Euodionosides A-G: Megastigmane glucosides from leaves of Euodia meliaefolia

Article abstract of DOI:10.1016/j.phytochem.2008.01.020

From a 1-BuOH-soluble fraction of the MeOH extract of leaves of Euodia meliaefolia, collected in Okinawa, seven megastigmane glucosides, named euodionosides A-G, were isolated together with three known megastgmane glucosides, and two aliphatic and three phenolic compounds. Their structures were elucidated through a combination of spectroscopic analyses and application of the modified Mosher's method.

Full text of DOI:10.1016/j.phytochem.2008.01.020

Available online at www.sciencedirect.com
PHYTOCHEMISTRY
Phytochemistry 69 (2008) 1586–1596
www.elsevier.com/locate/phytochem
Euodionosides A–G: Megastigmane glucosides from
leaves of Euodia meliaefolia
Miwako Yamamoto a, Takeyuki Akita a, Yuka Koyama a, Etsuko Sueyoshi a,
a
a,
b
*
Katsuyoshi Matsunami , Hideaki Otsuka , Takakazu Shinzato ,
Atsushi Takashima c, Mitsunori Aramoto d, Yoshio Takeda e
a Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
b Subtropical Field Science Center, Faculty of Agriculture, University of the Ryukyus, 1 Sembaru, Nishihara-cho,
Nakagami-gun, Okinawa 903-0213, Japan
c Yona Field, Subtropical Field Science Center, Faculty of Agriculture, University of the Ryukyus, 685 Aza Yona,
Kunigami-son, Kunigami-gun, Okinawa 905-1427, Japan
d Iriomote Station, Tropical Biosphere Research Center, University of the Ryukyus, 870 Aza Uehara,
Taketomi-cho, Yaeyama-gun, Okinawa 907-1541, Japan
e Faculty of Integrated Arts and Sciences, The University of Tokushima, 1 Minamijosanjima-cho, Tokushima 770-8502, Japan
Received 9 August 2007; received in revised form 28 December 2007
Available online 11 March 2008
Abstract
From a 1-BuOH-soluble fraction of the MeOH extract of leaves of Euodia meliaefolia, collected in Okinawa, seven megastigmane
glucosides, named euodionosides A–G, were isolated together with three known megastgmane glucosides, and two aliphatic and three
phenolic compounds. Their structures were elucidated through a combination of spectroscopic analyses and application of the modified
Mosher’s method.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Euodia meliaefolia; Rutaceae; Euodionoside; Megastigmane glucoside; Modified Mosher’s method
1. Introduction
ter, even an isolated compound was erroneously named
evodiamine.
The fruits of Evodia rutaecaepa (Rutaceae) are a well
known crude drug included in the Japanese Pharmaco-
poeia XV and contain characteristic indole alkaloids, i.e.
rutaecarpine and evodiamine. However, in reliable plant
name databases (IPNI), such as the Index Kewensis, Aus-
tralian Plant Name Index and Gray Card Index, the genus
name Evodia is not listed under the Rutaceae. Instead,
many Euodia species appear in the databases as Rutaceous
plants. Probably, some previous investigators made a mis-
take due to the resemblance of the letters u and v. Thereaf-
A closely related species, E. meliaefolia, is a tall deciduous
tree of about 15 m in height and found in southern Kyushu
through Okinawa, Taiwan and China (Hatusima, 1975).
Currently no chemical and medicinal investigations of this
plant have been reported. In a continuing study on Okina-
wan plants, the chemical constituents of E. meliaefolia,
collected in Okinawa, were investigated.
From a 1-BuOH-soluble fraction of the MeOH extract
of leaves of E. meliaefolia, 10 megastigmane glucosides
(1–10) were isolated together with two known aliphatic
[(Z)-3-hexenyl b-D-glucopyranoside (11) (Mizutani et al.,
1988) and 3,7-dimethylocte-1-en-3,6,7-triol 6-O-b-D-gluco-
pyranoside (12) (Manns, 1995)] and three phenolic [(+)-
catechin (13) (Nay et al., 2001), syringin (14) DellaGreca
*
Corresponding author. Tel./fax: +81 82 257 5335.
E-mail address: hotsuka@hiroshima-u.ac.jp (H. Otsuka).
0031-9422/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2008.01.020

M. Yamamoto et al. / Phytochemistry 69 (2008) 1586–1596
1587
et al., 1998) and 1-b-D-glucopyranosyloxy-3-methoxy-5-
hydroxybenzene (15) (Sakar et al., 1993)] compounds. Of
the ten megastigmane glucosides, three were known com-
pounds, namely spinoside A (8) (C¸ alißs et al., 2002), staph-
ylionoside D (9) (Yu et al., 2005), and corchoionoside C
(10) (Yoshikawa et al., 1997). This paper deals with struc-
tural elucidation of the seven new megastigmane
glucosides.
To clarify the relative arrangement of the substituents,
the phase-sensitive (PH)-NOESY spectrum was examined.
Judging from the key correlations between H-3 (dH 3.98)
and H3-11ax (dH 1.25), H3-11 and H-7 (dH 7.08), and H-
4eq (dH 2.38) and H3-13 (dH 1.17), H-3, the epoxy ring is
in an anti orientation, in contrast to in the case of icariside
B2, which was isolated from Epimedium grandiflorum var.
thunbergianum (Miyase et al., 1987). On enzymatic hydro-
lysis, an aglycone (1a) and D-glucose were obtained. From
the b-D-glucosylation-induced shift-trend between the 13C
NMR spectra of 1 and 1a, the absolute configuration of
the 3-position was determined to be R (Table 1, a) (Kasai
et al., 1977). This was further confirmed by the modified
Mosher’s method (Fig. 2) (Ohtani et al., 1991). Therefore,
the structure of euodionoside A (1) was elucidated to be
(3R,5R,6S,7E)-megastigman-7-en-5,6-epoxy-3-ol-9-one 3-
O-b-D-glucopyranoside, as shown in Fig. 1.
2. Results and discussion
Air-dried leaves of E. meliaefolia were extracted with
MeOH three times and the concentrated MeOH extract
was partitioned with solvents of increasing polarity. The
n-BuOH soluble fraction was separated by means of vari-
ous chromatographic procedures including column chro-
matography (CC) on a highly porous synthetic resin
(Diaion HP-20), then normal silica gel and reversed-phase
octadecyl silica gel (ODS) CC, droplet counter-current
chromatography (DCCC), and high-performance liquid
chromatography (HPLC) to afford 15 compounds (1–15).
The details and yields are given under Section 4. The struc-
tures of the new megastigmane glucosides (17) were eluci-
dated on the basis of spectroscopic evidence, that
obtained with the modified Mosher’s method (Ohtani
et al., 1991), and those of known compounds were identi-
fied by comparison of spectroscopic data with those
reported in the literature (Fig. 1). The 13C NMR spectro-
scopic data of spinoside A (8) in CD3OD are included
under Section 4 for the readers’ convenience.
25
26
Euodionosides B (2), ½aꢀD 24.8, and C (3), ½aꢀD ꢁ56.3,
were isolated as amorphous powders and their elemental
compositions were determined to be C19H32O8 by HR-
1
FAB-MS. The H and 13C NMR spectra indicated that
euodionosides B (2) and C (3) were compounds analogous
to euodionoside A (1), except for the presence of hydroxyl
groups at their 9-positions, instead of the carbonyl func-
tional group found in 1. The HMBC spectra showed a cor-
relation between the anomeric proton (dH 4.30) and C-3 (dC
72.2) in the case of 2, but between the former and (dH 4.28)
and C-9 (dC 74.8) in the case of 3. The stereochemistry of
the ring system of 2 was expected to be the same as that
of 1, since the 13C NMR spectrum of 2 showed essentially
the same chemical shifts as that of 1. On enzymatic hydro-
lysis of 2, an aglycone (2a) (½aꢀD +8.4) and D-glucose were
obtained. The stereochemistry of the 9-position of 2a was
found with the modified Mosher’s method to be S, and that
of the ring portion was also confirmed by the PH-NOESY
spectrum to comprise the 3R, 5R and 6S configurations
(Fig. 2). The stereochemistry of the 9-position of euodion-
oside C (3) can be deduced with the b-D-glucosylation-
induced shift-trend rule (Kasai et al., 1977) to be S, which
is the same as that of 2a, and that of the ring system was
also expected to be the same as that of 2. However, the ste-
reochemistry of the ring system must be determined inde-
pendently. Although the optical rotation value for the
aglycone (3a) (½aꢀD +5.2) of 3 was close to that of 2a, the
stereochemistry of 2a was similarly established with the
modified Mosher’s method (Fig. 2). Therefore, the struc-
tures of euodionosides B (2) and C (3) were elucidated to
be (3R,5R,6S,7E,9S)-megastigman-7-en-5,6-epoxy-3,9-diol
3- and 9-O-b-D-glucopyranosides, respectively.
