Bellisosides A-F, six novel acylated triterpenoid saponins from Bellis perennis (compositae)

Article abstract of DOI:10.1016/j.tet.2005.01.026

Six new acylated triterpenoid saponins, bellisosides A-F (1-6), were isolated from the roots of Bellis perennis (Compositae) together with a known saponin, bellissaponin BS2 (7). These saponins which bear polygalacic acid as sapogenin, are bisdesmosidic glycosides. The structures were elucidated on the basis of chemical and physicochemical evidence. The cytotoxic activities of 1-7 against HL-60 human promyelocytic leukemia cells are also reported.

Full text of DOI:10.1016/j.tet.2005.01.026

Tetrahedron 61 (2005) 2921–2929
Bellisosides A–F, six novel acylated triterpenoid saponins from
Bellis perennis (compositae)
a
Wei Li, Yoshihisa Asada, Kazuo Koike, Tamotsu Nikaido, Tsutomu Furuya
b,*
a
a
b
b
and Takafumi Yoshikawa
a
Faculty of Pharmaceutical Sciences, Toho University, Miyama 2-2-1, Funabashi, Chiba 274-8510, Japan
School of Pharmaceutical Sciences, Kitasato University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641, Japan
b
Received 22 December 2004; revised 11 January 2005; accepted 11 January 2005
Abstract—Six new acylated triterpenoid saponins, bellisosides A–F (1–6), were isolated from the roots of Bellis perennis (Compositae)
together with a known saponin, bellissaponin BS2 (7). These saponins which bear polygalacic acid as sapogenin, are bisdesmosidic
glycosides. The structures were elucidated on the basis of chemical and physicochemical evidence. The cytotoxic activities of 1–7 against
HL-60 human promyelocytic leukemia cells are also reported.
q 2005 Elsevier Ltd. All rights reserved.
1
. Introduction
eluate was chromatographed on an ODS column with 50%
MeOH to remove flavonoids, and then with MeOH to result
in a crude saponin fraction. Further purification of the
fraction by combination of reversed- and normal-phase
HPLC successively gave six novel acylated triterpenoid
saponins, termed bellisosides A–F (1–6), and a known
Bellis perennis L. (Compositae), the common daisy, is
widely distributed in Europe and North Africa. It has been
used in traditional medicine for the treatment of rheuma-
tism, and as an expectorant. The existence of triterpenoid
saponins in B. perennis has been known since 1931. To our
1
2
2a
saponin, bellissaponin BS2 (7) (Fig. 1).
best knowledge, to date, 12 triterpenoid saponins were
reported. But in all these reports, partial deacylation by
3
Bellisoside A (1) was obtained as an amorphous powder. It
revealed an [MCNa] ion peak at m/z 1343 in positive
C
alkaline hydrolysis was carried out during the isolation.
The potential medicinal importance and our interest in the
chemistry of saponins prompted us to investigate the
genuine acylated saponins of this plant. This paper deals
with the isolation and structure elucidation of six novel
acylated saponins, bellisosides A–F (1–6), from the root of
B. perennis. The cytotoxic activities of these saponins,
along with a known saponin, bellissaponin BS2 (7), against
HL-60 human promyelocytic leukemia cells are also
reported.
FABMS. The molecular formula of C H100O29 was
6
3
confirmed by HRFABMS. The compound displayed 63
carbon signals in its C NMR spectrum, of which 30 could
1
3
3
be assigned to the signals of aglycon. The six sp carbons at
2
d 15.3, 17.7, 17.8, 24.7, 27.3 and 33.3, and the two sp
carbons at 122.9 (d) and 144.4 (s), coupled with information
1
from the H NMR (six methyl proton singlets at d 0.93, 1.04,
1.18, 1.39, 1.58 and 1.78, and a broad triplet-like vinyl
proton signal at d 5.63), indicated that the aglycon had an
1
olean-12-ene skeleton. Also, in the H NMR spectrum,
signals assignable to carbinylic protons of the aglycon were
observed at d 4.81 (ddd, JZ3.5, 3.5, 3.5 Hz), 4.35 (d, JZ
2
. Results and discussion
3.5 Hz) and 5.15 (br.s), suggesting the carbinylic protons
could be placed at 2a, 3a and 16b, respectively.
Furthermore, the hydroxymethyl (dH 4.35 and 3.74, and
d 66.2) and ester carbonyl (d 176.3) groups were assigned
A methanolic extract of the roots of B. perennis was
suspended in H O and then partitioned successively with
EtOAc. The H O layer was then passed through a Diaion
2
2
C
C
to C-23 and C-28 after an extensive 2D NMR study. Thus,
the aglycon was identified as 2b,3b,16a,23-tetrahydroxy-
HP-20 column, followed by washing of MeOH. The MeOH
4
olean-12-en-28-oic acid (polygalacic acid). The aglycon
Keywords: Saponin; Bellis perennis; Compositae; Bellisoside.
*
was isolated after acid hydrolysis of 1 and confirmed by
comparison of the spectral and physical data with those of
Corresponding author. Tel.: C81 3 57916239; fax: C81 3 34446192;
e-mail: asaday@pharm.kitasato-u.ac.jp
0040–4020/$ - see front matter q 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2005.01.026

2922
W. Li et al. / Tetrahedron 61 (2005) 2921–2929
inner sugar chain at C-28 were further determined by
analysis of the HMBC spectrum. Namely, the HMBC
correlations were observed between d 6.03 (Fuc-H-1) and
H
d 176.3 (C-28), d 6.08 (iRha-H-1) and d 75.3 (Fuc-C-2),
C
C
H
d 5.17 (Xyl-H-1) and d 83.8 (iRha-C-4), d 6.15 (tRha-H-1)
H
C
H
and d 83.8 (Xyl-C-3). The b anomeric configurations for
C
the glucopyranose, fucopyranose and xylopyranose units
3
were determined from their large J
coupling constants
7.5–8.0 Hz). For the inner and terminal L-rhamnopyranose
H1,H2
(
moieties, the small J
3
coupling constants (1.0 Hz) and
H1,H2
the three bond strong HMBC correlations from the anomeric
proton to C-3 and C-5 of the rhamnose, indicated that the
anomeric protons were equatorial, thus possessing a
1
6
configuration in the C form. Besides the signals due to
4
1
the aglycon and component sugars, the H NMR spectrum
also showed two singlet methyl signals (d 1.98 and 2.00),
which could be assigned to those of two acetyl moieties.
1
Comparing the H NMR spectrum of 1 with that of 7, H-4 [d
5
1
.52 (d, JZ3.5 Hz)] of fucose and H-2 [d 6.01 (dd, JZ3.0,
.0 Hz)] of the inner rhamnose were observed to be shifted
downfield by 1.59 and 1.21 ppm, indicating that the binding
site of the acetyl moieties were at C-4 of fucose and C-2 of
inner rhamnose. This conclusion was supported by obser-
vation of correlations between dH 5.52 (Fuc-H-4) and dC
1
71.2 (Fuc-COCH ), and d 6.01 (iRha-H-2) and d 170.6
3 H C
(iRha-COCH ) in the HMBC spectrum. Based on the above
3
results, the structure of bellisoside A (1) was established as
-O-b-D-glucopyranosyl polygalacic acid 28-O-a-L-
3
rhamnopyranosyl(1/3)-b-D-xylopyranosyl(1/4)-2-O-
acetyl-a-L-rhamnopyranosyl(1/2)-4-O-acetyl-b-D-
fucopyranoside (Fig. 2, Tables 1–4).
