Article pubs.acs.org/jnp
Vetiverianines A, B, and C: Sesquiterpenoids from Vetiveria zizanioides Roots Yukiko Matsuo,* Saori Maeda, Chika Ohba, Haruhiko Fukaya, and Yoshihiro Mimaki School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, 1432-1, Horinouchi, Hachioji, Tokyo 192-0392, Japan S Supporting Information *
ABSTRACT: Three new sesquiterpenoidsvetiverianines A (1), B (2), and C (3)and a known eudesmane sesquiterpenoid (4) were isolated from the roots of Vetiveria zizanioides. Vetiverianine A (1) has a unique carbon framework comprising a rigid tricyclic ring system. Vetiverianines B (2) and C (3) are new eremophilane sesquiterpenoids. The structures of sesquiterpenoids 1−3, including the absolute configurations, were determined by NMR spectroscopic, X-ray crystallography, and vibrational circular dichroism data analysis. Vetiverianine C (3) exhibited weak cytotoxic activity against HL-60 cells.
Vetiveria zizanioides (Gramineae) is a perennial grass that is widely distributed in India and Indonesia. V. zizanioides has a deep complex root system, and it is widely used to prevent red soil erosion and soil contamination.1 The volatile matter obtained from the steam distillation of the roots of V. zizanioides, which is commonly called vetiver oil, exhibits antibacterial, antioxidant, and antifungal activities2−5 and is used in aromatherapy and perfumery. The roots of V. zizanioides contain sesquiterpenoids,6 such as α-vetivone, β-vetivone, and isovalencenol, and several flavonoids.7 However, no systematic phytochemical investigation has been conducted on the roots of V. zizanioides. During a continuing search for bioactive secondary metabolites from higher plants that yield essential oils,8,9 the methanol extract of V. zizanioides roots was examined and led to the isolation of three new sesquiterpenoidsvetiverianines A (1), B (2), and C (3)and a known eudesmane sesquiterpenoid (4). In this paper, the structural characterization of 1−3, including their absolute configurations, based on NMR, X-ray crystallography, and vibrational circular dichroism (VCD) data analysis, is described. The cytotoxic activity of 1−4 against HL-60 cells is also briefly discussed.
Figure 1. Compounds isolated from Vetiveria zizanioides.
C15H26O2 assigned based on HRESITOFMS (m/z 239.2001 [M + H]+, calcd for C15H27O2 239.2011) and 13C NMR data. The IR absorption band of the hydroxy group of 1 was found at 3395 cm−1. The 1H NMR spectrum of 1 displayed signals corresponding to four methyl groups at δH 1.26 (s, Me-16), 1.24 (s, Me-14), 1.19 (s, Me-15), and 0.82 (s, Me-13) and an oxymethine proton at δH 3.47 (dd, J = 11.5, 4.3 Hz, H-7). The 13 C NMR spectrum contained two oxygenated tertiary carbon signals at δC 73.8 (C-10) and 73.7 (C-2), a quaternary carbon signal at δC 44.9 (C-6), and four methyl carbon signals at δC 33.7 (C-15), 28.8 (C-16), 22.7 (C-14), and 14.1 (C-13). HMBC correlations from Me-13 (δH 0.82) to C-6 (δC 44.9), Me-14 (δH 1.24) to C-10 (δC 73.8), and Me-16 (δH 1.26) and Me-15 (δH 1.19) to C-2 (δC 73.7) revealed that C-6 and C-10 carried the Me-13 and Me-14 groups, respectively, and that C-2 carried the Me-15 and Me-16 groups (Figure 2). Furthermore, the COSY and HMQC data of 1 revealed that the structure of 1
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RESULTS AND DISCUSSION The roots of V. zizanioides (5.0 kg dry weight) were extracted with MeOH. After removing the solvent, the MeOH extract (480 g) was applied to a porous-polymer polystyrene resin (Diaion HP-20) column, and the MeOH-soluble fraction was subjected to column chromatography (CC) using silica gel and octadecylsilanized (ODS) silica gel, yielding compounds 1−4. Compound 4 was identified as (+)-1β,4β,6α-trihydroxyeudesmane10 (Figure 1). Vetiverianine A (1) was obtained as colorless prisms, with a specific rotation of −17 in CHCl3 and a molecular formula of © XXXX American Chemical Society and American Society of Pharmacognosy
Received: February 17, 2016
A
DOI: 10.1021/acs.jnatprod.6b00140 J. Nat. Prod. XXXX, XXX, XXX−XXX
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Figure 2. COSY and selected HMBC correlations of 1−3.
