Synthesis of neosaponins and neoglycolipids containing a chacotriosyl moiety

Article abstract of DOI:10.1016/j.carres.2007.06.008

α-l-Rhamnopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-β-d-glucopyranose (chacotriose) is the oligosaccharide moiety of dioscin. Chacotriosyl trichloroacetimidate was synthesized from d-glucose and l-rhamnose, and glycosylated to mevalonate (diosgenin, cholesterol, and glycyrrhetic acid) to yield dioscin and neosaponins. In order to simplify the structure of the aglycone part, the mevalonate moiety was replaced with double-chain neoglycolipids that mimicked glycosyl ceramides. A cytotoxicity test revealed the importance of the glycosidic linkage of the naturally occurring β-form and that dioscin and the neoglycolipid with the longest chain showed a moderate activity.

Full text of DOI:10.1016/j.carres.2007.06.008

Carbohydrate Research 342 (2007) 2182–2191
Synthesis of neosaponins and neoglycolipids containing a
chacotriosyl moiety
Hiroyuki Miyashita, Tsuyoshi Ikeda and Toshihiro Nohara*
Faculty of Medical and Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan
Received 21 February 2007; received in revised form 2 June 2007; accepted 4 June 2007
Available online 12 June 2007
Abstract—a-L-Rhamnopyranosyl-(1!4)-[a-L-rhamnopyranosyl-(1!2)]-b-D-glucopyranose (chacotriose) is the oligosaccharide
moiety of dioscin. Chacotriosyl trichloroacetimidate was synthesized from D-glucose and L-rhamnose, and glycosylated to mevalo-
nate (diosgenin, cholesterol, and glycyrrhetic acid) to yield dioscin and neosaponins. In order to simplify the structure of the agly-
cone part, the mevalonate moiety was replaced with double-chain neoglycolipids that mimicked glycosyl ceramides. A cytotoxicity
test revealed the importance of the glycosidic linkage of the naturally occurring b-form and that dioscin and the neoglycolipid with
the longest chain showed a moderate activity.
ꢀ
2007 Elsevier Ltd. All rights reserved.
Keywords: Solanaceous plant; Chacotriose; Glycoconjugate; Neosaponin; Neoglycolipid; Cytotoxicity
1
. Introduction
amounts of these compounds are available from natural
sources, and it is extremely difficult to obtain glycosides
with the desired structure to attain a comparable SAR.
Thus, the synthesis of chacotriosyl neoglycosides con-
taining many types of aglycones from commercial, easily
available starting materials is interesting. Although sev-
eral researches on the synthesis of chacotriose analogues
have been reported, our synthesis method may be use-
ful for directly transferring the chacotriose moiety to
various aglycones. In this paper, we have established a
facile synthesis method of producing chacotriosyl
donors by chemical and enzymatic reactions, and have
transferred the chacotriosyl moiety to some mevalonates
to afford neosaponins. Furthermore, to simplify the
aglycone structure, we converted mevalonate to a dou-
ble-chain lipid mimicking ceramide as the aglycone part
to obtain some neoglycolipids containing chacotriose.
Finally, we examined the cytotoxicity of the synthesized
glycoconjugates.
Several studies have reported that steroidal saponins
isolated from various natural products have pharmaceu-
tical and biological activities. We have studied steroidal
oligosaccharides in many solanaceous plants and clari-
fied some of their biological activities. In particular,
certain kinds of saponins from plants belonging to the
genus Solanum, for example, S. dulcamara, S. lyratum,
and S. nigrum, exhibited moderate to good anti-cancer
and anti-herpes activities. The results showed that spi-
rostane-type glycosides, particularly those containing
chacotriose as an oligosaccharide moiety represented
by dioscin, were highly effective. On the other hand,
other aglycone-type chacotriosyl glycosides had almost
no activity or weak activities. In addition, Kuo and
co-workers reported that the chacotriosyl moiety of sol-
amargine played a crucial role in triggering cell death by
apoptosis. Therefore, we desired to investigate the struc-
ture–activity relationship (SAR) of the aglycone part of
chacotriosyl glycosides. However, often only small
1
2
7
8
3
4
5
6
2. Results and discussion
*
Corresponding author. Tel.: +81 96 371 4380; fax: +81 96 362
799; e-mail: none@gpo.kumamoto-u.ac.jp
The chacotriosyl donor has 2 L-rhamnose units linked at
the 2- and 4-hydroxy groups of D-glucose; the 1-, 3-, and
7
0
008-6215/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2007.06.008

H. Miyashita et al. / Carbohydrate Research 342 (2007) 2182–2191
2183
6
-hydroxy groups must be protected, and the anomeric
Table 1. Glycosylation of 2 and 3 under various conditions
Entries Method Lewis acid (equiv) Temp (ꢁC) Yield (%)
group of the D-glucose unit must be reactivated for the
transglycosylation to aglycone. The anomeric position
of D-glucose was protected with allylalcohol, which
can also serve as a scaffold for the C–C bond.
1
2
3
4
5
6
N.P.
N.P.
N.P.
N.P.
I.P.
BF
3
ÆEt
2
O (2.0)
0
0
0
41
54
46
65
84
60
BF ÆEt O (4.3)
3
2
TMSOTf (0.8)
BF
BF
3
ÆEt
ÆEt
2
O (3.8)
O (4.4)
ꢀ78
ꢀ78
ꢀ78
D-Glucose was enzymatically converted to allyl-b-D-
9
3
2
glucopyranoside 1 (45%) by using almond b-glucosi-
dase and allylalcohol (Scheme 1). When compared with
I.P.
