Megastigmane glucosides and an unusual monoterpene from the leaves of Cananga odorata var. odorata, and absolute structures of megastigmane glucosides isolated from C. odorata var. odorata and Breynia officinalis

Article abstract of DOI:10.1007/s11418-010-0434-5

From a 1-BuOH-soluble fraction of a MeOH extract of Cananga odorata var. odorata, collected at the Botanical Garden of Chiang Mai University, a new megastigmane glucoside, named canangaionoside, and an irregular monoterpene were isolated. A known compound

Full text of DOI:10.1007/s11418-010-0434-5

J Nat Med (2010) 64:460–467
DOI 10.1007/s11418-010-0434-5
ORIGINAL PAPER
Megastigmane glucosides and an unusual monoterpene
from the leaves of Cananga odorata var. odorata, and absolute
structures of megastigmane glucosides isolated from C. odorata
var. odorata and Breynia officinalis
•
Katsuyoshi Matsunami Jiro Nagashima Sachiko Sugimoto
•
•
•
•
•
Hideaki Otsuka Yoshio Takeda Duangporn Lhieochaiphant
Sorasak Lhieochaiphant
Received: 5 March 2010 / Accepted: 26 May 2010 / Published online: 23 June 2010
Ó The Japanese Society of Pharmacognosy and Springer 2010
Abstract From a 1-BuOH-soluble fraction of a MeOH
extract of Cananga odorata var. odorata, collected at the
Botanical Garden of Chiang Mai University, a new meg-
astigmane glucoside, named canangaionoside, and an
irregular monoterpene were isolated. A known compound,
breyniaionoside A, which has been obtained from the
leaves of Breynia officinalis, was also isolated, and its
absolute structure was substantiated for the first time in this
study. On this occasion, the absolute stereochemistries of
structurally related megastigmane glucosides, breyniaion-
osides B and C, isolated from B. officinalis were examined.
globally used as an aroma therapy agent to remove
uneasiness, tension, shock and panic, while the aphrodisiac
qualities of the oil are useful for impotence and frigidity.
The origin of this oil is the flowers of some Cananga
species. Cananga odorata (Lam.) Hooker f. and Thomson
var. odorata (Annonaceae) are called fragrant cananga
or wild cananga, and C. odorata (Lam.) Hooker f. and
Thomson var. fruticosa (Craib) Corner are species closely
related to the former one and called shrubby cananga [1].
This paper deals with investigation of the constituents
(1, 2) of the leaves of C. odorata var. odorata, as well as
the absolute stereochemistries of a known compound (3)
[=breyniaionoside A (4)], isolated from the title plant, and
structurally related megastigmane glucosides, breyniaion-
osides B (5) and C (6), isolated from B. officinalis were
examined.
Keywords Cananga odorata var. odorata ꢀ Annonaceae ꢀ
Fragrant cananga ꢀ Megastigmane glucoside ꢀ
Breynia officinalis ꢀ Euphorbiaceae ꢀ Canangaionoside
Introduction
Results and discussion
Ylang-ylang is an essential oil, and has a euphoric and
sedative effect on the nervous system. Therefore, it is
Air-dried leaves of C. odorata var. odorata were extracted
with MeOH three times, and the concentrated MeOH
extract was partitioned with solvents of increasing polarity.
The 1-BuOH-soluble fraction was separated by means of
various chromatographic procedures including column
chromatography (CC) on a highly porous synthetic resin
(Diaion HP-20), and then normal silica gel and reversed-
phase octadecyl silica gel (ODS) CC and high-performance
liquid chromatography (HPLC) to afford four compounds
(1–3 and 7) (Fig. 1). The details and yields are given under
‘‘Experimental.’’ The structures of the new megastigmane
glucoside, named canangaionoside (1), the unusual mono-
terpene (2), and the two known megastigmane glucosides,
compound 3 [=breyniaionoside A (4)] [2] and citroside A
(7), were examined [3]. The structure of the aglycone of
K. Matsunami ꢀ J. Nagashima ꢀ S. Sugimoto ꢀ H. Otsuka (&)
Department of Pharmacognosy,
Graduate School of Biomedical Sciences, Hiroshima University,
1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
e-mail: hotsuka@hiroshima-u.ac.jp
Y. Takeda
Faculty of Pharmacy, Yasuda Women’s University,
6-13-1 Yasuhigashi, Asaminami-ku,
Hiroshima 731-0153, Japan
D. Lhieochaiphant ꢀ S. Lhieochaiphant
Department of Pharmaceutical Sciences,
Faculty of Pharmacy, Chiang Mai University,
239 Huaw Kaew Road, Chiang Mai 50200, Thailand
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461
Fig. 2 1H-1H COSY and diag-
nostic HMBC correlations of
canangaionoside (1). Dual
arrowheads denote HMBC cor-
relations were observed both
ways
Fig. 3 Results of the modified
Mosher’s method of 1 (DdS-R
)
with C-6, deshielded methyl protons (dH 1.93) with C-4 (dC
127.2), C-5 (dC 167.0), and C-6, and H-4 (dH 5.86) with C-
2 in the HMBC spectrum (Fig. 2). The above evidence
established the relative structure of the aglycone to be
megastigma-4,7-diene-6,9,10-triol-3-one. The position of
the sugar linkage was determined to be on the hydroxyl
group at the 9-position by the HMBC spectrum, in which
the anomeric proton (dH 4.29) showed a cross peak with C-
9 (dC 79.5). The absolute configuration at the 6-position
was determined to be S, judging from the positive and
negative Cotton effects at 241 and 329 nm, respectively, in
the CD spectrum [5]. The modified Mosher’s method was
applied to the aglycone (1a) of canangaionoside. Prior to a-
methoxy-a-trifluoromethylphenyl acetic acid (MTPA)
esterifications, the primary alcohol was protected as a
pivaloyl ester. Figure 3 shows the results of the modified
Mosher’s method. The structure of canangaionoside (1)
was elucidated to be (6S,9R,4Z,7E)-megastigma-4,7-diene-
6,9,10-triol-3-one 9-O-b-D-glucopyranoside, as shown in
Fig. 1. The aglycone of canangaionoside (1) was found to
be an epimer as to the 9-position of cucumegastigmane I,
which was isolated from Cucumis sativus [5].
