Novel megastigmanes with lipid accumulation inhibitory and lipid metabolism-promoting activities in HepG2 cells from Sedum sarmentosum

Article abstract of DOI:10.1016/j.tet.2009.03.040

Four novel megastigmanes, neosedumosides I (1), II (2), III (3), and IV (4) were isolated from the whole plant of Sedum sarmentosum (Crassulaceae). Absolute stereostructures of these constituents were determined on the basis of chemical and physicochemica

Full text of DOI:10.1016/j.tet.2009.03.040

Tetrahedron 65 (2009) 4142–4148
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Novel megastigmanes with lipid accumulation inhibitory and lipid metabolism-
promoting activities in HepG2 cells from Sedum sarmentosumq
Osamu Muraoka a, Toshio Morikawa a, Yi Zhang b, Kiyofumi Ninomiya a, Seikou Nakamura b,
,b,
Hisashi Matsuda b, Masayuki Yoshikawa a
*
a Pharmaceutical Research and Technology Institute, Kinki University, 3-4-1 Kowakae, Higashi-osaka, Osaka 577-8502, Japan
b Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8412, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Four novel megastigmanes, neosedumosides I (1), II (2), III (3), and IV (4) were isolated from the whole
plant of Sedum sarmentosum (Crassulaceae). Absolute stereostructures of these constituents were de-
termined on the basis of chemical and physicochemical evidence. Among them, 1–3 were found to show
lipid accumulation inhibitory activity in HepG2 cells. Furthermore, 2 and 3 were found to also show lipid
metabolism-promoting activity.
Received 29 January 2009
Received in revised form 16 March 2009
Accepted 18 March 2009
Available online 25 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Lipid accumulation inhibitory activity
Lipid metabolism-promoting activity
Sedum sarmentosum
Crassulaceae
Megastigmane
Neosedumoside
1. Introduction
glycosides, neosedumosides I (1, 25.4 mg), II (2, 12.3 mg), III (3,
54.2 mg), and IV (4, 9.2 mg) (Chart 1).
25
During the course of our characterization studies on bioactive
constituents from Chinese natural medicines,1–5 we have reported
the isolation and structural elucidation of 27 megastigmane
constituents, including sarmentoic acid, sarmentol A, sedumosides
A1–A6, B, C, D, E1–E3, F1, F2, and G–I, and four flavonol glycosides,
sarmenosides I–IV from the whole plant of Sedum sarmentosum
(Crassulaceae).2–5 As a continuing study on this herbal medicine,
we have isolated four novel bicyclic megastigmane glycosides,
neosedumosides I (1), II (2), III (3), and IV (4). This paper deals with
the isolation and structural elucidation of these new mega-
stigmanes (1–4) and their lipid accumulation inhibitory and lipid
metabolism-promoting activities.
Neosedumoside I (1), [
a
]
þ40.0 (MeOH), was obtained as an
D
amorphous powder. Its IR spectrum showed absorption bands at
3389, 1653, and 1036 cmꢀ1 ascribable to hydroxyl,
-unsaturated
olefin, and ether functions, respectively. In the UV spectrum, an
absorption maximum was observed at 241 nm (log 4.06 in MeOH)
ascribable to the enone moiety. The EIMS of 1 showed a molecular
ion peak at m/z 386 (Mþ), and the molecular formula was de-
termined as C19H30O8 by high-resolution EIMS measurement. The
1H and 13C NMR spectra of 1 (CD3OD, Tables 1 and 2) showed
a,b
3
11
6
12
2
1
7
8
9
13
10
2. Results and discussion
5
3
O
O
4
HO
HO
OGlc
OGlc
The MeOH-eluted fraction (72.0 g) from the whole plant of S.
sarmentosum3 was subjected to SiO2 and ODS column chromato-
graphies and finally HPLC (ODS column, eluted with CH3CN–
MeOH–H2O solvent system) to furnish five novel megastigmane
neosedumoside I (1)
neosedumoside II (2)
O
O
HO
OGlc
neosedumoside III (3)
OGlc
neosedumoside IV (4)
q
See Ref. 1.
* Corresponding author. Tel.: þ81 75 595 4633; fax: þ81 75 595 4768.
E-mail address: myoshika@mb.kyoto-phu.ac.jp (M. Yoshikawa).
Chart 1. Structures of neosedumosides I–IV (1–4).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.03.040

O. Muraoka et al. / Tetrahedron 65 (2009) 4142–4148
4143
Table 1
12
1
11
5
1H NMR data for neosedumosides I (1), II (2), III (3), and IV (4) (at 500 MHz in CD3OD,
J values in Hz)
6
2
12
2
8
H
11
H
H
H
H
1
2
3
4
3
10
9
O
7
13
4
H
H
6
HO
8
2
2
3
a
b
2.19 (d, 15.6)
2.25 (d, 15.6)
2.19 (d, 15.6)
2.26 (d, 15.6)
2.44 (d, 16.2)
2.17 (dd, 0.9, 16.2) 2.43 (d, 17.4)
1.88 (d, 17.4)
O
HO
13
O
10
O
4(
a
)
5.83 (br s)
5.83 (br s)
5.79 (br s)
2.09 (d, 17.7)
2.83 (d, 17.7)
1.80 (m)
OH
H
H
OH
1'
OGlc
4
6
7
7
8
b
1
2.23 (m)
1.95 (m)
1.43 (m)
1.61 (ddd,
4.0, 13.1, 13.4)
2.15 (m)
3.39, 3.78
(both d, 10.7)
0.95 (s)
2.19 (m)
1.89 (m)
1.78 (m)
1.74 (m)
2.50 (m)
2.05 (m)
1.45 (m)
2.40 (m)
HO
H
a
b
a
1.77 (2H, m)
OH
1, 2
H
H
1.64 (m)
H
H
H
H-H COSY :
HMBC :
H
H
8
b
1.81 (m)
3.40, 3.83
(both d, 10.1)
2.30 (m)
4.22, 4.43
(both d, 14.7)
0.88 (s)
1.71 (m)
3.48, 4.04
(both d, 10.5)
0.97 (s)
10
NOE :
O
OH
11
0.98 (s)
H
H
OGlc
12 1.06 (s)
1.07 (s)
1.15 (s)
1.05 (s)
2
13
2.37 (d, 13.9)
2.48 (d, 14.6)
6.45 (s)
1.10 (s)
Figure 1. 1H–1H COSY, HMBC, and NOE correlations of 1 and 2.
2.70 (dd, 2.1, 13.9) 2.54 (dd, 1.9, 14.6)
10
20
30
40
50
60
4.25 (d, 7.6)
4.29 (d, 7.7)
4.29 (d, 7.7)
4.22 (d, 7.7)
3.22 (dd, 7.7, 9.2)
3.22 (dd, 7.7, 9.2) 3.23 (dd, 7.7, 9.2) 3.23 (m)
3.36 (m) 3.36 (m)
3.26 (dd, 8.6, 8.6) 3.28 (m)
3.25 (m) 3.28 (m)
3.65 (dd, 5.5, 11.4) 3.65 (dd, 5.5, 11.9) 3.66 (dd, 5.5, 11.9) 3.65 (dd, 5.8, 11.9)
3.83 (dd, 1.8, 11.4) 3.87 (dd, 1.8, 11.9) 3.86 (dd, 2.1, 11.9) 3.86 (dd, 1.5, 11.9)
3.36 (dd, 8.5, 8.5) 3.35 (dd, 9.2, 9.2)
3.28 (m)
3.26 (m)
3.27 (m)
3.26 (m)
correlations were observed between the following proton pairs:
H-2 and H3-12; H-2 and H3-11; H-4 and H-13 ; H-6 and H-7
H3-12; H-7 and H-8 ; H-7 and H-8 , H3-11; H-8 and H-13 ; H-
and H2-10 (Fig. 1). On the basis of above-mentioned evidence,
a
b
b
a,
a
a
b
b
a
a
8b
the relative stereostructure of 1 was elucidated as shown in
Figure 1.