TMSOTf (1.0)
1
0
I.P. = Inverse procedure, N.P. = normal procedure.
the conventional chemical and enzymatic syntheses of
, the number of processes was decreased to one step,
1
although the process yield was the same as that obtained
by the chemical one. Next, the 3- and 6-hydroxy groups
of 1 were selectively protected using pivaloyl chloride in
pyridine at ꢀ15 ꢁC for 2 h to afford allyl 3,6-di-O-piva-
mevalonates (C27 steroid-type, cholestane-type, and tri-
terpenoid-type skeletons) as an aglycone part. Initially,
6 was transferred to diosgenin as an aglycone part of
dioscin in the presence of BF ÆEt O at 20 ꢁC to afford
3
2
1
1
loyl-b-D-glucopyranoside 2. Glycosylation of gluco-
protected neoglycosides (7 and 8) as a mixture of a/b
anomers (a:b = 1:0.44) in 64% yield (Scheme 2).
Although the stereoselectivity of this glycosylation was
not sufficient to predominantly obtain the naturally
occurring b anomer, the anomeric mixtures of the pro-
tected neoglycosides were easily separated using octad-
odecyl silica gel (ODS) column chromatography. In
the same manner, 6 was transferred to the other agly-
cones (cholesterol and glycyrrhetic acid) at room tem-
perature in the presence of BF ÆEt O to obtain
pyranoside
2
with 2,3,4-tri-O-acetyl-a-L-rhamnosyl
1
2
trichloroacetimidate 3 in the presence of boron trifluo-
ride etherate (BF ÆEt O) as a promoter provided the de-
3
2
sired fully protected chacotrioside 4 (Table 1, entry 1).
In this reaction, small amounts of undesirable 2- or 4-
O-glycosylated disaccharides were obtained; hence, we
1
3
attempted to compare the yields of chacotrioside 4
by using the ‘Normal Procedure’ (N.P.) and Schmidt’s
Inverse Procedure’ (I.P.) to determine, which procedure
‘
3
2
provided a greater yield. When I.P. was used instead of
N.P., the yield of 4 increased remarkably (Table 1,
entries 5 and 6). Moreover, the yield of glycosylation
when using BF ÆEt O as a promoter (Table 1, entry 5)
protected neoglycosides 9 and 10 in 77% and 11 and
12 in 64% yields, respectively (Scheme 2). The obtained
a/b anomeric mixture of protected neosaponins was sep-
arated using ODS column chromatography with 90%
MeOH to yield pure a- and b-protected neoglycosides
(9–12). Each protected neoglycoside (7–12) was depro-
tected in the usual manner to afford neosaponins (13–
18), respectively, except for 14 (dioscin). It is noteworthy
that a-chacotriosyl diosgenin cannot be obtained from a
natural source; therefore, compound 13 should be a
good candidate for comparison with dioscin in terms
of glycosidic linkages and biological activities. On the
other hand, the stepwise synthesis of b-chacotriosyl
derivatives from the glycosylation of some steroids and
3
2
was better than that obtained using trimethylsilyl triflu-
oromethanesulfonate (TMSOTf) (Table 1, entry 6). The
reason for the improved reaction yield might be the
prevention of the decomposition of donor 3 during the
glycosidic coupling reaction. The fully protected chaco-
trioside 4 was treated with tetrakis (triphenylphosphine)
palladium in acetic acid to give trisaccharide 5 in 78%
1
4
yield. The anomeric hydroxy group of 5 was converted
to an a-trichloroacetimidate derivative 6 (86%) in the
presence of trichloroacetonitrile and 1,8-diazabicyclo-
1
5
7
[
5.4.0]undec-7-ene (DBU).
In order to investigate their SAR, chacotriosyl a-tri-
glucose units was established; the transglycosylation
method of the chacotriosyl moiety described here
was useful for preparing various types of aglycone parts
chloroacetimidate 6 was transferred to several types of
OH
O
OPiv
O
a
b
HO
HO
HO
D-Glucose
O
O
PivO
OH
OH
1
2
OPiv
O
c
O
R2
PivO
O
AcO
OR1
NH
AcO
4 : R1= H, R2= OAllyl
OAc
AcO
d
e
O
CCl3
O
L-Rhamnose
1
2
5
6
: R = R = H or OH
AcO
O
AcO
3
OAc
1
2
: R = OC(NH)CCl , R = H
AcO
3
OAc
Scheme 1. Reagents and conditions: (a) b-glucosidase, allyl alcohol, H
2
O, 37 ꢁC; (b) pivaloyl chloride, pyridine, ꢀ15 ꢁC; (c) 3, BF
3 2
ÆEt O, MSAW
3
00, CH Cl , CH COOH, 80 ꢁC; (e) CCl CN, DBU, CH
2
2
, ꢀ78 ꢁC; (d) Pd[P(Ph)
3
]
4
3
3
2
2
Cl , 0 ꢁC.