Compound 2, [a]2D4 ?65.4, was isolated as an amorphous
powder, and its elemental composition was established to
be C12H18O5 by HR-ESI-MS. The IR spectrum exhib-
ited absorptions for hydroxyl groups (3381, 1097 and
1031 cm-1) and a conjugated ketonic functional group
(1685 cm-1). The UV absorption at 232 nm also supported
the presence of a conjugated enone system. In the 1H-NMR
spectrum, resonances for one aldehyde (dH 9.48), one
olefinic (dH 6.74), two acetalic [dH 5.13 (dd) and 4.93 (s)],
six aliphatic and one oxygenated methine (dH 4.00) protons
were observed, along with two methoxyl signals. Other
than two methoxyl signals, the 13C-NMR spectrum
exhibited ten signals for three methylene carbons, three
oxygenated methine carbons, one trisubstituted double
bond, one aldehyde carbon and one quaternary carbon. The
double bond must be conjugated with the aldehyde, and
from the elemental composition, compound 2 was pre-
sumed to possess a bicyclic system. Since a similar com-
pound, canangone (8), was isolated from a closely related
Fig. 1 Structures of compounds
breyniaionoside A (4), breyniaionol A, has been elucidated
to be (5R,6S,7E)-megstigman-7-ene-6,9,10-triol-3-one.
However, the absolute configuration at the 9-position
remained to be clarified. In this study, the absolute struc-
ture of breyniaionoside A (4) was examined by the modi-
fied Mosher’s method [4], and determination of the
absolute structures of two other megastigmane glucosides,
breyniaionosides B (5) and C (6), isolated from Breynia
officinalis (Euphorbiaceae) was also performed.
Canangaionoside (1), [a]2D5 ?52.0, was isolated as an
amorphous powder, and its elemental composition was
determined to be C19H30O9 by HR-electrospray ionization
(ESI)-MS. The IR spectrum exhibited absorptions for
hydroxyl groups (3364 and 1075 cm-1) and a conjugated
ketone functional group (1651 cm-1). In the 1H-NMR
spectrum, signals for three olefinic protons, two of which
were coupled to each other with a coupling constant of
16 Hz, an anomeric proton, two singlet methyls and one
doublet methyl (J = 1 Hz) were observed. The 13C-NMR
spectroscopic data showed the presence of six carbon sig-
nals assignable to b-glucopyranoside, and 13 carbon signal
for four sp2 carbons, three methyls, one methylene, one
oxymethylene, one oxymethine, two quaternary carbons
and one ketone. These carbons were expected to form a
magastigmane skeleton with a trans double bond between
the C-7 and C-8 positions. The H-1H COSY and HSQC
1
spectra indicated that the oxymethine carbon must be
adjacent to the trans double bond and that the oxymethine
proton was further correlated to oxymethylene protons.
Judging from the UV absorption at 232 nm, the carbonyl
group must be conjugated with the remaining trisubstituted
double bond. Two shielded methyl protons (dH 1.01 and
1.03) showed correlation cross peaks with C-2 (dC 50.7)
and C-6 (dC 80.2), two olefinic protons (dH 5.79 and 6.10)
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J Nat Med (2010) 64:460–467
Fig. 5 Results of the modified
Mosher’s method of 4 (DdS-R
Fig. 4 Diagnostic HMBC
correlations for 2
)
Fig. 6 Results of the modified
Mosher’s method of 5 (DdS-R
)
plant, C. odorata forma genuina Hook. f. et Thomson [6],
the structure of compound 2 was expected to be as shown
in Fig. 1. The planar structure was supported by significant
HMBC correlations, as shown in Fig. 4. Since, in the PS-
NOESY spectrum, a significant correlation was observed
between H-2 (dH 4.00) and H-8 (dH 1.99), the relative
structure of the spiro junction was established to be as
shown in Fig. 1. Although a mother compound has not yet
been isolated, compound 2 is probably an artifact, formed
through a dial. Thus, the geometry of methoxy groups and
the absolute structure of 2 were not further examined.
Compound 3, [a]2D5 -18.1, was isolated as an amorphous
powder, and its elemental composition was determined to
be C19H32O9 by HR-ESI-MS. The NMR spectral data were
essentially the same as those for breyniaionoside A, which
was isolated from Breynia officinalis [2]. The CD spectrum
of 3 was also superimposable on that of breyniaionoside A
(4). However, the absolute configuration at the 9-position
of breyniaionoside A (4) has not yet been determined.
On enzymatic hydrolysis, 3 and 4 gave identical agly-
cones, breyniaionol A (3a = 4a) and D-glucose. The b-D-
glucopyranosylation-induced shift-trend rule suggested
the absolute configuration at the 9-position to be R [7]
(Dd -3.2 ppm for C-8 and -1.4 ppm for C-10), which was
further confirmed by the modified Mosher’s method [4].