26
Euodionoside A (1), ½aꢀD ꢁ40.5, was isolated as an
amorphous powder and its elemental composition was
determined to be C19H30O9 by HR-ESI-TOF-MS. The IR
spectrum indicated the presence of a sugar moiety
(3395 cmꢁ1) and a ketonic functional group (1673 cmꢁ1),
and the UV spectrum a conjugated system (235 nm). The
1H and 13C MR spectra showed the presence of six signals
assignable to b-glucopyranose, the remaining 13 carbon
signals comprising four singlet methyls, two methylenes,
one methine with a hydroxyl substituent, and four quater-
nary carbons, and a disubstituted trans double bond, which
must form the megastigmane skeleton. Judging from the
chemical shifts, one (dH 2.29) of the methyl groups must
be adjacent to the carbonyl functional group, and one
(dC 200.1) and two (dC 72.1 and 67.0) of the quaternary
carbons must have a carbonyl group and oxygen substitu-
ents, respectively. When five degrees of unsaturation and a
deficiency of one oxygen atom for three hydroxyl groups
are considered, an epoxy ring must be required for one
more cyclic system between C-5 and C-6 of megastigmane
skeleton. In the H–H COSY spectrum, H2-2 through H2-4
showed a significant correlation, and the correlations
between H-7 (dH 7.08) and H-10 (dH 2.29), and the car-
bonyl carbon (dC 200.1) in the HMBC spectrum estab-
lished a structure as 1 (Fig. 1). The hydroxyl group at the
C-3 (dC 72.1) position is the sole site to be glucosylated.
Euodionoside D (4), ½aꢀD ꢁ42.7, was isolated as an
amorphous powder and its elemental composition was
determined to be C19H34O9 by HR-ESI-TOF-MS. On anal-
yses of the NMR spectroscopic data, 4 was also expected to
be a derivative of a megastigmane glucoside with four
hydroxyl groups at the C-3, 5, 6, and 9 positions. The
HMBC correlation between the anomeric proton (dH
4.38) and C-9 (dC 75.7) established that the sugar unit

1588
M. Yamamoto et al. / Phytochemistry 69 (2008) 1586–1596
OR2
12
11
O
7
1
2
10
O
O
GlcO
R1O
13
R1 R2
1
2
3
Glc
H
H
Glc
OGlc
OR1
OH
OR
OR2
O
HO
R
R1 R2
4
5
H
6
7
Glc
H
CH3
H
Glc
O
OGlc
OGlc
GlcO
H
OH
OH
OH
O
O
8
10
OH
9
OGlc
OGlc
HO
OH
11
OH
OH
OH
O
HO
14
GlcO
12
OCH3
OH
OH
OH
13
OH
GlcO
OH
15
Fig. 1. Structures.
was linked to the hydroxyl group at the 9-position, and the
coupling pattern of H-3 (dH 4.06, dddd, J = 12, 12, 6, 6 Hz)
indicated that the hydroxyl group at the 3-position was in
an equatorial orientation. From this evidence together with
the results of comparison of the 13C NMR spectroscopic
data, the relative orientations of the substitutents of the
ring system were found to be the same as those of
(3S,5R,6R,7E,9S)-megastigman-7-ene-3,5,6,9-tetrol 9-O-b-
D-glucopyranoside isolated from Glochidion zeylanicum.
According to the b-D-glucosylation-induced shift-trend rule
(Kasai et al., 1977) the absolute configuration of the 9-posi-
tion was the same in the megastigmanes from E. meliaefo-
lium and G. zeylanicum. However, although NMR data for
the ring systems of these two compounds were essentially
the same, the stereochemistries of the side chain and the
ring system must be determined independently (Otsuka
et al., 2003a). Thus, the modified Mosher’s method was
applied to the aglycone (4a) (Fig. 2), which was shown to
have opposite absolute configurations to that from G. zey-
lanicum. Therefore, the structure of euodionoside D (4)
was elucidated to be (3R,5S,6S,7E,9S)-megastigman-7-ene-
3,5,6,9-tetrol 9-O-b-D-glucopyranoside, as shown in Fig. 1.

M. Yamamoto et al. / Phytochemistry 69 (2008) 1586–1596
1589
Table 1
13C NMR spectroscopic data for euodionosides A–G (1–7) (CD3OD, 100 MHz)
C
1
2
3
4
5
6
7
1
2
3
4
5
6
7
8
35.9
35.8
41.4
72.2
38.6
66.2
72.2
124.3
140.1
68.7
24.0
25.1
27.2
21.8
35.9
44.4
63.8
40.2
66.2
72.1
128.2
136.9
74.8
22.5
25.3
27.2
22.2
40.7
46.4
65.3
45.7
77.7
79.1
135.5
133.3
75.7
22.6
27.5
26.3
27.7
40.9
46.3
65.1
38.7
83.2
79.2
136.3
132.7
75.7
22.5
27.6
26.3
20.3
48.7
37.5
48.6
202.4
121.7
172.3
48.4
27.2
37.4 (ꢁ2.5)b
77.7 (+8.8)b
21.9 (ꢁ4.1)b
27.7
37.3
48.7
202.2
123.3
167.8
47.9
27.6
39.8
68.9
23.6
27.7
28.9
70.9
41.0 (ꢁ3.3)a
72.1 (+8.2)a
38.4 (ꢁ1.6)a
67.0
72.1
143.7
134.6
200.1
27.3
25.0
27.5
9
10
11
12
13
–OCH3
28.9
65.2
21.0
10
20
30
40
50
60
a
103.0
75.1
78.1
71.8
78.0
63.0
102.9
75.1
78.1
71.8
77.9
62.9
101.4
75.1
78.4
71.8
78.1
63.0
100.7
75.1
78.4
71.9
78.0
63.0
100.5
75.0
78.4
71.7
78.1
62.8
104.0
75.4
78.3
71.7
77.9
62.9
103.5
75.1
78.3
71.7
78.2
62.9
Dd1–1a
.
b
Dd6–7a
.
+0.010
+0.018
—0.001
OMTPA
—0.010
—0.057
O
—
—0.037
—0.060
+0.069
—
—0.002
+0.063
O
OMTPA
O
MTPAO
—0.103
—0.060
—0.019
—0.038
—0.066
—0.103
7b,c
1b,c
OMTPA
OMTPA
+0.059
+0.059
+0.098
+0.057
+0.058
+0.098
+0.088
+0.094
+0.087
+0.092
+0.076
+0.010
—0.049
+0.073
—0.049
O
O
MTPAO
MTPAO
+0.010
—0.085
—0.047
—0.086
—0.048
3b,c
2b,c
OMPTA
OMPTA
+0.056
+0.038
+0.055
+0.057
—0.024
+0.079
+0.086
+0.082
+0.102
+0.083
+0.099
+0.088
—0.051
—0.043
OH
OH
OH
MTPAO
OCH3
—0.001
MTPAO
—0.060
—0.078
—0.076
—0.087
—0.014
0.000
5b,c
4b,c
Fig. 2. Results with the modified Mosher’s method (DdS–dR).

1590
M. Yamamoto et al. / Phytochemistry 69 (2008) 1586–1596
Euodionoside E (5), ½aꢀD ꢁ35.0, was isolated as an
for the H2-7 and eight protons could not be calculated,
due to overlapping of their signals, they obviously had
minus signs, whereas that for the H3-10 protons had a sig-
nificant plus value (+0.063) (Fig. 2). As a result, the struc-
ture of euodionoside G (7) was elucidated to be (6R,9R)-
megastigman-4-ene-9,13-diol 13-O-b-D-glucopyranoside.
A compound closely related to 6 has been isolated from
G. zeylanicum as glochidionionoside B, which has the 9S
configuration (Otsuka et al., 2003).
amorphous powder, and the one- and two-dimensional
NMR spectroscopic data showed that it was a compound
analogous to euodionoside D (4) with similar substituents
on the six-membered ring. In the NMR spectra, a methoxyl
signal was observed [dH 3.21 (3H, s) and dC 48.7 (q)], and
the presence of an extra carbon atom was also supported
by the HR-ESI-TOF-MS data (C20H36O9). From the
HMBC spectroscopic correlation between dH 3.21 and dC
83.2 (C-5), the position of the methoxyl group was assigned
as C-5, and from the PH-NOESY spectrum, the relative
orientations of the substituents were found to be the same
as those of 4. The absolute configurations of the ring sys-
tem were also determined by the application of the modi-
fied Mosher’s method to the aglycone (5a) (Fig. 2).
