Absolute configuration of (+)-pinoresinol 4-O-[6″-O-galloyl]-β-d-glucopyranoside, macarangiosides E, and F isolated from the leaves of Macaranga tanarius

Article abstract of DOI:10.1016/j.phytochem.2009.07.020

A lignan glucoside, (+)-pinoresinol 4-O-[6″-O-galloyl]-β-d-glucopyranoside (1), and two megastigmane glucosides, named macarangiosides E and F (2, 3), together with 15 known compounds (4-18) were isolated from leaves of Macaranga tanarius (L.) Muell.-Arg.

Full text of DOI:10.1016/j.phytochem.2009.07.020

Phytochemistry 70 (2009) 1277–1285
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Phytochemistry
journal homepage: www.elsevier.com/locate/phytochem
Absolute configuration of (+)-pinoresinol 4-O-[600-O-galloyl]-b-D-glucopyranoside,
macarangiosides E, and F isolated from the leaves of Macaranga tanarius
b
c
d
Katsuyoshi Matsunami a, Hideaki Otsuka a, , Kazunari Kondo , Takakazu Shinzato , Masatoshi Kawahata ,
*
Kentaro Yamaguchi d, Yoshio Takeda e
a Department of Pharmacognosy, Graduate School of Biomedical Sciences, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553, Japan
b Division of Novel Food and Immunochemistry, National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan
c Subtropical Field Science Center, Faculty of Agriculture, University of the Ryukyus, 1 Sembaru, Nishihara-cho, Nakagami-gun, Okinawa 903-0213, Japan
d Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Shido, Sanuki-shi, Kagawa 769-2193, Japan
e Faculty of Pharmaceutical Sciences, Yasuda Women’s University, 6-13-1 Ato, Asaminami-ku, Hiroshima 731-0153, Japan
a r t i c l e i n f o
a b s t r a c t
A lignan glucoside, (+)-pinoresinol 4-O-[600-O-galloyl]-b-
D-glucopyranoside (1), and two megastigmane
Article history:
Received 2 April 2009
Received in revised form 10 June 2009
Available online 13 August 2009
glucosides, named macarangiosides E and F (2, 3), together with 15 known compounds (4–18) were iso-
lated from leaves of Macaranga tanarius (L.) Müll.-Arg. (Euphorbiaceae). Their structures were elucidated
by spectroscopic and chemical analyses. In addition, the absolute stereochemistry of macarangiosides B
and C isolated previously from the same plant was also determined for the first time. Compounds 1 and 2
were galloylated on glucose and possessed potent DPPH radical-scavenging activity.
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Macaranga tanarius
Euphorbiaceae
Lignan
Megastigmane
DPPH
Cytotoxicity
1. Introduction
15 known compounds (4–18). Their structures were elucidated
on the basis of spectroscopic and chemical analyses. In addition,
In our continuing studies on the subtropical plants collected in
Okinawa Island, we have phytochemically investigated the chemi-
cal constituents of the leaves of Macaranga tanarius (L.) Müll.-Arg.
(Euphorbiaceae). It is well known as a pioneer tree that grows first
ahead of other plants on impoverished soil (Blattner et al., 2001),
and also known as an ant-plant defended by ants against herbi-
vores by producing the ant-attracting food body (Heil et al.,
2001). It is a small evergreen tree of about 4–5 m in the height
found in the bush layer throughout eastern and southern Asia,
especially in southern China, Korea and Okinawa, Japan. In China,
the root and bark of this plant are used as a folk medicine for hem-
optysis and dysentery, respectively (Primary Chinese Herbs Picto-
rial Illustrated Editorial Committee, 1986). In our previous
papers, we described the isolation and structure elucidations of
four new megastigmane glucosides, named macarangiosides A–D,
and seven prenylated flavanones, macaflavanones A–G, from the
leaves of M. tanarius (Matsunami et al., 2006; Kawakami et al.,
2008). As a result of further investigation of the same plant, we
successfully isolated three new glucosides (1–3), together with
the absolute stereochemistry of macarangiosides B and C was also
determined for the first time in this study by chemical conversion
and application of the modified Mosher’s method. Finally, DPPH
(2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity and
cytotoxicity of these compounds were also investigated.
2. Results
Air-dried leaves (12.1 kg) of M. tanarius were extracted with
MeOH at room temperature, then defatted with n-hexane, and
partitioned with EtOAc and 1-BuOH to give EtOAc- (801 g) and 1-
BuOH- (374 g) soluble fractions, respectively. Part of the 1-BuOH-
soluble fraction (181 g) was subjected to various kinds of column
chromatography to give three new glucosides (1–3) (Fig. 1), and
15 known compounds (4–18).
(+)-Pinoresinol 4-O-[600-O-galloyl]-b-
D-glucopyranoside (1) was
obtained as an off-white amorphous powder and its elemental
composition was determined to be C33H36O15 by HR-electro-
spray-ionization (ESI)-time-of-flight (TOF)-MS. Its IR spectrum
indicated the presence of hydroxyl groups (3367 cmꢀ1), carbonyl
functional group(s) (1703 cmꢀ1) and aromatic rings (1606 and
1515 cmꢀ1). Its UV spectrum also suggested the presence of aro-
* Corresponding author. Tel./fax: +81 82 257 5335.
E-mail address: hotsuka@hiroshima-u.ac.jp (H. Otsuka).
0031-9422/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.phytochem.2009.07.020

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K. Matsunami et al. / Phytochemistry 70 (2009) 1277–1285
HMBC spectra well supported the predicted structure shown in
HO
4'
6'
Fig. 1. The absolute stereochemistry was determined to be same
as (+)-pinoresinol by comparison of circular dichroism (CD) spec-
trum (Xie et al., 2003). Finally, mild alkaline methanolysis of 1
O
O
7'
3'
9
7
MeO
afforded (+)-pinoresinol 4-O-b-D-glucopyranoside (1a) (Abe and
H
H
Yamauchi, 1989). From these results, compound 1 was elucidated
to be (+)-pinoresinol 4-O-[600-O-galloyl]-b-
D
-glucopyranoside as a
9'
OMe
OR
3
R
new compound.
1: Glc(6'')Gall
1a: Glc
Gall = galloyl
Macarangioside E (2) was isolated as an off-white amorphous
powder and a molecular-related ion peak was observed at m/z
545.1990 [M+Na]+ (C26H34O11Na) on positive-ion HR-ESI-TOF-MS.
In the IR spectrum, strong absorptions at 3303 and 1706 cmꢀ1 sug-
gested the presence of hydroxyl and carbonyl functional groups,
respectively. The absorption maxima in the UV spectrum (244sh
and 275 nm) also suggested presence of aromatic ring and conju-
gated ketone moieties. The 13C NMR spectrum showed the pres-
ence of six signals assignable to a 6-O-acylated glucopyranose
and five resonances to a galloyl moiety (Table 2), which indicated
the presence a of 6-O-galloyl glucopyranose moiety in its structure.