The primary alcohol of 4a was protected as a pivalate, and
(R)- and (S)-MTPA esters were prepared. Figure 5 shows
the results of the modified Mosher’s method for brey-
niaionol A (4a). One of the methylene protons at the
10-position showed an irregular value. Kusumi et al. [8]
stated that this can be regarded as normal, since the cor-
responding protons of some compounds also exhibit
irregular values and the pivaloyl methyls exhibited regular
values. The structure of breyniaionoside A (3 and 4) was
elucidated to be (5R,6S,7E,9R)-megastigman-7-ene-6,9,10-
triol-3-one 9-O-b-D-glucopyranoside, as shown in Fig. 1.
From B. officinalis, structurally related megastigmane
glucosides, breyniaionosides B (5) and C (6), have also been
isolated (Fig. 1) [2]. However, their absolute structures
remained to be determined. Thus, they were enzymatically
hydrolyzed to give the common aglycone, breyniaionol B
(=C) (5a and 6a), and the modified Mosher’s method was
applied to breyniaionol B (5a) to establish the absolute
configuration at the 9-position. As a result, as shown in
Fig. 6, the absolute structures of breyniaionosides B (5) and
C (6) were elucidated to be (3S,5R,6S,7E,9R)-megastigman-
7-ene-3,6,9,10-tetrol 9- and 10-O-b-D-glucopyranosides,
respectively, as shown in Fig. 1.
Experimental
General experimental procedure
Optical rotations and CD spectrum were measured on
JASCO P-1030 and JASCO J-720 polarimeters, respec-
tively. IR and UV spectra were measured on Horiba FT-710
and JASCO V-520 UV/Vis spectrophotometers, respec-
1
tively. H- and 13C-NMR spectra were taken on a JEOL
JNM a-400 or ECA-600 spectrometer at 400 or 600 MHz
and 100 or 150 MHz, respectively, with tetramethylsilane as
an internal standard. Positive-ion HRESIMS was performed
with an Applied Biosystem QSTAR XL system ESI (Nano
Spray)-TOF-MS.
(R)- and (S)-MTPA, and b-glucosidase (emulsin) were
the products of Wako Pure Chemical Industry Co., Ltd.
(Tokyo, Japan).
Plant material
Leaves of C. odorata var. odorata (Annonaceae) were
collected in March 2005 from cultivated species in the
Botanical Garden, Faculty of Pharmacy, Chiang Mai
University, Chiang Mai, Thailand.
Extraction and isolation
Dried leaves of C. odorata var. odorata (1.17 kg) were
extracted three times with MeOH (4.5 l) at 25°C for
1 week and then concentrated to 3 l in vacuo. The extract
was washed with n-hexane (3 l, 20.1 g), and then the
MeOH layer was concentrated to a gummy mass. The latter
was suspended in water (3 l) and then extracted with
EtOAc (3 l) to give 14.0 g of an EtOAc-soluble fraction.
The aqueous layer was extracted with 1-BuOH (3 l) to give
a 1-BuOH-soluble fraction (27.4 g), and the remaining
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463
Table 1 13C-NMR spectroscopic data for canangaionoside (1)
(CD3OD, 100 MHz)
water layer was concentrated to furnish 70.3 g of a water-
soluble fraction.
The 1-BuOH-soluble fraction was subjected to Diaion
HP-20 (purchased from Mitsubishi Kagaku, Tokyo, Japan)
CC (U = 50 mm, L = 50 cm), using H2O-MeOH (4:1, 3 l),
(2:3, 3 l), (3:2, 3 l) and (1:4, 3 l), and MeOH (3 l), with
fractions of 500 ml being collected. The residue (2.08 g in
fractions 8-10) obtained on Diaion HP-20 (20–40% MeOH
eluate) was subjected to silica gel CC (125 g) with elution
with a linear gradient from CHCl3 (1 l) to CHCl3-MeOH (1:1,
1 l), with fractions of 10 g being collected. The residue
(78.6 mg) in fractions 46–50 was again separated by silica gel
CC (50 g) with an isocratic solvent system of CHCl3-MeOH
(18:1), with fractions of 1 ml being collected. The residue
(12.5 mg) in fractions 35–49 was purified by HPLC (MeOH-
H2O, 9:11) to give 1.4 mg of 2 from the peak at 12 min. The
residue (336 mg) in fractions 79–89 was separated by ODS
open CC to yield three subfractions, 143 mg in fractions 34–
53, 34.0 mg in fractions 54–70 and 55.6 mg in fractions
71–90. The first subfraction was purified by HPLC (MeOH-
H2O, 1:3) to obtain 28.3 mg of 1 from the peak at 14 min. The
second subfraction was purified by HPLC (MeOH-H2O, 3:7)
to obtain 3.0 mg of 3 from the peak at 15 min. The third
subfraction was also purified by HPLC (MeOH-H2O, 3:7) to
give 21.7 mg of 7 from the peak at 23 min.
C
C
1
42.4
50.7
10
20
30
40
50
60
101.5
74.9
78.2
71.6
78.1
62.7
2
3
201.2
127.2
167.0
80.2
4
5
6
7
135.8
128.9
79.5
8
9
10
11
12
13
66.0
23.5
24.7
19.5
on C-7), 3.36 (3H, s, –OCH3 on C-9), 2.33 (1H, m, H-5a),
2.02 (1H, m, H-5b), 1.99 (1H, dd, J = 14, 6 Hz, H-8a),
1.86 (1H, ddd, J = 14, 9, 6 Hz, H-6a), 1.77 (1H, dd,
J = 14, 5 Hz, H-8b), 1.77 (1H, m, H-6b); 13C-NMR
(CD3OD, 100 MHz) d: 195.9 (C-10), 149.6 (C-3), 143.6
(C-4), 107.8 (C-7), 107.4 (C-9), 70.6 (C-2), 56.0 (CH3O– at
C-7), 55.4 (CH3O– at C-9), 51.2 (C-1), 40.9 (C-8), 24.1
(C-5), 20.9 (C-6); HR-ESI-MS (positive-ion mode) m/z:
265.1047 [M?Na]? (Calcd for C12H18O5Na: 265.1046).