Therefore, the structure of euodionoside E (5) was eluci-
dated to be (3R,5S,6S,7E,9S)-megastigman-7-ene-5-meth-
3. Concluding remarks
Although fruits of closely related species, E. rutaecarpa,
are used for stomachic antipyretic and diuretic in tradi-
tional Chinese medicine, there has been no report on the
medicinal use of E. meloaefolia. Due to usage of leaves
for this experiment, alkaloids are so far not isolated. Bio-
logical evaluation and chemical investigation of fruits of
E. meloaefolia will be subject of a future study.
Megastigmane and its glycosides are currently an
expanding class of compounds. Even with only 13 carbon
atoms in the basic skeleton of megastigmane, several oxida-
tion steps and glycosylation afforded many kinds of meg-
astigmane derivatives and their glycosidic forms. In this
experiment, seven new megastigmane glucosides were iso-
lated from leaves of the title plant. Their stereostructures
were established by the modified Mosher’s method.
oxy-3,6,9-triol 9-O-b-D-glucopyranoside.
A
similar
megastigmane derivative, which has a methoxyl group at
the 6-position, was isolated as staphylionoside J from
Staphylea bumalda (Yu et al., 2005).
Euodionosides F (6), ½aꢀD ꢁ0.7 and G (7), ½aꢀD +5.9,
were isolated as amorphous powders and their elemental
compositions were determined to be C19H32O8 by HR-
ESI-TOF-MS. The IR (1649 and 1651 cmꢁ1, respectively)
and UV (238 and 237 nm, respectively) spectra showed
the presence of a conjugated ketone, their NMR spectra
being similar, with two singlet and one doublet methyls,
and one primary and one secondary alcohol. Thus, one
of the methyl groups was expected to be oxidized to a pri-
mary alcohol, whose proton signals (dH 4.16 and 4.32, and
dH 4.37 and 4.53, respectively) showed correlation to C-5
resonances (dC 172.3 and dC 167.8) in the HMBC spectra.
These facts were coincident with that C-13 was oxidized to
a primary alcohol. The HMBC spectra also indicated that 6
and 7 were isomers of each other as to the positions of
sugar linkages, the former had a sugar on the hydroxyl
group at the 9-position and the latter on that at the 13-
position, since correlation between anomeric protons and
C-9 (dC 77.3) in 6, and C-13 (dC 70.9) in 7 was observed,
respectively. The absolute configuration of the 9-position
of 6 was determined to be R on comparison with a similar
compound (Takeda et al., 1997) and that of the 6-position
was also assigned as R according to the circular dichroism
(CD) spectral data (Otsuka et al., 2003b). The absolute
configuration of the 9-position was further supported by
application of the b-D-glucosylation-induced shift-trend
rule (Table 1, b) (Otsuka et al. (1995)), Therefore, the struc-
ture of euodionoside F (6) was elucidated to be (6R,9R)-
4. Experimental
4.1. General experimental procedures
Optical rotations and CD spectra 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,
1
respectively. H and 13C NMR spectra were taken on a
JEOL JNM a-400 spectrometer at 400 MHz and
100 MHz, respectively, with tetramethylsilane as an inter-
nal standard. Negative-ion HR-MS was performed with a
JEOL SX-102 spectrometer in the FAB mode and posi-
tive-ion HR-MS with an Applied Biosystem QSTAR XL
system ESI (Nano Spray)-TOF-MS.
A highly-porous synthetic resin (Diaion HP-20) was
purchased from Mitsubishi Kagaku (Tokyo, Japan). Silica
gel CC and reversed-phase [octadecyl silica gel (ODS)]
open CC were performed on silica gel 60 (Merck, Darms-
tadt, Germany) and Cosmosil 75C18-OPN (Nacalai Tes-
que, Kyoto, Japan) [U = 50 mm, L = 25 cm, linear
gradient: MeOH–H2O (1:9, 1 L) ? (1:1, 1 L), fractions of
10 g being collected], respectively. Droplet counter-current
chromatography (DCCC) (Tokyo Rikakikai, Tokyo,
Japan) was equipped with 500 glass columns (U = 2 mm,
L = 40 cm), and the lower and upper layers of a solvent
mixture of CHCl3–MeOH–H2O–n-PrOH (9:12:8:2) were
megastigman-4-ene-9,13-diol
9-O-b-D-glucopyranoside.
Since euodionoside G (7) was an isomeric form of 6, the
absolute configuration of the 9-position was expected to
be the same as that of 6. However, for the same reason
as that for euodionoside D (4), the absolute configurations
of the 6- and 9-positions were independently established by
CD spectral analysis and the modified Mosher’s method
(Fig. 2), respectively. Although the exact DdS–dR values

M. Yamamoto et al. / Phytochemistry 69 (2008) 1586–1596
1591
used for the stationary and mobile phases, respectively.
Five-gram fractions were collected and numbered accord-
ing to their order of elution with the mobile phase. HPLC
was performed on an ODS column (Inertsil; GL Science,
Tokyo, Japan; U = 6 mm, L = 25 cm), and the eluate was
monitored with a UV detector at 254 nm and a refractive
index monitor.
Emulsin was purchased from Tokyo Chemical Indus-
tries Co. Ltd. (Tokyo, Japan), and crude hesperidinase
was a gift from Tokyo Tanabe Pharmaceutical Co. Ltd.
(Tokyo, Japan). (R)- and (S)-a-methoxy-a-trifluoromethyl-
phenylacetic acids (MTPA) were the products of Wako
Pure Chemical Industry Co. Ltd. (Tokyo, Japan).
fraction with CH3CN–H2O (3:17) gave 1.4 mg of 6 and
2.8 mg of 7 at Rts 16 min and 18 min, respectively. The lat-
ter was found to be a pure compound, 14.
The residue (1.50 g out of 3.03 g) in fractions 40–45
obtained on 15–17.5% MeOH eluate on silica gel CC was
subjected to RPCC to give five fractions, 172 mg in frac-
tions 37–47, 87.0 mg in fractions 62–65, 150 mg in fractions
81–89, 116 mg in fractions 98–104, and 101 mg in fractions
112–123. The first fraction was purified by DCCC to give
144 mg of 15 in fractions 21–29. The second fraction was
purified by DCCC (40.1 mg in fractions 47–57) and then
prep. HPLC with MeOH–H2O (1:4) to afford 5.4 mg of 8
at Rt 30 min. The third fraction was subjected to prep.
HPLC with MeOH–H2O (1:3) to give two partially purified
fractions at Rts 34 min (36.0 mg) and 42 min (59.4 mg). The
former was repeatedly purified by prep. HPLC with
CN3CN–H2O (1:9) to give 15.3 mg of 9 and 5.2 mg of 10
at Rts 56 min and 68 min, respectively. The latter gave
21.0 mg of 2 on prep. HPLC with CH3CN–H2O (13:87)
at Rt 44 min. The residue of the fourth fraction was found
to be a pure compound, 3. The fifth fraction was subjected
to prep. HPLC with MeOH–H2O (19:31) to give 8.5 mg of
6 at Rt 18 min.
4.2. Plant material
Leaves of E. meliaefolia Benth. (Rutaceae) were col-
lected in Okinawa, Japan, in August 2002, and a voucher
specimen was deposited in the Herbarium of Pharmaceuti-
cal Sciences, Graduate School of Biomedical Sciences,
Hiroshima University (02-EM-Okinawa-0704).
4.3. Extraction and isolation
The residue (1.61 g) in fractions 46–51 obtained from
the 17.5–20% MeOH eluate on silica gel CC was subjected
to RPCC to give two fractions, 130 mg in fractions 40–46
and 20.0 mg in fractions 146–153. The former was further
purified by DCCC to give 29.6 mg of 15 in fractions 53–
58 and the latter was a pure compound, 5.
Dried leaves of E. meliaefolia (15.7 kg) were extracted
three times with MeOH (45 l) at 25 °C for one week and
then concentrated to 6 l in vacuo. The extract was washed
with n-hexane (6 l, 531 g) and then the MeOH layer was
concentrated to a gummy mass. The latter was suspended
in water (6 l) and then extracted with EtOAc (6 l) to give
165 g of an EtOAc-soluble fraction. The aqueous layer
was extracted with 1-BuOH (6 l) to give a 1-BuOH-soluble
fraction (409 g), and the remaining water-layer was concen-
trated to furnish 971 g of a water-soluble fraction.
