Stereochemistry of megastigmane glucosides from Glochidion zeylanicum and Alangium premnifolium

Article abstract of DOI:10.1016/S0031-9422(02)00614-3

From Glochidion zeylanicum, two megastigmane glucosides, 3- and 9-O-β-D-glucopyranosides of (3S,5R,6R,7E,9S)-megastigman-7-ene-3,5,6,9-tetrol (1 and 2, respectively), were isolated. Their structures were different from those of kiwiionoside (3) and actinidioionoside (4), isolated from Actinidia chinensis and Actinidia polygama, respectively, in the stereochemistry at the 9-positions. Alangionosides E (5) and O (6), isolated from the leaves of Alangium premnifolium, are also megastigmane glucosides, and the latter is closely related to 1 and actinidioionoside (4). However, the absolute configurations of the 9-position remained to be determined. They were analyzed to be R by means of a modified Mosher's method. Alangionoside E (5) is identical with corchoionoside A in all aspects. The name of corchoionoside A must be retained thereafter.

Full text of DOI:10.1016/S0031-9422(02)00614-3

Phytochemistry 62(2003) 763–768
www.elsevier.com/locate/phytochem
Stereochemistry of megastigmane glucosides from Glochidion
zeylanicum and Alangium premnifolium
Hideaki Otsukaa,*, Eiji Hiratab, Takakazu Shinzatoc, Yoshio Takedad
aGraduate School of Biomedical Sciences, Hiroshima University, Hiroshima 734-8551, Japan
bFaculty of Agriculture, University of the Ryukyus, Okinawa 903-0129, Japan
cSubtropical Field Science Center, University of the Ryukyus, Okinawa 905-1427, Japan
dFaculty of Integrated Arts and Sciences, The University of Tokushima, Tokushima 770-8502, Japan
Received 23 August 2002; received in revised form 30 October 2002
Abstract
From Glochidion zeylanicum, two megastigmane glucosides, 3- and 9-O-b-d-glucopyranosides of (3S,5R,6R,7E,9S)-megastigman-
7-ene-3,5,6,9-tetrol (1 and 2, respectively), were isolated. Their structures were different from those of kiwiionoside (3) and actinidioio-
noside (4), isolated from Actinidia chinensis and Actinidia polygama, respectively, in the stereochemistry at the 9-positions. Alangiono-
sides E (5) and O (6), isolated from the leaves of Alangium premnifolium, are also megastigmane glucosides, and the latter is closely
related to 1 and actinidioionoside (4). However, the absolute configurations of the 9-position remained to be determined. They were
analyzed to be R by means of a modified Mosher’s method. Alangionoside E (5) is identical with corchoionoside A in all aspects.
The name of corchoionoside A must be retained thereafter.
# 2003 Elsevier Science Ltd. All rights reserved.
Keywords: Glochidion zeylanicum; Euphorbiaceae; Megastigmane glucoside; Alangionosides E and O; Stereochemistry
1. Introduction
remained to be determined. The absolute configurations
of the 9-position of 5 and 6 were also analyzed by the
same method. The NMR spectroscopic features of the
epimeric pairs of megastigmane glucosides are discussed.
In a previous paper (Otsuka et al., 2000), the isolation
of butenolide glucosides, glochidionolactones A–F,
from the leaves of Glochidion zeylanicum (Gaertn) A.
Juss (Euphorbiaceae) were reported. Further investiga-
tion of the same plant afforded two new megastigmane
glucosides (1,2). Their structures were elucidated on the
basis of spectroscopic evidence and the spectroscopic
analyses of the (R)- and (S)-a-methoxy-a-trifluoro-
methylphenylacetic acid (MTPA) esters (1b=2b,
1c=2c) of their common aglycones (1a=2a). It was
found that they are the diastereomers of kiwiionoside
(3) (Murai et al., 1992) and actinidioionoside (4) (Murai
and Tagawa, 1989) respectively, with alternate con-
figuration at the 9-positions. The absolute stereo-
structure of similar compounds, alangionosides E (5)
(Otsuka et al., 1995) and O (6) (60-O-b-d-xylopyrano-
side of 3) (Kijima et al., 1996), isolated from the leaves
of Alangium premnifolium Ohwi (Alangiaceae) have
2. Results and discussion
From the leaves of G. zeylanicum, megastigmane
glucosides 1 and 2, were isolated. Their structures were
elucidated by means of spectroscopic analyses, includ-
ing a modified Mosher’s method (Ohtani et al., 1991).