2184
H. Miyashita et al. / Carbohydrate Research 342 (2007) 2182–2191
O
e
O
7
13 (α-form)
OH
O
O
a, d
b, d
c, d
HO
O
α/β = 1 : 0.44
O
O
HO
HO
OH
HO
e
O
8
9
14 (β-form)
HO
OH
OPiv
O
e
OH
O
15 (α-form)
PivO
O
O
HO
O
O
O
CCl3
NH
AcO
AcO
O
O
O
HO
α/β = 1 : 0.73
OAc
AcO
HO
O
OH
HO
O
6
e
AcO
OAc
10
16 (β-form)
HO
OH
COOH
O
f
11
17 (α-form)
OH
O
O
HO
O
α/β = 1 : 0.56
O
O
HO
HO
OH
HO
f
O
12
18 (β-form)
HO
OH
Scheme 2. Reagents and conditions: (a) diosgenin, BF
3
ÆEt
2
2 2 3 2 2 2
O, CH Cl ; (b) cholesterol, BF ÆEt O, CH Cl ; (c) glycyrrhetic acid 30-methylester,
BF ÆEt O, CH Cl ; (d) ODS column chromatography (90% MeOH); (e) 3% KOH/MeOH, reflux; (f) 3% LiOH/MeOH, reflux.
3
2
2
2
of chacotriosides (e.g., compounds 13–18) because
the same synthesis procedure was not continually
repeated.
octanoyl chloride in a biphasic solution of tetrahydro-
1
8
furan (THF)/2 M NaOAc to afford N-octanoyl-1-octyl-
aminoethyl-2-O-a-L-rhamnopyranosyl-(1!4)-[a-L-rham-
nopyranosyl-(1!2)]-b-D-glucopyranoside 22 in 97%
yield; its reaction with tetradecyl chloride produced a
double-chain chacotrioside 23 in 73% yield and was
identical to the reaction of 20 with octanoyl chloride.
Similarly, allylchacotrioside 19 was converted to a sin-
gle-chain chacotrioside 21 (overall 71%), treated with
tetradecylamine, and acylated with octanoyl or tetradec-
anoyl chloride to yield double-chain chacotriosides 24
(79%) and 25 (83%), respectively. This series of reac-
tions, easy to carry out, appears suitable for the con-
struction of a library of chacotriosyl lipids of varying
lengths.
In order to simplify the structure of the aglycone part
and to avoid unnecessary glycosidic coupling reactions,
double-chain neoglycolipids that mimicked glycosyl
1
6
ceramides were designed and synthesized. As shown
in Scheme 3, fully protected chacotrioside 4 was depro-
tected with sodium hydroxide to yield allylchacotrioside
1
9 and ozonolyzed to yield an aldehyde. The aldehyde
was used without further purification because it was
unstable. It was treated with decylamine in the presence
1
7
of sodium cyanoborohydride to afford 1-octylamino-
ethyl-2-O-chacotrioside 20 (overall 73%). This single-
chain chacotrioside 20 was selectively N-acylated with
OH
O
OH
O
O
O
O
O
(CH2)nCH3
HO
HO
N
H
a
b, c
O
O
O
O
4
HO
HO
HO
HO
20 n = 7
1
9
OH
HO
OH
HO
O
O
21 n = 13
HO
O
HO
OH
OH
OH
O
O
(CH ) CH3
HO
2 n
N
d
22 n = 7, m = 6
O
O
HO
23 n = 7, m = 12
24 n = 13, m = 6
O
(CH2)mCH3
HO
OH
HO
2
5 n = 13, m = 12
O
HO
OH
Scheme 3. Reagents and conditions: (a) 2 N NaOH/dioxane; (b)
CH (CH COCl, 2 M NaOCOCH /THF.
3 3 2 2 2 n 3 3
O , (CH ) S, MeOH; (c) NH (CH ) CH , NaBH CN, MeOH; (d)
3
2
)
m
3

H. Miyashita et al. / Carbohydrate Research 342 (2007) 2182–2191
2185
1
13
Table 2. Cytotoxic activities of chacotriosyl derivatives
tures was based on H, C, COSY, HMQC, and
HMBC NMR experiments. Chemical shifts were
reported in parts per million (ppm) relative to residual
solvent peaks, and coupling constants (J) were reported
in Hertz. FABMS and HRFABMS were obtained
using a glycerol matrix in the positive ion mode by using
a JEOL JMS-DX-303HF spectrometer. The HRESIMS
was measured using a JMS-T100LC. Column chroma-
tography was carried out on Silica Gel 60 (Kanto Chem-
ical, Co, Inc., 70–230 mesh and 230–400 mesh);
Amberlite IR-120B; MB-3 (Organo Co., Ltd); and chro-
matorex ODS (Fuji Silysia Chemical Co., Ltd) columns.
TLC was performed on precoated Silica Gel 60 F254
plates (E. Merck).