4.4. Characterization data
4.4.1. Euodionoside A (1)
26
Amorphous powder; ½aꢀD ꢁ40.5 (c = 0.25, MeOH); IR
A portion (154 g) of the 1-BuOH-soluble fraction was
mmax (film) cmꢁ1: 3395, 2926, 1673, 1455, 1367, 1076; UV
1
subjected to
a
Diaion HP-20 column (U = 50 mm,
kmax (MeOH) nm (loge): 235 (3.75); H NMR (CD3OD,
L = 50 cm) using H2O–MeOH (4:1, 8 l), (2:3, 8 l), (3:2,
8 l), and (1:4, 8 l), and MeOH (6 l), 1 l fractions were being
collected. The residue (27.6 g in fractions 6–12) of the 20–
40% MeOH eluent was subjected to silica gel (1.50 kg)
CC, with elution with CHCl3 (6 l) and CHCl3–MeOH
[(99:1, 6 l), (97:3, 6 l), (19:1, 6 l), (37:3, 6 l), (9:1, 6 l), (7:1,
6 l), (17:3, 6 l), (33:7, 6 l), (4:1, 6 l), (31:9, 6 l), (3:1, 6 l),
and (7:3, 6 l)], 1 l fractions being collected. Combined frac-
tions 31–39 (1.20 g) were separated by RPCC. The residue
(383 mg) of fractions 62–78 was subjected to DCCC to give
25.4 mg of 11 in fractions 35–40, and the residue (44.5 mg)
in fractions 44–52 was further purified by prep. HPLC with
MeOH–H2O (3:7) to give 7.6 mg of 13 at Rt 10 min. The
residue (112 mg) in fraction 94–114 obtained on RPCC
was subjected to DCCC to give 3.9 mg of 1 in fractions
63–72, and the residue (10.6 mg in fractions 21–27) was fur-
ther purified by prep. HPLC with MeOH–H2O (3:7) to give
3.9 mg of 4 at Rt 13 min. The residue (85 mg) in fractions
115–132 obtained on RPCC was also subjected to DCCC
to give two fractions, 10.2 mg in fractions 90–105 and
37.3 mg in fractions 109–136. Prep. HPLC of the former
400 MHz) d: 7.08 (1H, d, J = 16 Hz, H-7), 6.20 (1H, d,
J = 16 Hz, H-8), 4.31 (1H, d, J = 8 Hz, H-10), 3.98 (1H,
dddd, J = 11, 10, 7, 7 Hz, H-3), 3.87 (1H, dd, J = 12,
2 Hz, H-60a), 3.65 (1H, dd, J = 12, 6 Hz, H-60b), 3.42–
3.25 (3H, m, H-30,40 and 50), 3.12 (1H, dd, J = 9, 8 Hz,
H-20), 2.38 (1H, dd, J = 15, 7 Hz, H-4pseudo-eq), 2.29
(3H, s, H3-10), 1.98 (1H, dd, J = 15, 10 Hz, H-4pseudo-
ax), 1.50 (1H, dd, J = 11, 7 Hz, H-2eq), 1.49 (1H, dd,
J = 11, 11 Hz, H-2ax), 1.25 (3H, s, H3-11), 1.17 (3H, s,
H3-13), 0.97 (3H, s, H3-12); 13C NMR (CD3OD,
100 MHz): Table 1; HR-ESI-TOF-MS (positive-ion mode)
m/z: 409.1856 [M+Na]+ (calcd for C19H30O8Na, 409.1832).
4.4.2. Euodionoside B (2)
25
Amorphous powder; ½aꢀD –24.8 (c = 0.79, MeOH); IR
1
mmax (film) cmꢁ1: 3367, 2965, 1653, 1453, 1379, 1075; H
NMR (CD3OD, 400 MHz) d: 5.84 (1H, dd, J = 16, 1 Hz,
H-7), 5.67 (1H, dd, J = 16, 6 Hz, H-8), 4.30 (1H, d,
J = 8 Hz, H-10), 4.29 (1H, qdd, J = 6, 6, 1 Hz, H-9), 3.94
(1H, dddd, J = 11, 10, 7, 6 HZ, H-3), 3.86 (1H, dd,
J = 12, 2 Hz, H-6b), 3.65 (1H, dd, J = 12, 6 Hz, H-60b),

1592
M. Yamamoto et al. / Phytochemistry 69 (2008) 1586–1596
3.36–3.25 (3H, m, H-30, 40 and 50), 3.12 (1H, dd, J = 9,
8 Hz, H-20), 2.32 (1H, ddd, J = 15, 7, 1 Hz, H-4pseudo-
eq), 1.89 (1H, dd, J = 15, 10 Hz, H-4pseudo-ax), 1.49
(2H, m, H2-2), 1.23 (3H, d, J = 6 Hz, H3-10), 1.20 (3H, s,
H3-13), 1.16 (3H, s, H3-11), 0.97 (3H, s, H3-12); 13C
NMR (CD3OD, 100 MHz): Table 1; HR-FAB-MS (nega-
tive-ion mode) m/z: 387.2028 [MꢁH]ꢁ (calcd for
C19H31O8, 387.2019).
4.4.6. Euodionoside F (6)
24
Amorphous powder; ½aꢀD ꢁ0.70 (c = 0.55, MeOH); IR
mmax (film) cmꢁ1: 3396, 2930, 1649, 1076, 1037; UV kmax
(MeOH) nm (loge): 238 (3.88); 1H NMR (CD3OD,
400 MHz) d: 6.60 (1H, br s, H-4), 4.33 (1H, d, J = 8 Hz,
H-10), 4.32 (1H, dd, J = 18, 1 Hz, H-13a), 4.16 (1H, dd,
J = 18, 1 Hz, H-13b), 3.84 (1H, m, H-60a), 3.82 (1H, m,
H-9), 3.67 (1H, dd, J = 11, 6 Hz, H-60b), 3.36–3.26 (3H,
m, H-30, 40 and 50), 3.15 (1H, dd, J = 9, 8 Hz, H-20), 2.58
(1H, d, J = 17 Hz, H-2a), 2.03 (1H, d, J = 17 Hz, H-2b),
1.92 (1H, m, H-6), 1.80-1.52 (4H, m, H2-7 and 8), 1.24
(3H, d, J = 6 Hz, H3-10), 1.11 (3H, s, H3-12), 1.02 (3H, s,
H3-11); 13C NMR (CD3OD, 100 MHz): Table 1; CD nm
(De): 219 (+2.24), 278 (ꢁ0.11), 335 (+0.51)
(c = 3.94 ꢂ 10ꢁ5 M, MeOH); HR-ESI-TOF-MS (positive-
ion mode) m/z: 411.1983 [M+Na]+ (calcd for C19H32O8Na,
411.1989).
4.4.3. Euodionoside C (3)
24
Amorphous powder; ½aꢀD ꢁ56.3 (c = 7.20, MeOH); IR
1
mmax (film) cmꢁ1: 3395, 2965, 1649, 1452, 1368, 1075; H
NMR (CD3OD, 400 MHz) d: 6.01 (1H, dd, J = 16, 1 Hz,
H-7), 5.57 (1H, ddd, J = 16, 7, 1 Hz, H-8), 4.52 (1H, br
dq, J = 7, 6 Hz, H-9), 4.28 (1H, d, J = 8 Hz, H-10), 3.86
(1H, dd, J = 12, 2 Hz, H-60a), 3.81 (1H, m, H-3), 3.64
(1H, dd, J = 12, 6 Hz, H-60b), 3.32–3.14 (4H, m, H-20, 30,
40 and 50), 2.21 (1H, dd, J = 15, 7 Hz, H-4pseudo-eq),
1.71 (1H, dd, J = 15, 10 Hz, H-4pseudo-ax), 1.46 (1H, dd,
J = 12, 12 Hz, H-2ax), 1.28 (3H, d, J = 6 Hz, H3-10),
1.27 (1H, m, H-2eq), 1.23 (3H, s, H3-13), 1.17 (3H, s, H3-
11), 0.96 (3H, s, H3-12); 13C NMR (CD3OD, 100 MHz):
Table 1; HR-FAB-MS (negative-ion mode) m/z: 387.2021
[MꢁH]ꢁ (calcd for C19H31O8, 387.2018).