Compound 1, [a]D À38.0ꢀ, was isolated as an amor-
phous powder and its elemental composition was deter-
mined to be C19H34O9 by negative-ion high-resolution
1
(HR) FAB mass spectrometry. The H and 13C NMR
(Table 1) spectra showed the presence of six signals
assignable to b-glucopyranoses, the remaining 13 signals
comprising three singlet and one doublet methyls, two
methylenes, two methines with hydroxyl substituents,
and three quaternary carbons, two of which bear a
hydroxyl substitutent and one disubstituted trans double
bond, which must form a megastigmane skeleton
(Naves, 1964). The functionalities and their relative
* Corresponding author. Tel.: +81-82-257-5292; fax: +81-82-257-
5294.
E-mail address: hotsuka@hiroshima-u.ac.jp (H. Otsuka).
0031-9422/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
PII: S0031-9422(02)00614-3

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H. Otsuka et al. / Phytochemistry 62 (2003) 763–768
arrangements on the six-membered ring, available from
the results of one- and two-dimensional NMR spectro-
scopy, were the same as those of kiwiinoside (3) (Murai
et al., 1992) and actinidioionoside (4) (Murai and
Tagawa, 1989), which were isolated as pentaacetates
from Actinidia spp. The absolute structure of kiwiiono-
side pentaacetate was determined to be 3S, 5R, 6R, and
9R by means of an X-ray crystallographic method in
which b-glucopyranose was used as a probe, supposing
that it is in d-series. The absolute configuration of the
3-position of 1 could be determined to be S by appli-
cation of b-d-glucopyranosylation-induced shift-trends
(see Table 1, 1 and 1a or 6a; Kasai et al., 1977), and
other chiral centers on the ring were simultaneously
determined to be 5R and 6R. However, the chirality of
the 9-position must be determined independently.
Therefore, 1 was enzymatically hydrolyzed, and then
the aglycone 1a, liberated was esterified with (R)- and
(S)-MTPAs. Analysis of 1b and 1c by the 1H NMR
showed that the 9-position had the S configuration,
opposite to that of kiwiionol (3a; Fig. 1), the aglycone of
kiwiionoside, and the same as that of a non-glycosylated
megastigmane, isolated from the leaves of Euscaphis
japonica (Takeda et al., 2000). Therefore, the structure of
1 was elucidated to be (3S,5R,6R,7E,9S)-megastigman-
7-ene-3,5,6,9-tetrol 3-O-b-d-glucopyranoside.
Compound 2, [a]D À56.3ꢀ, was isolated as an amor-
phous powder, and spectroscopic data indicated that 2
had the same elemental composition as that of 1 and
was the positional isomer regarding the glucopyranose
moiety, like actinidioionoside (4), which has a sugar
moiety on the hydroxyl group at the 9-position. Thus,
the absolute configuration of the 9-position was expected
to be S on the basis of b-d-glucopyranosylation-induced
shift-trends (Kasai et al., 1977), and the absolute con-
figurations of the ring carbons were assumed to be 3S,
5R, and 6R due to the co-occurrence of 1 in the same
plant. This assumption was confirmed by means of the
modified Mosher’s method, as shown in Fig. 1 (see fig-
ures in parentheses). Retrogressive application of
b-d-glucopyranosylation induced shift trends revealed
that the glucose is in d-series. Thus, the structure of 2
was elucidated to be (3S,5R,6R,7E,9S)-megastigman-7-
ene-3,5,6,9-tetrol 9-O-b-d-glucopyranoside. The abso-
lute structure of the aglycone 4a of actinidioionoside
was expected to be the same as that of kiwiionol (3a), as
both were isolated from the same genus. However,
except for the 9-position, there is some uncertainty
regarding the stereochemistry of the ring, since the
stereochemistry of the aglycone was not fully identical
with that of kiwiionol.
