Stereochemistry of the Black Tea Pigments Theacitrins A and C

Dec 21, 2015 - Theacitrins A–C are yellow pigments of black tea that are produced by ... sp (#HK18) culture inhabiting the topsoil in a Korean solar...
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Stereochemistry of the Black Tea Pigments Theacitrins A and C Yosuke Matsuo,* Keita Okuda, Hitomi Morikawa, Ryosuke Oowatashi, Yoshinori Saito, and Takashi Tanaka* Graduate School of Biomedical Sciences, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521, Japan S Supporting Information *

ABSTRACT: Theacitrins A−C are yellow pigments of black tea that are produced by oxidative coupling of gallocatechins, i.e., flavan-3-ols with pyrogallol-type B-rings. However, their stereostructures have not yet been determined. In this study, DFT calculations of NMR chemical shifts of theacitrin C (1) and TDDFT calculations of the ECD spectra of theacitrinin A (5), a degradation product of theacitrin C (1), were used to determine the stereostructure of the theacitrins. Furthermore, the preparation of theacitrins A (4) and C (1) by enzymatic oxidation of an epigallocatechin (7) and epigallocatechin-3-O-gallate (2) mixture confirmed their structural relationship.

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trione moiety (Figure 1); however, the configurations of these carbons have not yet been determined. To establish the absolute configuration, we first calculated the electronic circular dichroism (ECD) spectra and NMR chemical shifts of theacitrinin A (5), a degradation product of 1 with a 3a,8adihydrocyclopenta[a]indene-1,8-dione moiety, and the calculated data were compared with the experimental spectroscopic data. The methods were further applied to determine the absolute configurations of theacitrins C (1) and A (4).

lant polyphenols have attracted great interest worldwide due to their health-promoting properties.1 One important source of polyphenols is black tea, which accounts for almost 80% of world tea production. Black tea is produced by the crushing, tearing, and curling of Camellia sinensis leaves. In the process, flavan-3-ols (catechins) in the leaves are enzymatically oxidized to produce characteristic pigments, such as theaflavins, theacitrins, and thearubigins.2 Theacitrins A−C are characteristic yellow pigments originally isolated from commercial black tea (Figure 1).3 We recently reported that the enzymatic

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RESULTS AND DISCUSSION We previously demonstrated that the heat treatment of theacitrin C (1) in an acidic aqueous solution afforded theacitrinin A (5) and 2,3,5,7-tetrahydroxychroman-3-O-gallate (6) (Scheme 2).4 This reaction comprises the elimination of the A-, C-ring unit of the flavan-3-ol and the aromatization of the cyclohexanedienone moiety. The 3a,8a-dihydrocyclopenta[a]indene-1,8-dione moiety of 5 retained two stereogenic carbons, C-3a′ and C-8a′, and we first examined their configurations by experimental and calculated ECD spectra. There were four possible stereoisomers, (3a′R,8a′R), (3a′R,8a′S), (3a′S,8a′R), and (3a′S,8a′S); however, (3a′R,8a′S)-5 and (3a′S,8a′R)-5 were unlikely due to ring strain imposed by the trans-junction of two five-membered rings.6 Therefore, the ECD spectra for (3a′S,8a′S)-5 and (3a′R,8a′R)-5 were calculated. Low-energy conformers within 6 kcal/mol were initially obtained by a conformational search using the Monte Carlo method at the MMFF94 force field. The resulting conformers were subsequently optimized at the AM1 level and then reoptimized at the B3LYP/6-31G(d,p) level in MeOH (PCM). The lowest-energy conformers of (3a′S,8a′S)-5

Figure 1. Structures of theacitrins A−C.

oxidation of epigallocatechin-3-O-gallate (2), the major tea catechin (6−10% of dried leaves), afforded theacitrin C (1) along with dehydrotheasinensin A (3) (Scheme 1).4 Interestingly, compounds 1 and 3 were also produced stereoselectively by the nonenzymatic oxidation of 2 with K3[Fe(CN)6].5 Theacitrins A−C have three stereogenic carbons, C-3a″, C-3b″, and C-8a″, in the 3a,8a-dihydrocyclopenta[a]indene-1,6,8© XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 16, 2015

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DOI: 10.1021/acs.jnatprod.5b00832 J. Nat. Prod. XXXX, XXX, XXX−XXX