C NMR data of 2 were closely related to those of αvetivone,12 the molecular formula of 2 contained one more oxygen atom than that of α-vetivone. In addition, the two quaternary sp2 carbon signals in the 13C NMR spectrum of αvetivone were displaced by the two oxygenated tertiary sp3 carbon signals in the spectrum of 2. The HMBC correlations of 2 from Me-12 (δH 1.41) to C-7 (δC 64.2), C-11 (δC 63.5), and C-13 (δC 20.6) and from Me-13 (δH 1.42) to C-7 (δC 64.2), C11 (δC 63.5), and C-12 (δC 21.8) suggested that the epoxy functional group was located at C-7 and C-11 (Figure 2). In the NOESY spectrum, cross-peaks between Me-14 and Me-12, Me13, and Me-15 were consistent with the (4R*,5S*,7R*) relative configuration. The structure of 2, including its relative configuration, was confirmed by X-ray crystallography on an orthorhombic crystal obtained by crystallization from MeOH at room temperature (Figure 3). The absolute configuration of 2 was defined by VCD using the same method as for 1. The conformational and vibrational analyses of 2 with the (4R,5S,7R)-configuration were conducted by Monte Carlo analysis using the MMFF94S molecular mechanics program followed by DFT calculations at the B3PW91/DGDZVP2 level, yielding the three lowest energy conformers (2a−c) (Figure 4). Conformer 2a contributed 92.30% of the Boltzmann population, and the VCD spectrum of 2 was in strong agreement with the calculated data for 2a (Figure 6). Accordingly, the structure of 2 was defined as (4R,5S,7R)7,11-epoxy-α-vetivone. Vetiverianine C (3) was determined to have a molecular formula of C15H24O2 based on HRESITOFMS (m/z 259.1680 [M + Na]+, calcd for C15H24NaO2 259.1674) and 13C NMR data. An IR absorption band of 3 corresponding to a hydroxy group was identified at 3404 cm−1. The 1H and 13C NMR spectroscopic data of 3 were similar to those of 2. However, the carbonyl carbon signal observed in the 13C NMR spectrum of 2 was absent in that of 3. Instead, a methylene carbon at δC 25.7 (C-2) and methylene protons at δH 2.06 (m, H2-2a) and 1.95 (dddd, J = 17.8, 7.5, 5.1, 2.5 Hz, H2-2b) were observed. The olefinic proton at δH 5.39 (H-1) was coupled with the H2-2 methylene protons and appeared as a doublet of doublets (J = 2.5, 2.5 Hz). Furthermore, the C-13 methyl group of 2 was displaced by a hydroxymethylene group in 3, and its 1H and 13 C NMR signals were observed at δH 3.79 and 3.74 (each d, J = 11.4 Hz, H2-13) and δC 65.2, respectively. Two oxygenated tertiary carbon signals at δC 66.5 (C-11) and 66.4 (C-7) and HMBC correlations from Me-12 (δH 1.46) to C-7 (δC 66.4), C11 (δC 66.5), and C-13 (δC 65.2) and from H2-13 (δH 3.79 and 3.74, each d, J = 11.4 Hz) to C-7 (δC 66.4), C-11 (δC 66.5), and C-12 (δC 17.6) were attributable to the presence of a 7,11epoxy group (Figure 2). The (4R*, 5S*, 7R*, 11R*) relative configuration of 3 was defined by NOE correlations between Me-14 and both Me-12 and Me-15. X-ray analysis of an orthorhombic crystal of 3 obtained from a methanol solution 13