Table 1
13C NMR data for (3S,5R,6R,7E,9S)-megastigman-7-ene-3,5,6,9-
tetrol 3-O-b-d-glucopyranoside and 9-O-b-d-glucopyranoside (1 and
2, respectively), their common aglycone (1a), and alangionoside O (6)
and its aglycone (6a) (100 MHz, CD3OD)
C
1
1
2
6a
1a (= 2a)
6a
40.9
40.8
46.6
65.3
45.7
78.5
40.8
40.8
46.6
40.7
244.6 (
3
À2.0)b
44.5
73.3
42.7
77.8
46.5
65.3
45.7
77.8
73.4 (+8.1)
42.5 (À3.2)
77.8
65.4
45.8
4
5
77.8
79.0
6
79.279.3
131.0
79.1
79.0
131.1
7
135.9
131.2131.3
8
136.2133.1 (
69.275.5 (+5.9)
24.0
À3.1)c
136.2136.2
69.6 69.6
136.2
9
69.6
10
11
12
13
22.4 (À1.6)
28.1
26.5
24.3
27.6
26.4
27.3
24.1
27.6
26.2
27.1
24.2
27.5
26.1
27.1
27.6
26.3
27.2
27.2
10
20
30
40
50
60
102.3
100.8
75.1
102.2
75.275.1
78.278.278.0
71.8
71.8
77.8
62.8
71.5
77.0
69.6
77.9
62.8
a
Data from Kijima et al. (1996), and those for the xylopyranose
moiety were omitted.
The absolute configurations of the 9-positions of alan-
gionosides E (5) (Otsuka et al., 1995) and O (6) (Kijima et
al., 1996), isolated from A. premnifolium, have remained
b
Ád1À1a
Ád2À2a
.
c
.

H. Otsuka et al. / Phytochemistry 62 (2003) 763–768
765
Fig. 1. Results with the modified Mosher’s method for 1 and 2, and alangionosides E and O (5 and 6). Figures are Áꢀ values in Hz (ꢀS–ꢀR).
to be determined. Thus, from an aglycone 5a, previously
obtained through the hydrolysis of 5, (R)- and (S)-
MTPA esters were prepared. Based on H NMR spec-
(9:12:8:2) were used as the stationary and mobile phases,
respectively. Five-gram fractions were collected and
numbered according to the order of elution of the
mobile phase. (R)-(+)- and (S)-(À)-MTPAs were pur-
chased from Nacalai Tesque Co., Ltd.
1
tral analysis, the absolute configuration of the 9-posi-
tion was established to be R, which was the same as that
of corchoionol A (see Fig. 1) (Yoshikawa et al., 1997,
1998). This evidence regretfully indicates that com-
pound (5) has two different trivial names, as corchoio-
noside A isolated from Corchorus olitorius, whose
stereochemistry had fully been determined, and as
alangionoside E (5), whose stereochemical structure was
elucidated in the present experiment. Therefore, although
alangionoside E (5) first appeared in this journal, the
name of corchoionoside A must be retained thereafter,
since its structure was fully elucidated previously.
3.2. Plant materials
The leaves of G. zeylanicum were collected in Kuni-
gami-son, Kunigami-gun, Okinawa, Japan, in August,
1990. The plant was identified by Anki Takushi of the
Okinawa Prefectural Station of Forestry, whom the
authors acknowledge, and a voucher specimen was
deposited in the Herbarium of the Programs for Phar-
maceutical Sciences, Graduate School of Biomedical
Sciences, Hiroshima University (90-GZ-Okinawa-0822).
3. Experimental
3.3. Extraction and isolation
3.1. General experimental procedures
The air-dried leaves of G. zeylanicum (4.72kg) were
extracted with MeOH three times. The MeOH extract
was concentrated to 1.5 l and then 75 ml of H2O was
added to make a 95% aqueous solution. This solution
was washed with 1.5 l of n-hexane and the methanolic
layer was concentrated to a viscous gum. The gummy
residue was suspended in 1.5 l of H2O, and then extrac-
ted with 1.5 l each of EtOAc and n-BuOH, successively,
to give 75.5 g and 108 g of EtOAc and n-BuOH soluble
fractions, respectively. The n-BuOH soluble fraction
(107 g) was subjected to highly porous synthetic resin cc