Bellisoside B (2) was obtained as an amorphous powder.
The HRFABMS spectrum of 2 suggested the molecular
formula of C H O , which is the same as that of 1. On
6
3 100 29
acid hydrolysis, 2 afforded polygalacic acid as the aglycon,
and D-glucose, D-xylose, D-fucose and L-rhamnose in the
ratio of 1:1:1:2 as component sugars. The b-D-gluco-
pyranose and a-L-rhamnopyranosyl(1/3)-b-D-xylopyra-
nosyl(1/4)-a-L-rhamnopyranosyl(1/2)-b-D-fucopyra-
nose were connected to C-3 and C-28 of polygalacic acid,
respectively, which were established by a combination of
Figure 1. Structures of triterpenoid saponins 1–7.
an authentic sample. On acid hydrolysis, 1 also afforded
D-glucose, D-xylose, D-fucose and L-rhamnose in the ratio of
1
:1:1:2 as component sugars, which were identified by
HPLC analysis following their conversion to the 1-[(S)-N-
acetyl-a-methylbenzylamino]-1-deoxy-alditol acetate
5
1
derivatives. The H NMR spectrum showed five anomeric
proton signals at d 5.17 (d, JZ7.5 Hz, Glc-H-1), 5.17 (d,
JZ7.5 Hz, Xyl-H-1), 6.03 (d, JZ8.0 Hz, Fuc-H-1), 6.08 (d,
JZ1.0 Hz, iRha-H-1), and 6.15 (d, JZ1.0 Hz, tRha-H-1).
All protons signals due to sugars were then assigned by
1
1
careful analysis of the H- H COSY and 1D HOHAHA
1
3
spectra. When the C NMR data of 2 was compared with
that of authentic polygalacic acid, glycosylation shifts were
observed at C-2 (K1.2 ppm), C-3 (C9.9 ppm) and C-28
(K3.8 ppm), which suggested 2 was a 3,28-bisdesmoside.
The glucose was connected to C-3 of the aglycon, which
was deduced from the HMBC correlations between d 5.17
H
(
Glc-H-1) and dC 83.5 (C-3). Since the anomeric carbon
signal (d 94.5) due to fucose showed an ester-type glycoside
linkage, fucose was found to be linked at C-28 of
polygalacic acid. This connection and the sequence of the
Figure 2. Key HMBC correlations for bellisoside A (1).

W. Li et al. / Tetrahedron 61 (2005) 2921–2929
Table 1. C NMR data for aglycones and ester moieties of 1–6
2923
13
1
2
3
4
5
6
Sapogenin
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
44.4
70.6
83.5
43.0
48.1
18.5
33.5
40.4
47.7
37.2
24.2
122.9
144.4
42.5
36.3
74.3
49.6
41.8
47.5
30.9
36.1
32.2
66.2
15.3
17.8
17.7
27.3
176.3
33.3
24.7
44.4
70.7
83.4
43.0
48.1
18.5
33.5
40.4
47.7
37.2
24.2
123.1
144.3
42.4
36.3
74.3
49.6
41.8
47.5
30.9
36.1
32.0
66.0
15.3
17.8
17.7
27.3
176.4
33.3
24.8
45.0
71.2
81.6
43.2
47.8
18.6
33.4
40.4
47.7
37.4
24.2
122.9
144.5
42.5
36.5
74.2
49.6
41.8
47.5
30.9
36.1
32.2
65.5
15.1
17.9
17.8
27.3
176.3
33.3
24.7
45.0
71.2
81.6
43.2
47.8
18.5
33.4
40.4
47.7
37.2
24.2
122.9
144.5
42.5
36.4
74.0
49.6
41.8
47.5
30.9
36.1
32.2
65.5
15.1
17.9
17.8
27.2
176.4
33.3
24.7
45.0
71.2
81.6
43.2
47.8
18.6
33.4
40.4
47.7
37.4
24.2
122.9
144.5
42.5
36.4
74.0
49.6
41.8
47.5
30.9
36.1
32.2
65.5
15.1
17.9
17.8
27.2
176.4
33.3
24.7
45.0
71.2
81.6
43.2
47.8
18.5
33.4
40.4
47.7
37.4
24.3
122.9
144.4
42.5
36.3
74.3
49.7
41.8
47.5
30.9
36.1
32.2
65.4
15.1
17.9
17.9
27.2
176.3
33.3
24.7
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Ester moiety
Fuc-COCH
Fuc-COCH
3
171.2
20.9
171.2
20.9
3
Rha-COCH
Rha-COCH
3
170.6
21.1
171.1
21.5
170.6
21.1
3
A
B
C
C
1
2
3
4
1
2
172.5
45.1
65.0
24.1
170.9
40.8
67.6
19.9
170.2
21.3
170.8
40.7
68.1
19.9
170.1
41.2
68.0
20.0
169.8
41.2
67.8
20.1
171.6
45.3
64.1
24.1
170.8
40.7
68.1
20.0
170.1
41.1
68.0
20.0
169.8
41.1
67.8
20.0
171.6
45.3
64.6
24.1
0
0
0
0
3
4
0
0
0
0
0
0
0
0
0
1
2
3
4
1
2
3
4
0
0
0
00
00
00
00
Fuc, b-D-fucopyranosyl; Rha, a-L-rhamnopyranosyl.
1
1
the DEPT, H– H COSY, 1D HOHAHA, HMQC, HMBC
and phase-sensitive NOESY experiments, similarly as
carried out on 1. Besides the signals due to the aglycon
(iRha-H-3) and dC 171.1 (iRha-COCH ) in the HMBC
3
spectrum. Based on the above results, the structure of
bellisoside B (2) was established as 3-O-b-D-glucopyranosyl
polygalacic acid 28-O-a-L-rhamnopyranosyl(1/3)-b-D-
xylopyranosyl(1/4)-3-O-acetyl-a-L-rhamnopyranosyl-
(1/2)-4-O-acetyl-b-D-fucopyranoside.
1
13
and component sugars, the H- and C NMR spectra also
showed the presence of two sets of acetyl moieties.