comprised the following two fragments: C-3(H2)−C-4(H1)− [C-11(H2)−C-12(H2)]−C-5(H1) and C-7(H1) (OH)−C-8(H2)−C-9(H2); the 1H and 13C NMR signal assignments are collated in Table 1. The linkages of C-5 and C-9 through C-10 and of C-7 and C-12 through C-6 were verified by long-range correlations from H-9α (δH 1.75, dd, J = 8.2, 6.7 Hz) and H-9β (δH 1.38, ddd, J = 14.7, 8.2, 4.0 Hz) to C-10 (δC 73.8) and C-5 (δC 58.9); H-5 (δH 0.88, d, J = 12.5 Hz) to C-10 (δC 73.8), C-9 (δC 41.3), and C-14 (δC 22.7); H-7 (δH 3.47, dd, J = 11.5, 4.3 Hz) to C-6 (δC 44.9) and C-12 (δC 40.2); and H-12α (δH 1.65, ddd, J = 12.0, 8.9, 3.0 Hz) and H-12β (δH 1.45, overlapping) to C-6 (δC 44.9) and C-7 (δC 80.6), respectively. Further HMBC correlations from H-3α (δH 1.93, dd, J = 13.5, 3.7 Hz) and H3β (δH 1.15, dd, J = 13.5, 8.5 Hz) to C-2 (δC 73.7), Me-15 (δC 33.7), and Me-16 (δC 28.8) confirmed the C-2 and C-3 bonds. These data indicated that the C-4−C-5−C-6−C-12−C-11 unit formed a five-membered ring and that the C-5−C-6−C-7−C8−C-9−C-10 unit formed a six-membered ring. The three indices of hydrogen deficiency in 1 were deduced from the molecular formula C15H26O2. Because 1 contained no double bond, the structure was determined to include a tricyclic ring system. The third ring was presumed to be constructed from the C-2−C-3−C-4−C-5−C-10 unit, in which C-2 and C-10 were connected through an oxygen atom. Finally, the structure of 1, including its relative configuration, was determined by Xray crystallography (Figure 3). An orthorhombic crystal of 1 was obtained by slow evaporation of the n-hexane−acetone (1:2) solution of 1 at room temperature. The absolute configurations of 1 were established by VCD, which was recently used to directly determine the absolute configurations of natural compounds.11 Conformational studies of 1 with a (4S,5S,6S,7S,10S)-configuration were conducted by Monte Carlo analysis with the MMFF94S molecular mechanics program. Four conformers within 7 kcal/mol were obtained and subjected to geometry optimization by DFT at the B3PW91/DGDZVP2 level (1a−c) (Figure 4). A rigid tricyclic skeleton with three rotamers for the 7-hydroxy group were obtained in the ΔG range of 0.05 kcal/mol for 1, and their abundance ratios were 34.66%, 33.21%, and 32.12% (Table 2). When the experimental VCD spectrum of 1 was compared with the calculated data of the average of the lowest energy conformers (1a−c), they showed strong agreement (Figure 5). Accordingly, the absolute configuration of 1 was defined as (4S,5S,6S,7S,10S). Vetiverianine B (2) was isolated as colorless prisms, with a molecular formula of C15H22O2 based on HRESITOFMS (m/z 257.1512 [M + Na]+, calcd for C15H22NaO2 257.1517) and 13C NMR data. The IR absorption of 2 suggested the presence of a conjugated carbonyl group (1670 cm−1). Its 1H NMR data displayed signals attributable to three methyl groups at δH 1.42 (s, Me-13), 1.41 (s, Me-12), and 1.08 (s, Me-14); a secondary methyl group at δH 0.96 (d, J = 6.7 Hz, Me-15); and an olefinic proton at δH 5.81 (d, J = 1.5 Hz, H-1). Although the 1H and B
DOI: 10.1021/acs.jnatprod.6b00140 J. Nat. Prod. XXXX, XXX, XXX−XXX
1.24 1.65
1.45 0.82 1.24 1.19 1.26
β α
β
12
13 14 15 16
1.99
overlapping s s s s
(12.0, 8.9, 3.0)
(11.5, 4.3) (11.5, 7.6) (11.5, 4.0) (8.2, 6.7) (14.7, 8.2, 4.0)
(13.5, 3.7) (13.5, 8.5) (10.7) (12.5)
δH (J in Hz)
overlapping ddd
m
dd dd dd dd ddd
3.47 1.78 1.54 1.75 1.38
α
β α α β
dd dd br d d
1.93 1.15 1.99 0.88
10 11
9
4 5 6 7 8
α β
position
2 3
1 δc
14.1 22.7 33.7 28.8
40.2
73.8 28.2
41.3
28.5 58.9 44.9 80.6 30.6
73.7 47.0
type
CH3 CH3 CH3 CH3
CH2
C CH2
CH2
CH CH C CH CH2
C CH2
13 14 15
11 12
b
1.42 1.08 0.96
1.41
2.46
1.83 1.82 2.55
1.94 1.62
α β a b a
2.25 2.11
5.81 (2H)
position
10
9
7 8
1 2 3 4 5 6
s s d