GI50 (lM)
PC-12
HCT116
Cisplatin
Diosgenin
0.33
2.93
>5.76
2.29
2.85
>12.1
>5.76
3.17
1
1
1
1
1
1
2
2
2
2
2
2
2
3
4 (Dioscin)
5
>5.95
>5.95
>5.41
>5.41
>7.97
>7.03
>6.64
>5.97
>5.97
1.10
>5.95
>5.95
>5.41
>5.41
>7.97
>7.03
>6.64
>5.97
>5.97
>5.43
>10.6
6
7
8
0
1
2
3
4
5
6 (Chacotriose)
>10.6
3.2. Allyl 3,6-di-O-pivaloyl-b-D-glucopyranoside (2)
Pivaloyl chloride (1.3 mL, 10.6 mmol) was added to a
soln of 1 (910 mg, 4.1 mmol) in pyridine (4.0 mL)
at ꢀ15 ꢁC. After stirring for 2 h, the mixture was diluted
with EtOAc. Next, the soln was washed with water
and satd aq NaCl and concentrated under diminished
pressure. The residue was purified by silica gel column
chromatography (2:1 hexane–EtOAc) to yield 2 (1.1 g,
Dioscin 14, neosaponins (13, 15–18), neoglycolipids
20–25), diosgenin, and chacotriose 26 were prepared
from trisaccharide 5 by using alkaline hydrolysis; we
(
examined the cytotoxicity of these compounds toward
1
9
the PC-12 (human lung cancer) and HCT116 (human
2
0
colon cancer) cell lines (Table 2). Dioscin 14 showed
a moderate cytotoxicity despite having a lower activity
than cisplatin. Since neosaponin 13, which is an ano-
meric isomer of dioscin, did not show any activity, it
was suggested that the naturally occurring b-form might
be important. Because the other neosaponins (15–18)
did not exhibit any activity, the spirostane-type aglycone
part of 14 (diosgenin) could not be replaced by choles-
terol and glycyrrhetic acid. Furthermore, both diosgenin
and chacotriose 26, which is a component of dioscin, did
not show activity. Thus, these results suggest that the
cytotoxicity of chacotriosyl derivatives depends on the
b-glycosidic linkage of the chacotriosyl moiety and
appropriate aglycone parts. Interestingly, similar to
dioscin 14, the double-chain chacotrioside 25 showed a
moderate activity against PC-12 cell lines. Thus, a sim-
plified conversion of the aglycone part to double-chain
lipids instead of to diosgenin may provide a clue to
understanding the mechanism by which glycoconjugates
acquire cytotoxicity. Additionally, extending the lipid
portion of the mimicked glycosyl ceramides is expected
to improve their activity.
6
9%) as a colorless oil. [a]D ꢀ5.80 (c 0.13, CHCl );
3
HRFABMS: calcd for C H O 389.2175, found m/z
3
1
9
33
8
+
1
89.2208 [M+H] ; H NMR (in CDCl ): d 5.86 (1H,
3
m, H-2), 5.24 (1H, br d, J 17.2 Hz, H-3b), 5.15 (1H,
br d, J 10.2 Hz, H-3a), 4.84 (1H, dd, J 9.6, 9.6 Hz, Glc
H-3), 4.35 (1H, m, H-1b), 4.34 (1H, d, J 8.3 Hz, Glc
H-1), 4.32 (1H, m, Glc H-6b), 4.22 (1H, m, Glc H-6a),
4.05 (1H, m, H-1a), 3.51 (1H, m, Glc H-5), 3.47 (1H,
m, Glc H-2), 3.41 (1H, dd, J 9.6, 9.6 Hz, Glc H-4),
1
.17, 1.15 (each 9H, s, C(CH ) · 2).
3
3
3.3. Allyl (2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl)-
(
(
1!4)-[(2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl)-
1!2)]-3,6-di-O-pivaloyl-b-D-glucopyranoside (4)
BF ÆEt O (1.0 mL, 7.9 mmol) was added to a suspension
3
2
of 4 (700 mg, 1.8 mmol) and molecular sieves AW 300
4.0 g) in CH Cl (5.0 mL) at ꢀ78 ꢁC under nitrogen
(
2
2
gas. After stirring for 1 h, rhamnosyl imidate 3 (2.4 g,
.5 mmol) in CH Cl (5.0 mL) was added to the mixture
5
2
2
and stirred for 5 h at room temperature. The mixture
was diluted with CHCl and filtered using Celite. The fil-
3
trate was washed with satd aq NaHCO and satd aq
3
3. Experimental
NaCl, dried over MgSO , and concentrated under
4
diminished pressure. The residue was purified by silica
gel column chromatography (3:1 hexane–EtOAc) to
yield 4 (1.4 g, 84%) as a colorless oil. [a]D +1.26
(c 0.13, CHCl ); HRFABMS: calcd for C H NaO
22
3
.1. General methods
Optical rotations were measured using the JASCO DIP-
3
43 64
1
+ 1
1
000 KUY digital polarimeter (l = 0.5). H NMR and
C NMR spectra were recorded on JEOL EX-
70 MHz, JNM-GX 400 MHz, and JEOL a-500 MHz
955.3787, found m/z 955.3859 [M+Na] ; H NMR (in
1
3
CDCl ): d 5.91 (1H, m, H-2), 5.32–5.20 (3H, m, H-3,
3
0
2
Glc H-3), 5.19–5.16 (2H, m, Rha H-3, Rha H-3), 5.16
(1H, m, Rha H-2), 5.05 (1H, m, Rha H-2), 5.02 (1H,
0
NMR spectrometers. Elucidation of the chemical struc-

2186
H. Miyashita et al. / Carbohydrate Research 342 (2007) 2182–2191
0
m, Rha H-4), 5.01 (1H, m, Rha H-4), 4.89 (1H, br s,
1.16 (3H, d, J 6.6 Hz, Rha H-6), 1.16 (3H, d, J 6.1 Hz,
Rha H-6).