Canangaionoside (1)
Amorphous powder; [a]2D5 ?52.0 (c 1.81, MeOH); IR mmax
Compound 3 [=breyniaionoside A (4)]
(film) cm-1: 3364, 2934, 1651, 1075, 1037; UV (MeOH)
Amorphous powder; [a]2D5 -18.1 (c 0.20, MeOH); CD
(c 4.01 9 10-5 M, MeOH): De (nm): ?0.037 (294);
HR-ESI-MS (positive-ion mode) m/z: 427.1941 [M?Na]?
(Calcd for C19H32O9Na: 427.1938).
k
max nm (log e): 232 (4.16); 1H-NMR (CD3OD, 400 MHz)
d: 6.10 (1H, dd, J = 16, 1 Hz, H-7), 5.86 (1H, q, J = 1 Hz,
H-4), 5.79 (1H, dd, J = 16, 7 Hz, H-8), 4.45 (1H, m, H-9),
4.29 (1H, d, J = 8 Hz, H-10), 3.83 (1H, dd, J = 12, 2 Hz,
H-60a), 3.63 (1H, dd, J = 12, 6 Hz, H-60b), 3.63 (1H, dd,
J = 12, 4 Hz, H-10a), 3.57 (1H, dd, J = 12, 6 Hz, H-10b),
3.26 (3H, m, H-20, 30 and 40), 3.17 (1H, ddd, J = 9, 6, 2 Hz,
H-50), 2.60 (1H, d, J = 17 Hz, H-2a), 2.17 (1H, d,
J = 17 Hz, H-2b), 1.93 (3H, d, J = 1 Hz, H3-13), 1.03 (3H,
s, H3-11), 1.01 (3H, s, H3-12); 13C-NMR (CD3OD,
100 MHz): Table 1; CD (c 5.04 9 10-5 M, MeOH): De
(nm): -0.71 (329), ?17.3 (241); HR-ESI-MS (positive-ion
mode) m/z: 425.1774 [M?Na]? (C19H30O9Na requires
425.1782).
Enzymatic hydrolysis of canangaionoside (1)
to canangaionol (1a)
Canangaionoside (1) (10.5 mg) was hydrolyzed with
emulsin (10 mg) in 400 ll of 100 mM acetate buffer (pH
5.0) at 37°C for 18 h. The reaction solvent was evaporated
off, and the residue was purified by silica gel CC
(U = 10 mm, L = 20 cm) with elution with CHCl3
(50 ml) and CHCl3-MeOH [(19:1, 50 ml), (9:1, 50 ml),
(17:3, 50 ml), and (7:3, 50 ml)], with fractions of 5 ml
being collected. The aglycone (1a) (3.1 mg) was recovered
in fractions 34–38 and glucose (4.4 mg) in fractions 59–64.
Compound 2
Amorphous powder; [a]2D4 ?65.4 (c 0.11, MeOH); IR mmax
(film) cm-1: 3381, 2836, 1685, 1178, 1097, 1031, 994; UV
Canangaionol (1a)
1
(MeOH) kmax nm (log e): 232 (4.13); H-NMR (CD3OD,
Amorphous powder; [a]2D5 ?211 (c 0.20; MeOH); 1H-NMR
(CD3OD, 400 MHz): d 5.90 (1H, dd, J = 16, 1 Hz, H-7),
5.84 (1H, m, H-4), 5.80 (1H, dd, J = 16, 5 Hz, H-8), 4.20
400 MHz) d: 9.48 (1H, s, H-10), 6.74 (1H, ddd, J = 4, 2,
2 Hz, H-3), 5.13 (1H, dd, J = 6, 5 Hz, H-9), 4.93 (1H, s,
H-7), 4.00 (1H, br d, J = 4 Hz, H-2), 3.40 (3H, s, –OCH3
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(1H, br ddd, J = 6, 6, 6 Hz, H-9), 3.51 (1H, dd, J = 11,
5 Hz, H-10a), 3.46 (1H, dd, J = 11, 7 Hz, H-10b), 2.52
(1H, d, J = 17 Hz, H-2a), 2.16 (1H, d, J = 17 Hz, H-2b),
1.92 (1H, s, H3-13), 1.04 (3H, s, H3-11), 1.01 (3H, s, H3-
12); 13C-NMR (CD3OD, 100 MHz) d: 201.2 (C-3), 167.4
(C-5), 132.6 (C-7), 132.5 (C-8), 127.2 (C-4), 80.1 (C-6),
73.7 (C-9), 67.3 (C-10), 50.8 (C-2), 42.4 (C-1), 24.5
(C-12), 23.4 (C-11), 19.6 (C-13); HR-ESI-MS (positive-ion
mode) m/z: 263.1242 [M?Na]? (Cacld for C13H20O4Na:
263.1253).
CHCl3-MeOH (9:1)] to furnish an ester, 1c (0.63 mg).
Through a similar procedure, 1d (0.72 mg) was prepared
from 1b (0.5 mg) using (S)-MTPA (50 mg), EDC (32 mg)
and 4-DMAP (24 mg).