(Diaion HP-20, Mitsubishi Chemical Co. Ltd., È=80
mm, L=55 cm) using H2O–MeOH (4:1, 6 l), (2:3, 6 l),
(3:2, 6 l) and (1:4, 6 l), and MeOH (6 l), 2 l fractions
were being collected. The residue (28.7 g in fractions
4–6) of the 20% MeOH eluent was subjected to silica gel
(530 g) cc, with elution with CHCl3 (1.5 l) and CHCl3–
MeOH [(99:1, 3 l), (49:1, 6 l), (24:1, 6 l), (47:3, 6 l), (23:2,
6 l), (9:1, 6 l), (7:1, 6 l), (17:3, 4.5 l), (4:1, 3 l) and (7:3, 3
l)], 500 ml fractions being collected. Combined fractions
Optical rotations were measured on a Union Giken
PM-101 digital polarimeter. The FT–IR spectra were
recorded on a Horiba FT-710 spectrophotometer. H
1
and 13C NMR spectra were taken on a Jeol a-400
spectrometer (400 and 100 MHz, respectively) with
TMS as the internal standard. HR-FAB–MS were car-
ried out on a Jeol SX-102mass spectrometer using
PEG-400 as calibration matrix. Silica gel column chroma-
tography (CC) and reversed-phase [octadecyl silica
(ODS) gel] open CC (RPCC) were performed on silica
gel 60 (Merck, 70–230 mesh) and Cosmosil 75C18-OPN
(Nacalai Tesque Co., Ltd., Kyoto) [È=50 mm, L=25
cm, linear gradient: MeOH–H2O (1:9, 1 l)!(1:1, 1 l)],
fractions of 10 g being collected. The droplet counter-
current chromatograph (DCCC) (Tokyo Rikakikai,
Tokyo) was equipped with 500 glass columns (È=2
mm, L=40 cm), and the lower and upper layers of the
solvent mixture of CHCl3–MeOH–H2O–n-PrOH

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H. Otsuka et al. / Phytochemistry 62 (2003) 763–768
65–78 (2.18 g) were separated by RPCC. The residue (534
mg) of fractions 36–60 was subjected to DCCC and the
residue (77 mg) from fractions 12–18 was finally purified
by preparative HPLC [ODS (Inertsil, È=6 mm, L=25
cm, GL Science, Tokyo, Japan), MeOH–H2O (1:4), 1.6
ml/min, detection: refractive index] to give 10 mg of 2 and
19 mg of 1 at 5.8 and 8.0 min, respectively.
ddd, J=13, 5, 2Hz, H-4eq), 4.06 (1H, m, H-3), 4.34
(1H, quint.d, J=6, 1 Hz, H-9), 5.79 (1H, dd, J=16, 6
Hz, H-8), 6.07 (1H, dd, J=16, 1 Hz, H-7); 13C NMR
(CD3OD): see Table 1; HR-FAB–MS (negative-ion
mode) m/z: 243.1593 [MÀH]À (calc. for C13H23O4:
243.1596). d-Glucose, [a]2D1 +43.4ꢀ (c=0.25, H2O, 24 h
after being dissolved in the solvent).
3.3.1. (3S,5R,6R,7E,9S)-Megastigman-7-ene-3,5,6,9-
tetrol 3-O-ꢁ-d-glucopyranoside
3.3.4. Preparation of the (R)- and (S)-MTPA esters 1b
and 1c from 1a
An amorphous powder; [a]2D4 À38.0ꢀ (c=1.00,
MeOH); IR nmax (KBr) cmÀ1: 3433, 2929, 2879, 1637,
1457, 1371, 1076; 1H-NMR (CD3OD) ꢀ: 0.88 (3H, s, H3-
12), 1.12 (3H, s, H3-11), 1.22 (3H, s, H3-13), 1.27 (3H, d,
J=6 Hz, H3-10), 1.59 (1H, ddd, J=12, 4, 2 Hz, H-2eq),
1.73 (1H, t, J=12H, H-2ax), 1.77 (1H, dd, J=13, 12
Hz, H-4ax), 1.95 (1H, ddd, J=13, 4, 2Hz, H-4eq), 3.15
(1H, t, J=8 Hz, H-20), 3.68 (1H, dd, J=2, 5 Hz, H-60a),
3.86 (1H, dd, J=12, 2 Hz, H-60b), 4.19 (1H, tt, J=12, 4
Hz, H-4), 4.34 (1H, quint.d, J=6, 1 Hz, H-9), 4.41 (1H,
d, J=8 Hz, H-10), 5.79 (1H, dd, J=16, 6 Hz, H-8), 6.07
(1H, dd, J=16, 1 Hz, H-7); 13C NMR (CD3OD):
Table 1; HR-FAB–MS (negative-ion mode) m/z:
405.2151 [MÀH]À(calc. for C19H33O9: 405.2125).
A solution of 1a (2.6 mg) in 1 ml of dehydrated
CH2Cl2 was reacted with (R)-MTPA (36 mg) in the
presence of 4-dicyclohexylcarbodiimide (DCC) (29 mg)
and 4-dimethylaminopyridine (DMAP)ꢀ (18 mg), the
mixture being occasionally stirred at 25 C for 30 min.