signals assignable to two methyls [
11,12)], four methylenes, a methine, a methylene bearing an oxygen
function [
3.39, 3.78 (1H each, both d, J¼10.7 Hz, H2-10)], a qua-
ternary carbon bearing an oxygen function (dC 74.5), a tri-
substituted olefin [ 5.83 (1H, br s, H-4), dC 125.5 (C-4), 165.7 (C-5)],
d 0.95, 1.06 (3H each, both s, H3-
Neosedumoside II (2) was isolated as an amorphous powder
d
25
with positive optical rotation ([
a
]
D
þ39.6 (MeOH)). The EIMS of 2
showed a molecular ion peak at m/z 386 (Mþ), and the molecular
formula, C19H30O8, was found to be the same as that of 1 by high-
resolution EIMS measurement. The 1H and 13C NMR spectroscopic
properties of 2 (CD3OD, Tables 1 and 2) were quite similar to those
d
and a conjugated carbonyl carbon (dC 202.5) together with those of
a glucopyranosyl moiety [
drolysis of 1 with 1 M HCl liberated the
d
4.25 (1H, d, J¼7.6 Hz, H-10)]. Acid hy-
D-glucose, which was
of 1. That is, 2 showed signals due to two methyls [
each, both s, H3-11, 12)], four methylenes, a methine, a methylene
bearing an oxygen function [
3.40, 3.83 (1H each, both d, J¼10.1 Hz,
H2-10)], a quaternary carbon bearing an oxygen function (dC 79.0),
a trisubstituted olefin [ 5.83 (1H, br s, H-4), dC 125.6 (C-4), 167.1 (C-
d 0.98, 1.07 (3H
identified by HPLC analysis using an optical rotation detector.2–5
The bicyclic neomegastigmane skeleton of 1 was constructed on
the basis of various NMR experiments.6 Namely, the 1H–1H COSY
experiments on 1 indicated the presence of two partials written in
bold lines, while in the HMBC experiments, long range correlations
were observed between the following proton and carbon pairs: H2-
2 and C-1, 3, 4; H2-4 and C-2, 5, 6, 13; H-6 and C-1, 4, 5; H2-7 and C-
5; H2-8 and C-6; H2-10 and C-8, 9, 13; H3-11 and C-1, 2, 6, 12; H3-12
and C-1, 2, 6, 11; H-10 and C-10 (Fig. 1). Next, the relative stereo-
structure of 1 was clarified by the NOESY experiment, in which
d
d
5)], and a conjugated carbonyl carbon (dC 202.5) together with
those of a glucopyranosyl moiety. On acid hydrolysis with 1 M HCl,
2 liberated the D-glucose. The planar structure of 2 was character-
ized to be the same as that of 1 by means of 1H–1H COSY and HMBC
experiments as shown in Figure 1. The NOESY spectrum of 2
showed distinct correlation between H-13a and H2-10 (Fig.1). Thus,
2 was clarified to be the stereoisomer of 1 at the 10-position.
Table 2
Neosedumoside III (3) was isolated as a white powder with
13C NMR data for 1–4 (at 125 MHz)
27
negative optical rotation ([
a
]
ꢀ63.7 (MeOH)). The positive-ion
D
FABMS showed a quasimolecular ion peak at m/z 391 (MþNa)þ and
its molecular formula, C19H28O7, was determined by high-resolu-
tion FABMS measurement. In the UV spectrum, an absorption
1a
2a
3a
37.3
4a
34.7
4b
34.2
48.7
212.1
46.1
47.9
55.6
25.8
34.2
83.7
75.2
28.0
30.0
28.7
106.0
75.2
78.8
71.6
78.7
62.7
1
2
3
4
5
6
7
8
35.8
35.7
51.6
202.5
125.5
165.7
49.3
24.5
35.2
74.5
51.3
202.5
125.6
167.1
50.0
23.7
34.1
74.6
54.7
202.8
123.8
159.7
46.9
23.4
28.2
151.8
72.7
20.3
29.0
126.5
104.0
75.1
48.8
217.3
46.8
48.8
56.9
26.7
34.7
84.8
75.3
29.3
30.4
29.5
105.0
75.1
78.0
71.7
78.1
62.8
maximum was observed at 290 nm (log 3 4.12 in MeOH), which was
suggestive of a hetero-annular diene chromophore. The IR spec-
trum showed absorption bands due to hydroxyl, olefin, and ether
groups at 3649, 1653, and 1076 cmꢀ1, respectively. 1H and 13C NMR
spectra of 3 (CD3OD, Tables 1 and 2) showed signals assignable to
9
two methyls [
methylenes, a methine, a methylene bearing an oxygen function [
4.22, 4.43 (1H each, both d, J¼14.7 Hz, H2-10)], two trisubstituted
olefins [ 5.79, 6.45 (1H each, both br s, H-4, 13), dC 123.8 (C-4),
d 0.88, 1.15 (3H each, both s, H3-11, 12)], three
10
11
12
13
10
20
30
40
50
60
74.5
79.0
24.6
28.9
46.1
105.0
75.2
77.9
d
24.3
28.9
46.1
104.9
75.2
78.0
71.6
d
126.5 (C-13), 151.8 (C-9), 159.7 (C-5)], and a conjugated carbonyl
carbon (dC 202.8) together with those of a glucopyranosyl moiety.
78.1
71.6
78.0
62.8
The acid hydrolysis of 3 liberated the -glucose. Connectivities of
D
71.6
the quaternary carbons and the b-D-glucopyranosyl part were
77.9
62.7
78.0
62.7
elucidated on the basis of various NMR measurements, and the
relative stereostructure of 3 was unambiguously clarified as shown
in Figure 2 by the NOESY experiment.
a
In CD3OD.
In pyridine-d5.
b

4144
O. Muraoka et al. / Tetrahedron 65 (2009) 4142–4148
The 1H and 13C NMR spectra (CD3OD, Tables 1 and 2) showed
signals assignable to three methyls [ 0.97, 1.05, 1.10 (3H each, all s,
H3-11, 12, 13)], four methylenes, a methine [ 1.80 (1H, m, H-6)],
a methylene bearing an oxygen function [ 3.48, 4.04 (1H each, both
d, J¼10.5 Hz, H2-10)], and a carbonyl carbon (dC 217.3) together with
those of a -glucopyranosyl part. The acid hydrolysis of 4 liber-
ated the -glucose. The relative stereostructure was clarified by
various NMR experiments as shown in Figure 3. In order to de-
termine the absolute structure, following chemical modifications
were carried out. As shown in Scheme 3, treatment of the aglycone,
neosarmentol IV (4a), which was obtained by enzymatic hydrolysis
of 4, with NaBH4 gave the reductant (4b). Then, 4b was treated with
1,10-thiocarbonyldiimidazole in the presence of 4-DMAP to give
9,10-thiocarbonate (4c). Compound 4c showed a negative Cotton
12
2
11
1
12
2
d
H
H
11
6
H
H
H
8
d
7
H
H
6
8
10
d
3
5
9
O
4
13
O
O
10
HO
13
b-D
D
O
3
OGlc
OH
1'
HO
H
:
:
:
H-H COSY
HMBC
OH
3
NOE
Figure 2. 1H–1H COSY, HMBC, and NOE correlations of 3.
Finally, absolute stereostructures of 1–3 were determined by the
effect [305 nm (D3 ꢀ0.85) in MeOH] associated by the n/
p
*
CD excitation chirality method for allylic benzoates.7–9 As shown in
transition of cyclic thiocarbonate moiety.10,11 On the basis of these
evidences, the absolute configuration at the 9-position was de-
termined as R orientation. A variety of megastigmane constituents
have been isolated from many kinds of plant materials, however, as
far as we know, this is the first report on isolation and structural
elucidation as well as bioactivities of bicyclic megastigmanes hav-
ing a bicyclo[4.4.0]decane or bicyclo[4.3.0]nonane skeleton from
natural resources.