10-O-Pivaloyl canangaionol 9-O-(R)-MTPA ester (1c)
Amorphous powder; 1H-NMR (CDCl3, 400 MHz) d: 7.54–
7.51 (2H, aromatic protons), 7.41–7.37 (3H, m, aromatic
protons), 5.87 (1H, m, H-4), 5.86 (1H, d, J = 15 Hz, H-7),
5.80 (2H, m, H-8 and 9), 4.40 (1H, dd, J = 12, 4 Hz,
H-10a), 4.10 (1H, dd, J = 12, 7 Hz, H-10b), 3.57 (3H, q,
J = 1 Hz, –OCH3), 2.23 (1H, d, J = 17 Hz, H-2a), 2.18
(1H, d, J = 17 Hz, H-2b), 1.80 (3H, s, H3-13), 1.17 (9H, s,
CH3– 9 3), 1.04 (3H, s, H3-11), 0.89 (3H, s, H3-12); HR-
ESI-MS (positive-ion mode) m/z: 547.2281 [M?Na]?
(Calcd for C28H35O6F3Na: 547.2283).
D-Glucose: [a]2D4 ?50.4 (c 0.21, H2O, 24 h after being
dissolved in the solvent).
Preparation of 10-O-pivalate (1b)
from canangaionol (1a)
To a solution of the aglycone (1a) (3.0 mg) in 100 ll of
pyridine, 10 ll of pivaloyl chloride was added, and the
reaction was performed for 3 h at 25°C. One milliliter of
H2O was added, followed by extraction with 2 ml of
EtOAc three times. The organic layer was dried over
Na2SO4 and evaporated. The residue was purified by prep.
TLC [CHCl3-(CH3)2CO, 20:1] to give 1.2 mg of pivalate
(1b).
10-O-Pivaloyl canangaionol 9-O-(S)-MTPA ester (1d)
Amorphous powder; 1H-NMR (CDCl3, 400 MHz) d: 7.54–
7.49 (2H, m, aromatic protons), 7.41–7.37 (3H, m, aro-
matic protons), 5.99 (1H, d, J = 16 Hz, H-7), 5.90 (1H, m,
H-4), 5.85 (1H, dd, J = 16, 5 Hz, H-8), 5.74 (1H, m, H-9),
4.32 (1H, dd, J = 12, 3 Hz, H-10a), 4.13 (1H, dd, J = 12,
7 Hz, H-10b), 3.49 (3H, q, J = 1 Hz, –OCH3), 2.30 (1H, d,
J = 17 Hz, H-2a), 2.23 (1H, d, J = 17 Hz, H-2b), 1.83
(3H, s, H3-13), 1.14 (9H, s, CH3– 9 3), 1.06 (3H, s, H3-
11), 0.95 (3H, s, H3-12); HR-ESI-MS (positive-ion mode)
m/z: 547.2271 [M?Na]? (Calcd for C28H35O6F3Na:
547.2283).
10-O-Pivaloyl canangaionol (1b)
1
Amorphous powder; H-NMR (CDCl3, 400 MHz) d: 5.96
(1H, dd, J = 16, 1 Hz, H-7), 5.90 (1H, m, H-4), 5.84 (1H,
dd, J = 16, 5 Hz, H-8), 4.48 (1H, m, H-9), 4.20 (1H, dd,
J = 12, 4 Hz, H-10a), 4.03 (1H, dd, J = 12, 7 Hz, H-10b),
2.46 (1H, d, J = 17 Hz, H-2a), 2.26 (1H, dd, J = 17, 1 Hz,
H-2b), 1.89 (3H, d, J = 1 Hz, H3-13), 1.22 (9H, s, CH3–
9 3), 1.08 (3H, s, H3-11), 1.01 (3H, s, H3-12); HR-ESI-MS
(positive-ion mode) m/z: 347.1825 [M?Na]? (Calcd for
C18H28O5Na: 347.1834).
Enzymatic hydrolysis of compound 3
and breyniaionoside A (4) to its aglycone
and breyniaionol A (3a and 4a), respectively
Compound 3 (2.5 mg) was hydrolyzed in a similar manner
as for 1. The usual workup gave 0.27 mg of an aglycone
(3a). Breyniaionoisde A (4) (8.5 mg) was also hydrolyzed
enzymatically to give 4.9 mg of breyniaionol A (4a) and
3.2 mg of D-glucose.