The remaining 13 carbon signals corresponded to a compound hav-
ing a megastigmane skeleton. The 1H NMR spectra displayed two
olefinic protons at dH 5.69 (J = 15, 7, 1 Hz) and 5.53 (J = 15, 9,
1 Hz), indicative of the presence of a trans double bond (Table 3).
A ketonic carbonyl signal at dc 202.2 (s) and two olefinic carbons
[dc 166.0 (s) and 126.1 (d)] established the presence of a conju-
gated ketone functional group. The COSY spectrum clarified
connections from H-6 to H3-10, and HMBC correlations between
H3-13 and C-4, 5 and 6 confirmed the location of the conjugated
ketone function, and the correlations between H3-11 and 12 and
C-1, 2 and 6 also clarified the six-membered ring moiety of 2 as
in Fig. 2. The attachments of glucopyranosyl and galloyl moieties
were confirmed by analysis of HMBC correlations (Fig. 2). The 13C
chemical shift value of C-9 [dc 78.6 (d)] was diagnostic in assigning
the absolute configuration at C-9 as 9R, when compared with the
the reported deshielded signal of C-9 for 9R-configuration (Pabst
4
6
OR
OR
11
11
12
6
6
5
9
9
12
H
H
O
3
5
13
3
13
O
O
R
R
2: Glc(6')Gall
2a: Glc
3: Glc
3a:
H
Fig. 1. Structures of new compounds from Macaranga tanarius (L.) Müll.-Arg.
Table 1
13C and 1H NMR spectroscopic data for compound (1).
C
H
1
2
3
4
5
6
7
8
9
137.6
111.9
150.7
147.3
118.1
119.8
87.1
6.96
(1H, d, J = 1.9 Hz)
7.02
6.61
4.71
3.05
4.26
3.83
(1H, d, J = 8.4 Hz)
(1H, dd, J = 8.4, 1.9 Hz)
(1H, d, J = 5.2 Hz)
(1H, m)
(1H, dd, J = 9.1, 7.1 Hz)
(1H, overlapped with H-90)
55.5
73.0
et al., 1992). The Cotton effects observed in the CD spectrum [De
10
20
30
40
50
60
70
80
90
133.9
111.3
149.2
147.5
116.2
120.3
87.7
6.98
(1H, d, J = 1.9 Hz)
Table 2
13C NMR spectroscopic data for macarangiosides E (2), F (3), and degalloylmacaran-
gioside B (19a).
6.78
6.83
4.68
3.12
4.16
3.86
3.84
3.87
(1H, d, J = 8.1 Hz)
(1H, dd, J = 8.1, 1.9 Hz)
(1H, d, J = 5.8 Hz)
(1H, m)
(1H, dd, J = 9.1, 7.0 Hz)
(1H, overlapped with H-9)
(3H, s)
2
3
19a
55.4
72.6
1
2
3
4
5
6
7
8
37.0
48.5
44.9
49.6
212.4
50.7
85.6
54.9
22.2
37.8
76.0
20.1
20.9
79.5
25.2
45.4
49.9
211.6
51.1
85.0
59.6
124.9
141.3
76.9
21.2
20.6
80.0
24.4
202.2
126.1
166.0
56.3
129.5
138.0
78.6
21.6
27.4
27.9
23.7
3-OMe
30-OMe
56.9
56.6
(3H, s)
100
200
300
400
500
600
102.7
75.0
78.0
72.2
75.8
65.0
4.83
3.54
3.50
3.41
3.73
4.56
4.44
(1H, d, J = 7.6 Hz)
(1H, dd, J = 9.1, 7.6 Hz)
(1H, t, J = 9.1, 8.6 Hz)
(1H, dd, J = 9.7, 8.6 Hz)
(1H, ddd, J = 9.7, 7.8, 1.8 Hz)
(1H, dd, J = 11.8, 1.8 Hz)
(1H, dd, J = 11.8, 7.8 Hz)
9
10
11
12
13
1000
121.5
110.5
146.7
140.1
168.2
2
3
000,6000
000,5000
7.10
(2H, s)
Glc
10
103.3
75.3
78.1
72.1
75.6
65.2
102.4
75.3
78.3
72.0
78.0
63.1
102.6
75.3
78.2
71.7
78.1
62.8
20
4’’’
C@O
30
40
Measured in CD3OD.
50
60
Gall
100
matic rings (276 nm). Close inspection of 1H and 13C NMR spectra
(Table 1) suggested the presence of a pinoresinol aglycone (Inouye
et al., 1973), a 6-O-acylated glucopyranose (Matsunami et al.,
2006) and a galloyl moiety at the position-6 of glucose. From the
above evidence, compound 1 was proposed to be a pinoresinol
glucoside with a galloyl moiety on C-6’’. The COSY, HMQC and
121.7
110.4
146.7
140.0
168.3
200,600
300,500
400
C@O
Measured in CD3OD.

K. Matsunami et al. / Phytochemistry 70 (2009) 1277–1285
1279
Table 3
1H NMR spectroscopic data for macarangiosides E (2), F (3) and degalloylmacarangioside B (19a).
2
3
19a
2
4
1.94 (1H, d, J = 16.6 Hz)
2.31 (1H, d, J = 16.6 Hz)
5.79 (1H, br s)
2.16 (1H, dt, J = 17.3, 1.6 Hz, eq)
2.54 (1H, dd, J = 17.3, 2.4 Hz, ax)
2.21 (1H, dt, J = 17.1, 1.6 Hz, eq)
2.57 (1H, d, J = 17.1 Hz, ax)
1.74 (1H, m)
2.23 (1H, dt, J = 17.4, 1.7 Hz, eq)
2.57 (1H, dd, J = 17.4, 2.7 Hz, ax)
2.27 (1H, dt, J = 17.7, 1.7 Hz, eq)
2.56 (1H, d, J = 17.7 Hz, ax)
2.41 (1H, br dt, J = 9.5, 1.7 Hz)
5.80 (1H, ddd, J = 15.1, 9.5, 1.2 Hz)
6
7
2.41 (1H, br d, J = 9.0 Hz)
5.53 (1H, ddd, J = 15.4, 9.0, 0.7 Hz)
1.49 (1H, m, pro-S)
1.84 (1H, m, pro-R)
8
5.69 (1H, ddd, J = 15.4, 7.0, 0.5 Hz)
1.70 (1H, m)
5.95 (1H, ddd, J = 15.1, 6.3, 0.6 Hz)
1.78 (1H, m)
9
4.29 (1H, m)
1.28 (1H, d, J = 6.4 Hz)
0.87 (3H, s)
3.94 (1H, m)
1.22 (3H, d, J = 6.2 Hz)
1.09 (3H, s)
3.55 (1H, dd, J = 7.9, 2.4 Hz, pro-R)
3.57 (1H, d, J = 7.9 Hz, pro-S)
1.31 (3H, s)
4.45 (1H, qdd, J = 6.3, 6.3, 1.2 Hz)
1.32 (3H, d, J = 6.3 Hz)
1.04 (3H, s)
3.60 (1H, dd, J = 8.0, 2.7 Hz, pro-R)
3.68 (1H, d, J = 8.0 Hz, pro-S)
1.23 (3H, s)
10
11
12
0.86 (3H, s)
13
1.82 (3H, d, J = 1.1 Hz)
Glc
10
4.37 (1H, d, J = 7.9 Hz)
3.21 (1H, dd, J = 8.8, 7.9 Hz)
3.37 (1H, t, J = 8.8 Hz)
4.35 (1H, d, J = 7.8 Hz)
3.15 (1H, dd, J = 9.2, 7.8 Hz)
3.36 (1H, m)
3.26 (1H, overlapped)
3.26 (1H, overlapped)
3.63 (1H, dd, J = 11.6, 5.8 Hz)
3.87 (1H, dd, J = 11.6, 2.0 Hz)
4.38 (1H, d, J = 7.8 Hz)
3.19 (1H, dd, J = 9.1, 7.8 Hz)
3.35 (1H, t, J = 9.1 Hz)
20
30
40
3.35 (1H, t, J = 8.8 Hz)
3.18 (1H, t, J = 9.1 Hz)
50
3.51 (1H, ddd, J = 8.8, 6.8, 2.2 Hz)
4.57 (1H, dd, J = 11.8, 2.2 Hz)
4.30 (1H, dd, J = 11.8, 6.8 Hz)
3.23 (1H, ddd, J = 9.1, 5.6, 2.4 Hz)
3.65 (1H, dd, J = 11.8, 5.6 Hz)
3.82 (1H, dd, J = 11.8, 2.4 Hz)
60
Gall
200,600
7.10 (2H, s)
Measured in CD3OD.