After the addition of 1 ml each of H2O and CH2Cl2, the
solution was washed with 5% HCl (1 ml), NaHCO3
saturated H2O (1 ml), and brine (1 ml), successively.
The organic layer was dried over Na2SO4, filtered and
evaporated under reduced pressure. The residue was
purified by preparative TLC [silica gel (0.25 mm thick-
ness, and developed with CHCl3–(CH3)2CO (19:1) and
eluted with CHCl3–MeOH (9:1)] to furnish the ester, 1b
(3.3 mg, 46%). Through a similar procedure, 1c (4.5 mg,
63%) was prepared from 1a (2.6 mg) by use of
(S)-MTPA (40 mg), DCC (27 mg), and DMAP (14 mg).
3.3.2. (3S,5R,6R,7E,9S)-Megastigman-7-ene-3,5,6,9-
tetrol 9-O-ꢁ-d-glucopyranoside
An amorphous powder, [a]2D4 À56.3ꢀ (c=0.65,
MeOH); 1H NMR (CD3OD) ꢀ: 0.91 (3H, s, H3-12), 1.11
(3H, s, H3-11), 1.24 (3H, s, H3-13), 1.32(3H, d, J=6 Hz,
H3-10), 1.46 (1H, ddd, J=12, 4, 2 Hz, H-2eq), 1.66 (1H,
t, J=12H, H-2ax), 1.73 (1H, dd, J=13, 12Hz, H-4ax),
1.78 (1H, ddd, J=13, 4, 2Hz, H-4eq), 3.67 (1H, dd,
J=12, 6 Hz, H-60a), 3.85 (1H, dd, J=12, 2 Hz, H-60b),
4.06 (1H, ddt, J=13, 12, 4 Hz, H-4), 4.41 (1H, d, J=8 Hz,
H-10), 4.54 (1H, quint., J=6 Hz, H-9), 5.66 (1H, dd, J=16,
6 Hz, H-8), 6.18 (1H, d, J=16 Hz, H-7); 13C-NMR
(CD3OD): Table 1; HR-FAB–MS (negative-ion mode)
m/z: 405.2140 [MÀH]À (calc. for C19H33O9: 405.2125).
3.3.5. (3S,5R,6R,7E,9S)-Megastigman-7-ene-3,5,6,9-
tetrol 3,9-di-(R)-MTPA ester (1b)
Amorphous powder; 1H NMR (CDCl3) ꢀ: 0.80 (3H, s,
H3-12), 1.09 (3H, s, H3-13), 1.25 (3H, s, H3-11), 1.46
(3H, d, J=6 Hz, H3-10), 1.68 (1H, ddd, J=12, 4, 2 Hz,
H-2eq), 1.80 (1H, t, J=12Hz, H-2ax), 1.86 (1H, ddd,
J=13, 5, 2Hz, H-4eq), 1.88 (1H, dd, J=13, 11 Hz, H-
4ax), 3.56 (3H, q, J=1 Hz, –OCH3), 3.57 (3H, q, J=1
Hz, –OCH3), 5.47 (1H, m, H-3), 5.67 (1H, quint., J=6
Hz, H-10), 5.71 (1H, dd, J=16, 6 Hz, H-8), 6.18 (1H, d,
J=16 Hz, H-7), 7.35–7.41 (6H, m, aromatic protons),
7.51–7.55 (4H, m, aromatic protons); HR-FAB–MS
(negative-ion mode) m/z: 645.2271 [MÀCH3O]À (calc.
for C32H35O7F6: 645.2287).