Preparation of (R)- and (S)-9-O-MTPA esters
(1c and 1d) from 10-O-pivaloyl canangaionol (1b)
A solution of 1b (0.5 mg) in 1 ml of dehydrated CH2Cl2
was reacted with (R)-MTPA (38 mg) in the presence
of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydro-
chloride (EDC) (30 mg) and N,N-dimethyl-4-aminopyri-
dine (DMAP) (20 mg), with the mixture being occasionally
stirred at 25°C for 30 min. After the addition of 1 ml of
CH2Cl2, the solution was washed with H2O (1 ml), 5% HCl
(1 ml), NaHCO3-saturated H2O and then brine (1 ml),
successively. The organic layer was dried over Na2SO4
and then evaporated under reduced pressure. The residue
was purified by preparative TLC [silica gel (0.25 mm
thickness), being applied for 18 cm, developed with
CHCl3-(CH3)2CO (19:1) for 9 cm, and then eluted with
Aglycone (3a)
Amorphous powder; [a]2D4 ?5.2 (c 0.027, MeOH);
1H-NMR (CD3OD, 600 MHz) d: 5.82 (1H, dd, J = 16,
5 Hz, H-8), 5.77 (1H, dd, J = 16, 1 Hz, H-7), 4.22 (1H,
dddd, J = 7, 5, 5, 1 Hz, H-9), 3.54 (1H, dd, J = 11, 5 Hz,
H-10a), 3.48 (1H, dd, J = 11, 7 Hz, H-10b), 2.86 (1H, d,
J = 14 Hz, H-2a), 2.44 (1H, dd, J = 14, 13 Hz, H-4a),
2.28 (1H, dqd, J = 13, 7, 5 Hz, H-5), 2.12 (1H, ddd,
J = 14, 5, 2 Hz, H-4b), 1.82 (1H, dd, J = 14, 2 Hz, H-2b),
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465
10-O-Pivaloyl breyniaionol A 9-O-(R)-MTPA ester (4c)
0.96 (3H, s, H3-12), 0.92 (3H, d, J = 7 Hz, H3-13), 0.92
(3H, s, H3-11); 13C-NMR (CD3OD, 150 MHz) d: 215.0
(C-3), 134.8 (C-7), 132.2 (C-8), 78.2 (C-6), 74.1 (C-9),
67.7 (C-10), 52.5 (C-2), 46.3 (C-4), 43.9 (C-1), 38.0 (C-5),
25.1 (C-11), 25.0 (C-12), 16.5 (C-13); HR-ESI-MS
(positive-ion mode) m/z: 265.1413 [M?Na]? (Calcd for
C13H22O4Na: 265.1410).
Amorphous powder; 1H-NMR (CDCl3, 600 MHz) d: 7.55–
7.53 (2H, m, aromatic protons), 7.44–7.37 (3H, m,
aromatic protons), 5.89 (1H, dd, J = 15, 1 Hz, H-7), 5.81
(1H, br ddd, J = 7, 7, 3 Hz, H-9), 5.73 (1H, dd, J = 16,
7 Hz, H-8), 4.40 (1H, dd, J = 12, 3 Hz, H-10a), 4.15 (1H,
dd, J = 12, 7 Hz, H-10b), 3.59 (3H, br s, CH3O–), 2.78
(1H, d, J = 14 Hz, H-2a), 2.36 (1H, dd, J = 13, 13 Hz,
H-4a), 2.24 (1H, m, H-5), 2.21 (1H, br dd, J = 13, 4 Hz,
H-4b), 1.91 (1H, br d, J = 14 Hz, H-2b), 1.19 (9H, CH3–
9 3), 0.89 (3H, s, H3-12), 0.86 (3H, s, H3-11), 0.82 (3H, d,
J = 7, H3-13); HR-ESI-MS (positive-ion mode) m/z:
565.2380 [M?Na]? (Calcd for C28H37O7F3Na: 565.2383).
Breyniaionol A (4a)
Amorphous powder; [a]2D4 ?5.5 (c 0.49, MeOH); 1H-NMR
(CD3OD, 600 MHz) and 13C-NMR (CD3OD, 150 MHz):
essentially the same as those of 3a; HR-ESI-MS (positive-
ion mode) m/z: 265.1415 [M?Na]? (Calcd for
C13H22O4Na: 265.1410).
10-O-Pivaloyl breyniaionol A 9-O-(S)-MTPA ester (4d)
Preparation of 10-O-pivalate (4b)
from breyniaionol A (4a)
Amorphous powder; 1H-NMR (CDCl3, 600 MHz) d: 7.55–
7.53 (2H, m, aromatic protons), 7.44–7.38 (3H, m, aro-
matic protons), 5.98 (1H, dd, J = 15, 1 Hz, H-7), 5.82 (1H,
dd, J = 15, 7 Hz, H-8), 5.77 (1H, br ddd, J = 7, 7, 3 Hz,
H-9), 4.33 (1H, dd, J = 12, 3 Hz, H-10a), 4.15 (1H, dd,
J = 12, 7 Hz, H-10b), 3.53 (3H, br s, CH3O–), 2.80 (1H, d,
J = 14 Hz, H-2a), 2.38 (1H, dd, J = 13, 13 Hz, H-4a),
2.27 (1H, m, H-5), 2.22 (1H, dd, J = 13, 4 Hz, H-4b), 1.93
(1H, dd, J = 14, 2 Hz, H-2b), 1.69 (9H, CH3– 9 3), 0.93
(1H, s, H3-12), 0.91 (3H, s, H3-11), 0.82 (3H, d, J = 6 Hz,
H3-13); HR-ESI-MS (positive-ion mode) m/z: 565.2386
[M?Na]? (Calcd for C28H37O7F3Na: 565.2383).
In a similar manner as for the preparation of 1b from 1a, 4b
was prepared from 4a (4.9 mg) with 20 ll of pivaloyl
chloride. The usual workup gave 2.6 mg of pivaloyl ester
(4b).
10-O-Pivaloyl breyniaionol A (4b)
Amorphous powder; [a]2D4 -12.1 (c 0.07, MeOH);
1H-NMR (CDCl3, 600 MHz) d: 5.89 (1H, dd, J = 16,
1 Hz, H-7), 5.80 (1H, dd, J = 16, 5 Hz, H-8), 4.50 (1H,
dddd, J = 7, 5, 4, 1 Hz, H-9), 4.21 (1H, dd, J = 12, 4 Hz,
H-10a), 4.06 (1H, dd, J = 12, 7 Hz, H-10b), 2.83 (1H, d,
J = 14 Hz, H-2a), 2.41 (1H, dd, J = 14, 13 Hz, H-4a),
2.29 (1H, dqd, J = 13, 7, 5 Hz, H-5), 2.22 (1H, ddd,
J = 14, 5, 2 Hz, H-4b), 1.93 (1H, dd, J = 14, 2 Hz, H-2b),
1.24 (9H, s, CH3 9 3), 0.96 (3H, s, H3-12), 0.95 (3H, s,
H3-11), 0.91 (3H, d, J = 7 Hz, H3-13); 13C-NMR (CDCl3,
150 MHz) d: 210.9 (C-3), 178.8 (–CO–), 135.3 (C-7),
129.4 (C-8), 77.4 (C-6), 70.7 (C-9), 68.4 (C-10), 51.5
(C-2), 42.5 (C-1), 45.1 (C-4), 39.0 [(CH3)3C)–], 36.5 (C-5),
27.3 (CH3– 9 3), 24.4 (C-11), 24.5 (C-12), 16.0 (C-13);
HR-ESI-MS (positive-ion mode) m/z: 349.1991 [M?Na]?