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Scheme 1. Oxidation of Epigallocatechin-3-O-gallate (2) with Polyphenol Oxidase

Scheme 2. Formation of Theacitrinins A (5) and C (8) by Heat Treatment of Theacitrins C (1) and A (4), Respectively

much greater extent with the calculated values for (3a′S,8a′S)-5 rather than (3a′R,8a′R)-5 [R2 = 0.9924 (3a′S,8a′S), 0.9828 (3a′R,8a′R) for 1H NMR chemical shifts; R2 = 0.9978 (3a′S,8a′S), 0.9974 (3a′R,8a′R) for 13C NMR chemical shifts] (Figures S10−S12, Tables S9 and S10, Supporting Information). The DP4 probability analysis,9 which was developed to assign structure by comparing calculated and experimental NMR spectra, was also performed based on the calculated and experimental 1H and 13C NMR chemical shifts of 5. The DP4 analysis of the two possible structures gave 99.1% probability for the (3a′S,8a′S) structure (Table S11, Supporting Information). These results supported the (3a′S,8a′S) absolute configuration of 5. Theacitrin C (1) should have the same absolute configuration of the C-3a″ and C-8a″ stereocenters as 5, i.e., (3a″S,8a″S). Therefore, there are two possible stereostructures, (3a″S,3b″S,8a″S)-1 and (3a″S,3b″R,8a″S)-1. TDDFT calculations of the ECD spectra for (3a″S,3b″S,8a″S)-1 and (3a″S,3b″R,8a″S)-1 were performed; however, it was not possible to assign the absolute configuration, because the two calculated spectra showed similar Cotton effects (Figure S20, Supporting Information). Subsequently DFT calculations of the 1 H and 13C NMR chemical shifts of (3a″S,3b″S,8a″S)-1 and (3a″S,3b″R,8a″S)-1 were performed at the GIAOmPW1PW91/6-311+G(2d,p) level in DMSO (PCM) (Tables S15−18, Supporting Information). Experimental 1H and 13C NMR chemical shifts were in good agreement with the calculated data of (3a″S,3b″S,8a″S)-1 rather than those of (3a″S,3b″R,8a″S)-1 [R2 = 0.9639 (3a″S,3b″S,8a″S), 0.9503 (3a″S,3b″R,8a″S) for 1H NMR chemical shifts; R2 = 0.9962 (3a″S,3b″S,8a″S), 0.9948 (3a″S,3b″R,8a″S) for 13C NMR chemical shifts] (Figures 4A,C, S16, and S17, Tables S19 and S20, Supporting Information). It is noteworthy that the calculated chemical shift of C-7a″ in (3a″S,3b″R,8a″S)-1, which is adjacent to C-3b″, is significantly different from the experimental data, compared with that of (3a″S,3b″S,8a″S)-1 [Δδ = 11.4 ppm for (3a″S,3b″R,8a″S); Δδ = 5.0 ppm for (3a″S,3b″S,8a″S)]. NOE correlations of 1 were also examined

and (3a′R,8a′R)-5 are shown in Figure 2. ECD spectra of the low-energy conformers with Boltzmann populations (>1%)

Figure 2. Lowest-energy conformers of (3a′S,8a′S)-5 and (3a′R,8a′R)5. Geometrical optimization was performed at the B3LYP/6-31G(d,p) level in MeOH (PCM).

were calculated at the TD-CAM-B3LYP/6-31G(d,p) level in MeOH (PCM), and the results were averaged (Figure 3A).7 The experimental ECD spectrum of 5 showed a positive Cotton effect at 309 nm and negative Cotton effects at 371 and 244 nm. These Cotton effects resembled those of the calculated ECD spectrum of the (3a′S,8a′S)-5 diastereomer. In contrast, the calculated ECD spectrum of the (3a′R,8a′R)-5 diastereomer showed opposite signs of the Cotton effects at these wavelengths. From these data, the configurations of C-3a′ and C-8a′ of 5 were assigned as (3a′S,8a′S). The NMR chemical shifts of (3a′S,8a′S)-5 and (3a′R,8a′R)-5 were also calculated using the gauge-including atomic orbital (GIAO) method at the mPW1PW91/6-311+G(2d,p) level in acetone (PCM)8 (Tables S5−S8, Supporting Information). The experimental 1H and 13C NMR chemical shifts agreed to a B

