeLa 4.0 >30 7.9 9.0 >30 >30 0.22

± 0.4 ± 0.2 ± 0.6

± 0.01

HuH-7 1.9 >30 10.8 6.7 >30 27.0 0.22

± 0.02 ± 1.0 ± 1.1 ± 1.3 ± 0.01

HCT116 1.1 >30 15.4 5.5 >30 >30 0.25

± 0.01 ± 0.2 ± 0.3

± 0.01

HUVEC 1.8 5.8 7.7 2.3 >30 13.1 0.40

± ± ± ±

0.07 0.09 0.17 0.06

± 3.30 ± 0.01

a

The three human cancer cell lines and a human umbilical vein endothelial cell (HUVEC) line were incubated with each compound for 48 h. Cell viability was determined using the WST-8 assay; data, mean ± SD (n = 3). C

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In summary, new dihydronaphthoquinone derivatives, karuquinone A (1), karuquinone B (2), and karuquinone C (3), and known compounds javanicin (4), 2,3-dihydro-5hydroxy-8-methoxy-2,4-dimethylnaphtho[1,2-b]furan-6,9-dione (5), and 5-hydroxydihydrofusarubin C (6) were isolated from the culture broth of the fungus F. solani. The cytotoxic activities of isolated metabolites were evaluated, and karuquinone A was established to be the most potent of the compounds tested. The cytotoxicity of karuquinone A was found to be attributable to the induction of ROS. Moreover, our results indicated that apoptotic features were detected in cancer cells treated with karuquinone A.

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Figure 5. Effect of NAC on the viability of cells treated with compound 1. HeLa cells were treated with compound 1 in the presence or absence of 10 mM NAC for 48 h, and cell viability was determined using the WST-8 assay.

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were recorded on a Jasco P-1010 digital polarimeter at room temperature. UV spectra were obtained on a Hitachi U-3210 spectrophotometer. Infrared spectra (IR) were recorded on a Horiba FREEXACT-II FT720 spectrometer and reported as wavenumbers (cm−1). 1H and 13C NMR spectra were recorded on a Bruker 400 MHz spectrometer (Avance DRX-400), using CDCl3 (with TMS for 1H NMR and CDCl3 for 13C NMR as an internal reference). Chemical shifts are expressed in δ (ppm) relative to TMS or residual solvent resonance, and coupling constants (J) are expressed in Hz. Mass spectra (MS) were obtained on an Applied Biosystems mass spectrometer (APIQSTAR Pulsar i) under conditions of high resolution, using poly(ethylene glycol) as the internal standard. Analytical TLC was carried out on precoated silica gel 60 F254 plates (Merck, Darmstadt, Germany). Isolation and Cultivation of Fungi. Soil was collected in Karuizawa, Nagano, Japan, and suspended in sterilized H2O. The suspension was placed on potato dextrose agar (PDA) plates (Difco, Franklin Lakes, NJ), and the plates were incubated for 1−2 weeks at 37 °C. Fungi growing on this plate were transferred onto individual PDA plates and cultured. Cultures were repeated 2 to 5 times to obtain pure mycelium strains. The fungus producing the new compounds reported here was identified as Fusarium solani (Nectria hematococca) by Techno Suruga Laboratory Co., Ltd. (Shizuoka, Japan). The ITS-5.8S rDNA sequence of this strain showed 100% identity with N. hematococca 5133 (GenBank accession number HM054145). Extraction and Purification of Compounds. The isolated fungal strain was cultured by transferring a small piece of agar from the cultured plate into four 2 L Erlenmeyer flasks containing potato dextrose broth (Difco, 1 L). The culture (4 L) was grown under static conditions at room temperature in the dark for 21 days. The culture was then filtered through cheesecloth to remove fungal mycelia, and the resultant filtrate extracted using CH2Cl2. The organic layer was evaporated in vacuo to obtain a crude extract (222.6 mg). This crude extract was separated by silica gel column chromatography with CHCl3−MeOH (1:0−0:1) to give fractions 1−7. Fraction 1 was subjected to silica gel column chromatography with toluene−EtOAc (20:1−5:1) to give fractions 1-1−1-8. Fraction 1-3 was subjected to silica gel column chromatography with hexane− EtOAc (30:1−10:1) to give compound 1 (2.6 mg) as a red solid. Fraction 1-4 was subjected to silica gel column chromatography with hexane−EtOAc (30:1−6:1) to give compound 4 (1.1 mg). Fraction 15 was subjected to silica gel column chromatography with hexane− EtOAc (10:1−3:1) to give compound 2 (2.5 mg) as a brown solid. Fraction 1-7 was subjected to silica gel column chromatography with hexane−EtOAc (6:1−3:1) to give compounds 5 (1.5 mg) and 6 (1.2 mg). Fraction 4 was subjected to silica gel column chromatography with toluene−EtOAc (12:1−1:2) to give compound 3 (3.8 mg) as a red solid. Karuquinone A (1): red solid; UV (MeOH) λmax (log ε) 241 (4.12), 270 (3.77), 396 (3.75) nm; IR (film) νmax 2958, 2925, 2860, 1718, 1637, 1457, 1400, 1363, 1322, 1268, 1184 cm−1; HRESIMS m/z 285.0720 [M + Na]+ (calcd for C14H14O5Na, 285.0733); 13C and 1H data, see Table 1.

suggested that the cytotoxic activity of compound 1 was due to the generation of ROS. To examine whether compound 1-mediated ROS generation induced apoptosis, we analyzed the extent of DNA fragmentation in the cells before and after treatment using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method (Figure 6). Significant levels of DNA fragmentation were detected when the HeLa cells were treated with compound 1 at 4.0 μM for 48 h. The percentage of apoptotic cells produced upon treatment with compound 1 was almost the same as that observed after treatment with doxorubicin, a known apoptosis inducer and anticancer agent.