0
0
Rha H-1), 4.82 (1H, br s, Rha H-1), 4.55 (1H, d, J
6
.9 Hz, Glc H-1), 4.45 (1H, m, Glc H-6b), 4.35 (1H,
0
m, H-1), 4.30 (1H, m, Glc H-5), 4.11 (1H, m, Rha H-
3.6. General procedure for the preparation of compounds
7–12
5
), 4.08 (1H, m, H-1a), 3.93 (1H, m, Rha H-5), 3.77
(
2H, m, Glc H-4, 6a), 3.60 (1H, dd, J 7.6, 7.6 Hz, Glc
H-2), 2.12, 2.11, 2.05, 2.04, 1.98, 1.96 (each 3H, s,
Chacotriosyl imidate 6 (0.05 mmol), a glycosyl acceptor
(1.0 mmol), and molecular sieves AW 300 (800 mg)
were suspended in CH Cl (8.0 mL) at 20 ꢁC. After
COCH · 6), 1.23, 1.17 (each 9H, s, C(CH ) · 2), 1.17
3
3 3
0
13
(
6H, d, J 6.6 Hz, Rha H-6, Rha H-6); C NMR (in
2
2
CDCl ): d 133.4 (–CH–), 118.5 (–CH ), 70.8 (–OCH –
stirring for 30 min, BF ÆEt O (10% soln in CH Cl ,
3 2 2 2
3
2
2
)
, 99.5, 77.7, 72.0, 77.4, 75.4, 62.7 (Glc C-1–6), 98.0,
2.0 lmol) was added dropwise to the mixture. The mix-
6
6
9.9, 69.2, 70.5, 67.9, 17.1 (Rha [1!2] C-1–6), 97.3,
ture was stirred for 2 h at room temperature, diluted
9.5, 68.7, 70.2, 66.7, 17.1 (Rha [1!4] C-1–6).
with CHCl , and filtered using Celite. The filtrate was
3
washed with satd aq NaHCO and satd aq NaCl, dried
3
over MgSO , and concentrated under diminished pres-
4
3
.4. (2,3,4-Tri-O-acetyl-a-L-rhamnopyranosyl)-(1!4)-
sure. The residue was purified by column chromato-
graphy to yield an anomeric mixture of protected
glycosides. The mixture was separated by ODS column
chromatography to give a-glycoside and b-glycoside,
respectively.
[(2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl)-(1!2)]-3,6-di-
O-pivaloyl-D-glucopyranose (5)
Fully protected trisaccharide 4 (200 mg, 0.21 mmol) and
Pd[P(Ph )] (150 mg, 0.13 mmol) were dissolved in acetic
3
4
acid (3.0 mL) at 80 ꢁC under nitrogen gas. After stirring
for 1 h, the reaction solvent was removed, and the resi-
due was purified by silica gel column chromatography
3
.6.1. Diosgenin-3b-yl (2,3,4-tri-O-acetyl-a-L-rhamnopyr-
anosyl)-(1!4)-[(2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl)-
1!2)]-3,6-di-O-pivaloyl-D-glucopyranoside (7, 8). Gly-
cosylation of 6 (48 mg, 0.046 mmol) with diosgenin
400 mg, 0.97 mmol) was accomplished as described in
Section 3.6, and purified by column chromatography
2:1 hexane–EtOAc) to yield a mixture of 7 and 8 as a
(
(
3:2 n-hexane–EtOAc) to yield 5 (150 mg, 78%) as a col-
orless oil. [a] +7.81 (c 0.13, CHCl ); HRFABMS: calcd
D
3
(
for C H NaO
22
915.3474, found m/z 915.3580
4
0
60
1
+
[
M+H] ; H NMR (in CDCl ): d 5.55 (1H, dd, J 9.8,
3
(
9
.8 Hz, Glc H-3), 5.39 (1H, d, J 3.4 Hz, Glc H-1), 4.89
colorless oil. The mixture was separated by ODS column
chromatography (9:1 MeOH–water) to give both 7
0
(
1H, d, J 1.9 Hz, Rha H-1), 4.77 (1H, d, J 1.5 Hz,
Rha H-1), 3.73 (1H, dd, J 9.8, 9.3 Hz, Glc H-4), 3.47
(26 mg, 44%) and 8 (12 mg, 20%) as white solids. Data
(
1
1H, dd, J 9.8, 3.4 Hz, Glc H-2), 2.12 · 2, 2.04 · 2,
for a-glycoside (7): [a]D ꢀ2.9 (c 0.24, CHCl );
3
.98, 1.96 (each 3H, s, COCH · 6), 1.23 · 2 (each 9H,
3
HRFABMS: calcd for C H NaO 1311.6500, found
0
67 100
+ 1
24
s, C(CH ) · 2), 1.22 (6H, d, J 6.3 Hz, Rha H-6, Rha
H-6); C NMR (in CDCl ): d 91.8, 80.2, 70.7, 77.2,
3
3
m/z 1311.6210 [M+Na] ; H NMR (in CDCl ): d 4.96
1
3
3
3
(1H, d, J 3.7 Hz, Glc H-1), 4.81 (1H, s, Rha H-1),
7
0.8, 61.9 (Glc C-1–6), 99.9, 69.6, 68.7, 70.6, 67.9, 17.4
0
4
1
.64 (1H, s, Rha H-1), 2.03, 2.02, 1.95, 1.93, 1.89,
(
(
Rha [1!2] C-1–6), 97.5, 68.9, 68.7, 70.1, 67.2, 17.2
.86 (each 3H, s, COCH · 6), 1.14, 1.11 (each 9H, s,
3
Rha [1!4] C-1–6).