Comparing the H NMR spectrum of 2 with that of 7, H-4
1
[
9
d 5.48 (d, JZ3.5 Hz)] of fucose and H-3 [d 5.89 (dd, JZ
.0, 3.0 Hz)] of the inner rhamnose in 2 were observed to be
Bellisoside C (3) was obtained as an amorphous powder. It
revealed an [MCNa] ion peak at m/z 1329 in the positive
C
shifted downfield by 1.55 and 1.21 ppm, respectively, which
indicated that the acetyl moieties in 2 were located at C-4 of
fucose and C-3 of the inner rhamnose. This conclusion
FABMS. The molecular formula of C H102O28 was
6
3
confirmed by the HRFABMS spectrum. On acid hydrolysis,
3 afforded polygalacic acid as the aglycon, and D-xylose,
D-fucose and L-rhamnose in the ratio of 1:1:3 as component
was supported by the HMBC correlations between d 5.48
H
(
Fuc-H-4) and dC 171.2 (Fuc-COCH ), and d 5.89
3 H

2924
W. Li et al. / Tetrahedron 61 (2005) 2921–2929
13
Table 2. C NMR data for sugar moieties of 1–6
1
2
3
4
5
6
C-3 sugar
(Glc)
105.8
75.6
78.7
71.7
78.4
62.8
(Glc)
105.8
75.7
78.7
71.7
78.4
62.8
(Rha)
104.4
72.7
72.8
74.2
70.5
18.8
(Rha)
104.3
72.7
72.9
74.1
70.5
18.8
(Rha)
104.3
72.7
72.8
74.2
70.5
18.8
(Rha)
104.4
72.7
73.0
74.2
70.5
18.8
1
2
3
4
5
6
C-28 sugars
Fuc-1
Fuc-2
Fuc-3
Fuc-4
94.5
75.3
74.2
74.6
70.6
16.6
94.4
77.1
74.2
75.0
70.4
16.6
94.6
74.7
74.2
75.2
70.7
16.7
94.6
74.7
74.2
75.0
70.5
16.6
94.6
74.7
74.2
75.1
70.5
16.6
94.5
75.0
74.2
74.9
70.5
16.6
Fuc-5
Fuc-6
Inner
Rha-1
Rha-2
Rha-3
Rha-4
Rha-5
Rha-6
Xyl-1
Xyl-2
Xyl-3
Xyl-4
Xyl-5
99.0
73.4
70.4
83.8
68.7
18.7
106.8
76.3
83.8
69.5
67.4
102.9
70.0
75.7
78.1
69.1
19.4
106.0
75.1
83.8
69.7
67.3
102.1
71.9
72.7
84.9
68.8
18.9
107.3
76.6
83.6
69.4
67.5
102.0
71.9
72.8
84.9
68.8
18.8
107.3
76.6
83.6
69.4
67.5
102.0
72.0
72.7
84.9
68.8
18.8
107.3
76.6
83.6
69.4
67.5
98.8
73.4
70.2
83.9
68.7
18.7
106.9
76.4
83.5
69.4
67.5
Terminal
Rha-1
Rha-2
Rha-3
Rha-4
Rha-5
Rha-6
102.8
72.6
72.7
74.2
70.2
18.8
103.0
72.7
72.8
74.3
70.1
18.8
102.7
72.7
73.0
74.2
70.1
18.8
102.7
72.7
72.9
74.1
70.1
18.8
102.7
72.7
73.0
74.2
70.1
18.8
102.8
72.6
72.8
74.2
70.1
18.8
Glc, b-D-glucopyranosyl; Rha, a-L-rhamnopyranosyl; Xyl, b-D-xylpyranosyl; Fuc, b-D-fucopyranosyl.
sugars. Careful comparison of the NMR data of 3 and 2
suggested it is also a bisdesmosidic glycoside, and possesses
the same sugar chain as that of 2 at C-28. The a-L-
rhamnopyranose at C-3 was deduced from the HMBC
correlations between dH 5.78 (3-Rham-H-1) and dC 81.6
effect of the phenyl group in the MTPA ester (Fig. 3). Based
on the above results, the structure of bellisoside C (3) was
determined as 3-O-a-L- rhamnopyranosyl polygalacic acid
28-O-a-L-rhamnopyranosyl(1/3)-b-D-xylopyranosyl(1/
4)-a-L-rhamnopyranosyl(1/2)-4-O-3-(S)-3-hydroxy-1-
oxobutyl-b-D-fucopyranoside.
(
Besides the signals due to the aglycon and component
C-3), and dH 4.41 (H-3) and dC 104.4 (3-Rham-C-1).
1
sugars, the H- and C-NMR spectra also showed the
13
Bellisoside D (4) was obtained as an amorphous powder. In
the positive FABMS, it revealed an [MCNa] ion peak at
C
signals due to a secondary methyl [d 1.37 (d, JZ6.0 Hz),
H
d 24.1], a methylene [d 2.63 (dd, JZ14.0, 5.0 Hz), 2.73
m/z 1371 which is larger by 42 mass units than that of 3. The
molecular formula of C H104O29 was confirmed by
C
H
(
(
(
d, JZ14.0, 8.0 Hz), d 45.1], an oxymethylene [dH 4.57
C
65
ddd, JZ8.0, 6.0, 5.0 Hz), d 65.0], and an ester carbonyl
HRFABMS. On acid hydrolysis, 4 afforded polygalacic
acid as the aglycon, and D-xylose, D-fucose and L-rhamnose
in the ratio of 1:1:3 as component sugars. In comparing the
C
dC 172.5), suggesting the presence of a 3-hydroxybutyric
acid (HBA) moiety. The linkage of the HBA moiety to C-4
of fucose was determined by observation of the HMBC
correlations between d 5.57 (Fuc-H-4) and d 172.5 (HBA-
1
13
H and C NMR spectra of 4 and 3, all of the aglycon and
sugar signals of 4 were almost superimposable on those of 3,
while some differences were observed for the signals due to
the acyl moiety, indicating that 4 has the same aglycon and
sugar linkage and the same linkage position of the acylated
moiety as 3. Further analysis on the signals due to the acyl
H
C
C-1). The absolute configuration of the asymmetric center
of HBA was determined to be 3-S by the following chemical
correlation. Alkaline methanolysis of 3 with sodium
methoxide in dry methanol gave HBA methyl ester (8). 8
was treated with (S)-a-methoxy-a-trifuoromethyl-phenyl
acetate chloride (MTPA-Cl) to give its 3-O-MTPA ester (9),
which allows HPLC analysis with 9 and its diastereoisomer
moiety, suggested the presence of an HBA moiety [d 1.26
H
(3H, d, JZ6.0 Hz), 2.64 (1H, dd, JZ15.5, 5.5 Hz), 2.73 (d,
JZ15.5, 7.5 Hz) and 5.49 (1H, ddd, JZ7.5, 6.0, 5.5 Hz); dC
19.9, 40.8, 67.6 and 170.9] and an acetic acid moiety [dH
(
10) prepared from authentic 3-S- and 3-R-HBA methyl
ester, respectively (Scheme 1). Comparing the methyl,
methylene and carbomethoxy protons in 9 and 10, the
methyl proton in 9 was observed at 0.1 ppm downfield shift,
but the methylene and carbomethoxy protons were observed
at 0.04 and 0.09 ppm upfield shift. The difference in the
chemical shift in 9 and 10 is explained by the anisotropic
2.01 (3H, s); d 21.3 and 170.2]. The linkage of the HBA
C
moiety to C-4 of fucose and the acetyl moiety to C-3 of the
HBA moiety was determined by observation of the HMBC
correlations between d 5.52 (Fuc-H-4) and d 170.9 (HBA-
H
C
C-1), and dH 5.49 (HBA-H-3) and dC 170.2 (COCH3).