The whole plant of S. sarmentosum has traditionally been used
for the treatment of liver disease in China and South Korea.12 It is
recognized that fatty liver is a significant risk factor for serious liver
disease.13,14 There is a strong association between fatty liver disease
and hyperinsulinemic insulin-resistance.15,16 Thus, fatty liver is of-
ten associated with obesity and type 2 diabetes.15 As a part of our
characterization studies of antidiabetic constituents from natural
medicines, effects of the neosedumosides I–IV (1–4) on triglyceride
contents in HepG2 cells were examined in this study using two
methods; 1. triglyceride (TG) accumulation inhibitory activity in
HepG2 cells and 2. TG metabolism-promoting activity in high
glucose pre-treated HepG2 cells. Firstly, effects of 1–4 on oleic acid–
albumin-induced TG accumulation inhibitory activity in HepG2
cells were examined.17 As shown in Table 3, 1 (% of control at
Scheme 1, enzymatic hydrolysis of 1–3 with a b-glucosidase gave
the corresponding aglycones named neosarmentols I–III (1a–3a),
respectively. Dehydration of 1a and 2a with SOCl2/pyridine gave 3a,
thus, the stereochemistry of 1a–3a was found to be the same except
for the 9-position in 1a and 2a. Next, acetylation of 3a using Ac2O/
pyridine yielded 10-monoacetate (3b), which was then treated
with NaBH4 in the presence of CeCl3 to give the corresponding 3
hydroxy derivative (3c) as the main product [
4.30 (1H, br dd, J¼ca.
7, 11 Hz, H-3 )]. The orientation of the 3 -hydroxyl in 3c was also
clarified by NOE correlations observed between the following
proton pairs: H-3 and H3-12, H-2 . Treatment of 3c with benzoyl
b-
d
b
b
a
chloride in the presence of 4-DMAP yielded 3-benzoate (3d), which
showed a negative Cotton effect [225 nm (D3 ꢀ3.05) in MeOH]. The
above-mentioned evidence led us to conclude that the stereo-
chemistry at the 3-position in 3d was S orientation. The plausible
biogenetic pathways of 1–3 are shown in Scheme 2.
Neosedumoside IV (4) was isolated as an amorphous powder
27
with negative optical rotation ([
a
]
ꢀ27.6 (MeOH)). The EIMS
D
showed a molecular ion peak at m/z 388 (Mþ), and the molecular
formula, C19H32O8, was determined by high-resolution EIMS mea-
surement. Its IR spectrum showed absorption bands at 3389, 1708,
1076, and 1035 cmꢀ1due to hydroxyl, carbonyl, and ether functions.
100
m
M: 75.1ꢁ1.8), 2 (87.8ꢁ1.5), and 3 (81.6ꢁ4.0) were found to
5
3
O
O
O
RO
4
HO
OR
OR
OAc
OAc
e)
d)
1: R = Glc
3: R = Glc
3a: R = H
3c: R = H
3d: R = Bz
3b
a)
b)
a)
1a: R = H
c)
12
H
H
11
H
H
7
H
OBz
5
2
6
O
HO
3(4)
3
OR
HO
2: R = Glc
2a: R = H
a)
b)
H
OH
3d
NOE :
3d
Scheme 1. (a)
b
-glucosidase (Sigma, from almond)/H2O, 37 ꢂC, 16 h, 1a (21% from 1), 2a (64% from 2), 3a (48% from 3); (b) SOCl2/pyridine, rt, 1 h, 3a (48% from 1a, 62% from 2a);
(c) Ac2O/pyridine, rt, 18 h, 90%; (d) NaBH4, CeCl3/EtOH, rt, 20 min, 72%; (e) BzCl, 4-DMAP/pyridine, rt, 6 h, 64%.
O
O
9
9
CH2OGlc
CH2OGlc
6
5
— H2O
3
5
3
5
9
H
13
O
O
O
O
4
13
4
4
13
HO
sedumoside H4)
OGlc
OGlc
1: 9S
2: 9R
3
Scheme 2. Plausible biogenetic pathways of 1–3.

O. Muraoka et al. / Tetrahedron 65 (2009) 4142–4148
4145
Table 3
11
6
12
2
Effects of neosedumosides I–IV (1–4) on oleic acid–albumin-induced triglyceride
accumulation in HepG2 cells
1
12
2
11
H
H
8
H
H
5
9
7
H
Conc. (
m
M)
TG/protein (% of control)
0.1
H
H
3
6
O
8
4
10
13
HO
0
1
10
100
9
O
4
10
O
HO
Neosedumoside I (1) 100.0ꢁ3.5 80.4ꢁ1.5** 79.6ꢁ2.9** 78.3ꢁ2.0** 75.1ꢁ1.8**
13
H
H
O
Neosedumoside II (2) 100.0ꢁ2.9 97.8ꢁ0.9 91.6ꢁ4.8
Neosedumoside III (3) 100.0ꢁ3.1 94.3ꢁ4.2 98.3ꢁ2.5
91.2ꢁ2.8
96.6ꢁ2.7
87.8ꢁ1.5*
81.6ꢁ4.0**
HO
OGlc
OH
1'
4
Neosedumoside IV (4) 100.0ꢁ1.8 91.8ꢁ2.8 98.8ꢁ1.1 100.5ꢁ3.1 107.6ꢁ3.3
HO
H
Bezafibrate
100.0ꢁ1.1 90.5ꢁ1.3* 93.4ꢁ1.4
86.7ꢁ1.2** 72.7ꢁ0.6**
H-H COSY :
HMBC :
NOE :
OH
4
Significantly different from the control, *p < 0.05, **p < 0.01.
Figure 3. 1H–1H COSY, HMBC, and NOE correlations of 4.
1% Ce(SO4)2–10% aqueous H2SO4, followed by heating. All the organic
extracts were dried over anhydrous MgSO4 prior to evaporation.
inhibit TG accumulation in HepG2 cells. Lipid metabolism-pro-
moting activity8,9 of 1–4 on stored TG contents in high glucose pre-
treated HepG2 cells was also examined. As the result, 2 (% of control
3.2. Plant material
at 1
m
M: 92.6ꢁ1.8) and 3 (92.2ꢁ2.3) were found to show reduction
S. sarmentosum was cultivated at Huangshan, Anhui province,
China and plant material was identified by one of the authors
(M.Y.). A voucher specimen (2005.01. Eishin-02) of this plant is on
file in our laboratory.2
of triglyceride levels in HepG2 cells (Table 4). Thus, these mega-
stigmane constituents (2 and 3) were found to show not only TG
accumulation inhibitory activity but also TG metabolism-pro-
moting activity in the hepatocyte.
3. Experimental
3.1. General
3.3. Extraction and isolation
The hot H2O extract (1950 g) from the fresh whole plant of S.
sarmentosum (Huangshan, Anhui province, China, 1.25% from this
herbal medicine) was extracted three times with MeOH under reflux
for 3 h. Evaporation of the solvent under reduced pressure provided
a MeOH extract (887.5 g, 0.57%), and an aliquot (398.6 g) was sub-
jected to Diaion HP-20 CC (4.0 kg, H2O/MeOH, twice) to give H2O-
and MeOH-eluted fractions (305.0 and 93.6 g, respectively). The
MeOH-eluted fraction (72.0 g) was subjected to normal-phase silica
gel CC [2.0 kg, CHCl3–MeOH–H2O (10:3:0.5/7:3:1, v/v/v, lower
layer)/MeOH] to give five fractions [Fr.1 (12.1 g), Fr. 2 (19.2 g), Fr. 3
(10.4 g), Fr. 4 (8.7 g), and Fr. 5 (16.3 g)]. The fraction 1 (12.1 g) was
subjected to reversed-phase silica gel CC [300 g, MeOH–H2O
(5:95/10:90/20:80/30:70/50:50/70:30, v/v)/MeOH] to
afford 13 fractions [Fr. 1-1 (550 mg), Fr. 1-2 (980 mg), Fr. 1-3
(1460 mg), Fr. 1-4 (1230 mg), Fr. 1-5 (1510 mg), Fr. 1-6 (1800 mg), Fr.