Fig. 2. COSY and HMBC correlations of macarangiosides E (2) and F (3).
(nm) ꢀ0.9 (317) and +18.5 (245)] were well in accord with those of
(6R,9R)-3-oxo- -ionol b- -glucopyranoside. In addition, the degal-
loylated compound (2a) obtained in mild alkaline methanolysis
structure. The HMBC correlation between H2-12 and C-5 clarified
the presence of an oxirane ring system between C-5 and C-12
(Fig. 2). Thus the aglycone of macarangioside F (3) was considered
to be a 5,12-epoxymegastigmane. The COSY correlations from H-6
to H3-10, and the HMBC correlations between H3-13 and C-4, 5 and
6, and between H3-11 and C-1, 2, 6 and 12 established the struc-
ture of aglycone as shown in Fig. 2. The attachment of glucose
was also confirmed by analysis of the HMBC correlation. The rela-
tive stereochemistry of the aglycone moiety was suggested to be of
Fig. 2 and 3 from the proton–proton coupling pattern data espe-
cially ‘‘W”-type long-range coupling, i.e., H-2 eq was coupled with
H-4 eq and H-6, H-2ax with pro-R H-12, and H-4 eq also both with
H-2 eq and H-6. The NOESY experiment also supported these
observations (Fig. 3). The absolute stereochemistry at C-9 [dc 76.0
(d)] was suggested to be 9R by comparison of the chemical shift va-
lue of myrsinionoside A (dc 75.9) (Otsuka et al., 2001). The absolute
configuration of glucose was determined to be in D-series on HPLC
analysis with optical rotation detector after enzymatic hydrolysis
of 3. Thus the structure of macarangioside F (3) was tentatively
a
D
was fully identical with (6R,9R)-3-oxo-a-ionol b-D-glucopyrano-
side on the bases of 1H, and 13C NMR spectra and the optical rota-
tion value (Pabst et al., 1992; Mohamed et al., 1999). As a result,
macarangioside E (2) was elucidated to be (6R,9R)-3-oxo-
a-ionol
9-O-[60-O-galloyl]-b-
D
-glucopyranoside.
Macarangioside F (3) was obtained as a colorless amorphous
powder, whose molecular formula was determined to be
C19H32O8 based on its positive-ion HR-ESI-TOF-MS. Its IR spectrum
suggested the presence of hydroxyl groups (3396 cmꢀ1) and a car-
bonyl functional group (1712 cmꢀ1) in its structure. In the 1H and
13C NMR spectra (Tables 2 and 3), an anomeric proton signal and
six carbon signals assignable to a glucopyranose moiety were ob-
served. The chemical shift values of the remaining 13 carbon sig-
nals suggested
a 9-hydroxy-3-oxo-megastigmane skeleton, of
which one of the methyl signals was oxygenated [dc 79.5 (t)].
The index of hydrogen deficiency calculated from the molecular
formula indicated the presence of an additional ring system in its

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K. Matsunami et al. / Phytochemistry 70 (2009) 1277–1285
Fig. 4. Results with the modified Mosher’s method for 3c.
shown in ppm.
D
d values (dS ꢀ dR) were
identical to those of 3. Thus, macarangiosides B (19), C (20) and F
(3) had the same relative stereochemistry at their chiral centers.
Then, macarangioside F (3) was enzymatically hydrolyzed to afford
the aglycone (3a), and reduced with NaBH4 to give a mixture of 3
and 3b,9 -diols (3b and 3c). After preparative TLC separation, the
3b,9 -diol (3c) having an equatorial hydroxyl group was esterified
with (S)- and (R)- -methoxy- -trifluoromethylphenylacetic acids
a
a
a
Fig. 3. Phase-sensitive NOESY correlations of macarangioside F (3).
a
a
(MTPAs) to afford diesters, 3d and 3e, respectively. The
Dd values
from their 1H NMR spectra indicated that compound 3c had 3S
configuration (Fig. 4). Therefore, 3, 19 and 20 were elucidated to
*
*
*
elucidated to be (1S ,5R ,6R ,9R)-megastigman-3-on-5,12-epoxy-
9-ol 9-O-b- -glucopyranoside.
D
In our previous paper, closely related compounds, macarangio-
sides B (19) and C (20) (Scheme 1), were isolated from the same
plant (Matsunami et al., 2006). When comparing the structures
of 19 and 20 to that of 3, the differences were a galloyl moiety
on glucose for 19 and 20, and a trans double bond at C-7 and 8
for 19 (Scheme 1). However the absolute stereochemistry at C-1,
5 and 6 was not determined. Therefore, in this study, we have elu-
cidated the absolute stereochemistry of these compounds by
chemical conversion and application of the modified Mosher’s
method (Scheme 1). Macarangiosides B (19) and C (20) were first
hydrolyzed with 0.1 M NaOMe at room temperature to afford
19a and 20a, respectively. The physical data including optical rota-
tion value of 20a were essentially identical to those of 3. The dou-
ble bond at C-7 and 8 of 19a was then reduced by catalytic
hydrogenation to afford 19b. The physical data of 19b were fully
be (1S,5R,6R,9R)-megastigman-3-on-5,12-epoxy-9-ol 9-O-b-D-
glucopyranoside,
(1S,5R,6R,7E,9R)-megastigman-7-en-3-on-
5,12-epoxy-9-ol 9-O-[60-O-galloyl]-b-
D-glucopyranoside, and
(1S,5R,6R,9R)-megastigman-3-on-5,12-epoxy-9-ol 9-O-[60-O-
galloyl]-b-D-glucopyranoside, respectively.