Thus, the structure of bellisoside D (4) was determined as

W. Li et al. / Tetrahedron 61 (2005) 2921–2929
Table 3. H NMR data for aglycones and ester moieties of 1–6
2925
1
1
2
3
4
5
6
Sapogenin
2
4.81 ddd
(3.5, 3.5, 3.5)
4.35 d (3.5)
5.63 dd (3.0, 3.0)
5.15 br. s
4.83 ddd
4.75 ddd
4.75 ddd
4.75 ddd
4.76 m
(3.5, 3.5, 3.5)
4.37 d (3.5)
5.63 dd (3.0, 3.0)
5.18 br. s
3.41 dd (13.5, 3.5)
2.73 dd
(3.5, 3.5, 3.5)
4.41 d (3.5)
5.66 dd (3.0, 3.0)
5.20 br. S
3.42 dd (13.5, 4.0)
2.75 dd
(3.5, 3.5, 3.0)
4.41 d (3.5)
5.68 dd (3.5, 3.5)
5.23 br. S
3.43 dd (13.0, 4.0)
2.76 dd
(3.5, 3.5, 3.5)
4.40 d (3.5)
5.68 dd (3.0, 3.0)
5.22 br. S
3.43 dd (13.5, 3.5)
2.76 dd
3
4.40 d (3.5)
5.66 dd (3.0, 3.0)
5.16 br. S
3.41 dd (13.5, 4.0)
2.75 dd
1
1
1
1
2
6
8
9
3.40 dd (13.5, 3.5)
2.74 dd
(
13.5, 13.5)
4.35 d (11.0)
.74 d (11.0)
(13.5, 13.5)
4.34 d (10.0)
3.76 d (10.0)
1.42 s
1.54 s
1.16 s
1.77 s
0.92 s
1.05 s
(13.5, 13.5)
3.72 s
(13.0, 13.0)
3.72 br.s
(13.5, 13.5)
3.72 s
(13.5, 13.5)
3.71 br.s
23
3
2
2
2
2
2
3
4 (CH
5 (CH
6 CH
3
3
)
)
1.39 s
1.58 s
1.18 s
1.78 s
0.93 s
1.04 s
1.23 s
1.58 s
1.20 s
1.76 s
0.93 s
1.02 s
1.23 s
1.58 s
1.21 s
1.77 s
0.93 s
1.04 s
1.23 s
1.58 s
1.20 s
1.76 s
0.93 s
1.04 s
1.24 s
1.59 s
1.19 s
1.76 s
0.93 s
1.04 s
3
)
7 (CH
9 (CH
0 (CH
3
3
3
)
)
)
Ester moiety
Fuc-COCH
Rha-COCH
3
2.00 s
1.98 s
2.00 s
2.04 s
3
2.00 s
C
A
B
C
a
2
3
2.63 dd (14.0, 5.0)
.73 dd (14.0, 8.0)
2.64 dd (15.5, 5.5)
2.73 dd (15.5, 7.5)
2.61w2.74*
2.63 dd (15.5, 5.5)
2
b
2
.75w2.84*
2.77 dd (15.5, 7.5)
5.47w5.62*
4.57 ddd
8.0, 6.0, 5.0)
1.37 d (6.0)
5.49 ddd
5.48w5.60*
(
(7.5, 6.0, 5.5)
1.26 d (6.0)
2.01 s
4
2
(CH
(CH
3
)
1.30 d (6.0)
2.61w2.74*
1.33 d (6.0)
2.63 dd (15.0, 5.5)
0
b
a
3
)
2
.75w2.84*
2.79 dd (15.0, 7.5)
5.47w5.62*
1.33d (6.0)
0
0
0
3
4
2
5.48w5.60*
1.32 d (6.0)
2.61w2.74*
(CH
3
)
0
a
2.64 dd (15.5, 5.5)
b
2
.75w2.84*
2.82 dd (15.5, 7.5)
5.47w5.62*
1.33 d (6.0)
0
0
0
0
3
4
2
5.48w5.60*
1.34 d (6.0)
2.61w2.74*
0
00
(CH
3
)
a
2.64 dd (15.5, 5.5)
b
2
.75w2.84*
4.57 m
2.82 dd (15.5, 7.5)
4.56 m
1.40 d (6.0)
0
0
00
00
3
4
(CH
3
)
1.40 d (6.0)
Fuc, b-D-fucopyranosyl; Rha, a-L-rhamnopyranosyl.
Overlapped signals.
a,b
Signals maybe exchangable.
*
3
-O-a-L-rhamnopyranosyl polygalacic acid 28-O-a-L-
absolute configuration of the asymmetric center of HBA in 5
was determined to be 3-S by the same method as that of 3.
Thus, the structure of bellisoside E (5) was determined as
3-O-a-L-rhamnopyranosyl polygalacic acid 28-O-a-L-
rhamnopyranosyl(1/3)-b-D-xylopyranosyl(1/4)-a-L-
rhamnopyranosyl(1/2)4-O-3-(S)-3-{3-(S)-3-[3-(S)-3-(3-
(S)-3-hydroxy-1-oxobutoxy)-1-oxobutoxy]-1-oxobuty}-1-
oxobutyl-b-D-fucopyranoside.
rhamnopyranosyl(1/3)-b-D-xylopyranosyl(1/4)-a-L-
rhamnopyranosyl(1/2)-4-O-3-(S)-3-acetoxy-1-oxobutyl-
b-D-fucopyranoside.