1-7 (540 mg), Fr.1-8 (600 mg), Fr.1-9 (710 mg), Fr.1-10 (220 mg), Fr.
1-11 (1170 mg), Fr.1-12 (1030 mg), andFr.1-13 (150 mg)]as reported
previously.2 The fraction 1-7 (540 mg) was subjected to Sephadex
LH-20 CC [150 g, CHCl3–MeOH (1:1, v/v)], and then by HPLC [MeOH–
H2O (42:58, v/v)] to furnish neosedumoside III (3, 21.3 mg,
0.00004%) together with myrsinionoside A2 (48.5 mg, 0.00009%),
secoisolariciresinol3 (56.3 mg, 0.00010%). The fraction 2 (19.2 g) was
subjected to reversed-phase silica gel CC [600 g, MeOH–H2O
(20:80/30:70/40:60/70:30, v/v)/MeOH] to afford 12 frac-
tions [Fr. 2-1 (200 mg), Fr. 2-2 (4630 mg), Fr. 2-3 (1160 mg), Fr. 2-4
The following instruments were used to obtain spectral and
physical data: specific rotations, Horiba SEPA-300 digital polari-
meter (l¼5 cm); CD spectra, JASCO J-720WI spectrometer; UV
spectra, Shimadzu UV-1600 spectrometer; IR spectra, Shimadzu
FTIR-8100 spectrometer; 1H and 13C NMR spectra, JEOL JNM-LA500
(500 and 125 MHz) spectrometer with tetramethylsilane as an in-
ternal standard; EIMS, CIMS, high-resolution EIMS, and high-res-
olution CIMS, JEOL JMS-GCMATE mass spectrometer; FABMS and
high-resolution FABMS, JEOL JMS-SX 102A mass spectrometer;
HPLC detector, Shimadzu RID-6A refractive index, Shimadzu SPD-
10A UV–vis, and Shodex OR-2 optical rotation detectors. HPLC
column, Cosmosil 5C18-MS-II (Nacalai Tesque Inc., 250ꢃ4.6 mm
i.d.) and (250ꢃ20 mm i.d.) columns were used for analytical and
preparative purposes, respectively.
The following experimental conditions were used for chroma-
tography: normal-phase silica gel column chromatography (CC),
silica gel BW-200 (Fuji Silysia Chemical, Ltd., 150–350 mesh); re-
versed-phase silica gel CC, Chromatorex ODS DM1020T (Fuji Silysia
Chemical, Ltd., 100–200 mesh); normal-phase TLC, pre-coated TLC
plates with silica gel 60F254 (Merck, 0.25 mm); reversed-phase TLC,
pre-coated TLC plates with silica gel RP-18 F254S (Merck, 0.25 mm);
reversed-phase HPTLC, pre-coated TLC plates with silica gel RP-18
WF254S (Merck, 0.25 mm), detection was achieved by spraying with
8
5
3
c)
9
10
O
HO
HO
HO
4: R = Glc
HO
O
OR
OH
O
4b
a)
b)
4a: R = H
S
4c
12
11
H
H
8
H
2
H
6
8
H
9
9
C
4
10
HO
5
13
H
H
O
O
S
HO
OH
CH2
4b NOE :
10
Scheme 3. (a) b
-Glucosidase/H2O, 37 ꢂC, 16 h, 93%; (b) NaBH4/MeOH, rt, 2 h, 60%; (c) 1,10-thiocarbonyldiimidazole, 4-DMAP/toluene, 60 ꢂC, 3 h, 19%.

4146
O. Muraoka et al. / Tetrahedron 65 (2009) 4142–4148
Table 4
CD (MeOH, nm, D3): 282 (ꢀ0.73), 317 (þ0.02). IR (KBr): 3389, 1708,
1076, 1035 cmꢀ1. 1H NMR (500 MHz, CD3OD) : given in Table 1. 1H
NMR (500 MHz, pyridine-d5) : 0.88, 0.97, 1.06 (3H each, all s, H3-11,
12, 13), 1.65 (1H, m, H-6), 1.65 (1H, m, H-7 ), 1.84 (1H, m, H-8 ), 1.88
(1H, m, H-7 ), 1.94 (1H, m, H-8 ), 2.28
), 2.01 (1H, d, J¼16.8 Hz, H-2
(1H, d, J¼17.4 Hz, H-4 ), 2.60 (1H, d, J¼16.8 Hz, H-2 ), 3.16 (1H, d,
J¼17.4 Hz, H-4
Effects of neosedumosides I–IV (1–4) on triglyceride contents in high glucose
pre-treated HepG2 cells
d
d
Conc. (
m
M)
TG/protein (% of control)
a
a
a
a
b
0
0.01
0.1
1
10
a
b
Neosedumoside I (1)
100.0ꢁ1.4
96.6ꢁ1.0 99.3ꢁ1.6
97.2ꢁ1.2
Neosedumoside II (2) 100.0ꢁ0.8
Neosedumoside III (3) 100.0ꢁ1.0
Neosedumoside IV (4) 100.0ꢁ2.0
95.3ꢁ1.8 92.6ꢁ1.8** 89.7ꢁ2.2**
b
), 3.80, 4.28 (1H each, both d, J¼10.4 Hz, H2-10), 3.96
95.3ꢁ0.3* 92.2ꢁ2.3* 94.2ꢁ3.0
(1H, m, H-50), 4.04 (1H, dd, J¼7.6, 8.0 Hz, H-20), 4.22 (1H, m, H-30),
4.23 (1H, m, H-40), [4.38 (1H, dd, J¼4.9, 11.3 Hz), 4.55 (1H, br d, J¼ca.
11 Hz), H2-60], 4.90 (1H, d, J¼7.6 Hz, H-10). 13C NMR (125 MHz,
CD3OD and pyridine-d5) dC: given in Table 2. EIMS m/z: 388 (Mþ, 4),
226 (45), 208 (96), 195 (60), 151 (100).
92.0ꢁ3.1 93.4ꢁ2.3
92.8ꢁ2.7
Metformin
100.0ꢁ2.0 94.0ꢁ2.1 90.0ꢁ2.7* 89.3ꢁ3.1*
Significantly different from the control, *p < 0.05, **p < 0.01.
(1950 mg), Fr. 2-5 (3300 mg), Fr. 2-6 (650 mg), Fr. 2-7 (700 mg), Fr. 2-
8 (1800 mg), Fr. 2-9 (810 mg), Fr. 2-10 (1360 mg), Fr. 2-11 (2270 mg),
and Fr. 2-12 (770 mg)] as reported previously.2 The fraction 2-4
(1950 mg) was subjected to normal-phase silica gel CC [100 g,
CHCl3/CHCl3–MeOH (50:1/20:1/10:1, v/v)/CHCl3–MeOH–
H2O (20:3:1, v/v/v, lower layer)/MeOH] to give seven fractions [Fr.