The known compounds (4–18) were identified by comparison of
spectroscopic data with those reported in the literature as follows.
Roseoside (4), amorphous powder, ½a D25
ꢁ
+135 (c 0.45, MeOH)
(Otsuka et al., 1995), icariside B5 (5), amorphous powder, ½a D24
ꢁ
ꢀ1.0 (c 0.32, MeOH), CD (c 4.12 ꢂ 10ꢀ5 M, MeOH)
De (nm):+0.59
(329), ꢀ1.47 (254), +3.06 (219) (Andersson and Lundgren, 1988),
(6R,9R)-3-oxo-
a
-ionol b-
D
-glucoside (6), amorphous powder, ½a D25
ꢁ
+154 (c 1.08, MeOH), CD (c 2.92 ꢂ 10ꢀ5 M, MeOH)
D
e
a
(nm):
-ionol
+27.7 (c 0.84, MeOH)
ꢀ0.64 (319),+20.0 (244) (Pabst et al., 1992), (6R,9S)-3-oxo-
b-
D
-glucoside (7), amorphous powder, ½a D26
ꢁ
Scheme 1. (a) 0.1 M NaOMe in MeOH, rt, 2 h, (b) PtO2/H2, rt, 1 h, (c) crude hesperidinase, 37 °C, 12 h, (d) NaBH4 in MeOH, 0–25 °C, 30 min, (e) EDC, DMAP, (R) and (S)-MTPA in
CH2Cl2, rt, 30 min.

K. Matsunami et al. / Phytochemistry 70 (2009) 1277–1285
1281
Fig. 5. X-ray crystallographic analysis of 18. (a) ORTEP drawing of 18 with crystallographic numbering, (b) lateral view, (c) bird’s-eye view and (d) inter-molecule hydrogen
bonding.
(Pabst et al., 1992), (2S,3R)-dihydrodehydrodiconiferyl alcohol b-
D
-
3-keto-type 5,12-epoxymegastigmane, such as asysgangoside
(Kanchanapoom and Ruchirawat, 2007), spionoside B (Calis et al.,
2002), and annuionone E (Macías et al., 2004) have never been fully
determined, because of a lack of reliable reference data such as CD
spectra. In this study, both macarangioside F (3) and degalloylmac-
arangioside B (19a) showed similar CD values to those reported for
asysgangoside and spionoside B. Thus, asysgangoside and spiono-
side B are considered to must have the same absolute stereochem-
istry as that of 3 and 19a. In addition, the physical data of
annuionone E including its optical rotation value were superim-
posable on those of the aglycone of macarangioside F (3a), which
meant that annuionone E also has the same absolute stereostruc-
ture as that of 3a.
The plant hormone, abscisic acid (ABA), is mainly oxidized at C-
80 (equivalent to C-12 of megastigmane) by ABA 80-hydroxylase
(cytochrome P450 monooxygenase) in plant (Krochko et al.,
1998). 80-Hydroxy ABA spontaneously isomerizes to phaseic acid
(PA) by formation of a 5-membered ether ring between C-80 and
C-20 (equivalent to C-5 of megastigmane). Thus the same mecha-
nism may contribute to the biosynthesis of 5,12-epoxymegastig-
mane. However, another oxidation pathway of ABA has
discovered recently (Zhou et al., 2004). This minor pathway in-
volved the hydroxylation of 90-methyl group (equivalent to C-11
methyl group of megastigmane) of ABA resulting in 9’-hydroxy
ABA, which isomerizes to neo-PA. Therefore, 5,11-epoxymegastig-
mane might be isolated in future.
glucoside (8), amorphous powder, ½a D25
ꢀ27.6 (c 0.9, MeOH), CD (c
ꢁ
1.72 ꢂ 10ꢀ5 M, MeOH)
D
e
(nm): ꢀ0.48 (289), ꢀ0.92 (242), +1.16
(223) (Matsuda et al., 1996), (+)-pinoresinol 4-O-b-D-glucopyrano-
side (9), amorphous powder, ½a D25
ꢁ
+0.25 (c 0.8, MeOH) (Deyama,
1983), scopolin (10), amorphous powder, ½a D26
ꢀ45.2 (c 0.65,
ꢁ
MeOH) (Tsukamoto et al., 1985), quercetin 3-O-rutinoside (11),
amorphous powder, ½a D25
ꢀ23.1 (c 6.65, MeOH) (Harborne and
ꢁ
Mabry, 1982), quercetin 3-O-galactopyranoside (12), amorphous
powder, ½a 2D5
ꢀ30.3 (c 0.07, MeOH) (Harborne and Mabry, 1982),
ꢁ
quercetin 3-O-arabinopyranoside (13), amorphous powder, ½a D25
ꢁ
ꢀ102 (c 0.20, MeOH) (Harborne and Mabry, 1982), isovitexin
(14), amorphous powder, ½a D24
ꢁ
+19.6 (c 0.51, MeOH) (Harborne
and Mabry, 1982), methyl gallate (15), amorphous powder, (Z)-3-
hexenyl b-
D
-glucoside (16), amorphous powder, ½a D25
ꢁ
ꢀ13.1 (c
0.32, MeOH) (Mizutani et al., 1988), (E)-2-hexenyl b-
D
-glucoside
(17), amorphous powder, ½a D25
ꢀ39.2 (c 0.53, MeOH) (Mizutani
ꢁ
et al., 1988), malloapeltine (18), colorless rods, mp 203–205
(Cheng et al., 1998).
The pyridine N-oxide 18 is rare and its isolation was the second
time from Nature. It is noteworthy that the calculated specific den-
sity value from the X-ray crystallographic analysis was unusually
large (1.461 g/cm3) due to tight stacking of molecules by
interaction of the pyridine rings as shown in Fig. 5.
p–p
The MeOH extract of M. tanarius possessed anti-oxidative activ-
ity (Lim et al., 2009). Thus, the free radical-scavenging activity of
new compounds 1–3 was evaluated using 1,1-diphenyl-2-picryl-
hydrazyl (DPPH). Trolox was used as a reference compound. Com-
pounds 1 and 2 showed potent radical-scavenging activities. The
activity of compounds 1 and 2 were 1.07 and 0.55 times of Trolox,
respectively. Considering the structural feature, the radical-scav-
enging activity was interpreted as dependent on their galloyl and
phenolic hydroxyl moieties. Cytotoxicity of the new compounds
(1–3) was also assayed against human lung adenocarcinoma cell
4. Concluding remarks
Three new glucosides, (+)-pinoresinol 4-O-[600-O-galloyl]-b-
D-
glucopyranoside (1), macarangioside E and F (2, 3), and 15 known
compounds (4–18) were isolated from the leaves of Macaranga
tanarius. The absolute stereochemistries of them were fully
established using both spectroscopic and chemical approaches.