C(CH ) · 2), 1.09 (3H, d, J 5.7 Hz, Rha H-6), 1.08
3
3
0
(3H, d, J 6.7 Hz, Rha H-6), 0.98 (3H, s, H-19), 0.88
3
.5. (2,3,4-Tri-O-acetyl-a-L-rhamnopyranosyl)-(1!4)-
(3H, d, J 7.3 Hz, H-21), 0.79 (3H, s, H-18), 0.70 (3H,
1
3
[
2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl-(1!2)]-3,6-di-
d, J 6.1 Hz, H-27); C NMR (in CDCl ): d 37.1, 28.8,
3
O-pivaloyl-D-a-glucopyranosyl trichloroacetimidate (6)
79.1, 40.1, 140.1, 122.2, 32.0, 31.4, 50.1, 36.8, 20.8,
3
9.8, 40.2, 56.5, 32.1, 80.8, 62.1, 16.3, 19.4, 41.6, 14.5,
Trichloroacetonitrile (0.40 mL, 3.9 mmol) was added to
a soln of trisaccharide 5 (150 mg, 0.17 mmol) in
CH Cl (3.0 mL) at 0 ꢁC under nitrogen gas. After stir-
ring for 30 min, DBU (0.050 mL, 0.32 mmol) was added
to the mixture and stirred for 1 h. The mixture was con-
centrated under diminished pressure, and the residue
was purified by silica gel column chromatography (2:1
109.3, 31.8, 28.0, 30.3, 66.8, 17.1 (C-1–27), 97.5, 80.0,
71.0, 77.4, 70.9, 62.7 (Glc C-1–6), 99.8, 70.2, 68.8,
71.0, 67.8, 17.6 (Rha [1!2] C-1–6), 96.2, 69.6, 68.0,
70.9, 66.8, 17.2 (Rha [1!4] C-1–6). Data for b-glycoside
(8): [a] ꢀ32.7 (c 0.19, CHCl ); HRFABMS: calcd for
2
2
D
3
C H NaO
24
1311.6500, found m/z 1311.6690
6
7
100
+
1
[M+Na] ; H NMR (in CDCl ): d 4.77 (1H, s, Rha
3
0
hexane–EtOAc) to yield 6 (150 mg, 86%) as a colorless
H-1), 4.70 (1H, s, Rha H-1), 4.47 (1H, d, J 7.3 Hz,
1
oil. H NMR (in CDCl ): d 8.77 (1H, s, NH), 6.42
Glc H-1), 1.99, 1.97, 1.91, 1.89, 1.84, 1.82 (each 3H, s,
3
(
1H, d, J 3.7 Hz, Glc H-1), 5.64 (1H, dd, J 9.8, 9.1 Hz,
COCH · 6), 1.09, 1.04 (each 9H, s, C(CH ) · 2), 1.06
3
3 3
0
0
Glc H-3), 4.91 (1H, d, J 1.8 Hz, Rha H-1), 4.81 (1H,
s, Rha H-1), 2.12, 2.04 · 2, 1.98, 1.97, 1.94 (each 3H,
s, COCH · 6), 1.23, 1.21 (each 9H, s, C(CH ) · 2),
(3H, d, J 6.1 Hz, Rha H-6), 1.03 (3H, d, J 6.1 Hz, Rha
H-6), 0.88 (3H, s, H-19), 0.85 (3H, d, J 6.7 Hz, H-21),
1
3
0.67 (3H, d, J 6.1 Hz, H-27), 0.66 (3H, s, H-18);
C
3
3 3

H. Miyashita et al. / Carbohydrate Research 342 (2007) 2182–2191
2187
NMR (in CDCl ): d 37.4, 29.0, 79.1, 38.5, 140.3, 122.3,
1.7 mmol) was accomplished as described in Section
3.6, and purified by column chromatography (2:1:1 hex-
3
32.1, 31.7, 50.3, 37.1, 20.9, 40.0, 40.5, 56.7, 32.3, 81.0,
62.4, 16.5, 19.5, 41.9, 14.7, 109.5, 31.6, 27.4, 30.5, 67.0,
17.4 (C-1–27), 99.1, 76.4, 75.4, 77.2, 72.5, 63.3 (Glc C-
1–6), 97.6, 70.2, 69.3, 71.3, 68.3, 17.5 (Rha [1!2] C-1–
6), 97.3, 69.7, 69.0, 70.8, 66.7, 17.4 (Rha [1!4] C-1–6).
ane–EtOAc–CHCl ) to yield a mixture of 11 and 12 as a
3
colorless oil. The mixture was separated by ODS column
chromatography (9:1 MeOH–water) to give both 11
(71 mg, 41%) and 12 (39 mg, 23%) as white solids. Data
for a-glycoside (11): [a]D +73.1 (c 0.26, CHCl3);
HRFABMS: calcd for C H106NaO25 1381.6920, found
3
.6.2. Cholesterol-3b-yl (2,3,4-tri-O-acetyl-a-L-rhamno-
7
+
1
1
pyranosyl)-(1!4)-[(2,3,4-tri-O-acetyl-a-L-rhamnopyran-
osyl)-(1!2)]-3,6-di-O-pivaloyl-D-glucopyranoside (9, 10).