s
ddd
dddd
(6.7)
m (15.3, 12.8, 6.1, 1.5) (15.3, 4.6, 3.2)
(13.6, 2.3) (13.6)
(9.8)
(1.5)
δH (J in Hz)
overlapping
dd d
d m
d
2
Table 1. 1H and 13C NMR (500 and 125 MHz, CDC13) Spectroscopic Assignments for 1−3 δc
20.6 17.1 15.0
63.5 21.8
168.1
31.6
64.2 30.8
124.9 199.0 41.6 40.0 40.5 41.4
type
CH3 CH3 CH3
C CH3
C
CH2
C CH2
CH C CH2 CH C CH2
C
14 15
11 12 13
10
9
7 8
4 5 6
3
b
b a
a
α β
position 1 2
1.46 3.79 3.74 0.94 0.88
2.17
1.70 2.37
1.79
1.87 1.48
5.39 2.06 1.95 1.43 1.39 1.48
s d d s d
ddd
ddd ddddd
dddd
dd d
dd m dddd m m overlapping
(11.4) (11.4)
(15.9, 13.0, 4.6) (14.4, 14.1, 2.8, 1.7, 1.4) (14.4, 4.5, 2.8)
(13.0, 4.6, 2.6, 2.4)
(12.8, 2.7) (12.8)
(17.8, 7.5, 5.1, 2.5)
(2.5, 2.5)
δH (J in Hz)
3 δc
18.1 15.7
66.5 17.6 65.2
141.2
31.6
66.4 31.7
40.4 39.2 42.8
26.6
121.0 25.7
type
CH3 CH3
C CH3 CH2
C
CH2
C CH2
CH C CH2
CH2
CH CH2
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Figure 3. Oak Ridge thermal ellipsoid plot (ORTEP) drawings of 1−3 based on single X-ray crystallography.
Figure 4. Conformers 1a−c, 2a−c, and 3a−c.
and etoposide (1.8 ± 0.01 and 0.3 ± 0.01 μM, respectively). Compounds 1, 2, and 4 showed no cytotoxicity. In conclusion, three new sesquiterpenoidsvetiverianines A (1), B (2), and C (3)and a known eudesmane sesquiterpenoid (4) were isolated from the roots of V. zizanioides. Vetiverianine A (1) has a unique carbon framework comprising a rigid tricyclic ring system. The absolute configurations of 1−3 were determined by comparing their experimental and calculated VCD spectra obtained by a Monte Carlo search with the MMFF94S molecular mechanics program followed by geometry optimization using DFT calculations. Vetiverianine C (3) exhibited weak selective cytotoxic activity against HL-60 cells.
confirmed its structure and relative configuration (Figure 3). Finally, the absolute configurations of 3 were established by VCD, as for 1 and 2. The difference in the conformations of the three lowest energy conformers (3a−c) was found in the rotating 13-OH group (Figure 4). Of these three conformers, 3a and 3b within a ΔG 1.46 kcal/mol energy window contributed 99.35% of the Boltzmann population according to conformational optimization using DFT at the B3PW91/ DGDZVP2 level (Table 2). The calculated VCD spectra of 3a and 3b were in good agreement with the experimental VCD spectrum (Figure 7). Thus, the structure of 3 was defined as (4R,5S,7R,11R)-2-deoxo-7,11-epoxy-13-hydroxy-α-vetivone. The cytotoxic activities of 1−4 against HL-60 human promyelocytic leukemia cells and TIG-3 normal human diploid fibroblast cells were evaluated. Compound 3 exhibited weak selective cytotoxicity against HL-60 cells with an IC50 value of 57.7 ± 0.08 μM compared with the positive controls cisplatin
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EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were recorded on a Yanaco micromelting point apparatus (Japan). A PD
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Table 2. Calculated Relative Energies (kcal/mol) at the DFT/B3PW91/DGDZVP2 Level and Populations (%) of the Three Lowest Energy Conformers of 1−3 conformer
ΔG
P (%)d
1aa 1b 1c 2ab 2b 2c 3ac 3b 3c
0.00 0.03 0.05 0.00 1.73 2.14 0.00 1.46 2.92
34.66 33.21 32.12 92.30 4.99 2.47 91.55 7.80 0.65