Galloylated compounds (1 and 2) possessed potent anti DPPH rad-
ical-scavenging activity.
line A549. Even at the highest concentration employed (100 lM),
compounds 1–3 did not exhibit any significant cytotoxicity
(30.3%, 11.3% and 23.8% inhibition, respectively). Thus, compounds
1 and 2 can be considered as non-toxic safe antioxidant agents.
5. Experimental
3. Discussion
5.1. General experimental procedures
There were several literature references concerning 5,12-epoxy-
megastigmane. However, the absolute stereochemistry of the
A highly porous synthetic resin (Diaion HP-20) was purchased from
Mitsubishi Chemical Co., Ltd. (Tokyo, Japan). Column chromatography

1282
K. Matsunami et al. / Phytochemistry 70 (2009) 1277–1285
(CC) was performed using silica gel 60 (Merck, Darmstadt, Germany),
and reversed-phase [octadecyl silica gel (ODS)] open CC (RPCC) on Cos-
to give five fractions. The residue (23.8 mg in fractions 85–100 of
DCCC) was further purified by HPLC (H2O–CH3CN, 4:1) to give
compound 15 (5.2 mg). The residue (23.1 g in fractions 13–17) of
the 60% MeOH eluate obtained on Diaion HP-20 CC was subjected
mosil 75C18-OPN (Nacalai Tesque, Kyoto, Japan) [U = 5 cm, L = 25 cm,
linear gradient: MeOH–H2O (1:9, 1 L) ? (1:1, 1 L), then (1:1,
0.5 L) ? (7:3, 0.5 L), fractions of 10 g being collected]. Droplet Coun-
ter-Current Chromatography (DCCC) (Tokyo Rikakikai, Tokyo, Japan)
to silica gel (U = 5 cm, L = 50 cm) CC with increasing amounts of
MeOH in CHCl3 [CHCl3–MeOH (100:1, 6 L), (25:1, 3 L), (10:1, 3 L),
(5:1, 3 L), (2:1, 3 L), (1:1, 3 L)]. The residue (7.66 g) of the CHCl3–
MeOH (2:1) eluate obtained on silica gel CC was subsequently sub-
jected to RPCC with gradient mixture of MeOH–H2O (1:9, 1 L; 1:1,
1 L, then 1:1, 500 mL; 3:7, 500 mL). 10 g fractions were collected.
The residue (2.47 g in fractions 126–144 of RPCC) was then sub-
jected to DCCC to give five fractions. The residue (670 mg in frac-
tions 26–36 of DCCC) was further purified by HPLC (H2O–CH3CN,
41:9) to afford compound 11 (66.5 mg). The residue (206 mg in
fractions 54–64 of DCCC) was further purified by HPLC (H2O–
CH3CN, 83:17) to afford compounds 12 (13.1 mg) and 14
(20.3 mg). The residue (154 mg in fractions 86–100 of DCCC) was
further purified by HPLC (H2O–CH3CN, 4:1) to afford compounds
8 (9.0 mg) and 2 (0.9 mg). The residue (3.54 g) of the CHCl3-MeOH
(1:1) eluate obtained on silica gel CC was subsequently subjected
to RPCC with gradient mixture of MeOH–H2O (1:9, 1 L; 1:1, 1 L,
then 1:1, 500 mL; 3:7, 500 mL). 10 g fractions were collected. The
residue (306 mg in fractions 51–105 of RPCC) was then subjected
to DCCC to afford five fractions, whereas residue (82.8 mg in frac-
tions 32–42 of DCCC) was further purified by HPLC (H2O–CH3CN,
4:1) to give compound 13 (40.8 mg). The residue (97.6 mg in frac-
tions 43–60 of DCCC) was further purified by HPLC (ODS, H2O–
CH3CN, 4:1, v/v) to give compound 1 (7.6 mg), residue (114 mg in
fractions 61–85 of DCCC) was further purified by HPLC (H2O–
CH3CN, 4:1) to afford compound 9 (8.0 mg).
was equipped with 300 glass columns (
U = 2 mm, L = 40 cm), and
the lower and upper layers of a solvent mixture of CHCl3–MeOH–
H2O-1–PrOH (9:12:8:2) were used as stationary and mobile phases,
respectively. Five-gram fractions were collected and numbered
according to their order of elution with the mobile phase. HPLC was
performed on ODS (Inertsil ODS-3; GL Science, Tokyo, Japan;
U
= 6 mm, L = 250 mm), and the eluate was monitored with a UV
detector at 254 nm and a refractive index monitor. Optical rotations
were measured on a JASCO P-1030 polarimeter. IR spectra were re-
corded on a Horiba FT-710 Fourier transform infrared spectrophotom-
eter and UV spectra on a JASCO V-520 UV/Vis spectrophotometer. 1H
and 13C NMR spectra were obtained on JEOL JNM
a-400, k-500 and
ECA-600 spectrometers at 400, 500, and 600 MHz for 1H, and 100,
125 and 150 MHz for 13C, respectively, with tetramethylsilane as an
internal standard. Positive-ion HR-ESI-TOF-MS was recorded on an Ap-
plied Biosystem QSTAR XL spectrometer. CD spectra were obtained on
a JASCO J-720 spectropolarimeter. VERSA max (Molecular Device) was
used as a microplate reader.
5.2. Plant material
Leaves of M. tanarius Müll.-Arg (Euphorbiaceae) were collected
in Okinawa, Japan, in June, 2003, and a voucher specimen was
deposited in the herbarium of the Department of Pharmacognosy,
Division of Medicinal Chemistry, Graduate School of Biomedical
Sciences, Hiroshima University (03-MT-OKINAWA-0630).
5.4. Characterization data
5.3. Extraction and isolation
5.4.1. (+)-Pinoresinol 4-O-[600-O-galloyl]-b-
D
-glucopyranoside (1)
Amorphous powder; ½a D25
ꢁ
+18.1 (c 0.44, MeOH); IR mmax (film)
The 1-BuOH soluble material (801 g) was prepared by the pro-
cedure described previously (Matsunami et al., 2006). A portion of
the 1-BuOH-soluble fraction (181 g) was subjected to highly por-
ous synthetic resin (Diaion HP-20) CC (Mitsubishi Chemical Co.,
cmꢀ1: 3367, 2961, 1703, 1606, 1515, 1266, 1226, 1073, 1034; UV
kmax (MeOH) nm (log e
): 218 (4.42), 276 (4.05); for 13C- and 1H
NMR (CD3OD) spectroscopic data, see Table 1; CD
De (nm): +1.74
(280), +1.79 (256), ꢀ0.23 (235), +1.27 (223), (c 1.18 ꢂ 10ꢀ5 M,
MeOH); HR-ESI-TOF-MS (positive-ion mode) m/z: 695.1949
[M+Na]+ (Calcd. for C33H36O15Na: 695.1946).