Bellisoside E (5) was obtained as an amorphous powder. It
revealed an [MCNa] ion peak at m/z 1587 in the positive
FABMS. The molecular formula of C H102O28 was
6
C
3
confirmed by HRFABMS. The superimposable signals due
1
13
to the aglycon and component sugars in H and C NMR
spectra between 5 and 4, together with the results of acid
hydrolysis of 5, suggested 5 is only different by the acyl
moiety from 4. Although, except the four secondary methyl
Bellisoside F (6) was obtained as an amorphous powder. It
revealed an [MCNa] ion peak at m/z 1629 in the positive
C
FABMS. The molecular formula of C H O35 was
7
7 122
1
13
confirmed by HRFABMS. Comparing the H, C NMR
and FAB-MS data of 6 with those of 5, 6 had one more
acetyl group than 5. The position of the acetyl group in 6
was suggested to be located at C-2 of the inner rhamnose
signals observed at d 1.30, 1.32, 1.34 and 1.40, the proton
H
signals due to the acyl moiety were very complicated
1
13
overlapping in the H NMR spectrum, the C NMR
spectrum suggested the acyl moiety in 6 was constructed by
four sets of the HBA moiety, which was also supported by
the FABMS data. Assignment of the carbon of the acyl
moiety was deduced from a combinational analysis of 1D
from a clear downfield shift of H-2 of the inner rhamnose at
1
d 5.98 by 1.20 ppm, comparing with the H NMR data of 5
H
1
3
and 6. Furthermore, in comparing the C NMR spectra of 5
and 6, acylation shifts were observed for the signals due to
C-1 (K3.2 ppm), C-2 (C1.4 ppm) and C-3 (K2.5 ppm) of
1
1
HOHAHA, H– H COSY, HMQC and HMBC spectra. The

2926
W. Li et al. / Tetrahedron 61 (2005) 2921–2929
1
Table 4. H NMR data for sugars of 1–6
1
2
3
4
5
6
C-3 sugar
(Glc)
5.17 d (7.5)
4.03 dd (8.5, 7.5)
4.19 dd (8.5, 8.0)
4.24 dd (9.0, 8.0)
3.92 ddd
(Glc)
5.17 d (7.5)
4.13 dd (8.5, 7.5)
4.18 dd (8.5, 8.0)
4.24 dd (8.5, 8.0)
3.91 dd (8.5, 4.0)
(Rha)
5.78 d (1.0)
4.72 dd (3.0, 1.0)
4.60 dd (9.0, 3.0)
4.30 dd (9.0, 9.0)
4.94 dq (9.0, 6.0)
(Rha)
5.79 d (1.0)
4.73 dd (3.5, 1.0)
4.60 dd (9.0, 3.5)
4.31 dd (9.0, 9.0)
4.61 dq (9.0, 6.0)
(Rha)
5.78 d (1.0)
4.72 dd (3.5, 1.0)
4.60 dd (9.0, 3.5)
4.30 dd (9.0, 9.0)
4.61 dq (9.0, 6.0)
(Rham)
5.78 d (1.0)
4.73 dd (3.5, 1.0)
4.59 dd (9.0, 3.5)
4.30 dd (9.0, 9.0)
4.94 dq (9.0, 6.0)
1
2
3
4
5
(
9.0, 5.0, 2.0)
4.33 dd (11.5, 5.0)
.48 dd (11.5, 2.0)
6
4.36 dd (11.5, 4.0)
4.49 br.d (11.5)
1.62 d (6.0)
1.62 d (6.0)
1.62 d (6.0)
1.62 d (6.0)
4
C-28 sugars
Fuc-1
Fuc-2
Fuc-3
Fuc-4
6.03 d (8.0)
4.50 dd (9.0, 8.0)
4.28 dd (9.0, 3.5)
5.52 d (3.5)
4.02*
6.08 d (8.0)
6.06 d (8.0)
6.06 d (8.0)
6.06 d (8.0)
6.03 d (8.0)
4.47 dd (8.0, 8.0)
4.32 dd (8.0, 3.5)
5.48 d (3.5)
4.00 q (6.0)
1.25 d (6.0)
4.54 dd (9.0, 8.0)
4.32 dd (9.0, 3.5)
5.57 d (3.5)
4.05 q (6.0)
1.32 d (6.0)
4.53 dd (9.0, 8.0)
4.33 dd (9.0, 3.5)
5.52 d (3.5)
4.02 q (6.5)
1.25 d (6.5)
4.53 dd (9.0, 8.0)
4.33 dd (9.0, 3.5)
5.51 d (3.5)
4.03 q (6.5)
1.25 d (6.5)
4.49 dd (9.0, 8.0)
4.31 dd (9.0, 3.5)
5.54 d (3.5)
4.03 q (6.5)
1.26 d (6.5)
Fuc-5
Fuc-6
1.25 d (6.0)
Inner
Rha-1
Rha-2
Rha-3
Rha-4
Rha-5
Rha-6
Xyl-1
Xyl-2
Xyl-3
Xyl-4
6.08 d (1.0)
6.08 d (1.0)
6.23 d (1.0)
6.27 d (1.5)
6.26 d (1.5)
6.10 d (1.5)
6.01 dd (3.0, 1.0)
4.80 dd (9.0, 3.0)
4.20 dd (9.0, 9.0)
4.48 dq (9.0, 6.0)
1.76 d (6.0)
5.02 dd (3.0, 1.0)
5.89 dd (9.0, 3.0)
4.57 dd (9.0, 9.0)
4.48*
1.76 d (6.0)
4.98 d (7.5)
3.93 dd (8.0, 7.5)
4.23 dd (8.0, 8.0)
4.09 ddd
4.80 dd (3.0, 1.0)
4.66 dd (9.0, 3.0)
4.34 dd (9.0, 9.0)
4.47 dq (9.0, 6.0)
1.73 d (6.0)
4.79 dd (3.0, 1.5)
4.66 dd (9.0, 3.0)
4.36 dd (9.0, 9.0)
4.52 dq (9.0, 6.0)
1.77 d (6.0)
4.78 dd (3.0, 1.5)
4.66 dd (9.0, 3.0)
4.36 dd (9.0, 9.0)
4.53 dd (9.0, 6.0)
1.76 d (6.0)
5.98 dd (3.5, 1.5)
4.78 dd (9.0, 3.5)
4.25 dd (9.0, 9.0)
4.48 dd (9.0, 6.0)
1.78 d (6.0)
5.17 d (7.5)
5.03 d (8.0)
5.04 d (7.8)
5.04 d (7.8)
5.16 d (7.5)
4.06 dd (8.0, 7.5)
4.21 dd (8.0, 8.0)
4.12 ddd
4.08 dd (8.0, 8.0)
4.23 dd (8.0, 8.0)
4.10 ddd
4.09 dd (8.0, 8.0)
4.24 dd (9.0, 8.0)
4.09 ddd
4.09 dd (8.0, 8.0)
4.23 dd (9.0, 8.0)
4.09 ddd
4.06 dd (8.0, 8.0)
4.22 dd (9.0, 8.0)
4.13 ddd
(
10.5, 8.0, 4.5)
3.52 dd
10.5, 10.5)
.21 dd (10.5, 4.5)
(10.0, 8.0, 5.0)
3.54dd
(10.5, 10.0)
(10.5, 8.0, 5.0)
3.46 dd
(10.5, 10.5)
(10.0, 9.0, 5.0)
3.46 dd
(10.5, 10.0)
(10.0, 9.0, 5.5)
3.46 dd
(10.5, 10.0)
(10.0, 9.0, 5.0)
3.53 dd
(11.0, 10.0)
Xyl-5
(
4
4.10 dd (10.5, 5.0)
4.16 dd (10.5, 5.0)
4.16 dd (10.5, 5.0)
4.16 dd (10.5, 5.5)
4.22 dd (11.0, 5.0)
Terminal
Rha-1
Rha-2
Rha-3
Rha-4
Rha-5
Rha-6
6.15 d (1.0)
6.20 br.s
6.23 d (1.0)
6.24 d (1.0)
6.23 d (1.5)
6.18 d (1.0)
4.76 dd (3.0, 1.0)
4.59 dd (9.0, 3.0)
4.31 dd (9.0, 9.0)
4.93 dq (9.0, 6.0)
1.66 d (6.0)
4.82 br.d (3.5)
4.62 dd (9.0, 3.5)
4.33 dd (9.0, 9.0)
4.98 dq (9.0, 6.0)
1.69 d (6.0)
4.80 dd (3.0, 1.0)
4.60 dd (9.0, 3.0)
4.32 dd (9.0, 9.0)
4.50 dq (9.0, 6.0)
1.67 d (6.0)
4.81 dd (3.5, 1.0)
4.61 dd, 9.0, 3.0)
4.34 dd (9.0, 9.0)
4.96 dq (9.0, 6.0)
1.67 d (6.0)
4.80 dd (3.5, 1.5)
4.60 dd (9.0, 3.5)
4.33 dd (9.0, 9.0)
4.95 dq (9.0, 6.0)
1.67 d (6.0)
4.76 dd (3.5, 1.0)
4.61 dd (9.0, 3.5)
4.32 dd (9.0, 9.0)
4.60 dq (9.0, 6.0)
1.66 d (6.0)
Glc, b-D-glucopyranosyl; Rha, a-L-rhamnopyranosyl; Xyl, b-D-xylpyranosyl; Fuc, b-D-fucopyranosyl.