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Figure 3. Experimental and calculated ECD spectra of theacitrinins A (5) (A) and C (8) (B). The experimental ECD spectra were measured in MeOH. The calculations for the ECD spectra were performed at the TD-CAM-B3LYP/6-31G(d,p) level in MeOH (PCM). The calculated spectra were red-shifted by 20 nm.

experimental spectrum (Figure 3B). Tannase hydrolysis of 5 was also performed to afford 8. Thus, the stereostructure of 8 (theacitrinin C) was defined as shown in Scheme 2. Furthermore, NMR chemical shifts of the two possible structures of theacitrin A (4), that is, (3a″S,3b″S,8a″S)-4 and (3a″S,3b″R,8a″S)-4, were calculated at the GIAOmPW1PW91/6-311+G(2d,p) level in acetone (PCM) (Tables S28−S31, Supporting Information). Experimental 1H and 13C NMR chemical shifts of 4 were in good agreement with calculated data of (3a″S,3b″S,8a″S)-4 rather than those of (3a″S,3b″R,8a″S)-4 [R2 = 0.9793 (3a″S,3b″S,8a″S), 0.9760 (3a″S,3b″R,8a″S) for 1H NMR chemical shifts; R2 = 0.9974 (3a″S,3b″S,8a″S), 0.9958 (3a″S,3b″R,8a″S) for 13C NMR chemical shifts] (Figures 4B,D, S25, and S26, Tables S32, S33, Supporting Information). These results are similar to those of 1. The calculated chemical shift of C-7a″ in (3a″S,3b″R,8a″S)-4 was also largely different from the experimental data, compared with that of (3a″S,3b″S,8a″S)-4 [Δδ = 9.1 ppm for (3a″S,3b″R,8a″S); Δδ = 2.6 ppm for (3a″S,3b″S,8a″S)]. These data permitted assignment of the absolute configuration of the 3a,8a-dihydrocyclopenta[a]indene-1,6,8-trione moiety of 4 as (3a″S,3b″S,8a″S). The mechanisms of the formation of the theacitrins had been proposed as shown in Scheme 3, except for the stereochemistry of the process.3,4 Our results clearly showed the stereoselective C−C bond formation between the two epigallocatechin moieties and the subsequent formation of bicyclo[3.2.1]octane-type intermediates. In summary, the absolute configuration of the 3a,8adihydrocyclopenta[a]indene-1,6,8-trione moiety of theacitrins A (4) and C (1), yellow pigments of black tea, was determined by experimental and calculated spectroscopic data. In addition, 4 was prepared in low yield by enzymatic oxidation of a mixture of 2 and 7 for the first time. The results also showed stereoselectivity in the dimerization process of 2 and 7. The mechanism of the formation of theacitrins is related to those of theaflavins and proepitheaflagallins, catechin dimers produced by oxidative coupling between two B-rings via bicyclo[3.2.1]octane-type intermediates.4,10,11 Interestingly, theacitrin B, a regioisomeric galloyl ester of 4, was not detected in this study, and a previous study indicated that a theacitrin-type product was not generated by enzymatic oxidation of 7.4,10 These experimental results suggest that the presence of the free C-3 hydroxy or galloyl group is a crucial factor in the selectivity of the oxidative dimerization reactions of tea catechins. An investigation of the relationship between the production