Ltd.;
U = 8 cm, L = 40 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), 1 L fractions were col-
lected. The residue (24.9 g in fractions 9–12) of the MeOH–H2O
(40:60, v/v) eluate obtained on Diaion HP-20 CC was subjected to
5.4.2. Macarangioside E (2)
silica gel (
U
= 6 cm, L = 50 cm) CC with increasing amounts of
Amorphous powder; ½a D25
ꢁ
+78.4 (c 0.19, MeOH); IR mmax (film)
MeOH in CHCl3 [CHCl3 (2 L), and CHCl3–MeOH (49:1, 3 L), (19:1,
3 L), (47:3, 3 L), (23:2, 3 L), (9:1, 3 L), (17:3, 3 L), (4:1, 3 L), (3:1,
3 L), and (7:3, 3 L)], CHCl3–MeOH–H2O (70:30:4, 3 L) and MeOH
(3 L). 500 mL fractions were collected. The residue (0.45 g in frac-
tions 22–31) of the MeOH–CHCl3 (1:9) eluate obtained on silica
gel CC was subsequently subjected to RPCC with gradient mixture
of MeOH–H2O (1:9, 1 L; 1:1, 1 L, and then 1:1, 500 mL; 3:7,
500 mL). 10 g fractions were collected. The residue (24.0 mg in
fractions 1–55 of RPCC) was then purified by HPLC (H2O–CH3CN,
19:1) to give compound 18 (10.2 mg), whereas the residue
(42.2 mg in fractions 65–88 of RPCC) was purified by HPLC (H2O–
CH3CN, 9:1) to afford compound 10 (6.5 mg). The residue
(65.4 mg in fractions 89–102 of RPCC) was purified by HPLC
(H2O–CH3CN, 19:1) to give compounds 3 (5.1 mg), 4 (4.5 mg) and
5 (3.2 mg), with residue (77.2 mg in fractions 123–145 of RPCC)
purified by HPLC (H2O–CH3CN, 41:9) to afford compounds 6
(10.8 mg), 7 (12.6 mg), 16 (3.2 mg) and 17 (5.3 mg). The residue
(3.15 g in fractions 45–59) of the MeOH–CHCl3 (1:3, v/v) eluate ob-
tained on silica gel CC was subsequently subjected to RPCC with a
gradient mixture of MeOH–H2O (1:9, 1 L; 1:1, 1 L, and then 1:1,
500 mL; 3:7, 500 mL). 10 g fractions were collected. The residue
(1.44 g in fractions 71–110 of RPCC) was then subjected to DCCC
cmꢀ1: 3303, 2961, 1706, 1648, 1514, 1449, 1259, 1076, 1032; UV
kmax (MeOH) nm (log e): 219 (4.54), 244sh (4.25), 275 (4.12); for
13C and 1H NMR (CD3OD) spectroscopic data, see Tables 2 and 3;
CD
D
e
(nm): ꢀ0.90 (317), +18.5 (245) (c 1.85 ꢂ 10ꢀ5 M, MeOH);
HR-ESI-TOF-MS (positive-ion mode) m/z: 545.1990 [M+Na]+
(Calcd. for C26H34O11Na: 545.1993).
5.4.3. Macarangioside F (3)
Amorphous powder; ½a D26
ꢀ6.0 (c 0.34, MeOH); IR mmax (film)
ꢁ
cmꢀ1: 3396, 2928, 1712, 1380, 1275, 1077, 1037; for 13C and 1H
NMR (CD3OD) spectroscopic data, see Table 2 and 3; CD
De (nm):
+0.31 (362), ꢀ0.06 (298), +0.53 (250) (c 5.03 ꢂ 10ꢀ5 M, MeOH);
HR-ESI-TOF-MS (positive-ion mode) m/z: 411.1987 [M+Na]+
(Calcd. for C19H32O8Na: 411.1989).
5.4.4. Mild alkaline methanolysis of 1
A mixture of 1 (3.3 mg) and 0.1 M NaOMe in MeOH (1.0 mL)
was allowed to stand at room temperature for 1 h under N2. Liber-
ation of degalloylated compound 1a was monitored by TLC analy-
sis (CHCl3: MeOH: H2O, 15:6:1, Rf values, 1: 0.48 and 1a: 0.57). The
reaction mixture was neutralized with Amberlite IR-120B (Organo)
and subjected to silica gel CC [7 g,
U = 1 cm, L = 15 cm, linear gra-

K. Matsunami et al. / Phytochemistry 70 (2009) 1277–1285
1283
dient, CHCl3–MeOH (10:1, 100 mL) ? CHCl3–MeOH–H2O (15:6:1,
100 mL), 8 g fractions being collected]. Degalloylated compound
1a (1.7 mg) was recovered in fractions 10–14 as (+)-pinoresinol
(positive-ion mode) m/z: 409.1835 [M+Na]+ (Calcd. for
C19H30O8Na: 409.1832).
O-b-
D
-glucopyranoside, which was identified analysis of 1H- and
5.4.8. Catalytic hydrogenation of 19a
13C NMR spectroscopic data in C5D5N (Abe and Yamauchi, 1989)
An aliquot of 19a (1.1 mg) was dissolved in MeOH (0.5 mL),
then 1 mg of Adams’ catalyst (PtO2) was added and stirred at room
temperature for 1 h under H2 atmosphere. Hydrogenation of 19a
was monitored by TLC analysis [CHCl3: MeOH: H2O, 15:6:1, Rf val-
ues, 19a: 0.45 and dihydrodegalloylmacarangioside B (19b): 0.49].
The spot of 19b was confirmed to be identical to 3 by TLC co-chro-
matography. The reaction mixture was filtered and concentrated to
afford 19b (0.82 mg). The 1H and 13C NMR, MS and specific optical
and optical rotation value, ½a D26
ꢁ
+13.1 (c 0.11, MeOH) [Ref. for
(+)-pinoresinol O-b-D-glucopyranoside, +8.0 (c 0.1, MeOH) (Dey-
ama, 1983)]. 13C NMR (CD3OD, 150 MHz) dC: 151.2 (C-3), 149.2
(C-30), 147.6 (2 ꢂ C, C-4 & 40), 137.7 (C-1), 133.9 (C-10), 120.1 (C-
60), 119.9 (C-6), 118.3 (C-5), 116.2 (C-50), 111.9 (C-2), 111.2 (C-
20), 103.1 (C-100), 87.6 (C-70), 87.2 (C-7), 78.3 (C-300), 78.0 (C-500),
75.0 (C-200), 72.8 (2 ꢂ C, C-9 & 90), 71.5 (C-400), 62.6 (C-600), 56.9
(3-OMe), 56.6 (30-OMe), 55.6 (C-8), 55.4 (C-80).
rotation were also identical to 3 [19b: ½a D25
ꢀ8.4 (c = 0.055,
ꢁ
MeOH)].