Overlapped signals.
*
the inner rhamnose. This ester linkage was also supported
by the HMBC correlations between d 5.98 (Inner-Rham-
H-2) and dC 170.6 (COCH ). Thus, the structure of
3
moiety. A triterpenoid saponin with an acyl side chain
containing the HBA moiety is rare in nature, which has only
H
7
been reported from Solidago virga-aurea. To our knowl-
bellisoside F was determined as 3-O-a-L-rhamnopyranosyl
polygalacic acid 28-O-a-L-rhamnopyranosyl(1/3)-b-D-
xylopyranosyl(1/4)-3-acetoxyl-a-L-rhamnopyrano-
syl(1/2)-4-O-3-(S)-3-{3-(S)-3-[3-(S)-3-(3-(S)-3-hydroxy-
1-oxobutoxy)-1-oxobutoxy]-1-oxobuty}-1-oxobutyl-b-D-
fucopyranoside.
edge, however, bellisosides E (5) and F (6) F are the first
example of the triterpenoid saponin with the acyl side chain
constructed by four sets of the HBA moiety. Further, the
absolute stereochemistry of the HBA moiety in the
triterpenoid saponins was firstly determined. A wide range
of bacteria synthesize (R)-3-hydroxybutiric acid as an
intracellular storage material, and the poly[(R)-3-hydro-
xybutiric acid] (PHB) accumulates as granules within the
Bellisosides C–F (3–6) are newly described triterpenoid
saponins bearing an acyl side chain containing the HBA
8
cytoplasm. PHB is synthesized from acetyl-coenzyme A
Scheme 1. Stereochemistry of the acyl moieties in 3–6.

W. Li et al. / Tetrahedron 61 (2005) 2921–2929
2927
Table 5. Cytotoxic activity of 1–7 and cisplatine against HL-60 cells
Compound
IC50 (mM)
1
2
3
4
5
6
26.0G0.5
14.0G5.7
3.9G1.0
20.0G2.8
1.4G0.5
0.5G0.1
4.6G1.9
1.8G0.06
7
Cisplatine
The data shown represent the meanGSEM of two independent experiments
with three determinations in each.
1.4 and 0.5 mM, while cytotoxic activity of the other
saponins was moderate (IC50 3.9–26.0 mM) compared with
that of cisplatine used as a positive control (IC50 1.8 mM).
The cytotoxic activity in 1–7 may be related to the size of
the acyl groups constructed from HBA attached at C-4 of
fucose.
Figure 3. Computer-generated 3D structures and Dd values [Dd (in ppm)Z
Kd10] of 9 and 10.
d
9
(
CoA) by a sequence of three enzymatic reactions.
-Ketothiolase catalyzes the reversible condensation
3
reaction of two acetyl-CoA molecules into acetoacetyl-
CoA; the intermediate is reduced to (R)-3-hydroxybutyryl-
CoA by NADPH-linked acetoacetyl-CoA reductase,
and PHB is then produced by polymerization of
3
. Experimental
3
.1. General
(R)-3-hydroxybutyryl-CoA with PHB synthase. It has been
shown that at least two different pathways exist for the
The IR spectra were measured with a JASCO FT/IR-300E
(by a KBr disk method) spectrometer. The optical rotations
were measured with a JASCO DIP-370 digital polarimeter
in a 0.5 dm length cell. The FABMS and HRFABMS were
taken on a JEOL JMS-700 MStation. The H and C NMR
were measured with a Varian XL-400 spectrometer in
pyridine-d solution and chemical shifts are expressed in d
synthesis of (R)-3-hydroxybutyryl-CoA. One of these is the
9
pathway found in Rhodospirillum rubrum in which
acetoacetyl-CoA is first reduced to (S)-3-hydroxybutyryl-
CoA by an NAD-dependent dehydrogenase. This inter-
mediate is then converted to (R)-3-hydroxybutyryl-CoA.
The other pathway for PHA synthesis is that found in
1
13
1
0
11
5
Azotobacter beijerinckii, and Alcaligenes eutrophusin in
which acetoacetyl-CoA is converted directly into (R)-3-
hydroxybutyryl-CoA by NADPH-linked acetoacetyl-CoA
reductase. A possible biogenetic pathway of 3–6 is proposed
as shown in Figure 4.
(
ppm) referring to TMS. For HPLC, a Waters model 510
HPLC system, equipped with a Shimadzu SPD-6A and
Waters Differential Refractometer R401 detector was used
for preparation.
Isolation. B. perennis L. (Compositae) was grown in the
cultivation garden in Narita, Japan. Its roots (536.04 g) were
extracted with MeOH (10 L!5. Evaporation of the solvent
under reduced pressure from the combined extract gave the
MeOH extract (85.34 g). The extract was then partitioned
between EtOAc and H O. The H O phase was evaporated
under reduced pressure below 40 8C to remove the EtOAc,
and then subjected to a Diaion HP-20 column and further
washed by MeOH to give the crude saponin fraction (BPS,
The isolated saponins 1–7 were evaluated for their cytotoxic
activities against HL-60 human promyelocytic leukemia
cells. The cells were continuously treated with each sample
for 72 h, and the cell growth was measured by an MTT
1
2
reduction assay procedure (Table 5). The bellisosides E
5) and F (6) with a long chain acyl group, which contain
2
2
(
four molecular HBA moieties, showed strong cytotoxic
activity against HL-60 cells, with respective IC50 values of
Figure 4. Hypothetical biogenetic pathway for 3–6.