Calculated relative energies to 1a with ΔG = −462 857.07 kcal/mol. Calculated relative energies to 2a with ΔG = −461 319.98 kcal/mol. c Calculated relative energies to 3a with ΔG = −462 070.43 kcal/mol. d Boltzman population at 298 K and 1 atm. a b
Figure 7. Experimental VCD spectrum (top) and calculated VCD spectrum (bottom) of 3. (Waters-Micromass, UK). VCD spectra were obtained with a DualPEM Chiral IR-2X FT-VCD spectrometer (BioTools, Inc., FL, USA) in a 75 μm cell with BaF2. For CC, Diaion HP-20 (50 mesh, Mitsubishi-Chemical, Japan), BW-300 silica gel (300 mesh, Fuji-Silysia Chemical, Japan), and the ODS silica gel COSMOSIL 75C18-OPN (75 μM, Nacalai Tesque, Japan) were used. Thin-layer chromatography (TLC) was conducted on precoated 60 F254 or RP18 F254S silica gel plates (0.25 mm thick, Merck, Germany), and the spots of the compounds were colored with 10% H2SO4(aq). The following materials and biochemical-grade reagents were used for the cell culture assays: a Spectra Classic microplate reader (Tecan, Austria); a 96-well flat-bottom plate (Iwaki Glass, Japan); JCRB 0085 HL-60 cells and JCRB 0506 TIG-3 cells (Human Science Research Resources Bank, Japan); fetal bovine serum (Bio-Whittaker, MD, USA); 0.25% trypsin-EDTA solution, RPMI-1640 medium, minimum essential medium, cisplatin, etoposide, and 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl-2H-tetrazolium bromide (MTT; Sigma, MO, USA); and penicillin G sodium salt and streptomycin sulfate (Gibco, NY, USA). Plant Material. The dried roots of V. zizanioides (5.0 kg) were obtained from Akaru Co., Ltd. (Fukuoka, Japan). A voucher specimen has been deposited at the herbarium of this university (KS-2011-004). The roots of V. zizanioides were identified based on their morphological characteristics. The photograph is available in the Supplementary Information. Extraction and Isolation. The dried roots of V. zizanioides (5.0 kg) were extracted with MeOH (3 × 20 L). After evaporating the solvent in vacuo, the MeOH extract (480 g) was applied to a Diaion HP-20 column (2200 g, 85 mm i.d. × 600 mm) and successively partitioned with MeOH−H2O (3:7, 1:1), MeOH, EtOH, and EtOAc (each 10 L). The CC of the MeOH-eluted fraction (295 g) on silica gel (2000 g, 85 mm i.d. × 600 mm) was eluted with a stepwise gradient mixture of n-hexane−EtOAc (7:1; 5:1; 2:1; 1:1) and MeOH and produced nine fractions (A−I). The third fraction was separated by a silica gel column (1800 g, 80 mm i.d. × 400 mm) and eluted sequentially with n-hexane−EtOAc (7:1; 4:1; 1:1) to yield nine subfractions (Frs. C-1 to C-9). Fraction C-5 was passed through silica gel (1600 g, 80 mm i.d. × 300 mm) with CHCl3−MeOH (30:1) and purified by ODS preparative TLC (20 cm × 20 cm) using MeCN− H2O (1:1) to yield 2 (17.6 mg). Fraction C-6 was fractionated by ODS silica gel (1800 g, 80 mm i.d. × 400 mm) with MeCN−H2O (3:1) and purified by repeated ODS preparative TLC (20 cm × 20 cm) using MeCN−H2O (3:1) to yield 1 (7.1 mg) and 3 (7.9 mg). Fraction F was partitioned with a silica gel column (1500 g, 75 mm i.d. × 350 mm) and eluted with n-hexane−CHCl3−MeOH (1:19:1) to yield nine fractions (Frs. F-1 to F-9). The seventh fraction, F-7, was further purified by ODS silica gel (600 g, 35 mm i.d. × 300 mm) using MeCN−H2O (1:2) to yield 4 (12.3 mg). Compound 1: colorless prisms; mp 63−70 °C; [α]25D −17 (c 0.3, CHCl3); IR νmax (film) cm−1: 3395 (OH), 2927, 2863 (CH); 1H and
Figure 5. Experimental VCD spectrum (top) and calculated VCD spectrum (bottom) of 1.