5.4.5. Mild alkaline methanolysis of 2
In a similar to that described above, compound 2 (1.5 mg) liber-
ated degalloylated compound 2a (0.72 mg) (TLC, CHCl3: MeOH:
H2O, 15:6:1, Rf values, 2: 0.42 and 2a: 0.58). 2a: Amorphous pow-
5.4.9. NaBH4 reduction of 3a
A solution of 3a (1.62 mg) in EtOH (0.5 mL), 1.5 mg of NaBH4
dissolved in EtOH (0.5 mL) was added dropwise at 0 °C and then
stirred for 1 h at rt with the progress of the reaction was monitored
der; ½a 2D6
ꢁ
+84.2 (c 0.048, MeOH) and CD
D
e
(nm): ꢀ0.9 (311),+11.0
(244) (c 0.97 ꢂ 10ꢀ5 M, MeOH) [Ref. for (6R,9R)-3-oxo-
a
-ionol 9-O-
by TLC analysis [CHCl3: MeOH, 5:1, Rf values, 3a: 0.58, 3
a,9a-diol
b-D
-glucopyranoside; ½a D22
ꢁ
+95.2 (c 1.33, MeOH), (Mohamed et al.,
(3b): 0.49, and 3b,9 -diol (3c): 0.37]. The reaction mixture was
a
1999), CD rel.int. (nm): ꢀ8 (327), +207 (243) (EtOH) (Pabst et al.,
quenched by addition of 0.5 mL of 1% CH3COOH at 0 °C, concen-
trated and then purified by preparative TLC [CHCl3: MeOH, 5:1]
1992)].
to afford 3
3a,9a
a,9a-diol (3b) (0.64 mg) and 3b,9a-diol (3c) (0.78 mg).
5.4.6. Enzymatic hydrolysis of 3
-diol (3b): Amorphous powder; 1H NMR (CD3OD, 500 MHz)
Compound 3 (4.1 mg) was hydrolyzed with crude hesperidinase
at 37 °C in H2O (1 mL) with reciprocal shaking for 12 h. Liberation
of glucose was monitored by TLC analysis (CHCl3: MeOH: H2O,
dH: 3.99 (1H, d, J = 7 Hz, pro-R H-12), 3.97 (1H, m, H-3), 3.71 (1H,
m, H-9), 3.40 (1H, dd, J = 7, 2 Hz, pro-S H-12), 1.88–1.81 (2H, m,
H-2ax and H-4ax), 1.61–1.54 (2H, m, H-2 eq and H-4 eq), 1.54–
1.51 (3H, m, H2-8 and pro-R H-7), 1.37 (1H, m, H-6), 1.23 (3H, s,
H3-13), 1.18 (1H, m, pro-S H-7), 1.16 (3H, d, J = 6 Hz, H3-10), 0.97
(3H, s, H3-11); 1H NMR (CDCl3, 500 MHz) dH: 3.95 (1H, d, J = 8 Hz,
pro-R H-12), 3.90 (1H, m, H-3), 3.78 (1H, m, H-9), 3.47 (1H, dd,
J = 8, 2 Hz, pro-S H-12), 1.84 (1H, dd, J = 15, 5 Hz, H-4ax), 1.78
(1H, ddd, J = 15, 5, 2 Hz, H-2ax), 1.68 (1H, br d, J = 15 Hz, H-2 eq
or H-4 eq), 1.66 (1H, br d, J = 15 Hz, H-2 eq or H-4 eq), 1.56–1.49
(3H, m, H2-8 and pro-R H-7), 1.41 (1H, m, H-6), 1.27 (3H, s, H3-
13), 1.22 (3H, d, J = 6 Hz, H3-10), 1.15 (1H, m, pro-S H-7), 0.97
(3H, s, H3-11); HR-ESI-TOF-MS (positive-ion mode) m/z:
15:6:1, Rf values, 3: 0.47, aglycone 3a: 0.75, and
The reaction mixture was concentrated and subjected to silica gel
CC [7 g, = 1 cm, L = 15 cm, linear gradient, CHCl3–MeOH (10:1,
D-glucose: 0.12).
U
100 mL) ? CHCl3–MeOH–H2O (15:6:1, 100 mL) ? EtOH 30 mL,
8 g fractions were collected]. The aglycone 3a (1.8 mg, 80%) and
glucose were recovered in fractions 6–11 and 28–29, respectively.
(1S,5R,6R,9R)-megastigman-3-on-5,12-epoxy-9-ol (3a): Amor-
phous powder; ½a D24
ꢁ
+6.1 (c 0.11, MeOH) [Ref. for annuionone E:
[a]D + 4.2 (c 0.1, MeOH) (Macías et al., 2002)], IR mmax (film)
cmꢀ1: 3423, 2925, 1712, 1457, 1377, 1261, 1019; 13C NMR (CDCl3,
100 MHz) dC: 209.3 (C-3), 83.5 (C-5), 78.5 (C-12), 68.1 (C-9), 53.8
(C-6), 49.5 (C-4), 48.8 (C-2), 43.5 (C-1), 38.7 (C-8), 24.9 (C-13),
23.9 (C-10), 21.5 (C-7), 20.9 (C-11); 1H NMR (CDCl3, 400 MHz)
dH: 3.83 (1H, sextet, J = 6 Hz, H-9), 3.64 (1H, d, J = 8 Hz, pro-S H-
12), 3.58 (1H, dd, J = 8, 2 Hz, pro-R H-12), 2.42–2.32 (3H, over-
251.1614 [M + Na]+ (Calcd. for C13H24O3Na: 251.1617). 3b,9
a-diol
(3c): Amorphous powder; [
a
]
D
24 + 18.5 (c 0.052, MeOH); 1H NMR
(CD3OD, 500 MHz) dH: 3.95 (1H, tt, J = 10, 7 Hz, H-3), 3.62 (1H, d,
J = 8 Hz, pro-R H-12), 3.73 (1H, sextet, J = 6 Hz, H-9), 3.42 (1H, dd,
J = 8, 2 Hz, pro-S H-12), 1.80 (1H, m, H-4ax), 1.66 (1H, m, pro-R
H-7), 1.64 (1H, m, H-2ax), 1.54 (2H, m, H2-8), 1.46 (2H, m, H-
2 eq and H-4 eq), 1.35 (1H, m, H-6), 1.32 (1H, m, pro-S H-7), 1.23
(3H, s, H3-13), 1.18 (3H, d, J = 6 Hz, H3-10), 1.00 (3H, s, H3-11);
HR-ESI-TOF-MS (positive-ion mode) m/z: 251.1616 [M+Na]+
(Calcd. for C13H24O3Na: 251.1617).
lapped, H-2b, 4
a and 4b), 2.23 (1H, dt, J = 18, 2 Hz, H-2a), 1.74
(1H, dq, J = 16, 6 Hz, pro-R H-7), 1.68 (1H, m, H-6), 1.63 (2H, q,
J = 6 Hz, H2-8), 1.37 (1H, dq, J = 16, 6 Hz, pro-S H-7), 1.33 (3H, s,
H3-13), 1.24 (3H, d, J = 6 Hz, H3-10), 1.08 (3H, s, H3-11); CD
De
(nm): +0.04 (357), ꢀ0.25 (281), ꢀ0.70 (211) (c = 4.78 ꢂ 10ꢀ5 M,
MeOH); HR-ESI-TOF-MS (positive-ion mode) m/z: 249.1464
[M+Na]+ (Calcd. for C13H22O3Na: 249.1461).