4.4.7. Euodionoside G (7)
27
Amorphous powder; ½aꢀD +5.9 (c = 0.19, MeOH); IR
mmax (film) cmꢁ1: 3367, 2927, 1651, 1077, 1040; UV kmax
(MeOH) nm (loge): 237 (3.57); 1H NMR (CD3OD,
400 MHz) d: 6.17 (1H, br s, H-4), 4.53 (2H, dd, J = 18,
2 Hz, H-13a), 4.37 (1H, dd, J = 18, 2 Hz, H-13b), 4.33
(1H, d, J = 8 Hz, H-10), 3.88 (1H, dd, J = 12, 2 Hz, H-60a),
3.68 (1H, m, H-9), 3.62 (1H, dd, J = 12, 6 Hz, H-60b),
3.36–3.15 (4H, m, H-20, 30, 40 and 50), 2.54 (1H, d,
J = 18 Hz, H-2a), 2.03 (1H, t, J = 6 Hz, H-6), 2.03 (1H, d,
J = 18 Hz, H-2b), 1.80–1.52 (4H, m, H2-7 and 8), 1.60 (3H,
d, J = 6 Hz, H3-10), 1.11 (3H, s, H3-11), 1.03 (3H, s, H3-
12); 13C NMR (CD3OD, 100 MHz): Table 1; CD nm (De):
235 (+1.78), 276 (ꢁ0.21), 337 (+0.29) (c = 3.22 ꢂ 10ꢁ5 M,
MeOH); HR-ESI-TOF-MS (positive-ion mode) m/z:
411.1995 [M+Na]+ (calcd for C19H32O8Na, 411.1989).
4.4.4. Euodionoside D (4)
24
Amorphous powder; ½aꢀD ꢁ42.7 (c = 0.28, MeOH); IR
1
mmax (film) cmꢁ1: 3395, 2927, 1649, 1372, 1072, 1027; H
NMR (CD3OD, 400 MHz) d: 6.16 (1H, d, J = 16 Hz, H-
7), 5.63 (1H, dd, J = 16, 8 Hz, H-8), 4.52 (1H, dq, J = 8,
7 Hz, H-9), 4.38 (1H, d, J = 8 Hz, H-10), 4.06 (1H, dddd,
J = 12, 12, 6, 6 Hz, H-3), 3.87 (1H, dd, J = 12, 2 Hz, H-
60a), 3.65 (1H, dd, J = 12, 6 Hz, H-60b), 3.35–3.18 (4H,
m, H-20, 30, 40 and 50), 1.76 (2H, m, H2-4), 1.65 (1H, dd,
J = 12, 12 Hz, H-2ax), 1.45 (1H, ddd, J = 12, 4, 2 Hz, H-
2eq), 1.31 (3H, d, J = 7 Hz, H3-10), 1.173 (3H, s, H3-12),
1.165 (3H, s, H3-13), 0.85 (3H, s, H3-11); 13C NMR
(CD3OD, 100 MHz): Table 1; HR-ESI-TOF-MS (posi-
tive-ion mode) m/z: 429.2098 [M+Na]+ (calcd for
C19H34O9Na, 429.2095).
4.4.8. Enzymatic hydrolysis of euodionosides A (1)–E (5)
and G (7)
Euodionoside A (1) (3.8 mg) in 2 ml of H2O was hydro-
lyzed with emulsin (6.0 mg) and crude hesperidinase
(6.0 mg) for 15 h at 37 °C. The reaction mixture was evap-
orated to dryness, and then the methanolic solution was
absorbed on silica gel and subjected to silica gel CC
(20 g, U = 18 mm, L = 18 cm) with CHCl3 (100 ml) and
CHCl3–MeOH (19:1, 100 ml, 9:1, 100 ml, 17:3, 100 ml
and 7:3, 300 ml), 12 ml fractions being collected. An agly-
cone (1a) (1.8 mg, 82%) and D-glucose (1.3 mg, 77%) were
recovered in fractions 15–25 and 37–47, respectively. Agly-
4.4.5. Euodionoside E (5)
24
Amorphous powder; ½aꢀD ꢁ35.0 (c = 1.13, MeOH); IR
1
mmax (film) cmꢁ1: 3395, 2931, 1635, 1370, 1077, 1035; H
NMR (CD3OD, 400 MHz) d: 6.13 (1H, d, J = 16 Hz, H-
7), 5.59 (1H, dd, J = 16, 9 Hz, H-8), 4.49 (1H, dq, J = 9,
6 Hz, H-9), 4.37 (1H, d, J = 8 Hz, H-10), 3.85 (1H, dd,
J = 12, 2 Hz, H-60a), 3.83 (1H, dddd, J = 12, 12, 4, 4 Hz,
H-3), 3.67 (1H, dd, J = 12, 6 Hz, H-60b), 3.33–3.18 (4H,
m, H-20, 30, 40 and 50), 3.21 (3H, s, –OCH3), 2.10 (1H,
ddd, J = 12, 4, 2 Hz, H-4eq), 1.65 (1H, dd, J = 12, 12 Hz,
H-2ax), 1.57 (1H, dd, J = 14, 12 Hz, H-4ax), 1.45 (1H,
ddd, J = 12, 4, 2 Hz, H-2eq), 1.30 (3H, d, J = 6 Hz, H3-
10), 1.11 (3H, s, H3-13), 1.10 (3H, s, H3-12), 0.85 (3H, s,
H3-11); 13C NMR (CD3OD, 100 MHz): Table 1; HR-
ESI-TOF-MS (positive-ion mode) m/z: 443.2244
[M+Na]+ (calcd for C20H36O9Na: 443.2251).
25
cone (1a): An amorphous powder, ½aꢀD +27.9 (c = 0.12,
MeOH); 1H NMR (CD3OD, 400 MHz) d: 7.08 (1H, d,
J = 16 Hz, H-7), 6.20 (1H, d, J = 16 Hz, H-8), 3.85 (1H,
dddd, J = 12, 10, 7, 4 Hz, H-3), 2.28 (3H, s, H3-10), 2.24
(1H, ddd, J = 15, 7, 2 Hz, H-4pseudo-eq), 1.73 (1H, dd,
J = 15, 10 Hz, H-4pseudo-ax), 1.48 (1H, dd, J = 12,
12 Hz, H-2ax), 1.33 (1H, ddd, J = 12, 4, 2 Hz, H-2eq),
1.24 (3H, s, H3-11) 1.17 (3H, s, H3-13), 0.96 (3H, s, H3-
12); 13C NMR (CD3OD, 100 MHz) d: 143.8 (C-7), 134.6
(C-8), 63.9 (C-3), 44.3 (C-2), 40.0 (C-4), 36.0 (C-1), 27.6

M. Yamamoto et al. / Phytochemistry 69 (2008) 1586–1596
1593
27
(C-12), 27.3 (C-10), 25.2 (C-11), 21.4 (C-13), singlet car-
bons (C-5, 6 and 9) were not observed; HR-ESI-TOF-MS
D-Glucose, ½aꢀD +25.0 (c = 0.073, H2O, 24 h after being
dissolved in the solvent).
From euodionoside B (5) (10.4 mg), 5.3 mg (83%) of an
aglycone (5a) and 3.5 mg (78%) of D-glucose were obtained
(positive-ion mode) m/z: 247.1307 [M+Na]+ (calcd for
25
C13H20O3Na, 247.1304). D-Glucose: ½aꢀD
+32.2°
25
(c = 0.087, H2O, 24 h after being dissolved in the solvent).
From euodionoside B (2) (12 mg), 6.8 mg (91%) of an
aglycone (2a) and 4.9 mg (83%) of D-glucose were obtained:
Aglycone (5a): An amorphous powder; ½aꢀD +25.5°
1
(c = 0.35, MeOH); H NMR (CD3OD, 400 MHz) d: 5.95
(1H, d, J = 16 Hz, H-7), 5.65 (1H, dd, J = 16, 9 Hz, H-8),
4.21 (1H, m, H-9), 3.73 (1H, m, H-3), 3.09 (3H, s, -OMe),
1.99 (1H, ddd, J = 14, 4, 2 Hz, H-4eq), 1.55 (1H, dd,
J = 12, 12 Hz, H-2ax), 1.45 (1H, dd, J = 14, 12 Hz, H-
4ax), 1.35 (1H, ddd, J = 12, 4, 2 Hz, H-2eq), 1.15 (3H, d,
J = 6 Hz, H3-10), 1.02 (3H, s, H3-12), 0.97 (3H, s, H3-
13), 0.74 (3H, s, H3-11); 13C NMR (CD3OD, 100 MHz):
d 135.6 (C-7), 131.5 (C-8), 83.3 (C-5), 79.0 (C-6), 69.7 (C-
9), 65.2 (C-3), 48.4 (–OCH3), 46.5 (C-2), 40.9 (C-1), 38.8
(C-4), 27.7 (C-11), 26.4 (C-12), 24.2 (C-10), 19.7 (C-13);
HR-ESI-TOFMS (positive-ion mode) m/z: 281.1729
[M+Na]+ (calcd for C14H26O4Na, 281.1723). D-Glucose,
25
Aglycone (2a): an amorphous powder; ½aꢀD +8.4 (c = 0.31,
1
MeOH); H NMR (CD3OD, 400 MHz) d: 5.85 (1H, dd,
J = 16, 1 Hz, H-7), 5.67 (1H, dd, J = 16, 6 Hz, H-8), 4.30
(1H, qd, J = 6, 1 Hz, H-9), 3.81 (1H, dddd, J = 12, 10, 7,
4 Hz, H-3), 2.20 (1H, ddd, J = 15, 7, 2 Hz, H-4pseudo-
ax), 1.71 (1H, dd, J = 15, 10 Hz, H-4pseuo-eq), 1.46 (1H,
dd, J = 12, 12 Hz, H-2ax), 1.28 (1H, ddd, J = 12, 4, 2 Hz,
H-2eq), 1.23 (3H, d, J = 6 Hz, H3-10), 1.20 (3H, s, H3-
13), 1.15 (3H, s, H3-11), 0.95 (3H, s, H3-12); 13C NMR
(CD3OD, 100 MHz) d: 140.1 (C-8), 124.4 (C-7), 72.0 (C-
6), 68.7 (C-9), 66.1 (C-5), 63.8 (C-3), 44.5 (C-2), 40.2 (C-
4), 36.0 (C-1), 27.2 (C-12), 25.3 (C-11), 23.9 (C-10), 21.8
(C-13); HR-ESI-TOFMS (positive-ion mode) m/z:
249.1466 [M+Na]+ (calcd for C13H22O5Na, 249.1461). D-
25
½aꢀD +49.7 (c = 0.23, H2O, 24 h after being dissolved in
the solvent).