2-4-1 (90.5 mg), Fr. 2-4-2 (50.1 mg), Fr. 2-4-3 (284.0 mg), Fr. 2-4-4
(153.8 mg), Fr. 2-4-5 (348.2 mg), Fr. 2-4-6 (721.1 mg), and Fr. 2-4-7
(300.0 mg)]. The fraction 2-4-5 (348.2 mg) was further purified by
HPLC [CH3CN–MeOH–H2O (10:8:82, v/v/v) and MeOH–H2O (30:70
or 32:68, v/v)] to furnish neosedumosides I (1, 25.4 mg, 0.00005%)
and II (2,12.3 mg, 0.00002%) together with sedumoside D2 (43.0 mg,
0.00008%), staphylionoside D2 (3.2 mg, 0.00001%), 3-hydroxy-5,6-
3.4. Acid hydrolysis of 1–4
A solution of 1–4 (each 1.0 mg) in 1 M HCl (1.0 mL) was heated
under reflux for 3 h. After being cooled, the reaction mixture was
washed with EtOAc. The aqueous layer was subjected to HPLC
[column, Kaseisorb LC NH2-60-5, 4.6 mm i.d.ꢃ250 mm (Tokyo
Kasei Co., Ltd., Tokyo, Japan); detector, Shodex OR-2 optical rotator
(Showa Denko Co., Ltd., Tokyo, Japan); mobile phase, CH3CN–H2O
(85:15, v/v); flow rate 0.8 mL/min]. Identification of
present in the aqueous layer was carried out by comparison of its
retention time and optical rotation with those of an authentic
D-glucose
sample, tR: 13.9 min (
D
-glucose, positive optical rotation).
epoxy-
p-menth-1-ene-7,8-diol
0.00006%), and 4R-p-menth-1-ene-7,8-diol 8-O-
b
-ionol 9-O-
b
-
D
-glucopyranoside2 (22.0 mg, 0.00004%), 4R-
7-O- (31.3 mg,
-glucopyranoside3
-glucopyrano-
3.5. Enzymatic hydrolysis of 1–4 with b
-glucosidase
b-D
b-
D
A solution of 1–4 (19.6,11.8, 9.6, and 6.3 mg, respectively) in H2O
(2.0 mL) was treated with a -glucosidase (12.1, 7.8,10.2, 5.0, and
side3 (22.9 mg, 0.00004%). The fraction 2-8 (1800 mg) was purified
by Sephadex LH-20 CC [150 g, MeOH–H2O (30:70, v/v)] and then by
HPLC [CH3CN–MeOH–H2O (20:8:72, v/v/v) and MeOH–H2O (40:60,
v/v)] to furnish neosedumosides III (3, 32.9 mg, 0.00006%) and IV (4,
9.2 mg, 0.00002%) together with sarmentoic acid2 (429.8 mg,
0.00080%), sarmentoic acid methyl ester2 (24.5 mg, 0.00005%),
b
4.5 mg, respectively, from almond, Oriental yeast Co., Ltd., Tokyo,
Japan), and the solution was stirred at 37 ꢂC for 16 h. EtOH was
added to the reaction mixture, and the solvent was removed under
reduced pressure. The residue obtained starting from 1 and 2 was
purified by HPLC [MeOH–H2O (50:50, v/v)] to furnish neo-
sarmentols I (1a, 2.4 mg, 21% from 1) and II (2a, 4.2 mg, 64% from 2),
respectively. The residue obtained starting from 3 was purified by
HPLC [MeOH–H2O (60:40, v/v)] to furnish neosarmentol III (3a,
2.6 mg, 48%). The residue obtained starting from 4 was purified by
HPLC [MeOH–H2O (55:45, v/v)] to furnish neosarmentol IV (4a,
3.4 mg, 93% from 4).
4
alangioside J2 (80.9 mg, 0.00015%), sedumosides A4 (4.9 mg,
0.00001%), F13 (82.5 mg, 0.00015%), F23 (22.6 mg, 0.00004%), and G3
(2.5 mg, 0.00001%).
3.3.1. Neosedumoside I (1)
An amorphous powder, [
EIMS: calcd for C19H30O8 (M)þD: 386.1940. Found: 386.1936. UV [MeOH,
25
a]
þ40.0 (c 1.35, MeOH). High-resolution
nm (log
3
)]: 241 (4.06). CD (MeOH, nm, D3): 242 (þ2.92), 283 (ꢀ0.02),
3.5.1. Neosarmentol I (1a)
23
319 (þ0.37). IR (KBr): 3389, 1653, 1036 cmꢀ1
.
1H NMR (500 MHz,
Colorless oil, [
a
]
þ126.8 (c 0.06, MeOH). High-resolution EIMS:
D
CD3OD)
d
:giveninTable 1.13C NMR (125 MHz, CD3OD) dC:giveninTable
calcd for C13H20O3 (M)þ: 224.1412. Found: 224.1415. UV [MeOH, nm
2. EIMS m/z: 386 (Mþ, 3), 206 (86), 190 (92), 178 (13), 148 (100).
(log
3
)]: 243 (4.02). CD (MeOH, nm, D3): 241 (þ2.10), 283 (ꢀ0.11),
321 (þ0.47). IR (film): 3397, 1653, 1287, 1082 cmꢀ1
.
1H NMR
3.3.2. Neosedumoside II (2)
An amorphous powder, [
EIMS: calcd for C19H30O8 (M)þD: 386.1940. Found: 386.1934. UV [MeOH,
(500 MHz, CDCl3) d: 0.94, 1.07 (3H each, both s, H3-11, 12), 1.30 (1H,
25
a]
þ39.6 (c1.90, MeOH). High-resolution
m, H-7
), 2.08 (1H, ddd, J¼2.8, 5.5, 12.5 Hz, H-8
13.4 Hz, H-6), 2.21 (2H, s, H2-2), 2.37 (1H, d, J¼13.8 Hz, H-13
b
), 1.64 (1H, ddd, J¼4.0, 12.5, 13.4 Hz, H-8
), 2.14 (1H, dd, J¼4.9,
), 2.65
a), 1.97 (1H, m, H-
7a
b
nm (log
3
)]: 241 (4.10). CD (MeOH, nm, D3): 239 (þ2.35), 278 (ꢀ0.05),
a
321 (þ0.42). IR (KBr): 3389, 1653, 1037 cmꢀ1
.
1H NMR (500 MHz,
(1H, dd, J¼2.2, 13.8 Hz, H-13
b), 3.44, 3.47 (1H each, both d,
CD3OD)
d
: given in Table 1. 13C NMR (125 MHz, CD3OD) dC: given in
J¼11.0 Hz, H2-10), 5.83 (1H, br s, H-4). 13C NMR (125 MHz, CDCl3) dC
:
Table 2. EIMS m/z: 386 (Mþ, 1), 206 (85), 190 (100), 178 (12), 148 (4).
34.8 (C-1), 50.7 (C-2),199.4 (C-3), 125.2 (C-4), 161.4 (C-5), 48.1 (C-6),
23.5 (C-7), 34.3 (C-8), 73.7 (C-9), 66.1 (C-10), 24.1 (C-11), 28.7 (C-
12), 45.0 (C-13). EIMS m/z: 224 (Mþ, 18), 206 (53), 193 (37), 151 (81),
123 (100).
3.3.3. Neosedumoside III (3)
27
An amorphous powder, [
a
]
ꢀ63.7 (c 1.00, MeOH). High-reso-
D
lution positive-ion FABMS: calcd for C19H28O7Na (MþNa)þ:
391.1733. Found: 391.1738. UV [MeOH, nm (log
3)]: 290 (4.12). CD
3.5.2. Neosarmentol II (2a)
(MeOH, nm, D3): 283 (ꢀ2.63). IR (KBr): 3649,1653, 1576,1076 cmꢀ1
.
Colorless oil, [
a
]
D
þ116.0 (c 0.22, MeOH). High-resolution EIMS:
22
1H NMR (500 MHz, CD3OD)
d
: given in Table 1. 13C NMR (125 MHz,
calcd for C13H20O3 (M)þ: 224.1412. Found: 224.1419. UV [MeOH, nm
CD3OD) dC: given in Table 2. Positive-ion FABMS m/z: 391 (MþNa)þ.
(log
3
)]: 242 (4.10). CD (MeOH, nm, D3): 239 (þ1.96), 283 (ꢀ0.01),
314 (þ0.42). IR (film): 3391, 1659, 1258, 1042 cmꢀ1
.