5.4.10. Preparation of (S)- and (R)-MTPA diesters (3d and 3e) from 3c
A solution of 3c (0.39 mg) in dry CH2Cl2 (0.5 mL) was reacted
with (S)-MTPA (14.3 mg) in the presence of 1-ethyl-3-(3-dimethyl-
aminopropyl) carbodiimide hydrochloride (EDC) (7.7 mg) and 4-
N,N’-dimethylaminopyridine (DMAP) (5.7 mg), and stirred at
25 °C for 30 min, then 40 °C for 5 min. After addition of H2O
(0.5 mL) and CHCl3 (0.5 mL) the solution was successively washed
with 1 M HCl (1 mL), NaHCO3-saturated H2O (1 mL), and saturated
brine (1 mL). The organic layer was dried (Na2SO4), and evaporated
under reduced pressure. The resulting residue was then purified by
preparative TLC (silica gel (0.25 mm thickness, applied for 18 cm
and developed with CHCl3–(CH3)2CO (20: 1) for 9 cm and eluted
with CHCl3–MeOH (2:1)) to furnish a diester, 3d (0.7 mg, 66%).
Through a similar procedure, 3e (0.8 mg, 75%) was also prepared
from 3c (0.39 mg) using (R)-MTPA (10.3 mg), EDC (7.9 mg) and
DMAP (3.9 mg). (1S,3S,5R,6R,9R)-megastigman-5,12-epoxy-3,9-
diol 3,9-di-(S)-MTPA ester (3d): Amorphous powder; 1H NMR
The absolute configuration of glucose was determined to be
D
by the sign of its positive optical rotation and retention time
(14.8 min) following HPLC separation [JASCO OR-2090 plus: Opti-
cal rotation detector, SHODEX Asahipak NH2P-50:
L = 25 cm, 80% CH3CN aq., 1 mL/min]. Peaks were identified by
co-chromatography with authentic -glucose.
U = 4.5 mm,
D
5.4.7. Mild alkaline methanolysis of macarangioside B (19)
In a similar manner to that described above, compound 19
(2.0 mg) liberated degalloylated compound 19a (1.2 mg) (TLC,
CHCl3: MeOH: H2O, 15:6:1, Rf values, 19: 0.25 and 19a: 0.51). Deg-
alloylmacarangioside B (19a): Amorphous powder; ½a D24
ꢀ29.5
ꢁ
(c = 0.078, MeOH); IR mmax (film) cmꢀ1: 3395, 2925, 1712, 1652,
1455, 1266, 1072, 1037; 13C NMR (CD3OD): Table 2; 1H NMR
(CD3OD): Table 3; CD
D
e
(nm): +0.33 (354), ꢀ0.72 (284), +0.62
(243), ꢀ0.73 (211) (c = 2.02 ꢂ 10ꢀ5 M, MeOH); HR-ESI-TOF-MS

1284
K. Matsunami et al. / Phytochemistry 70 (2009) 1277–1285
(CDCl3, 500 MHz) dH: 7.52–7.49 (4H, m, aromatic protons), 7.42–
7.32 (6H, m, aromatic protons), 5.33 (1H, m, H-3), 5.12 (1H, m,
H-9), 3.72 (1H, d, J = 8 Hz, pro-S H-12), 3.54 (3H, m, OMe), 3.52
(3H, m, OMe), 3.44 (1H, dd, J = 8, 2 Hz, pro-R H-12), 1.97 (1H, m,
H-4 eq), 1.75 (1H, m, H-2 eq), 1.72 (2H, m, H2-8), 1.50 (1H, m, H-
4ax), 1.36 (3H, d, J = 6 Hz, H3-10), 1.31 (1H, m, H-2ax), 1.15 (1H,
m, H-7a), 1.20 (3H, s, H3-13), 0.97 (3H, s, H3-11); HR-ESI-TOF-MS
(positive-ion mode) m/z: 683.2406 [M+Na]+ (Calcd. for C33H38O7F6-
Na: 683.2413). (1S,3S,5R,6R,9R)-megastigman-5,12-epoxy-3,9-diol
3,9-di-(R)-MTPA ester (3e): Amorphous powder; 1H NMR (CDCl3,
500 MHz) dH: 7.52–7.49 (4H, m, aromatic protons), 7.41–7.35
(6H, m, aromatic protons), 5.35 (1H, m, H-3), 5.11 (1H, m, H-9),
3.74 (1H, d, J = 8 Hz, pro-S H-12), 3.52 (3H, m, OMe), 3.49 (3H, m,
OMe), 3.47 (1H, dd, J = 8, 2 Hz, pro-R H-12), 1.94 (1H, m, H-2 eq),
1.84 (1H, m, H-4 eq), 1.77 (2H, m, H-8), 1.47 (1H, m, H-4ax), 1.38
(1H, m, H-2ax), 1.29 (3H, d, J = 6 Hz, H3-10), 1.27 (1H, m, H-7a),
1.21 (3H, s, H3-13), 0.97 (3H, s, H3-11);HR-ESI-TOF-MS (positive-
ion mode) m/z: 683.2419 [M+Na]+ (Calcd. for C33H38O7F6Na:
683.2413).
amount of compounds (final concentrations were ranging from
100 to 1 M with 1% DMSO). Etoposide (3 M) was used as a posi-
tive control. Negative control cells were incubated in the same
medium only with 1% DMSO (vehicle). After 72 h incubation, the
medium in each well was replaced by fresh medium (100 lL) con-
taining 0.5 mg/mL of 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-
2H-tetrazolium bromide (MTT). Two hours later, the medium
was removed, and the precipitated MTT formazan was dissolved
in DMSO. The absorbance at 540 nm was measured using a micro-
plate reader. The viability was calculated by the following
equation.
l
l
% cytotoxicity ¼ 1ꢀðAsample ꢀAbackgroundÞ=ðAvehicle ꢀAbackgroundÞꢂ100
Acknowledgments
This research was supported by Grant-in Aid for Scientific Re-
search (C) (No. 20590103), for Scientific Research on Priority Areas
(No. 18032050), the Research Foundation for Pharmaceutical Sci-
ences, and Takeda Science Foundation. We are also grateful for
the use of NMR, UV and MS spectrometers at Research Center for
Molecular Medicine, the Analysis Center of Life Science, and the
Natural Science Center for Basic Research and Development (N-
BARD), Hiroshima University.
5.4.11. Single-crystal X-ray structure analysis of 18
A suitable crystal (0.30 ꢂ 0.10 ꢂ 0.10 mm) was used for analy-
sis. The data were measured using a Bruker SMART 1000 CCD dif-
fractometer, using MoK
a graphite-monochromated radiation
(k = 0.71073 Å). The structure was solved by a direct method using
the program SHELXTL-97 (Sheldrick, 2008). The refinement and all
further calculations were carried out using SHELXTL-97. The
absorption correction was carried out utilizing the SADABS routine
(Sheldrick, 1996). The H atoms were included at calculated posi-
tions and treated as riding atoms using the SHELXTL default
parameters. The non-H atoms were refined anisotropically, using
weighted full-matrix least-squares on F2. Fig. 5 was drawn with
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