Figure 6. Experimental VCD spectrum (top) and calculated VCD spectrum (bottom) of 2. 1030 (Jasco, Japan) instrument was used to measure the optical rotations. UV spectra were recorded on a Jasco V-630. IR absorptions were measured with a Jasco FT-IR 410 spectrophotometer. 1H NMR spectroscopic data were recorded with a DRX-500 spectrometer using standard Bruker pulse programs at 300 K (Bruker, Germany). Chemical shift values are given as δ with reference to tetramethylsilane as an internal standard. HRESITOFMS data were recorded using a liquid chromatography time-of-flight (LCT) mass spectrometer E
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(7) Champagnat, P.; Heitz, A.; Carnat, A.; Fraisse, D.; Carnat, A. P.; Lamaison, J. L. Biochem. Syst. Ecol. 2008, 36, 68−70. (8) Matsuo, Y.; Mimaki, Y. Phytochemistry 2012, 77, 304−311. (9) Matsuo, Y.; Sakagami, H.; Mimaki, Y. Chem. Pharm. Bull. 2014, 62, 1192−1199. (10) De Menezes, J. E. S.; Machado, F. E. A.; Lemos, T. L. G.; Silveira, E. R.; Pessoa, O. D. L. Z. Naturforsch., C: J. Biosci. 2004, 59, 19−22. (11) Nafie, L. A. Nat. Prod. Commun. 2008, 3, 451−466. (12) Revial, G.; Jabin, I.; Pfau, M. Tetrahedron: Asymmetry 2000, 11, 4975−4983. (13) Matsuo, Y.; Akagi, N.; Hashimoto, C.; Tachikawa, F.; Mimaki, Y. Phytochemistry 2013, 96, 244−256. (14) Fukaya, H.; Hitotsuyanagi, Y.; Aoyagi, Y.; Shu, Z.; Komatsu, K.; Takeya, K. Chem. Pharm. Bull. 2013, 61, 1085−1089.
C NMR (500 and 125 MHz, CDCl3), see Table 1; HRESITOFMS (m/z 239.2001 [M + H]+, calcd for C15H27O2 239.2011). Compound 2: colorless prisms; mp 109−113 °C; [α]25D +166 (c 0.1, CHCl3); IR νmax (film) cm−1 3440 (OH), 2967 (CH), 1670 (C O), 1618 (CC); UV λmax (MeOH) nm (log ε) 235 (4.27); CD λmax (MeOH) nm (Δε) 232.7 (+111.604); 1H and 13C NMR (500 and 125 MHz, CDCl3), see Table 1; HRESITOFMS (m/z 257.1512 [M + Na]+, calcd for C15H22NaO2 257.1517). Compound 3: colorless prisms; mp 95−101 °C; [α]25D +121 (c 0.1, CHCl3); IR νmax (film) cm−1 3404 (OH), 2966, 2922, 2861 (CH); 1H and 13C NMR (500 and 125 MHz, CDCl3), see Table 1; HRESITOFMS (m/z 259.1680 [M + Na]+, calcd for C15H24NaO2 259.1674). Compound 4: amorphous solid; [α]25D +14 (c 0.3, CH3OH); IR νmax (film) cm−1 3310 (OH), 2931, 2864 (CH); 1H and 13C NMR (500 and 125 MHz, CDCl 3 ), see Supporting Information; HRESITOFMS (m/z 279.1938 [M + Na]+, calcd for C15H28NaO3 279.1936). Cell Culture Assays. Cell growth was determined using the MTT reagent according to a published method.13 HL-60 (4 × 104 cells/mL) and TIG-3 (1 × 104 cells/mL) cells were incubated with each sample (up to 80 μM) for 72 h. Data are represented as the means ± standard error of the mean (SEM) of three experiments performed in triplicate. Molecular Modeling and VCD Calculations. The conformational analysis of 1−3 was performed as previously described.14 VCD Measurements. VCD spectra were measured as previously described.14 Compounds 1−3 were dissolved in CDCl3 at 0.12 M (3.6 mg/150 μL), 0.16 M (5.7 mg/150 μL), and 0.17 M (6.1 mg/150 μL), respectively.
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ASSOCIATED CONTENT
* Supporting Information S
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00140. Crystallographic data for 1 (CIF) Crystallographic data for 2 (CIF) Crystallographic data for 3 (CIF) 1D and 2D NMR spectroscopic data, X-ray crystallography, and VCD data for 1−3 (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel: +81 42 676 4577. E-mail:
[email protected]. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS The authors are grateful to Professor Yasuo Shida, Tokyo University of Pharmacy and Life Sciences, for providing the mass spectra.
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REFERENCES
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