From euodionoside G (7) (2.5 mg), 1.2 mg (82%) of an
aglycone (7a) and 1.0 mg (86%) of D-glucose were obtained.
24
Glucose, ½aꢀD +41.3° (c = 0.33, H2O, 24 h after being dis-
25
solved in the solvent).
Aglycone (7a): An amorphous powder; ½aꢀD +61.7°
1
From euodionoside B (3) (17 mg), 5.2 mg (49%) of an
aglycone (3a) and 5.8 mg (69%) of D-glucose were obtained.
Aglycone (3a): An amorphous powder; ½aꢀ2D4 +5.2 (c = 0.41,
(c = 0.08, MeOH); H NMR (CD3OD, 400 MHz) d: 6.06
(1H, br s, H-4), 4.30 (1H, dd, J = 18, 2 Hz, H-13), 4.15
(1H, dd, J = 18, 2 Hz, H-13), 3.66 (1H, qd, J = 6, 6 Hz,
H-9), 2.54 (1H, dd, J = 18, 1 Hz, H-2), 2.03 (1H, ddd,
J = 18, 1, 1 Hz, H-2), 1.94 (1H, dd, J = 5, 5 Hz, H-6),
1.821.49 (4H, m, H2-7, 8), 1.16 (3H, d, J = 6 Hz, H3-10),
1.11 (3H, s, H3-11),1.02 (3H, s, H3-12); 13C NMR
(CD3OD, 100 MHz) d: 172.3 (C-5), 121.7 (C-4), 68.9
(C-9), 65.1 (C-13), 48.7 (C-2), 48.4 (C-6), 39.9 (C-8), 37.4
(C-1), 28.9 (C-12), 28.0 (C-7), 27.6 (C-11), 23.6 (C-10);
HR-ESI-TOF-MS (positive-ion mode) m/z: 249.1465
[M+Na]+ (calcd for C13H22O2Na, 249.1461). D-Glucose,
1
MeOH); H NMR (CD3OD, 400 MHz) d: 5.85 (1H, dd,
J = 16, 1 Hz, H-7), 5.67 (1H, dd, J = 16, 6 Hz, H-8), 4.30
(1H, qd, J = 6, 1 Hz, H-9), 3.81 (1H, dddd, J = 12, 10, 7,
4 Hz, H-3), 2.20 (1H, ddd, J = 15, 7, 2 Hz, H-4pseudo-
eq), 1.71 (1H, dd, J = 15, 10 Hz, H-4pseudo-ax), 1.46
(1H, dd, J = 12, 12 Hz, H-2ax), 1.28 (1H, ddd, J = 12, 4,
2 Hz, H-2eq), 1.23 (3H, d, J = 6 Hz, H3-10), 1.20 (3H, s,
H3-13), 1.15 (3H, s, H3-11), 0.95 (3H, s, H3-12); 13C
NMR (CD3OD, 100 MHz) d: 140.1 (C-8), 124.4 (C-7),
72.0 (C-6), 68.7 (C-9), 66.1 (C-5), 63.8 (C-3), 44.5 (C-2),
40.3 (C-4), 36.0 (C-1), 27.2 (C-12), 25.3 (C-11), 23.9 (C-
10), 21.8 (C-13); HR-ESI-TOFMS (positive-ion mode) m/
z: 249.1466 [M+Na]+ (calcd for C13H22O5Na, 249.1464).
27
½aꢀD +20.4 (c = 0 .07, H2O, 24 h after being dissolved in
the solvent).
4.4.9. Preparation of (R)- and (S)-MPTA esters
27
D-Glucose, ½aꢀD +51.6 (c = 0.38, H2O, 24 h after being dis-
A solution of 1a (0.9 mg) in 1 ml of dehydrated
CH2Cl2 was reacted with (R)-MTPA (27 mg) in the pres-
ence of 1-ethyl-3-(3-dimethylaminopropyl)cardodiimide
hydrochloride (EDC) (16 mg) and N,N-dimethyl-4-ami-
nopyridine (4-DMAP) (11 mg), and then the mixture
was 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 CHCl3–MeOH (9:1)] to fur-
nish an ester, 1b (1.2 mg, 68%). Through a similar proce-
dure, 1c (1.3 mg, 73%) was prepared from 1a (0.9 mg)
using (S)-MTPA (27 mg), EDC (17 mg), and 4-DMAP
(12 mg).
solved in the solvent).
From euodionoside B (4) (3.8 mg), 1.8 mg (79%) of an
aglycone (4a) and 1.1 mg (65%) of D-glucose were
30
obtained. Aglycone (4a): An amorphous powder; ½aꢀD
1
+18.6 (c = 0.19, MeOH); H NMR (CD3OD, 400 MHz)
d: 6.06 (1H, d, J = 16 Hz, H-7), 5.63 (1H, dd, J = 16,
6 Hz, H-8), 4.34 (1H, qd, J = 6, 1 Hz, H-9), 4.05 (3H,
m, H-3), 1.78 (1H, ddd, J = 13, 5, 2 Hz, H-4eq), 1.73
(1H, dd, J = 13, 12 Hz, H-4ax), 1.65 (1H, dd, J = 12,
12 Hz, H-2ax), 1.44 (1H, ddd, J = 12, 4, 2 Hz, H-2eq),
1.27 (3H, d, J = 6 Hz, H3-10), 1.20 (3H, s, H3-12), 1.14
(3H, s, H3-13), 0.84 (3H, s, H3-11); 13C NMR (CD3OD,
100 MHz) d: 136.2 (C-7), 131.3 (C-8), 83.6 (C-6), 77.8
(C-5), 69.6 (C-9), 65.3 (C-3), 46.5 (C-2), 45.8 (C-4),
40.8 (C-1), 27.5 (C-11), 27.1 (C-13), 26.3 (C-12), 24.3
(C-10); HR-ESI-TOFMS (positive-ion mode) m/z:
267.1554 [M+Na]+ (calcd for C13H24O4Na, 247.1566).

1594
M. Yamamoto et al. / Phytochemistry 69 (2008) 1586–1596
(3R,5R,6S,7E)-Megastigman-7-en-5,6-epoxy-3-ol-9-one
(3R,5R,6S,7E,9S)-Megastigman-7-en-5,6-epoxy-3,9-diol
3,9-O-(R)-MTPA diester (3b): An amorphous powder; H
3-O-(R)-MTPA ester (1b): An amorphous powder; 1H
NMR (CDCl3, 400 MHz) d: 7.72–7.70 (2H, m, aromatic
protons), 7.45–7.40 (3H, m, aromatic protons), 6.94 (1H,
d, J = 16 Hz, H-7), 6.30 (1H, d, J = 16 Hz, H-8), 5.17
(1H, m, H-3), 3.53 (3H, br s, –OCH3), 2.41 (1H, ddd,
J = 15, 8, 2 Hz, H-4pseudo-eq), 2.28 (3H, s, H3-10), 2.03
(1H, dd, J = 15, 10 Hz, H-4pseudo-ax), 1.70 (1H, dd,
J = 12, 12 Hz, H-2ax), 1.41 (1H, m, H-2eq), 1.29 (3H, s,
H3-11), 1.20 (3H, s, H3-13), 0.99 (3H, s, H3-12); HR-ESI-
TOF-MS (positive-ion mode) m/z: 463.1700 [M+Na]+
(calcd for C26H27O5F3Na, 463.1708).