Glycosylation of 6 (50 mg, 0.048 mmol) with cholesterol
m/z 1381.6860 [M+Na] ; H NMR (in CDCl ): d 5.68
3
(1H, s, H-12), 5.00 (1H, d, J 3.7 Hz, Glc H-1), 4.92
0
(1H, s, Rha H-1), 4.74 (1H, s, Rha H-1), 3.69 (3H, s,
(
180 mg, 0.47 mmol) was accomplished as described in
Section 3.6, and purified by column chromatography
2:1 hexane–EtOAc) to yield a mixture of 9 and 10 as
COOCH ), 2.34 (1H, s, H-9), 2.12 · 2, 2.04, 2.01, 1.97,
3
1.94 (each 3H, s, COCH · 6), 1.24, 1.20 (each 9H, s,
3
(
C(CH ) · 2), 1.16 · 2, 1.15, 1.13 · 2, 0.97, 0.81 (each
3
3
1
3
a colorless oil. The mixture was separated by ODS col-
umn chromatography (9:1 MeOH–water) to give both 9
(
3H, s, tert-CH · 7); C NMR (in CDCl ): d 38.6,
3
3
26.5, 91.2, 39.1, 55.4, 17.4, 32.8, 44.1, 61.8, 37.0, 200.2,
128.5, 169.3, 45.5, 26.5, 26.5, 31.9, 48.4, 41.1, 43.2,
31.2, 37.8, 28.3, 16.4, 16.4, 18.7, 23.3, 28.7, 28.5, 176.9
27 mg, 45%) and 10 (20 mg, 32%) as white solids. Data
for a-glycoside (9): [a]D +33.1 (c 0.18, CHCl3);
HRFABMS: calcd for C H104NaO22 1283.6920, found
(C-1–30), 51.8 (COOCH ), 96.1, 79.6, 71.2, 78.9, 70.1,
63.1 (Glc C-1–6), 99.5, 69.5, 68.7, 71.2, 67.9, 17.6 (Rha
6
7
3
+
1
m/z 1283.6700 [M+Na] ; H NMR (CDCl ): d 5.05
3
(
4
1
1H, d, J 3.7 Hz, Glc H-1), 4.90 (1H, s, Rha H-1),
.73 (1H, s, Rha H-1), 2.12, 2.11, 2.04, 2.02, 1.98,
[1!2] C-1–6), 97.1, 68.8, 68.1, 70.6, 66.8, 17.2 (Rha
0
[1!4] C-1–6). Data for b-glycoside (12): [a] +26.4 (c
D
.95 (each 3H, s, COCH · 6), 1.22, 1.20 (each 9H, s,
0.21, CHCl ); HRFABMS: calcd for C H106NaO25
3
3
71
+
1
C(CH ) · 2), 1.18 (3H, d, J 6.7 Hz, Rha H-6), 1.17
1381.6920, found m/z 1381.6940 [M+Na] ; H NMR
3
3
0
(
(
3H, d, J 6.7 Hz, Rha H-6), 1.05 (3H, s, H-19), 0.92
3H, d, J 6.1 Hz, H-21), 0.87 (3H, d, J 6.7 Hz, H-27),
(in CDCl ): d 5.68 (1H, s, H-12), 4.92 (1H, s, Rha H-
3
0
1), 4.89 (1H, s, Rha H-1), 4.68 (1H, d, J 6.1 Hz, Glc
1
3
0
.86 (3H, d, J 6.7 Hz, H-26), 0.69 (3H, s, H-18);
C
H-1), 3.69 (3H, s, COOCH ), 2.33 (1H, s, H-9), 2.13,
3
NMR (in CDCl ): d 37.2, 28.2, 79.2, 40.1, 140.4,
2.11, 2.04, 2.02, 1.97, 1.95 (each 3H, s, COCH · 2),
3
3
1
3
2
22.5, 31.9, 31.9, 50.2, 36.6, 21.1, 28.0, 42.3, 56.8, 24.3,
9.8, 56.2, 11.9, 19.4, 35.8, 18.7, 36.2, 23.8, 39.5, 20.8,
2.8, 22.6 (C-1–27), 97.5, 80.0, 71.1, 77.5, 70.9, 62.7
1.22, 1.19 (each 9H, s, C(CH ) ), 1.16, 1.15 · 2, 1.13,
3
3
1
3
1.12, 0.84, 0.81 (each 3H, s, tert-CH · 7); C NMR
(in CDCl ): d 39.4, 26.5, 89.2, 39.4, 55.5, 17.4, 32.8,
3
3
(
Glc C-1–6), 99.9, 69.6, 68.8, 71.1, 67.8, 17.6 (Rha
44.1, 61.8, 36.9, 200.1, 128.6, 169.1, 45.5, 26.5, 26.5,
31.9, 48.4, 41.1, 43.2, 31.2, 37.8, 28.1, 16.5, 16.5, 18.7,
23.4, 28.5, 28.4, 176.9 (C-1–30), 51.8 (COOCH3),
102.2, 76.7, 75.0, 77.2, 72.4, 63.4 (Glc C-1–6), 97.3,
69.9, 68.9, 71.1, 67.9, 17.4 (Rha [1!2] C-1–6), 96.0,
69.5, 68.8, 70.6, 66.6, 17.2 (Rha [1!4] C-1–6).