3.3.3. Enzymatic hydrolysis of 1 to 1a
Compound 1 (14 mg) was hydrolyzed with hesper-
ꢀ
idinase (20 mg) in 2 ml of H2O at 37 C for 24 h. The
3.3.6. (3S,5R,6R,7E,9R)-Megastigman-7-ene-3,5,6,9-
tetrol 3,9-di-(S)-MTPA ester (1c)
reaction mixture was concentrated, and then subjected to
silica gel column (20 g, È=15 mm, L=20 cm) chroma-
tography with C6H6 (40 ml), C6H6–CHCl3 (1:1, 40 ml),
CHCl3 (100 ml), and CHCl3–MeOH (19:1, 100 ml, 9:1
100 ml, 17:3, 100 ml and 7:3, 300 ml), 10 ml fractions
being collected. The aglycone (1a) and d-glucose were
recovered from fractions 40–46 (5.3 mg, 63%) and 51–
59 (3.8 mg, 62%), respectively. Aglycone (1a): Amor-
phous powder; [a]D22 À21.1ꢀ (c=0.38, MeOH); 1H NMR
(CD3OD) ꢀ: 0.88 (3H, s, H3-12), 1.10 (3H, s, H3-13),
1.22 (3H, s, H3-10), 1.27 (3H, d, J=6 Hz, H3-10), 1.45
(1H, ddd, J=12, 4, 2 Hz, H-2eq), 1.65 (1H, t, J=12Hz,
H-2ax), 1.73 (1H, dd, J=11, 2Hz, H-4ax), 1.78 (1H,
Amorphous powder; 1H NMR (CDCl3) ꢀ: 0.82(3H, s,
H3-12), 1.15 (3H, s, H3-13), 1.27 (3H, s, H3-11), 1.41
(3H, d, J=6 Hz, H3-10), 1.62(1H, ddd, J=12, 5, 2 Hz, ,
H-2eq), 1.74 (1H, t, J=12Hz, H-2ax), 1.95 (1H, ddd,
J=13, 5, 2Hz, H-4eq), 2.02(1H, dd, J=13, 11 Hz, H-
4ax), 3.53 (3H, q, J=1 Hz, -OCH3), 3.55 (3H, q, J=1
Hz, –OCH3), 5.48 (1H, tt, J=12, 5 Hz, H-3), 5.66 (1H,
quint.d, J=6, 1 Hz, H-9), 5.79 (1H, dd, J=16, 6 Hz, H-
8), 6.28 (1H, dd, J=16, 1 Hz, H-7), 7.34–7.42(6H, m,
aromatic protons), 7.51–7.56 (4H, m, aromatic protons);
HR-FAB–MS (negative-ion mode) m/z: 645.2286
[MÀCH3O]À (calc. for C32H35O7F6: 645.2287).

H. Otsuka et al. / Phytochemistry 62 (2003) 763–768
767
3.3.7. Enzymatic hydrolysis of 2 to 2a, and preparation
of the (R)- and (S)-MTPA esters 2b and 2c
aromatic protons); HR-FAB–MS (positive-ion mode)
m/z: 681.2280 [M+Na]+ (+NaI) (calcd. for
C33H36O7F6Na: 681.2263).
Compound 2 (9.1 mg) was hydrolyꢀzed with hesper-
idinase (10 mg) in 2ml of H O at 37 C for 12h. The
2
reaction mixture was concentrated and subjected to
silica gel cc with the same solvent system as used for
purification of 1a to give 3.9 mg (69%) of an aglycone,
2a. Using a similar procedure to that used for the pre-
paration of 1b and 1c from 1a, 2b (2.9 mg, 58%) and 2c
(3.1 mg, 62%) were prepared from 2a (1.8 mg each) by
use of the respective amounts of (R)- and (S)-MTPA
(26 and 24 mg, respectively), DCC (19 and 20 mg,
respectively), and DMAP (9 and 11 mg, respectively).
The esters, 2b and 2c, gave essentially the same 1H
NMR spectra as those of 1b and 1c, respectively. The
ÁꢀS–ꢀR values are included in Fig. 1 (see figures in
parentheses).
3.3.11. Preparation of (R)- and (S)-MTPA esters 6b
and 6c from 6a
Using a similar procedure to that used for the pre-
paration of 1b and 1c from 1a, 6b (10.6 mg, 77%) and
6c (10.7 mg, 77%) were prepared from 6a (5.0 mg each)
by use of the respective amount of (R)- and (S)-MTPA
(58 mg and 57 mg), DCC (40 mg and 39 mg), and
DMAP (19 mg each).
3.3.12. (3S,5R,6R,7E,9R)-Megastigman-7-ene-3,5,6,9-
tetrol 3,9-di-(R)-MTPA ester (6b)
Amorphous powder; 1H NMR (CDCl3) ꢀ: 0.86 (3H, s,
H3-12), 1.09 (3H, s, H3-13), 1.28 (3H, s, H3-11), 1.42
(3H, d, J=7 Hz, H3-10), 1.70 (1H, ddd, J=12, 4, 2 Hz,
H-2eq), 1.81 (1H, t, J=12Hz, H-2ax), 1.87 (1H, ddd,
J=13, 5, 2Hz, H-4eq), 1.91 (1H, dd, J=13, 11 Hz, H-
4ax), 3.53 (3H, q, J=1 Hz, –OCH3), 3.55 (3H, q, J=1
Hz, –OCH3), 5.49 (1H, dddd, J=13, 11, 5, 4 Hz, H-3),
5.65 (1H, quint., J=7 Hz, H-9), 5.77 (1H, dd, J=16, 7
Hz, H-8), 6.30 (1H, dd, J=16, 1 Hz, H-7), 7.34–7.41
(6H, m, aromatic protons), 7.50–7.55 (4H, m, aromatic
protons); HR-FAB–MS (negative-ion mode) m/z:
645.2271 [MÀCH3O]À (calc. for C32H35O7F6:
645.2287).