1H NMR
3.3.4. Neosedumoside IV (4)
(500 MHz, CDCl3) d: 1.00, 1.07 (3H each, both s, H3-11, 12), 1.70 (1H,
27
An amorphous powder, [
a]
ꢀ27.6 (c 0.70, MeOH). High-reso-
m, H-7
J¼3.1, 6.1, 13.5 Hz, H-8
b
), 1.53 (1H, ddd, J¼4.0, 12.8, 13.5 Hz, H-8
a), 1.89 (1H, ddd,
), 2.05 (1H, dd, J¼4.6,
D
lution EIMS: calcd for C19H32O8 (M)þ: 388.2097. Found: 388.2099.
b
), 1.93 (1H, m, H-7
a

O. Muraoka et al. / Tetrahedron 65 (2009) 4142–4148
4147
12.8 Hz, H-6), 2.18 (1H, d, J¼15.6 Hz, H-2
H-2 ), 2.50 (1H, dd, J¼2.5, 13.7 Hz,
), 2.30 (1H, d, J¼13.7 Hz, H-13
H-13
a
), 2.28 (1H, d, J¼15.6 Hz,
3.7.1. Compound 3b
24
b
b
a
Colorless oil, [
a
]
ꢀ72.9 (c 0.14, CHCl3). High-resolution EIMS:
D
), 3.51, 3.54 (1H each, both d, J¼11.0 Hz, H2-10), 5.89 (1H, br s,
calcd for C15H20O3 (M)þ: 248.1412. Found: 248.1409. UV [MeOH, nm
H-4). 13C NMR (125 MHz, CDCl3) dC: 34.8 (C-1), 50.1 (C-2), 199.5 (C-
3), 125.5 (C-4), 162.2 (C-5), 49.2 (C-6), 23.2 (C-7), 33.3 (C-8), 74.1 (C-
9), 70.8 (C-10), 24.8 (C-11), 28.6 (C-12), 44.5 (C-13). EIMS m/z: 224
(Mþ, 18), 206 (44), 193 (39), 151 (83), 123 (100).
(log
1663, 1592, 1372, 1310, 1289, 1225, 1138, 1032 cmꢀ1
(500 MHz, CDCl3) : 0.90,1.13 (3H each, both s, H3-11,12),1.44 (1H, m,
H-7 ), 2.02 (1H, m, H-7
), 2.13 (3H, s, Ac), 2.25 (1H, d, J¼16.2 Hz, H-
), 2.25 (1H, m, H-8 ), 2.31 (1H, m, H-8
), 2.37 (1H, d, J¼16.2 Hz, H-
3)]: 287 (4.16). CD (MeOH, nm, D3): 283 (ꢀ3.13). IR (film): 1748,
.
1H NMR
d
b
a
2b
b
a
3.5.3. Neosarmentol III (3a)
2
a), 2.37 (1H, m, H-6), 4.62 (2H, s, H2-10), 5.83 (1H, s, H-4), 6.23 (1H, s,
24
Colorless oil, [
a
]
ꢀ125.3 (c 0.13, MeOH). High-resolution EIMS:
H-13).13CNMR(125 MHz,CDCl3)dC:36.2(C-1),53.9(C-2),199.8(C-3),
124.4 (C-4), 155.1 (C-5), 45.5 (C-6), 22.1 (C-7), 27.0 (C-8), 145.6 (C-9),
66.5 (C-10), 20.1 (C-11), 28.8 (C-12), 125.8 (C-13), 20.9, 170.6 (C–Ac).
EIMS m/z: 248 (Mþ, 3),188 (47),160 (77),145 (66),132 (100),104 (48).
D
calcd for C13H18O2 (M)þ: 206.1307. Found: 206.1308. UV [MeOH, nm
(log
)]: 291 (4.32). CD (MeOH, nm, D3): 285 (ꢀ3.80). IR (film):
3422, 1659, 1586, 1370, 1312, 1289, 1138 cmꢀ1. 1H NMR (500 MHz,
CDCl3) : 0.90, 1.13 (3H each, both s, H3-11, 12), 1.44 (1H, m, H-7 ),
2.03 (1H, m, H-7 ), 2.20 (1H, m, H-8
), 2.25 (1H, d, J¼16.2 Hz, H-
), 2.31 (1H, m, H-8 ), 2.38 (1H, m,
3
d
b
a
b
3.8. NaBH4–CeCl3 reduction of 3b
2
b
a
), 2.38 (1H, d, J¼16.2 Hz, H-2
a
H-6), 4.22 (2H, s, H2-10), 5.83 (1H, s, H-4), 6.30 (1H, s, H-13). 13C
NMR (125 MHz, CDCl3) dC: 36.3 (C-1), 54.0 (C-2), 199.9 (C-3), 123.7
(C-4), 155.8 (C-5), 45.8 (C-6), 22.2 (C-7), 27.0 (C-8), 151.1 (C-9), 65.8
(C-10), 20.1 (C-11), 28.9 (C-12), 123.5 (C-13). EIMS m/z: 206 (Mþ,
84), 191 (40), 173 (12), 163 (9), 150 (100), 122 (56).
To a solution of 3b (2.8 mg) in EtOH (2.0 mL) was added sodium
borohydride (NaBH4, 4.0 mg) in the presence of cerium chloride
(CeCl3, 11.0 mg), and the mixture was stirred at room temperature
for 20 min. The reaction was quenched by acetone, and the solvent
was removed under reduced pressure. To the residue was added
ice-water and the mixture was extracted with EtOAc. The extract
was washed with brine, and filtered. Removal of the solvent under
reduced pressure gave a residue, which was purified by normal-
phase silica gel CC (500 mg, CHCl3) to furnish 3c (2.0 mg, 72%).
3.5.4. Neosarmentol IV (4a)
23
Colorless oil, [
a]
ꢀ49.4 (c 0.17, CHCl3). High-resolution EIMS:
D
calcd for C13H22O3 (M)þ: 226.1569. Found: 226.1577. CD (MeOH,
nm, D3): 281 (ꢀ0.88), 317 (þ0.03). IR (film): 3397, 1712, 1287,
1082 cmꢀ1. 1H NMR (500 MHz, CDCl3)
d
: 0.96, 1.05,1.06 (3H each, all
3.8.1. Compound 3c
22
s, H3-11, 13, 12), 1.65 (2H, m, H2-8), 1.76 (1H, m, H-6), 1.78 (2H, m,
H2-7), 1.93 (1H, d, J¼17.7 Hz, H-2 ), 2.10 (1H, d, J¼17.4 Hz, H-4 ),
2.17 (1H, t, J¼4.0 Hz, OH), 2.42 (1H, d, J¼17.7 Hz, H-2 ), 2.72 (1H, d,
J¼17.4 Hz, H-4 ), 3.55, 3.65 (1H each, both dd, J¼4.0, 11.0 Hz, H2-
10). 1H NMR (500 MHz, C6D6)
: 0.69, 0.73, 0.76 (3H each, all s, H3-
11, 13, 12), 1.28 (1H, m, H-8 ), 1.31 (1H, m, H-6), 1.40 (1H, m, H-7
1.49 (1H, m, H-8 ), 1.59 (1H, m, H-7
1.99 (1H, d, J¼17.4 Hz, H-4
d, J¼17.4 Hz, H-4
Colorless oil, [
a
]
D
ꢀ40.0 (c 0.10, CHCl3). High-resolution EIMS:
a
a
calcd for C15H22O3 (M)þ: 250.1569. Found: 250.1565. UV [MeOH,
nm (log
)]: 241 (3.98). CD (MeOH, nm, D3): 241 (ꢀ3.26). IR (film):
3406, 1742, 1372, 1368, 1238, 1028 cmꢀ1. 1H NMR (500 MHz, CDCl3)
: 0.81, 1.03 (3H each, both s, H3-11, 12), 1.22 (1H, m, H-7 ), 1.33 (1H,
dd, J¼11.3, 11.3 Hz, H-2 ), 1.89 (1H, dd, J¼6.9, 11.3 Hz, H-2 ), 1.91
(1H, m, H-7 ), 1.95 (1H, m, H-6), 2.09 (3H, s, Ac), 2.17 (2H, m, H2-8),
4.30 (1H, br dd, J¼ca. 7, 11 Hz, H-3), 4.54 (2H, s, H2-10), 5.54 (1H, br
s, H-4), 6.06 (1H s, H-13). 13C NMR (125 MHz, CDCl3) dC: 33.9 (C-1),
48.1 (C-2), 66.5 (C-3), 126.9 (C-4), 136.7 (C-5), 44.7 (C-6), 22.6 (C-7),
27.3 (C-8), 135.9 (C-9), 67.6 (C-10), 20.1 (C-11), 29.2 (C-12), 127.7 (C-
13), 21.0, 170.9 (C–Ac). EIMS m/z: 250 (Mþ, 3), 232 (49), 190 (100),
175 (10), 172 (49).