to confirm the absolute configuration of the 3a,8adihydrocyclopenta[a]indene-1,6,8-trione moiety, and the correlation between H-3a″ and H-8′ was observed (Figure 5). H3a″ in (3a″S,3b″S,8a″S)-1 is oriented in the same direction as the A′/C′-ring of the flavan-3-ol moiety; however, H-3a″ in (3a″S,3b″R,8a″S)-1 is oriented in the opposite direction. The distances of H-3a″/H-8′ for (3a″S,3b″S,8a″S)-1 and (3a″S,3b″R,8a″S)-1 in the lowest-energy conformers were 3.5 and 5.8 Å, respectively. These data strongly supported the absolute configuration of the 3a,8a-dihydrocyclopenta[a]indene-1,6,8trione moiety as (3a″S,3b″S,8a″S). On the basis of these results, the absolute configuration of the 3a,8a-dihydrocyclopenta[a]indene-1,6,8-trione moiety in 1 was defined as (3a″S,3b″S,8a″S). Theacitrins A (4) and B, which have a galloyl group at C-3′ or C-3, have also been isolated from black tea (Figure 1).3 These pigments should be produced by enzymatic oxidation of a mixture of epigallocatechin-3-O-gallate (2) and epigallocatechin (7). Therefore, the oxidation of a mixture of 2 and 7 using polyphenol oxidase was next evaluated. Theacitrin C (1) shows a characteristic UV/vis spectrum, with a gradual slope from ca. 350 to 430 nm;4 therefore, theacitrins A (4) and B should also show similar UV/vis spectra. Photodiode-arraydetected HPLC analysis of the oxidation mixture showed many peaks derived from the oxidation products of 2 and 7; however, only two peaks showed the aforementioned characteristic UV/ vis absorption. The retention time and UV absorption of one of the two peaks coincided with those of 1. The other product having the characteristic UV/vis absorption was purified to afford theacitrin A (4) in low yield (0.17%). Theacitrin A (4) was prepared for the first time from the mixture of 2 and 7; however, the peak attributable to theacitrin B was not detected in this experiment. The absolute configuration of the 3a,8a-dihydrocyclopenta[a]indene-1,6,8-trione moiety of theacitrin A (4) was determined in a manner similar to that for 1: first, 4 was heated in an acidic solution to afford the new compound 8 (Scheme 2). Its 1H and 13C NMR spectra were similar to those of 5 except for the absence of the galloyl group. Therefore, the 2D structure of 8 was determined as a degalloyl derivative of 5. The experimental ECD spectrum of 8 showed similar Cotton effects to 5 (Figure 3), thus confirming that the absolute configuration of the 3a,8a-dihydrocyclopenta[a]indene-1,8dione moiety of 8 is the same as in 5, i.e., (3a′S,8a′S). To confirm this, the ECD spectra of (3a′S,8a′S)-8 and (3a′R,8a′R)8 were calculated. The calculated ECD spectrum of (3a′S,8a′S)8 showed Cotton effects similar to those observed in the C

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Figure 4. Differences between experimental and calculated 1H NMR chemical shifts of theacitrin C (1) (A) and theacitrin A (4) (B) and 13C NMR chemical shifts of 1 (C) and 4 (D). Δδ (ppm) = δcalcd − δexptl. G: galloyl group. Experimental spectra were recorded in DMSO-d6 + CF3COOD (9:1) (1)4 or in acetone-d6 (4).3 Calculations of NMR chemical shifts were performed at the mPW1PW91/6-311+G(2d,p) level in DMSO (1) or in acetone (4) (PCM) and linearly corrected for the experimental data. spectrophotometer. Ultraviolet (UV) spectra were obtained on a JASCO V-560 UV/vis spectrophotometer. The ECD spectra were measured with a JASCO J-725N spectrophotometer. 1H NMR and NOESY spectra were recorded on a Varian Unity Plus 500 spectrometer (Varian, Palo Alto, CA, USA) operating at 500 MHz. 1 H and 13C NMR spectra were also recorded on a JEOL JNM-AL400 spectrometer (JEOL Ltd., Tokyo, Japan) operating at 400 and 100 MHz for the 1H and 13C nuclei, respectively. Coupling constants (J)

mechanisms of these three types of catechin dimers theacitrins, theaflavins, and proepitheaflagallinsis in progress.

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EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured on a JASCO P-1020 digital polarimeter (Jasco, Tokyo, Japan). IR spectra were measured on a JASCO FT/IR 410 D

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Figure 5. Lowest-energy conformers of (3a″S,3b″S,8a″S)-1 and (3a″S,3b″R,8a″S)-1 and NOE correlations. Geometrical optimization was performed at the B3LYP/6-31G(d,p) level in DMSO (PCM).

Scheme 3. Proposed Mechanism of the Formation of Theacitrins from Epigallocatechins