(Calcd for C18H30O5Na: 349.1985).
Enzymatic hydrolysis of breyniaionoside B (5)
and breyniaionoside C (6) to their aglycones (5a = 6a)
Breyniaionoside B (5) (10.2 mg) and breyniaionoside C (6)
(4.0 mg) were hydrolyzed in a similar manner as for 1. The
usual workup gave 5.5 and 1.8 mg of the common agly-
cone, breyniaionol B (5a and 6a, respectively), and 3.2 and
0.61 mg of D-glucose, respectively.
Breyniaionol B (5a)
Amorphous powder; [a]2D4 -9.9 (c 0.55, MeOH); 1H-NMR
(CD3OD, 600 MHz) d: 5.72–5.66 (2H, m, H-7 and 8), 4.18
(1H, dddd, J = 7, 5, 5, 1 Hz, H-9), 3.80 (1H, dddd, J = 12,
12, 4, 4 Hz, H-3), 3.51 (1H, dd, J = 11, 5 Hz, H-10a), 3.45
(1H, dd, J = 11, 7 Hz, H-10b), 1.95 (1H, dqd, J = 12, 7,
4 Hz, H-5), 1.68 (1H, dddd, J = 12, 4, 4, 2 Hz, H-4a), 1.65
(1H, dd, J = 12, 12 Hz, H-2a), 1.41 (1H, ddd, J = 12, 4,
2 Hz, H-2b), 1.39 (1H, ddd, J = 12, 12, 12 Hz, H-4b), 0.97
(3H, s, H3-12), 0.86 (3H, s, H3-11), 0.84 (3H, d, J = 7 Hz,
H3-13); 13C-NMR (CD3OD, 150 MHz) d: 136.5 (C-7),
131.0 (C-8), 78.4 (C-6), 74.3 (C-9), 67.8 (C-10), 67.5
(C-3), 46.0 (C-2), 40.5 (C-1), 40.0 (C-4), 35.5 (C-5), 25.9
Preparation of (R)- and (S)-9-O-MTPA esters
(4c and 4d) from 10-O-pivaloyl breyniaionol A (4b)
In a similar manner as for the preparation of 1c and 1d
from 1b, 4c and 4d were prepared from 4b (0.75 mg each)
with the respective amounts of the reagents, (R)- and (S)-
MPTA (10.9 and 10.8 mg), EDC (16.5 and 16.2 mg) and
DMAP (6.6 and 7.3 mg). The usual workup gave 0.83 mg
(4c) and 0.72 mg (4d) of esters, respectively.
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J Nat Med (2010) 64:460–467
(C-11), 25.2 (C-12), 6.5 (C-13); HR-ESI-MS (positive-ion
mode) m/z: 267.1571 [M?Na]? (Calcd for C13H24O4Na:
267.1566).
protons), 5.81 (1H, dd, J = 16, 1 Hz, H-7), 5.78 (1H, m,
H-9), 5.64 (1H, dd, J = 16, 7 Hz, H-8), 5.20 (1H, dddd,
J = 12, 12, 5, 5 Hz, H-3), 4.37 (1H, dd, J = 12, 3 Hz,
H-10a), 4.14 (1H, dd, J = 12, 6 Hz, H-10b), 3.58 (3H, br s,
CH3O–), 3.55 (3H, br s, CH3O–), 2.01 (1H, m, H-5), 1.89
(1H, m, H-4a), 1.76 (1H, dd, J = 12, 12 Hz, H-2a), 1.57
(1H, m, H-4b), 1.53 (1H, m, H-2b), 1.19 (9H, s, CH3– 9 3),
0.97 (3H, s, H3-11), 0.82 (3H, s, H3-12), 0.77 (3H, d,
J = 7 Hz, H3-13); HR-ESI-MS (positive-ion mode) m/z:
783.2941 [M?Na]? (Calcd for C38H46O9F6Na: 783.2938).
Breyniaionol B (6a)
Amorphous powder; [a]2D4 -12.2 (c 0.18, MeOH); 1H- and
13C-NMR spectra were indistinguishable from those of 5a;
HR-ESI-MS (positive-ion mode) m/z: 267.1570 [M?Na]?
(Calcd for C13H24O4Na: 267.1566).
Preparation of 10-O-pivalate (5b)
from breyniaionol B (5a)
10-O-Pivaloyl breyniaionol B 3,9-di-O-(S)-MTPA
ester (5d)
In a similar manner as for the preparation of 1b from 1a, 5b
was prepared from 5a (3.2 mg) with 16 ll of pivaloyl
chloride. The usual workup gave 1.62 mg of ester (5b).