b
3
b
d
d
b
a
a
a
),
),
b
a
b
b
), 1.85 (1H, d, J¼17.4 Hz, H-2
a
a
), 2.39 (1H, d, J¼17.4 Hz, H-2
b
), 2.85 (1H,
b
), 3.25, 3.35 (1H each, both d, J¼10.7 Hz, H2-10).
13C NMR (125 MHz, CDCl3) dC: 33.7 (C-1), 47.7 (C-2), 213.9 (C-3),
45.5 (C-4), 47.0 (C-5), 55.6 (C-6), 25.6 (C-7), 34.0 (C-8), 84.2 (C-9),
65.9 (C-10), 28.1 (C-11), 30.1 (C-12), 28.7 (C-13). 13C NMR (125 MHz,
C6D6) dC: 33.5 (C-1), 47.8 (C-2), 212.1 (C-3), 45.8 (C-4), 47.1 (C-5),
55.6 (C-6), 25.9 (C-7), 34.0 (C-8), 84.2 (C-9), 65.9 (C-10), 28.1 (C-11),
30.0 (C-12), 28.7 (C-13). EIMS m/z: 226 (Mþ, 16), 208 (41), 195 (44),
151 (100).
3.9. Benzoylation of 3c
To a solution of 3c (2.0 mg) in dry pyridine (1.0 mL) was added
3.6. Dehydration of 1a and 2a
benzoyl chloride (10 mL) in the presence of 4-DMAP (2.0 mg), and
the mixture was stirred at room temperature for 6 h. The reaction
mixture was poured into ice-water and extracted with EtOAc. The
extract was successively washed with 5% aqueous HCl, saturated
aqueous NaHCO3, brine, and filtrated. Removal of the solvent under
reduced pressure yielded a residue, which was purified by normal-
phase silica gel CC [500 mg, hexane–EtOAc (3:1, v/v)] to give 3d
(1.8 mg, 64%).
A solution of 1a (1.0 mg) in dry pyridine (0.5 mL) was treated
with thionyl chloride (SOCl2, 20 mL). The mixture was stirred at
room temperature for 1 h and was poured into ice-water, then
extracted with EtOAc. The extract was successively washed with
saturated aqueous NaHCO3 and brine, and filtrated. After removal
of the solvent under reduced pressure, the resulting residue was
purified by HPLC [MeOH–H2O (60:40, v/v)] to give 3a (0.4 mg, 48%).
Through the similar procedure, the same product 3a (0.6 mg, 62%)
was obtained from 2a (1.0 mg).
3.9.1. Compound 3d
25
Colorless oil, [
a
]
þ3.8 (c 0.10, CHCl3). High-resolution EIMS:
D
calcd for C22H26O4 (M)þ: 354.1829. Found: 354.1831. UV [MeOH, nm
(log
)]: 237 (4.08). CD (MeOH, nm, D3): 225 (ꢀ3.05). IR (film): 1742,
1717, 1603, 1451, 1368, 1269, 1113, 1026 cmꢀ1 1H NMR (500 MHz,
CDCl3) : 0.92, 1.08 (3H each, both s, H3-11, 12), 1.30 (1H, m, H-7 ),
1.63 (1H, dd, J¼11.0, 11.0 Hz, H-2 ), 1.95 (1H, m, H-7 ), 2.01 (1H, m,
H-2 ), 2.02 (1H, m, H-6), 2.09 (3H, s, Ac), 2.18 (2H, m, H2-8), 4.55
3.7. Acetylation of 3a
3
.
To a solution of 3a in dry pyridine (1.0 mL) was added acetic
anhydride (0.8 mL), and the mixture was stirred at room temper-
ature for 18 h. The reaction mixture was poured into ice-water and
extracted with EtOAc. The extract was successively washed with 5%
aqueous HCl, saturated aqueous NaHCO3, and brine, and filtrated.
After removal of the solvent under reduced pressure, the residue
was purified by normal-phase silica gel CC (500 mg, CHCl3) to give
3b (2.8 mg, 90%).
d
b
b
a
a
(2H, s, H2-10), 5.60 (1H, br s, H-4), 5.68 (1H, m, H-3), 6.09 (1H, s, H-
13), 7.43 (2H, dd, J¼7.7, 7.7 Hz, H-30,50), 7.55 (1H, m, H-50), 8.04 (2H,
dd, J¼1.3, 7.7 Hz, H-20,60). 13C NMR (125 MHz, CDCl3) dC: 33.9 (C-1),
43.4 (C-2), 70.5 (C-3), 122.6 (C-4), 138.3 (C-5), 44.7 (C-6), 22.5 (C-7),
27.2 (C-8), 136.4 (C-9), 67.4 (C-10), 20.1 (C-11), 29.1 (C-12), 127.3

4148
O. Muraoka et al. / Tetrahedron 65 (2009) 4142–4148
(C-13), 20.9, 170.9 (C–Ac), 128.3, 128.3ꢃ2, 129.6ꢃ2, 132.8, 166.4 (C–
Bz). EIMS m/z: 294 (Mþ, 59), 232 (57), 172 (63), 157 (100).
3.13. Lipid metabolism-promoting activity of stored
triglyceride in HepG2 cells induced by high glucose
HepG2 cells were inoculated in a 48-well tissue culture plate
(105 cells/well in 200
mL/well in MEM). After 20 h, the medium was
3.10. NaBH4 reduction of 4a
replaced with 200
mL/well of DMEM containing high glucose
A mixture of 4a (1.5 mg) and NaBH4 (1.0 mg) in MeOH (1.0 mL)
was stirred at room temperature for 2 h. The reaction was
quenched by acetone. After removal of the solvent under reduced
pressure, the residue was purified by HPLC [Shiseido Chiral CD-Ph,
4.6ꢃ250 mm i.d. (Shiseido Co., Ltd., Tokyo, Japan), MeOH–H2O
(50:50, v/v)] to give 4b (0.9 mg, 60%).
(4500 mg/L) and cultured for 6 days with replacement with the
fresh medium every 2 days. After accumulation of the lipid, the
medium was exchanged to 200 mL/well of DMEM containing low-
glucose (1000 mg/L) and a test sample, and the cells were cultured.
After 20 h, the TG and protein contents in the cells were de-
termined by the same manner as described above. An antidiabetic
agent, metformin was used as a reference compound.14,15
3.10.1. Compound 4b
23
Colorless oil, [
a
]
þ28.0 (c 0.04, MeOH). High-resolution posi-
3.14. Statistics
D
tive-ion FABMS: calcd for C13H24O3Na (MþNa)þ: 251.1623. Found:
251.1628. IR (film): 3405, 1045 cmꢀ1 1H NMR (500 MHz, C6D6)
. d:
Values were expressed as meansꢁS.E.M. One-way analysis of
variance (ANOVA) followed by Dunnett’s test was used for statis-
tical analysis. Probability (p) values less than 0.05 were considered
significant.