2928
W. Li et al. / Tetrahedron 61 (2005) 2921–2929
1
9.90 g). BPS (10.00 g) was subjected to ODS chromato-
EtOAc (1 mL!3) to remove the aglycone. The aqueous
layer was neutralized by passing through an ion-exchange
resin (Amberlite MB-3, Organo, Tokyo, Japan) column,
concentrated under reduced pressure to dryness, to give a
sugar fraction. The sugar fraction was dissolved in 1 mL
graphy and eluted by aqueous MeOH with the ratios from 0
to 100% to give four fractions A–D. Further purification of
fractions B (1.6 g) was achieved by repeated RP-HPLC (m
˚
Bondasper 5 mm C -300 A, 19 mm!150 mm) with sol-
1
8
vent as 70% MeOH and NP-HPLC (Senshu Pak. Aquasil
SS-5151, 19 mm!150 mm) with solvent as CHCl3–
H O, to which (K)-a-methylbenzylamine (5 mg) and
2
NaBH CN (3 mg) in EtOH (1 mL) was added. After being
3
MeOH–H O (60:18:2) to give 1 (18.5 mg), 2 (15.5 mg), 3
2
set aside at 40 8C for 4 h followed by addition of glacial
acetate (0.2 mL) and evaporated to dryness, the resulting
solid was acetylated with acetic anhydride (0.3 mL) in
pyridine (0.3 mL) for 24 h at room temperature. The
(21.5 mg), 4 (12.3 mg), 5 (54.6 mg), 6 (26.9 mg) and 7
(21.5 mg).
3.1.1. Bellisoside A (1). Powder, [a] K19.68 (c 0.45,
MeOH, 24 8C). IR nmax cm : 3423, 2934, 1733, 1637,
reaction mixture was evaporated 5 times by adding H
to remove pyridine, and then passed through a Sep-Pak C18
cartridge (Waters) with 20% and 50% CH CN (each 10 ml)
as solvents. The 50% CH CN eluate, which is the mixture of
the 1-[(S)-N-acetyl-a-methylbenzylamino]-1-deoxy-alditol
acetate derivatives of the monosaccharides, was then
analyzed by HPLC under the following conditions: column,
2
O
D
K1
C
379, 1251, 1048. FABMS (positive) m/z: 1343 [MCNa] .
1
HRFABMS (positive): observed 1343.6182, calcd for
3
3
C
C H O Na [MCNa] , 1343.6248.
1
H
400 MHz, pyridine-d ), see Tables 1 and 2. C NMR
NMR
6
3
100 29
1
3
(
(
5
100 MHz, pyridine-d ), see Tables 3 and 4.
5
Inertsil ODS-3 (4.6!250 mm); solvent: 40% CH CN; flow
3
3
.1.2. Bellisoside B (2). Powder, [a]D K22.18 (c 0.51,
rate, 0.8 mL/min; column temperature, 40 8C; detection, UV
230 nm. The derivatives of D-xylose, D-galactose, D-fucose,
D-glucose and L-rhamnose were detected as follows: t
R
(min) 21.1 (derivative of D-xylose), 23.6 (derivative of
D-galactose), 24.6 (derivative of D-fucose), 27.7 (derivative
of D-glucose), and 31.6 (derivative of L-rhamnose).
K1
MeOH, 24 8C). IR nmax cm : 3443, 2924, 1735, 1637,
458, 1378, 1255, 1049. FABMS (positive) m/z: 1343 [MC
Na] . HRFABMS (positive): observed 1343.6217, calcd
1
C
C
1
for C H O Na [MCNa] , 1343.6248. H NMR
6
3
100 29
1
400 MHz, pyridine-d ), see Tables 1 and 2. C NMR
100 MHz, pyridine-d ), see Tables 3 and 4.
5
3
(
(
5
3.1.8. Stereochemistry of 3-hydroxybutric acid moiety.
Each compound of 3–6 (each 1.0 mg) was treated with
0.28% NaOCH in dry MeOH (100 mL) for 2 h at room
3
3
.1.3. Bellisoside C (3). Powder, [a] K27.88 (c 0.47,
D
K1
MeOH, 24 8C). IR nmax cm : 3442, 2933, 1733, 1637,
456, 1384, 1241, 1048. FABMS (positive) m/z: 1329 [MC
Na] . HRFABMS (positive): observed 1329.6438, calcd
1
temperature. The solution was neutralized with Dowex
50W-X8 (H ) resin, and then filtered through a filter paper
C
C
C
1
for C H O Na [MCNa] , 1329.6455. H NMR
3
to remove the resin. The filtrate was evaporated. A part
of the residue was trimethylsilylated with 1-trimethylsilyl-
imidazole, analyzed by GLC–MS analysis to confirm the
product of HBA methyl ester. GLC conditions: capillary
column, EQUITYe-1 (30 m!0.25 mm !0.25 mm,
Supelco), oven temperature program, an initial temperature
of 60 8C for 5 min, to 240 8C at 3 8C/min, hold for 5 min;
injection temperature, 250 8C; carrier N2 gas. 3-[(Tri-
methylsilyl)oxy]butanoic acid methyl ester was detected at
9.67 min. The residue obtained from methanolysis was
treated with (S)-MTPA-Cl and then subjected to HPLC
analysis under the following conditions: column, YMC
6
102 28
1
400 MHz, pyridine-d ), see Tables 1 and 2. C NMR
100 MHz, pyridine-d ), see Tables 3 and 4.
5
3
(
(
5
3
.1.4. Bellisoside D (4). Powder, [a] K39.08 (c 0.44,
D
K1
MeOH, 24 8C). IR nmax cm : 3423, 2933, 1733, 1637,
456, 1383, 1262, 1049. FABMS (positive) m/z: 1371 [MC
Na] . HRFABMS (positive): observed 1371.6475, calcd
1
C
C
1
for C H O Na [MCNa] , 1371.6561. H NMR
6
5
104 29
1
400 MHz, pyridine-d ), see Tables 1 and 2. C NMR
100 MHz, pyridine-d ), see Tables 3 and 4.
5
3
(
(
5
3.1.5. Bellisoside E (5). Powder, [a]D K32.08 (c 0.85,
MeOH, 24 8C). IR nmax cm : 3441, 2936, 1736, 1453,
ODS-A (4.6!150 mm); solvent: 40% CH CN; flow rate,
3
K1
0.8 mL/min; column temperature, 40 8C; detection, UV
230 nm. The peak was detected at 8.86 min, which was
identified as 3-(S)-3-[2-(R)-2-methoxy-2-phenyl-2-
trifluoromethyl]-acetoxy butyric acid methyl ester (9),
while under the same conditions, synthetic 3-(R)-3-[2-(R)-
2-methoxy-2-phenyl-2-trifluoromethyl]-acetoxy butyric
acid methyl ester (10) was detected at 9.55 min. 3-(S)-3-
C
384, 1260, 1054. FABMS (positive) m/z: 1587 [MCNa] .
1
HRFABMS (positive): observed 1587.7582, calcd for
C
C H O Na [MCNa] , 1587.7559.