330 (+0.65), 317 (0), 297 (−1.9), 283 (0), 263 (+7.8), 243 (0), 228 (−8.0) nm. Theacitrinin A (5). Theacitrinin A (5) was prepared by degradation of 1 as previously reported.4 ECD (MeOH) λmax (Δε) 371 (−4.5), 346 (0), 309 (+8.8), 284 (0), 244 (−10.0) nm. Preparation of Theacitrin A (4) from Epigallocatechin (7) and Epigallocatechin-3-O-gallate (2). Japanese pear (Pyrus pyrifolia) fruits (150 g), which have a high polyphenol oxidase activity,13 were homogenized in H2O (150 mL) and filtered through four layers of gauze. The filtrate (200 mL) was mixed with an aqueous solution (200 mL) of a mixture of epigallocatechin (7) (1.0 g) and epigallocatechin-3-O-gallate (2) (1.5 g) and vigorously stirred for 60 min at room temperature. After stirring, the solution was acidified with trifluoroacetic acid (TFA) and filtered. The solution obtained was directly applied to a column of MCI-gel CHP20P (3 × 21 cm) and eluted with 0−100% aqueous MeOH containing 0.1% TFA to afford five fractions: fr.1 (209 mg), fr.2 (783 mg), fr.3 (1008 mg), fr.4 (438 mg), and fr.5 (110 mg). HPLC-DAD analysis of each fraction revealed the presence of a product showing a characteristic UV/vis spectrum similar to theacitrin C (1) in fr.2 and 1 in fr.3. Purification of fr.2 by a column of Cosmosil 40C18-PREP (2 × 30 cm, 4−40% aqueous CH3CN containing 0.1% TFA), Sephadex LH-20 (2.5 × 13 cm, 0− 100% aqueous MeOH containing 0.1% TFA), and Cosmosil 40C18PREP (2 × 30 cm, 4−50% aqueous MeOH) afforded theacitrin A (4)3

are expressed in hertz, and chemical shifts (δ) are reported in ppm with the solvent signal used as a standard (acetone-d6: δH 2.04, δC 29.8). FABMS data were recorded on a JMS700N spectrometer (JEOL Ltd.) using m-nitrobenzyl alcohol or glycerol as the matrix. Column chromatography was performed using Sephadex LH-20 (25− 100 μm, GE Healthcare UK Ltd., Little Chalfont, UK), MCI-gel CHP20P (75−150 μm, Mitsubishi Chemical Co., Tokyo, Japan), Chromatorex ODS (Fuji Silysia Chemical Ltd., Kasugai, Japan), and Cosmosil 40C18-PREP (Nacalai Tesque Inc., Kyoto, Japan) columns. TLC was performed on precoated Kieselgel 60 F254 plates (0.2 mm thick, Merck, Darmstadt, Germany) with toluene−ethyl formate− formic acid (1:7:1 or 1:7:2, v/v) mixtures being used as the eluents. The spots were detected using UV illumination and by spraying with a 5% H2SO4 solution followed by heating. Analytical HPLC was performed on a Cosmosil 5C18-ARII (Nacalai Tesque) column (250 × 4.6 mm, i.d.) with a gradient elution of 4−30% (39 min) and 30−75% (15 min) CH3CN in 50 mM H3PO4 at 35 °C (flow rate, 0.8 mL/min; detection, Jasco photodiode array detector MD-2010). Epigallocatechin-3-O-gallate (2) and epigallocatechin (7) were isolated from a commercial tea catechin mixture according to reported methods.12 The resulting material was purified by crystallization from H2O. Theacitrin C (1). Theacitrin C (1) was prepared by enzymatic oxidation of epigallocatechin-3-O-gallate (2) as previously reported.4 ECD (MeOH) λmax (Δε) 418 (+2.7), 394 (0), 370 (−2.9), 343 (0), E