3.3.8. Preparation of the (R)- and (S)-MTPA esters 5b
and 5c
Using a similar procedure to that used for the pre-
paration of 1b and 1c from 1a, 5b (12.0 mg, 81%) and
5c (14.0 mg, 95%) were prepared from 5a (5.1 mg each)
by use of the respective amounts of (R)- and (S)-MTPA
(49 and 48 mg), DCC (35 and 34 mg), and DMAP (18
and 19 mg). The developing solvent for preparative
TLC was C6H6–(CH3)2CO (9:1).
3.3.9. (3S,5R,6S,7E,9R)-Megastigman-7-en-5,6-epoxy-
3,9-diol 3,9-di-(R)-MTPA ester (5b)
3.3.13. (3S,5R,6R,7E,9R)-Megastigman-7-ene-3,5,6,9-
tetrol 3,9-di-(S)-MTPA ester (6c)
Amorphous powder; 1H NMR (CDCl3) ꢀ: 0.95 (3H, s,
H3-11), 1.03 (3H, s, H3-12), 1.10 (3H, s, H3-13), 1.36
(3H, d, J=7 Hz, H3-10), 1.39 (1H, dd, J=14, 9 Hz, H-2
pseudo-ax), 1.74 (1H, ddd, J=13, 3, 1 Hz, H-2pseudo-
eq), 1.77 (1H, dd, J=15, 7 Hz, H-4 pseudo-ax), 2.39
(1H, ddd, J=15, 4, 1 Hz, H-4 pseudo-eq), 3.53 (3H, s,
–OCH3), 3.55 (3H, s, –OCH3), 5.12(1H, m, H-3), 5.59
(1H, quint.d, J=6, 1 Hz, H-9), 5.71 (1H, dd, J=16, 6
Hz, H-8), 5.98 (1H, dd, J=16, 1 Hz, H-7), 7.35–7.43
(6H, m, aromatic protons), 7.49–7.55 (4H, m, aromatic
protons); HR-FAB–MS (positive-ion mode) m/z:
681.2286 [M+Na]+ (+NaI) (calc. for C33H36O7F6Na:
681.2263).
Amorphous powder; 1H NMR (CDCl3) ꢀ: 0.80 (3H, s,
H3-12), 1.11 (3H, s, H3-13), 1.24 (3H, s, H3-11), 1.46
(3H, d, J=7 Hz, H3-10), 1.61 (1H, ddd, J=12, 4, 2 Hz,
H-2eq), 1.72 (1H, t, J=12Hz, H-2ax), 1.94 (1H, ddd,
J=13, 5, 2Hz, H-4eq), 1.99 (1H, dd, J=13, 11 Hz, H-
4ax), 3.55 (3H, q, J=1 Hz, –OCH3), 3.56 (3H, q, J=1
Hz, –OCH3), 5.48 (1H, dddd, J=13, 11, 5, 4 Hz, H-3),
5.67 (1H, quint., J=7 Hz, H-9), 5.68 (1H, dd, J=16, 7
Hz, H-8), 6.22 (1H, d, J=16 Hz, H-7), 7.34–7.41 (6H,
m, aromatic protons), 7.50–7.55 (4H, m, aromatic pro-
tons); HR-FAB–MS (negative-ion mode) m/z: 645.2299
[MÀCH3O]À (calc. for C32H35O7F6: 645.2287).