1
H
400 MHz, pyridine-d ), see Tables 1 and 2. C NMR
NMR
7
5
120 34
1
3
(
(
5
100 MHz, pyridine-d ), see Tables 3 and 4.
5
3
.1.6. Bellisoside F (6). Powder, [a]D K27.98 (c 0.55,
[2-(R)-2-methoxy-2-phenyl-2-trifluoromethyl]-acetoxy
butyric acid methyl ester (9): H NMR (400 MHz, CDCl ) d
K1
1
MeOH, 24 8C). IR nmax cm : 3443, 2931, 1734, 1637,
457, 1382, 1258, 1050. FABMS (positive) m/z: 1629 [MC
Na] . HRFABMS (positive): observed 1629.7677, calcd
3
1
1.39 (3H, d, JZ6.4 Hz, HBA-H -4), 2.62 (1H, dd, JZ16.2,
3
C
8.0 Hz, HBA-H-2), 2.66 (1H, dd, JZ16.2, 5.1 Hz, HBA-H-
C
1
for C H O Na [MCNa] , 1629.7664. H NMR
7
2), 3.56 (3H, s, OCH ), 3.50 (3H, s, MTPA-OCH ), 5.52
(1H, m, HBA-H-3), 7.40–7.42 (3H, m, MTPA-Ph), 7.49
7
122 35
3
3
1
400 MHz, pyridine-d ), see Tables 1 and 2. C NMR
100 MHz, pyridine-d ), see Tables 3 and 4.
5
3
(
(
5
(2H, dd, JZ8.0, 2.0 Hz, MTPA-Ph). 3-(R)-3-[2-(R)-2-
methoxy-2-phenyl-2-trifluoromethyl]-acetoxy butyric acid
1
methyl ester (10): H NMR (400 MHz, CDCl ) d 1.29 (3H,
3
1
.1.7. Sterochemistry of sugars of 1–6. Each solution of
–6 (each 1.0 mg), in 1 M HCl (dioxane–H O, 1:1, 200 mL)
3
d, JZ6.2 Hz, HBA-H -4), 2.66 (1H, dd, JZ16.5, 8.5 Hz,
2
3
was heated at 100 8C for 1 h under an Ar atmosphere. After
dioxane was removed, the solution was extracted with
HBA-H-2), 2.70 (1H, dd, JZ16.5, 5.2 Hz, HBA-H-2), 3.65
(3H, s, OCH ), 3.49 (3H, s, MTPA-OCH ), 5.51 (1H, m,
3
3

W. Li et al. / Tetrahedron 61 (2005) 2921–2929
2929
HBA-H-3), 7.40–7.42 (3H, m, MTPA-Ph), 7.49 (2H, dd,
JZ8.0, 2.0 Hz, MTPA-Ph).
sample necessary to inhibit the growth to 50% of the
control.
3
.2. Cytotoxic assay
HL-60 leukemia cells, which were obtained from RIKEN
Cell Bank (Tsukuba, Japan), were maintained in RPMI 1640
medium (GIBCO RBL Co., Grand Island, NY, USA)
containing heat-inactivated 10% fetal bovine serum (Bio-
Whittaker, Walkersville, MD) supplemented with L-gluta-
mine, 100 units/mL penicillin and 100 mg/mL streptomycin
References and notes
1. Hansel, R.; Keller, K.; Rimpler, H.; Schneider, G. Hagers
Handbuch der Pharmazeutischen Praxis; Springer: Berlin,
1992.
2. Kroeber, L. Pharmazeutische Zentralhalle fuer Deutschland
1931, 72, 745.
(
washed and resuspended in the above medium to 3!
Meiji-Seika, Tokyo, Japan). The leukemia cells were
3. (a) Hiller, K.; Schoepke, T.; Wray, V.; Schulten, H. R.
Pharmazie 1988, 43, 850–852. (b) Schoepke, T.; Wray, V.;
Kunath, A.; Hiller, K. Pharmazie 1990, 45, 870–871. (c)
Schoepke, T.; Wray, V.; Rzazewska, B.; Hiller, K. Phyto-
chemistry 1991, 30, 627–631. (d) Schoepke, T.; Wray, V.;
Kunath, A.; Hiller, K. Phytochemistry 1992, 31, 2555–2557.
(e) Schoepke, T.; Hiller, K. Sci. Pharm. 1996, 64, 663–668. (f)
Glensk, M.; Wray, V.; Nimtz, M.; Schoepke, T. Sci. Pharm.
2001, 69, 69–73.
4
0 cells/mL, and 180 mL of this cell suspension was placed
1
in each well of a 96-well flat-bottom plate (Iwaki Glass,
Chiba, Japan). The cells were incubated in 5% CO /air for
2
24 h at 37 8C. After incubation, 20 mL of EtOH–H O (1:9)
2
solution containing the sample was added to give the final
concentrations of 0.1–100 mM/mL; 20 mL of EtOH–H O
2
(
1:9) was added into control wells. The cells were further
incubated for 72 h in the presence of each agent, and then
cell growth was evaluated by an MTT assay procedure. At
the end of incubation, 10 mL of 5 mg/mL MTT (Sigma, St.
Louis, MO, USA) in phosphate-buffered saline was added to
every well, and the plate was further incubated in 5%
4. Asada, Y.; Ueoka, T.; Furuya, T. Chem. Pharm. Bull. 1989, 37,
2139–2146.
5. Hara, S.; Okabe, H.; Mihashi, K. Chem. Pharm. Bull. 1987, 35,
501–506.
CO /air for 4 h at 37 8C. The plate was then centrifuged at
2
6. Jia, Z.; Koike, K.; Nikaido, T. J. Nat. Prod. 1998, 61,
1368–1373.
1
500g for 5 min to precipitate cells and formazan. An
aliquot of 150 mL of the supernatant was removed from
every well, and 175 mL of DMSO was added to dissolve the
MTT formazan crystals. The plate was mixed on a
microshaker for 10 min and then read on a microplate
reader (Spectra Classic, Tecan, Salzburg, Austria) at
7. (a) Inose, Y.; Miyase, T.; Ueno, A. Chem. Pharm. Bull. 1991,
39, 2037–2042. (b) Inose, Y.; Miyase, T.; Ueno, A. Chem.
Pharm. Bull. 1992, 40, 946–953. (c) Inose, Y.; Miyase, T.;
Ueno, A. Chem. Pharm. Bull. 1994, 42, 617–624.
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450–472.
5
50 nm. T/C (%) score was calculated by the formulae as
described below and the graph on the concentration of the
samples and T/C (%) was made. The value of concentration
of samples that crossed the T/C (50%) was measured as the
IC50 values. Data are mean values of two experiments
performed in triplicate. T/C (%)Z(TKS)/(CKS)!100. T:
OD550 values of the cell with samples after 3 days
incubation. C: OD550 values of the cell without samples.
S: OD550 values of the cell before samples were added.
The IC50 value was defined as the concentration of
9. Moskowitz, M. J.; Merrick, J. M. Biochemistry 1969, 8,
2748–2755.
10. (a) Ritchie, G. F. A.; Senior, P. J.; Dawes, E. A. Biochem. J.
1971, 121, 309–316. (b) Senior, P. J.; Dawes, E. A. Biochem. J.
1973, 134, 225–238.
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FEMS Microbiol. Lett. 1988, 52, 259–264. (b) Oeding, V.;
Schlegel, H. G. Biochem. J. 1973, 134, 239–248.
12. Sargent, J. M.; Taylor, C. G. Br. J. Cancer 1989, 60, 206–210.