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(4.3 mg, 0.17%). Fr.3 was purified using a column of Sephadex LH-20 (2 × 21 cm, 0−100% aqueous MeOH and 50% aqueous acetone containing 0.1% TFA), Cosmosil 40C18-PREP (2 × 30 cm, 4−30% aqueous CH3CN containing 0.1% TFA), and Cosmosil 40C18-PREP (2 × 30 cm, 4−50% aqueous MeOH) to afford theacitrin C (1)4 (5.0 mg, 0.33%). At all stages of purification, obtained fractions were evaporated under reduced pressure below 40 °C, then lyophilized. Heat Treatment of Theacitrin A (4) in Aqueous Solution Containing 0.1% TFA. An aqueous solution of theacitrin A (4; 4.3 mg/1.0 mL) containing 0.1% TFA was heated for 1 h at 80 °C. The solution was directly applied to a column of Chromatorex ODS (1 × 15 cm, 0−50% aqueous MeOH containing 0.1% TFA) to afford theacitrinin C (8) (1.6 mg, 66%) along with 2,3,5,7-tetrahydroxychroman-3-O-gallate (6)14 (1.6 mg, 81%). Theacitrinin C (8): yellow, amorphous powder; [α]21D −8 (c 0.1, MeOH); FABMS m/z 429 [M + H]+; HRFABMS m/z 429.0820 [M + H]+ (calcd for 429.0822, C21H17O10); IR νmax cm−1 3220, 1714, 1604, 1505, 1469, 1339; UV (MeOH) λmax (log ε) 354 sh (3.24), 305 sh (3.69), 276 (3.84); ECD (MeOH) λmax (Δε) 370 (−3.8), 348 (0), 308 (+8.6), 279 (0), 246 (−10.1), 226 (−7.4); 1H NMR (400 MHz, acetone-d6 + D2O) δ 6.92 (s, H-4′), 6.18 (br s, H-2′), 6.08, 6.07 (each 1H, d, J = 2.4 Hz, H-6, 8), 4.94 (br s, H-2), 4.49 (m, H-3), 4.36 (s, H3a′), 2.83 (dd, J = 4.8, 16.8 Hz, H-4), 2.71 (dd, J = 4.4, 16.8 Hz, H-4); 13 C NMR (100 MHz, acetone-d6 + D2O) δ 202.0 (C-1′), 198.8 (C-8′), 177.1 (C-3′), 157.8, 156.3, 155.5 (C-5, 7, 8a), 146.7 (C-7′), 143.7 (C3b′), 133.1 (C-6′), 126.9 (C-2′), 114.0 (C-7a′), 106.9 (C-4′), 100.1 (C-4a), 97.0, 95.6 (C-6, 8), 85.1 (C-8a′), 75.4 (C-2), 64.5 (C-3), 55.9 (C-3a′), 28.1 (C-4). Tannase Hydrolysis of Theacitrinin A (5). Theacitrinin A (5) was dissolved in H2O (4.6 mg/5.0 mL). Then tannase (Wako, Japan) was added, and the mixture stirred. After 2 h, the solution was acidified with TFA and directly applied to a column of MCI-gel CHP20P (1 × 20 cm, 0−50% aqueous MeOH) to afford theacitrinin C (8) (2.4 mg, 71%) along with gallic acid (1.3 mg, 96%). Calculations of ECD Spectra and NMR Chemical Shifts. A conformational search was performed using the Monte Carlo method at the MMFF94 force field with Spartan’10 (Wavefunction, Irvine, CA, USA). The resulting low-energy conformers within 6 kcal/mol were optimized at the AM1 level with MOPAC2012,15 to afford low-energy conformers within 6 kcal/mol that were reoptimized at the B3LYPSCRF/6-31G(d,p) level (PCM). The vibrational frequencies were also calculated at the same level to confirm their stability, and no imaginary frequencies were found. The 1H and 13C NMR chemical shifts of the low-energy conformers with Boltzmann populations greater than 1% were calculated using the GIAO method at the mPW1PW91-SCRF/6311+G(2d,p)//B3LYP-SCRF/6-31G(d,p) level in acetone or DMSO (PCM).8 Calculated NMR chemical shifts were linearly corrected for the experimental data. The energies, oscillator strengths, and rotational strengths of the low-energy conformers were calculated using TDDFT at the CAM-B3LYP-SCRF/6-31G(d,p)//B3LYP-SCRF/6-31G(d,p) level in MeOH (PCM).7 The ECD spectra were simulated by the overlapping Gaussian function with a 0.3 eV exponential half-width. The calculated data for each conformer were averaged according to the Boltzmann distribution theory at 298 K based on their relative Gibbs free energies. All DFT calculations were performed using Gaussian 09.16 GaussView was used to draw the 3D molecular structures.17

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +81-95-819-2434. E-mail: [email protected]. *Tel: +81-95-819-2432. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported in part by JSPS KAKENHI Grant Nos. 25870532 and 26460125. The authors are grateful to Mr. K. Inada, Mr. N. Yamaguchi, and Mr. N. Tsuda (Center for Industry, University and Government Cooperation, Nagasaki University) for collecting NMR and MS data. The computation was partly carried out using the computer facilities at the Research Institute for Information Technology, Kyushu University.

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.5b00832. 1

H NMR and NOESY spectra for compound 1, 1D NMR spectra for compound 8, and computational results (PDF) F

DOI: 10.1021/acs.jnatprod.5b00832 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

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DOI: 10.1021/acs.jnatprod.5b00832 J. Nat. Prod. XXXX, XXX, XXX−XXX