0.87, 1.01, 1.10 (3H each, all s, H3-11, 13, 12), 1.13 (1H, dd, J¼5.5,
13.7 Hz, H-2 ), 1.32 (1H, dd,
), 1.27 (1H, dd, J¼5.5, 14.4 Hz, H-4
J¼7.6, 8.2 Hz, H-6), 1.37 (1H, m, H-8 ), 1.43 (2H, m, H2-7), 1.52 (1H,
dd, J¼4.8, 13.7 Hz, H-2 ), 1.68 (1H, ddd, J¼5.5, 8.9, 15.1 Hz, H-8 ),
a
a
b
b
a
1.76 (1H, dd, J¼5.5, 14.4 Hz, H-4
b), 3.09, 3.31 (1H each, both d,
Acknowledgements
J¼10.3 Hz, H2-10), 4.01 (1H, m, H-3). 13C NMR (125 MHz, C6D6) dC
:
32.7 (C-1), 43.5 (C-2), 67.2 (C-3), 37.8 (C-4), 46.1 (C-5), 55.3 (C-6),
23.4 (C-7), 33.8 (C-8), 84.5 (C-9), 66.7 (C-10), 28.0 (C-11), 32.4 (C-
12), 26.7 (C-13). Positive-ion FABMS m/z: 251 (MþNa)þ.
O.M., T.M., andK.N. were supported bya Grant-in Aid for Scientific
Research from ‘High-tech Research Center’ Project for Private Uni-
versities: matching fund subsidy from a Grant-in Aid for Scientific
Research from the Ministry of Education, Culture, Sports, Science and
Technology of Japan (MEXT), 2007–2011 and also supported by
a Grant-in Aid for Scientific Research by Japan Society for the Pro-
motion of Science (JSPS). M.Y., H.M., and S.N. were supported by the
21st COE Program, Academic Frontier Project, and MEXT.
3.11. Thiocarbonylation of 4b
To a solution of 4b (0.9 mg) in dry toluene (1.0 mL) was added
1,10-thiocarbonyldiimidazole (3.5 mg) in the presence of 4-dime-
thylaminopyridine (4-DMAP, 1.0 mg), and the mixture was stirred
at 60 ꢂC for 3 h. The reaction mixture was poured into ice-water and
extracted with EtOAc. The extract was successively washed with 5%
aqueous HCl, saturated aqueous NaHCO3, brine, and filtrated. Re-
moval of the solvent under reduced pressure gave a residue, which
was purified by normal-phase silica gel CC [500 mg, hexane–EtOAc
(1:2/1:3, v/v)] to give 4c (0.2 mg, 18.8%).
References and notes
1. This paper is number 34 in the series ‘Bioactive constituents from Chinese
natural medicines’. The number 33: Matsuda, H.; Kiyohara, S.; Sugimoto, S.;
Ando, S.; Nakamura, S.; Yoshikawa, M. Biol. Pharm. Bull. 2009, 32, 147–149.
2. Yoshikawa, M.; Morikawa, T.; Zhang, Y.; Nakamura, S.; Muraoka, O.; Matsuda, H.
J. Nat. Prod. 2007, 70, 575–583.
3. Morikawa, T.; Zhang, Y.; Nakamura, S.; Matsuda, H.; Muraoka, O.; Yoshikawa, M.
Chem. Pharm. Bull. 2007, 55, 435–441.
4. Ninomiya, K.; Morikawa, T.; Zhang, Y.; Nakamura, S.; Matsuda, H.; Muraoka, O.;
Yoshikawa, M. Chem. Pharm. Bull. 2007, 55, 1185–1191.
5. Zhang, Y.; Morikawa, T.; Nakamura, S.; Ninomiya, K.; Matsuda, H.; Muraoka, O.;
Yoshikawa, M. Heterocycles 2007, 71, 1565–1576.
3.12. Inhibitory effects on oleic acid–albumin-induced
triglyceride accumulation in HepG2 cells
6. The 1H and 13C NMR spectra of 1–4 and related compounds (1a, 2a, 3a–3d, 4a–
4c) were assigned with the aid of distortionless enhancement by polarization
transfer (DEPT), homocorrelation spectroscopy (1H–1H COSY), heteronuclear
multiple-quantum coherence (HMQC), and HMBC experiments.
7. Harada, N.; Iwabuchi, J.; Yokota, Y.; Uda, H.; Nakanishi, K. J. Am. Chem. Soc. 1981,
103, 5590–5591.
HepG2 cells (Dainippon Pharmaceuticals, Osaka, Japan) were
maintained in Minimum Essential Medium Eagle (MEM, Sigma–
Aldrich) containing 10% fetal calf serum, 1% MEM non-essential
amino acids (Invitrogen), penicillin G (100 units/mL), and strepto-
8. Morikawa, T.; Xu, F.; Kashima, Y.; Matsuda, H.; Ninomiya, K.; Yoshikawa, M. Org.
Lett. 2004, 6, 869–872.
9. Morikawa, T.; Xu, F.; Ninomiya, K.; Matsuda, H.; Yoshikawa, T. Chem. Pharm.
Bull. 2004, 52, 494–497.
mycin (100
m
g/mL) at 37 ꢂC under 5% CO2 atmosphere. The cells
were inoculated in 48-well tissue culture plate [105 cells/well in
200
200
m
m
L/well in MEM]. After 20 h, the medium was replaced with
L/well of Dulbecco’s Modified Eagle’s Medium (DMEM) con-
10. Haines, A. H.; Jenkins, C. S. P. J. Chem. Soc. C 1971, 1438–1442.
11. De Pascual Teresa, J.; Barrero, A. F.; San Feliciano, A.; Grande, M.; Medarde, M.
Tetrahedron Lett. 1978, 43, 4141–4144.
12. Kang, T. H.; Pae, H. O.; Yoo, J. C.; Kim, N. Y.; Kim, Y. C.; Ko, G. I.; Chung, H. T.
J. Ethnopharmacol. 2000, 70, 177–182.
13. El-Hassan, A. Y.; Ibrahim, E. M.; Al-Mulhim, F. A.; Nabhan, A. A.; Chammas, M. Y.
Br. J. Radiol. 1992, 65, 774–778.
14. Bellentani, S.; Tiribelli, C.; Saccoccio, G.; Sodde, M.; Fratti, N.; De Martin, C.;
Cristianini, G. Hepatology 1994, 20, 1442–1449.
taining low-glucose (1000 mg/L), 5% (v/v) oleic acid–albumin
(Sigma–Aldrich), and a test sample. The cells were cultured for 4
days with replacement with the fresh medium every 2 days. Then
the medium was removed, and the cells were homogenized in
distilled water (105 mL/well) with sonication. The triglyceride (TG)
15. Marchesini, G.; Brizi, M.; Morselli-Labate, A. M.; Bianchi, G.; Bugianesi, E.;
McCullough, A. J.; Forlani, G.; Melchionda, N. Am. J. Med. 1999, 107, 450–455.
16. Marceau, P.; Biron, S.; Hould, F. S.; Marceau, S.; Simard, S.; Thung, S. N.; Kral, J.
G. J. Clin. Endocrinol. Metab. 1999, 84, 1513–1517.
and protein contents in the homogenate were determined using
commercial kits (Triglyceride E-test Wako and BCA protein assay
kit, respectively). Data were expressed as % of control of TG/protein
17. Okamoto, Y.; Tanaka, S.; Haga, Y. Hepatology Res. 2002, 23, 138–144.
18. Lin, H. Z.; Yang, S. Q.; Chuckaree, C.; Kuhajda, F.; Ronnet, G.; Diehl, A. M. Nat.
Med. 2000, 6, 998–1003.
19. Hawley, S. A.; Gadalla, A. E.; Olsen, G. S.; Hardie, D. G. Diabetes 2002, 51,
2420–2425.
(
m
g/mg). Each test compound was dissolved in DMSO, and the so-
lution was added to the medium (final DMSO concentration was
0.5%). A PPAR-
a agonist, bezafibrate was used as a reference
compound.18,19