3.3.10. (3S,5R,6S,7E,9R)-Megastigman-7-en-5,6-epoxy-
3,9-diol 3,9-di-(S)-MTPA ester (5c)
Acknowledgements
Amorphous powder, 1H NMR (CDCl3) ꢀ: 0.93 (3H, s,
H3-11), 0.908 (3H, s, H3-12), 1.14 (3H, s, H3-11), 1.30
(1H, dd, J=13, 9 Hz, H-2pseudo-ax), 1.41 (3H, d, J=6
Hz, H3-10), 1.68 (1H, ddd, J=13, 3, 1 Hz, H-2pseudo-
eq), 1.85 (1H, dd, J=15, 7 Hz, H-4 pseudo-ax), 2.46
(1H, ddd, J=15, 6, 1 Hz, H-4 pseudo-eq), 3.53 (3H, q,
J=1 Hz, –OCH3), 3.55 (3H, q, J=1 Hz, –OCH3), 5.16
(1H, m, H-3), 5.60 (1H, quint., J=6 Hz, H-9), 5.66 (1H,
dd, J=16, 6 Hz, H-8), 5.89 (1H, d, J=16 Hz, H-7),
7.34–7.44 (6H, m, aromatic protons), 7.49–7.54 (4H, m,
The authors are grateful for the access to the NMR
facilities of the Analytical Center of Molecular Medi-
cine of Hiroshima University Faculty of Medicine. This
work was supported, in part, by a Grant-in-Aid from
the Ministry of Education, Science, Sports, Culture
and Technology of Japan (No. 13672216). Thanks are
also due to the Okinawa Foundation for financial sup-
port from an Okinawa Research Promotion Award
(H.O.).

768
H. Otsuka et al. / Phytochemistry 62 (2003) 763–768
1995. Alangionosides C-F: Megastigmane glycosides derivatives
References
from Alangium premnifolium. Phytochemistry 38, 1431–1435.
Otsuka, H., Hirata, E., Takushi, A., Shinzato, T., Takeda, Y., Bando,
M., Kido, M., 2000. Glochidionolactones A-F: Butenolide gluco-
sides from leaves of Glochidion zeylanicum (Gaertn) A. Juss. Chem.
Pharm. Bull. 48, 547–551.
Kasai, R., Suzuno, M., Asakawa, I., Tanaka, O., 1977. Carbon-13
chemical shifts of isoprenoid-b-d-glucopyranosides and -b-d-manno-
pyranosides. Stereochemical influence of aglycone alcohols. Tetra-
hedron Lett. 175–178.
Kijima, H., Otsuka, H., Ide, T., Ogimi, C., Hirata, E., Takushi, A.,
Takeda, Y., 1996. Glycosides of megastigmane and of the simple
alcohols from Alangium premnifolium. Phytochemistry 42, 723–727.
Naves, Y.-R., 1964. The ionones in products of plant origin. Perfum.
Essent. Oil Rec. 55, 658–667.
Takeda, Y., Okada, Y., Masuda, T., Hirata, E., Shinzato, T., Takushi,
A., Yu, Q., Otsuka, H., 2000. New megastigmane and tetraketide
from the leaves of Euscaphis japonica. Chem. Pharm. Bull. 48, 752–
754.
Yoshikawa, M., Murakami, T., Shimada, H., Yoshimizu, S., Saka,
M., Yamahara, J., Matsuda, H., 1998. Medicinal foodstuffs. XIV.
On the bioactive constituents of moroheiya. (2): New fatty acids,
corchorifatty acids A, B, C, D, E, and F, from the leaves of Corch-
orus olitorius L. (Tiliaceae): structures and inhibitory effect on NO
production in mouse peritoneal macrophages. Chem. Pharm. Bull.
46, 1008–1014.
Murai, F., Tagawa, M., 1989. Relationship between ionone glycosides
and terpenoids in Actinidia polygama. In: Abstract Papers of the
33rd Symposium on the Chemistry of Terpenes, Essential Oils, and
Aromatics (TEAC), Sendai, pp. 68–70.
Murai, F., Tagawa, M., Ohishi, H., 1992. Absolute structure of
kiwiionoside as a precursor of loliolide and actinidiolide from Acti-
nidia chinensis. Planta Med. 58, 112–114.
Yoshikawa, M., Shimada, H., Saka, M., Yoshizumi, S., Yamahara, J.,
Matsuda, H., 1997. Medicinal foodstuffs. V. Moroheiya. (1): Abso-
lute stereostructures of corchoionosides A, B, and C, histamine
release inhibition from the leaves of Vietnamese Corchorus olitorius
L. (Tiliaceae). Chem. Pharm. Bull. 45, 464–469.
Ohtani, I., Kusumi, T., Kashman, Y., Kakisawa, H., 1991. High-field
FT NMR application of Mosher’s method. The absolute configur-
ation of marine terpenoids. J. Amer. Chem. Soc. 113, 4092–4096.
Otsuka, H., Kamada, K., Yao, M., Yuasa, K., Kida, I., Takeda, Y.,