Figure 6. Apoptotic effect of compound 1 on HeLa cells. (A) Apoptotic cell number. Apoptotic cells treated with compound 1 (4.0 μM, based on the IC50 value) or 0.2 μM doxorubicin (positive control) were individually counted from a total of at least 200 cells (for each set of conditions). Values are shown as the mean ± SD from three independent experiments. (B) Photographs of apoptotic cells stained and detected by the TUNEL assay using an ApopTag Red in situ apoptosis detection kit. The HeLa cells were incubated for 48 h in the absence (control) or presence of 4.0 μM compound 1. The white bar is 10 μm. D

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Karuquinone B (2): brown solid; [α]24D +5.1 (c 0.078, CHCl3); UV (MeOH) λmax (log ε) 226 (4.09), 274 (3.63), 300 (3.58), 390 (3.56) 501 (3.21) nm; IR (film) νmax 3267, 2925, 2856, 1712, 1628, 1456, 1415, 1356, 1263, 1219, 1169 cm−1; HRESIMS m/z 287.0881 [M + Na]+ (calcd for C14H16O5Na, 287.0889); 13C and 1H data, see Table 1. Karuquinone C (3): red solid; UV (MeOH) λmax (log ε) 239 (3.76), 275 (3.74), 370 (3.40) nm; IR (film) νmax 2924, 2856, 1714, 1626, 1599, 1460, 1433, 1396, 1365, 1300, 1274, 1207, 1163 cm−1; HRESIMS m/z 315.0831 [M + Na]+ (calcd for C15H16O6Na, 315.0839); 13C and 1H data, see Table 1. 2,3-Dihydro-5-hydroxy-8-methoxy-2,4-dimethylnaphtho[1,2-b]furan-6,9-dione (5): [α]23D +61.8 (c 0.034, acetone) [lit. [α]20D −125 (c 0.2, acetone)].15 5-Hydroxydihydrofusarubin C (6): [α]22D −156.3 (c 0.027, acetone) [lit. [α]24D −172.5 (c 0.03, acetone)].16 Cell Culture. HeLa and HuH-7 cells were cultured in Dulbecco’s modified Eagle medium, and HCT116 cells were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum, penicillin (100 units/mL), and streptomycin (100 μg/mL). HUVEC cells were cultured in endothelial cell growth medium (Cell Applications Inc., San Diego, CA, USA), supplemented with penicillin (100 units/mL) and streptomycin (100 μg/mL). All cells were cultured at 37 °C in a humid atmosphere of 5% CO2−95% air. Cytotoxicity Assay. The viability of each cell was evaluated using a cell counting kit (Cell Count Reagent SF; Nakalai Tesque, Kyoto, Japan) according to the manufacturer’s instructions based on the WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] assay. Cells were cultured in a 96-well plate, with each well containing 1 × 104 or 5 × 103 cells in a total volume of 100 μL. The cells were treated with various concentrations of each compound for 48 h. A 10 μL amount of the solution provided with the kit was then added, and the resulting mixture incubated for 30−180 min at 37 °C. The absorbance values were then measured at 450 nm with a microplate reader (Wallac 1420 ARVO sx; PerkinElmer, Waltham, MA, USA). Doxorubicin (SigmaAldrich, St. Louis, MO, USA) was used as positive control. Analysis of ROS Generation. Intracellular levels of ROS were measured by staining cells with CM-H2DCFDA (Molecular Probes, Eugene, OR, USA). Cells following treatment with 1 (0−10 μM) for 1 h were incubated with 10 μM CM-H2DCFDA for 20 min at 37 °C and then washed. The fluorescent intensity was recorded by excitation at 490 nm and emission at 535 nm using a microplate reader (Wallac 1420 ARVO sx). Apoptosis Assay Using Immunofluorescence Microscopy. Aliquots of 2.5 × 104 cells were plated in each well of an eight-well chamber slide (Nunc, Rochester, NY, USA). HeLa cells were incubated with 1 (4.0 μM, based on their IC50 value) or 0.2 μM doxorubicin as a positive control, for 48 h at 37 °C. TUNEL is a method for detecting DNA fragmentation by labeling the terminal end of nucleic acids. The percentage of apoptotic cells was determined using the ApopTag Red in situ apoptosis detection kit (CHEMICON, Temecula, CA, USA). The culture dishes were stained, and the percentage of apoptotic cells was examined under an Olympus IX70 fluorescence microscope (Olympus, Tokyo, Japan).

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ACKNOWLEDGMENTS S.K. acknowledges a Grant-in-Aid for Young Scientists (B) (No. 25750389) from MEXT (Ministry of Education, Culture, Sports, Science and Technology). Y.M. acknowledges Grantsin-Aids for Scientific Research (C) (no. 24580205) from MEXT (Japan).

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR, DEPT, COSY, HMQC, and HMBC spectra for 1−3 and the NOESY spectrum for 3 are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +81-4-7124-1501, ext. 6326. Fax: +81-4-7123-9767. Email: [email protected]. Notes

The authors declare no competing financial interest. E

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