1
NMR (CDCl3, 400 MHz) d: 7.517.50 (4H, m, aromatic
protons), 7.407.36 (6H, m, aromatic protons), 5.98 (1H,
d, J = 15 Hz, H-7), 5.74 (1H, dd, J = 15, 7 Hz, H-8), 5.64
(1H, dq, J = 7, 7 Hz, H-9), 5.12 (1H, dddd, J = 12, 10, 8,
4 Hz, H-3), 3.55 (3H, br s, –OCH3), 3.52 (3H, br s,
–OCH3), 2.35 (1H, ddd, J = 15, 8, 2 Hz, H-4eq), 1.98
(1H, dd, J = 15, 10 Hz, H-4ax), 1.66 (1H, dd, J = 12,
12 Hz, H-2ax), 1.43 (3H, d, J = 7 Hz, H3-10), 1.35 (1H,
ddd, J = 12, 4, 2 Hz, H-2eq), 1.13 (3H, s, H3-11), 1.13
(3H, s, H3-13), 0.91 (3H, s, H3-12); HR-ESI-TOF-MS
(positive-ion mode) m/z: 681.2250 [M+Na]+ (calcd for
C33H36O7F6Na, 681.2257).
(3R,5R,6S,7E)-Megastigman-7-en-5,6-epoxy-3-ol-9-one
3-O-(S)-MTPA ester (1c): An amorphous powder, 1H
NMR (CDCl3, 400 MHz) d: 7.70–7.69 (2H, m, aromatic
protons), 7.54–7.51 (3H, m, aromatic protons), 6.94 (1H,
d, J = 16 Hz, H-7), 6.30 (1H, d, J = 16 Hz, H-8), 5.17
(1H, m, H-3), 3.53 (3H, q, J = 1 Hz, -OCH3), 2.35 (1H,
ddd, J = 15, 8, 2 Hz, H-4pseudo-eq), 2.28 (3H, s, H3-10),
1.92 (1H, dd, J = 15, 9 Hz, H-4pseudo-ax), 1.77 (1H, dd,
J = 12, 12 Hz, H-2ax), 1.46 (1H, m, H-2eq), 1.30 (3H, s,
H3-11), 1.18 (3H, s, H3-13),1.00 (3H, s, H3-12); HR-ESI-
TOF-MS (positive-ion mode) m/z: 463.1705 [M+Na]+
(calcd for C26H27O5F3Na, 463.1708).
(3R,5R,6S,7E,9S)-Megastigman-7-en-5,6-epoxy-3,9-diol
1
3,9-O-(S)-MTPA diester (3c): An amorphous powder; H
NMR (CDCl3, 400 MHz) d: 7.537.49 (4H, m, aromatic
protons), 7.427.35 (6H, m, aromatic protons), 5.98 (1H,
d, J = 15 Hz, H-7), 5.74 (1H, dd, J = 15, 7 Hz, H-8), 5.61
(1H, dq, J = 7, 7 Hz, H-9), 5.14 (1H, dddd, J = 12, 10, 8,
4 Hz, H-3), 3.54 (3H, q, J = 1 Hz, –OCH3), 3.52 (3H, q,
J = 1 Hz, -OCH3), 2.30 (1H, ddd, J = 15, 8, 2 Hz, H-
4pseudo-eq), 1.89 (1H, dd, J = 15, 10 Hz, H-4pseudo-ax),
1.75 (1H, dd, J = 12, 12 Hz, H-2ax), 1.45 (1H, ddd,
J = 12, 4, 2 Hz, H-2eq), 1.38 (3H, d, J = 7 Hz, H3-10),
1.19 (3H, s, H3-11), 1.14 (3H, s, H3-13), 0.97 (3H, s, H3-
12); HR-ESI-TOF-MS (positive-ion mode) m/z: 681.2268
[M+Na]+ (calcd for C33H36O7F6Na, 681.2257).
From 2a (4.3 mg and 2.5 mg), 2b (5.0 mg, 40%) and 2c
(2.3 mg, 36%), respectively, were prepared.
(3R,5R,6S,7E,9S)-Megastigman-7-en-5,6-epoxy-3,9-diol
1
3,9-O-(R)-MTPA diester (2b): An amorphous powder; H
NMR (CDCl3, 400 MHz) d: 7.517.49 (4H, m, aromatic
protons), 7.417.36 (6H, m, aromatic protons), 5.98 (1H,
d, J = 15 Hz, H-7), 5.74 (1H, dd, J = 15, 7 Hz, H-8), 5.64
(1H, dq, J = 7, 7 Hz, H-9), 5.12 (1H, dddd, J = 12, 10, 8,
4 Hz, H-3), 3.55 (3H, br s, –OCH3), 3.52 (3H, br s,
–OCH3), 2.35 (1H, ddd, J = 15, 8, 2 Hz, H-4pseudo-eq),
1.98 (1H, dd, J = 15, 10 Hz, H-4pseudo-ax), 1.66 (1H, dd,
J = 12, 12 Hz, H-2ax), 1.43 (3H, d, J = 7 Hz, H3-10),
1.35 (1H, ddd, J = 12, 4, 2 Hz, H-2eq), 1.131 (3H, s, H3-
13), 1.126 (3H, s, H3-11), 0.91 (3H, s, H3-12); HR-ESI-
TOF-MS (positive-ion mode) m/z: 681.2250 [M+Na]+
(calcd for C33H36O7F6Na, 681.2257).
From 4a (0.9 mg each), 4b (1.6 mg, 64%) and 4c (1.2 mg,
48%) were prepared.
(3R,5S,6S,7E,9S)-Megastigman-7-ene-3,5,6,9-tetrol 3,9-
O-(R)-MTPA diester (4b): An amorphous powder; 1H
NMR (CDCl3, 400 MHz) d: 7.547.51 (4H, m, aromatic
protons), 7.417.37 (6H, m, aromatic protons), 6.22 (1H,
d, J = 15 Hz, H-7), 5.68 (1H, dd, J = 16, 7 Hz, H-8), 5.66
(1H, q, J = 6 Hz, H-9), 5.48 (1H, m, H-3), 3.56 (3H, q,
J = 1 Hz, -OCH3), 3.55 (3H, q, J = 1 Hz, -OCH3), 1.99
(1H, ddd, J = 13, 5, 2 Hz, H-4eq), 1.94 (1H, dd, J = 13,
12 Hz, H-4ax), 1.72 (1H, dd, J = 12, 12 Hz, H-2ax), 1.62
(1H, ddd, J = 12, 4, 2 Hz, H-2eq), 1.52 (3H, s, H3-13),
1.46 (3H, d, J = 6 Hz, H3-10), 1.11 (3H, s, H3-12), 0.80
(3H, s, H3-11); HR-ESI-TOF-MS (positive-ion mode) m/z:
699.2383 [M+Na]+ (calcd for C33H38O8F6Na, 699.2363).
(3R,5S,6S,7E,9S)-Megastigman-7-ene-3,5,6,9-tetrol 3,9-
O-(S)-MTPA diester (4c): An amorphous powder; 1H
NMR (CDCl3, 400 MHz) d: 7.537.51 (4H, m, aromatic
protons), 7.427.36 (6H, m, aromatic protons), 6.30 (1H,
d, J = 15 Hz, H-7), 5.77 (1H, dd, J = 16, 7 Hz, H-8), 5.65
(1H, q, J = 6 Hz, H-9), 5.49 (1H, m, H-3) 3.56 (3H, q,
J = 1 Hz, -OCH3), 3.53 (3H, q, J = 1 Hz, –OCH3), 1.92
(1H, dd, J = 13, 12 Hz, H-4ax), 1.86 (1H, ddd, J = 13, 5,
2 Hz, H-4eq), 1.82 (1H, dd, J = 12, 12 Hz, H-2ax), 1.70
(1H, ddd, J = 12, 4, 2 Hz, H-2eq), 1.52 (3H, s, H3-13),
1.42 (3H, d, J = 6 Hz, H3-10), 1.09 (3H, s, H3-12, 0.86
(3H, s, H3-11); HR-ESI-TOF-MS (positive-ion mode) m/z:
699.2373 [M+Na]+ (calcd for C33H38O8F6Na, 681.2257).