Amorphous powder; 1H-NMR (CDCl3, 600 MHz) d: 7.55–
7.52 (4H, m, aromatic protons), 7.42–7.38 (6H, m, aro-
matic protons), 5.90 (1H, dd, J = 15, 1 Hz, H-7), 5.75 (1H,
m, H-8), 5.74 (1H, m, H-9), 5.22 (1H, dddd, J = 12, 12, 5,
5 Hz, H-3), 4.30 (1H, dd, J = 12, 3 Hz, H-10a), 4.14 (1H,
dd, J = 12, 6 Hz, H-10b), 3.56 (3H, br s, CH3O–), 3.52
(3H, br s, CH3O–), 2.03 (1H, m, H-5), 1.83 (1H, m, H-4a),
1.86 (1H, dd, J = 12, 12 Hz, H-2a), 1.62 (1H, br d,
J = 12 Hz, H-2b), 1.48 (1H, ddd, J = 12, 12, 12 Hz,
H-4b), 1.15 (9H, s, CH3– 9 3), 1.02 (3H, s, H3-11), 0.88
(3H, s, H3-12), 0.75 (3H, d, J = 7 Hz, H3-13); HR-ESI-MS
(positive-ion mode) m/z: 783.2932 [M?Na]? (Calcd for
C38H46O9F6Na: 783.2938).
10-O-Pivaloyl breyniaionol B (5b)
Amorphous powder; [a]2D3 -16.0 (c 0.16, MeOH); 1H-NMR
(CDCl3, 600 MHz) d: 5.77 (1H, d, J = 15 Hz, H-7), 5.73
(1H, dd, J = 15, 5 Hz, H-8), 4.46 (1H, m, H-9), 4.18 (1H,
dd, J = 11, 4 Hz, H-10a), 4.04 (1H, dd, J = 11, 7 Hz,
H-10b), 3.87 (1H, dddd, J = 12, 12, 5, 5 Hz, H-3), 1.96 (1H,
m, H-5), 1.80 (1H, ddd, J = 12, 12, 12 Hz, H-4a), 1.63 (1H,
dd, J = 12, 12 Hz, H-2a), 1.51 (1H, br d, J = 12 Hz, H-2b),
1.36 (1H, br d, J = 12 Hz, H-4b), 1.23 (9H, CH3 9 3), 0.98
(3H, s, H3-11), 0.87 (3H, s, H3-12), 0.83 (3H, d, J = 7 Hz,
H3-13); 13C-NMR (CDCl3, 150 MHz) d: 178.8 (CO), 136.5
(C-7), 128.5 (C-8), 77.2 (C-6), 70.8 (C-9), 68.5 (C-10), 66.7
(C-3), 45.3 (C-2), 39.5 (C-1), 39.3 (C-4), 39.0 [(CH3)3C–],
34.1 (C-5), 27.3 (CH3– 9 3), 25.1 (C-11), 24.6 (C-12), 15.9
(C-13); HR-ESI-MS (positive-ion mode) m/z: 351.2148
[M?Na]? (Calcd for C18H32O5Na: 351.2141).
Sugar analysis
Glucose obtained from compound 3, breyniaionosides A (4),
B (5) and C (6) by enzymatic hydrolysis was analyzed with a
chiral detector (JASCO OR-2090plus) on an amino column
[Asahipak NH2P-50 4E, CH3CN-H2O (4:1), 1 ml/min] to
give peaks for D-glucose at 13.7 min. All peaks showed
positive optical rotation signs. Peaks were identified by
co-chromatography with authentic D-glucose.
Preparation of (R)- and (S)-3,9-di-O-MTPA esters
(5c, 5d) from 10-O-pivaloyl breyniaionol B (5b)
Known compounds isolated
In a similar manner as for the preparation of 1c and 1d
from 1b, 5c and 5d were prepared from 5b (0.81 mg each)
with the respective amounts of the reagents, (R)- and
(S)-MPTA (22.2 and 21.0 mg), EDC (21.0 and 25.8 mg)
and DMAP (23.4 and 16.4 mg). To complete the esterifi-
cation, a prolonged reaction time (24 h) and an increased
reaction temperature (40°C) were used. The usual workup
gave 0.92 mg (5c) and 1.01 mg (5d) of esters, respectively.
Citroside A (7): amorphous powder; [a]2D5 -102 (c 1.45,
MeOH) [3].
Acknowledgments The authors are grateful for access to the
superconducting NMR instrument (JEOL a-400) at the Analytical
Center of Molecular Medicine and an Applied Biosystem QSTAR XL
system ESI (Nano Spray)-MS at the Analysis Center of Life Science
of the Graduate School of Biomedical Sciences, Hiroshima Univer-
sity. The authors are also grateful for the NMR instrument (JEOL
ECA-600) at the Natural Science Center for Basic Research and
Development, Hiroshima University. The authors express sincere
thanks to the Japan Society for the Promotion of Science (JSPS)-
National Research Council of Thailand (NRCT) Core University
Program on Natural Medicine for allowing them to conduct joint
work with Thai scientists. This work was financially supported in part
10-O-Pivaloyl breyniaionol B 3,9-di-O-(R)-MTPA
ester (5c)
Amorphous powder; 1H-NMR (CDCl3, 600 MHz) d: 7.55–
7.52 (4H, m, aromatic protons), 7.42–7.36 (6H, m, aromatic
123

J Nat Med (2010) 64:460–467
467
by Grants-in-Aid from the Ministry of Education, Culture, Sports,
Science and Technology of Japan, and the Japan Society for the
Promotion of Science. Thanks are also due to the Astellas Foundation
for Research on Medicinal Resources and the Takeda Science
Foundation for the financial support.
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FT NMR application of Mosher’s method. The absolute config-
urations of marine terpenoids. J Am Chem Soc 113:4092–4096
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