(3R,5R,6S,7E,9S)-Megastigman-7-en-5,6-epoxy-3,9-diol
1
3,9-O-(S)-MTPA diester (2c): An amorphous powder; H
NMR (CDCl3, 400 MHz) d: 7.527.49 (4H, m, aromatic
protons), 7.417.35 (6H, m, aromatic protons), 5.98 (1H,
d, J = 15 Hz, H-7), 5.74 (1H, dd, J = 15, 7 Hz, H-8), 5.61
(1H, dq, J = 6, 6 Hz, H-9), 5.14 (1H, dddd, J = 12, 10, 7,
4 Hz, H-3), 3.53 (3H, br s, –OCH3), 3.52 (3H, br s,
–OCH3), 2.30 (1H, ddd, J = 15, 7, 1 Hz, H-4pseudo-eq),
1.89 (1H, dd, J = 15, 10 Hz, H-4pseudo-ax), 1.75 (1H, dd,
J = 12, 12 Hz, H-2ax), 1.45 (1H, ddd, J = 12, 4, 1 Hz, H-
2eq), 1.38 (3H, d, J = 6 Hz, H3-10), 1.19 (3H, s, H3-11),
1.14 (3H, s, H3-13), 0.97 (3H, s, H3-12); HR-ESI-TOF-
MS (positive-ion mode) m/z: 681.2242 [M+Na]+ (calcd
for C33H36O7F6Na, 681.2257).
From 3a (2.9 and 2.3 mg), 3b (5.5 mg, 65%) and 3c
(2.7 mg, 32%), respectively were prepared.

M. Yamamoto et al. / Phytochemistry 69 (2008) 1586–1596
1595
From 5a (2.6 mg each), 5b (3.4 mg, 49%) and 5c (3.9 mg,
56%) were prepared.
(positive-ion mode) m/z: 681.2264 [M+Na]+ (calcd for
C33H36O7F6Na, 681.2262).
(3R,5S,6S,7E,9S)-Megastigman-7-ene-5-methoxy-3,5,6,
9-tetrol 3,9-O-(R)-MTPA diester (5b): An amorphous pow-
der; H NMR (CDCl3, 400 MHz) d: 7.547.51 (4H, m, aro-
4.4.10. Known compounds isolated
22
1
Spinoside A (8): Amorphous powder; ½aꢀD +26.5
matic protons), 7.427.34 (6H, m, aromatic protons), 6.25
(1H, d, J = 16 Hz, H-7), 5.67 (1H, qdd, J = 6, 6, 6 Hz, H-
9), 5.63 (1H, dd, J = 16, 6 Hz, H-8), 5.21 (1H, dddd,
J = 12, 12, 4, 4 Hz, H-3), 3.56 (3H, q, J = 1 Hz,
–OCH3), 3.55 (3H, q, J = 1 Hz, –OCH3), 3.26 (3H, s,
–OCH3 on C-5), 2.28 (1H, ddd, J = 14, 4, 2 Hz, H-4eq),
1.76 (1H, dd, J = 12, 12 Hz, H-2ax), 1.73 (1H, dd, J = 14,
12 Hz, H-4ax), 1.57 (1H, ddd, J = 12, 4, 2 Hz, H-2eq),
1.43 (3H, d, J = 6 Hz, H3-10), 1.15 (3H, s, H3-12), 1.00
(3H, s, H3-13), 0.80 (3H, s, H3-11); HR-ESI-TOF-MS
(positive-ion mode) m/z: 713.2516 [M+Na]+ (calcd for
C34H40O8F6Na, 713.2519).
(3R,5S,6S,7E,9S)-Megastigman-7-ene-5-methoxy-3,6,9-
triol 3,9-O-(S)-MTPA diester (5c): An amorphous powder;
1H NMR (CDCl3, 400 MHz) d: 7.547.52 (4H, m, aromatic
protons), 7.427.34 (6H, m, aromatic protons), 6.30 (1H, d,
J = 15 Hz, H-7), 5.73 (1H, dd, J = 15, 7 Hz, H-8), 5.66
(1H, qdd, J = 6, 6, 6 Hz, H-9), 5.23 (1H, dddd, J = 12, 12,
4, 4 Hz, H-3), 3.55 (3H, q, J = 1 Hz, –OCH3), 3.53 (3H, q,
J = 1 Hz, –OCH3), 3.26 (3H, s, –OCH3 on C-5), 2.22 (1H,
ddd, J = 14, 4, 2, Hz, H-4eq), 1.85 (1H, dd, J = 12, 12 Hz,
H-2ax), 1.67 (1H, dd, J = 14, 12 Hz, H-4ax), 1.63 (1H, ddd,
J = 12, 4, 2 Hz, H-2eq), 1.40 (3H, d, J = 6 Hz, H3-10), 1.19
(3H, s, H3-12), 0.98 (3H, s, H3-13), 0.85 (3H, s, H3-11);
HR-ESI-TOF-MS (positive-ion mode) m/z: 713.2523
[M+Na]+ (calcd for C34H40O8F6Na, 713.2519).
(c = 0.21, MeOH); 13C NMR (CD3OD, 100 MHz) d: 201.3
(C-3), 169.1 (C-5), 133.8 (C-7 and 8), 123.2 (C-4), 101.3 (C-
10), 79.4 (C-6), 78.3 (C-30), 78.1 (C-50), 75.0 (C-20), 74.7 (C-
9), 71.8 (C-40), 62.9 (C-60), 61.4 (C-13), 50.8 (C-2), 42.8 (C-
1), 24.3 (C-12), 23.4 (C-11), 22.3 (C-10); CD nm (De) 238
(+12.0), 320 (ꢁ0.70) (C¸ alißs et al., 2002). Staphylionoside D
25
(9): Amorphous powder; ½aꢀD ꢁ40.0 (c = 0.25, MeOH)
(Yu et al., 2005). Corchoionoside C (10): Amorphous pow-
25
der; ½aꢀD +61.5 (c = 0.39, MeOH) (Yoshikawa et al.,
1997). (Z)-3-Hexenyl b-D-glucopyranoside (11): Amorphous
25
powder; ½aꢀD ꢁ30.2 (c = 2.39, MeOH) (Mizutani et al.,
1988). 3,7-Dimethyloct-1-ene-3,6,7-triol 6-O-b-D-glucopy-
25
ranoside (12): Amorphous powder; ½aꢀD ꢁ3.8 (c = 0.41,
MeOH) (Manns, 1995). (+)-Catechin (13): Amorphous
30
powder; ½aꢀD +30.8 [c = 0.49, H2O: (CH3)2CO = 1:1] (Nay
25
et al., 2001). Syringin (14): Amorphous powder; ½aꢀD ꢁ40.8
(c = 0.37, MeOH) (DellaGreca et al., 1998). 1-b-D-Gluco-
pyranosyloxy-3-methoxy-5-hydroxybenzene (15): An off-
25
white amorphous powder; ½aꢀD ꢁ59.7 (c = 0.71, MeOH)
(Sakar et al., 1993).
Acknowledgements
The authors are grateful for access to the superconduc-
ting NMR instrument and ESI-TOF-MS at the Analytical
Centers of Molecular Medicine and Life Science, respec-
tively, of the Hiroshima University Faculty of Medicine.
This work was supported in part by Grants-in-Aid from
the Ministry of Education, Science, Sports, Culture and
Technology of Japan, the Japan Society for the Promotion
of Science, and the Ministry of Health, Labour and Wel-
fare. Thanks are also due to the Astellas Foundation for
Research on Medicinal Resources and the Takeda Science
Foundation for the financial support.
From 7a (0.7 mg and 0.5 mg), 7b (1.5 mg, 74%) and 7c
(1.4 mg, 69%), respectively, were prepared.
(4Z,6R,9R)-Megastigman-4-ene-9,13-diol-3-one 9,13-O-
(R)-MTPA diester (7b): An amorphous powder; 1H
NMR (CDCl3, 400 MHz) d: 7.447.42 (4H, m, aromatic
protons), 7.367.29 (6H, m, aromatic protons), 5.82 (1H,
br s, H-4), 5.00 (1H, m, H-9), 4.81 (1H, dd, J = 15, 1 Hz,
H-13), 4.67 (1H, dd, J = 15, 1 Hz, H-13), 3.47 (3H, q,
J = 1 Hz, –OCH3), 3.41 (3H, q, J = 1 Hz, –OCH3), 2.26
(1H, d, J = 18 Hz, H-2), 2.00 (1H, d, J = 18 Hz, H-2),
1.77 (1H, dd, J = 5, 5 Hz, H-6), 1.551.64 (4H, m, H2-7
and 8), 1.20 (3H, d, J = 6 Hz, H3-10), 0.92 (3H, s, H3-
11), 0.86 (3H, s, H3-12); HR-ESI-TOF-MS (positive-ion
mode) m/z: 681.2270 [M+Na]+ (calcd for C33H36O7F6Na,
681.2262).
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