[
[
0
1!2] C-1–6), 96.2, 69.8, 68.0, 70.2, 66.9, 17.2 (Rha
1!4] C-1–6). Data for b-glycoside (10): [a] ꢀ33.3 (c
D
.16, CHCl ); HRFABMS: calcd for C H NaO
3 67 104 22
+
1
1
283.6920, found m/z 1283.6800 [M+Na] ; H NMR
0
(
in CDCl ): d 4.89 (1H, s, Rha H-1), 4.83 (1H, s, Rha
3
H-1), 4.60 (1H, d, J 7.3 Hz, Glc H-1), 2.12, 2.10, 2.04,
2
.01, 1.97, 1.95 (each 3H, s, COCH · 6), 1.21, 1.17
3.7. General procedure for the preparation of compounds
13–18
3
(
each 9H, s, C(CH ) · 2), 1.11 (3H, d, J 6.7 Hz, Rha
3
3
0
H-6), 1.10 (3H, d, J 6.7 Hz, Rha H-6), 0.99 (3H, s, H-
1
6
9), 0.92 (3H, d, J 6.7 Hz, H-21), 0.87 (3H, d, J
.7 Hz, H-27), 0.86 (3H, d, J 6.7 Hz, H-26), 0.67 (3H,
The protected neoglycoside (0.1 mmol) was dissolved in
3% KOH/MeOH (5.0 mL) and heated at 65 ꢁC. After
stirring for 20 h, the mixture was purified by column
chromatography (MCI gel, CHP P; water, 250 mL;
MeOH, 150 mL), and the organic solvent was concen-
trated under diminished pressure to afford a neosaponin.
1
3
s, H-18); C NMR (in CDCl ): d 37.4, 28.2, 78.8,
3
39.8, 139.9, 122.4, 31.9, 31.9, 50.2, 36.7, 21.1, 28.2,
42.3, 56.8, 24.3, 39.5, 56.2, 11.9, 19.2, 35.8, 18.7, 36.2,
23.8, 39.6, 20.8, 22.8, 22.6 (C-1–27), 98.8, 76.2, 75.1,
78.8, 72.2, 63.1 (Glc C-1–6), 97.4, 69.9, 69.1, 71.1,
68.0, 17.3 (Rha [1!2] C-1–6), 97.1, 69.5, 68.7, 70.6,
66.5, 17.2 (Rha [1!4] C-1–6).
2
0
3.7.1. Diosgenin-3b-yl a-L-rhamnopyranosyl-(1!4)-
[a-L-rhamnopyranosyl-(1!2)]-a-D-glucopyranoside (13).
Compound 7 (102 mg, 0.079 mmol) was deprotected as
described in Section 3.7 to give 13 (67 mg, 98%) as a
white solid: [a]D ꢀ26.1 (c 0.11, MeOH); HRESIMS:
calcd for C H NaO 891.4718, found m/z 891.4733
3
.6.3. 30-Methyl glycyrrhetinate-3b-yl (2,3,4-tri-O-
acetyl-a-L-rhamnopyranosyl)-(1!4)-[(2,3,4-tri-O-acetyl-
a-L-rhamnopyranosyl)-(1!2)]-3,6-di-O-pivaloyl-D-gluco-
pyranoside (11, 12). Glycosylation of 6 (130 mg,
4
5
72
16
+
1
[M+Na] ; H NMR (in C D N): d 5.95 (1H, s, Rha
H-1), 5.86 (1H, s, Rha H-1), 5.52 (1H, d, J 3.7 Hz,
5
5
0
0
.13 mmol) with 30-methyl glycyrrhetinate (870 mg,

2188
H. Miyashita et al. / Carbohydrate Research 342 (2007) 2182–2191
Glc H-1), 1.70 (3H, d, J 6.1 Hz, Rha H-6), 1.68 (3H, d, J
32.1, 50.5, 37.0, 21.3, 28.5, 42.5, 56.9, 24.6, 40.0, 56.4,
12.0, 19.5, 36.1, 18.9, 36.5, 24.2, 39.8, 20.8, 23.0, 22.7
(C-1–27), 100.3, 78.2, 76.9, 78.0, 77.8, 61.3 (Glc C-1–
6), 102.2, 72.6, 72.7, 74.2, 69.5, 18.7 (Rha [1!2] C-1–
6), 102.9, 72.6, 72.9, 73.9, 70.4, 18.5 (Rha [1!4] C-1–6).
0
6
.1 Hz, Rha H-6), 1.15 (3H, d, J 6.7 Hz, H-21), 0.88
(
3H, s, H-19), 0.84 (3H, s, H-18), 0.70 (3H, d, J
1
3
5
7
3
1
7
7
7
.5 Hz, H-27); C NMR (in C D N): d 37.3, 29.3,
5 5
9.6, 40.8, 141.1, 121.6, 32.2, 31.6, 50.2, 37.0, 21.1,
9.9, 40.5, 56.7, 32.3, 81.1, 62.9, 16.4, 19.3, 42.0, 15.0,
09.3, 31.8, 28.7, 30.6, 66.9, 17.3 (C-1–27), 98.2, 79.4,
2.8, 78.7, 72.3, 61.5 (Glc C-1–6), 104.2, 72.8, 72.8,
4.0, 70.5, 18.7 (Rha [1!2] C-1–6), 103.0, 72.6, 72.8,
4.0, 70.3, 18.5 (Rha [1!4] C-1–6).
3.7.5. Glycyrrhetic acid-3b-yl a-L-rhamnopyranosyl-
(1!4)-[a-L-rhamnopyranosyl-(1!2)]-a-D-glucopyran-
oside (17). Compound 11 (60 mg, 0.044 mmol) was
deprotected as described in Section 3.7 to give 17
(39 mg, 97%) as a white solid: [a]D +56.5 (c 0.13,
3
.7.2. Diosgenin-3b-yl a-L-rhamnopyranosyl-(1!4)-[a-L-
MeOH); HRESIMS: calcd for C H NaO 947.4980,
found m/z 947.5025 [M+Na] ; H NMR (in C D N):
5 5
4
8
76
17
2
1
+ 1
rhamnopyranosyl-(1!2)]-b-D-glucopyranoside
(14).
Compound 8 (41 mg, 0.032 mmol) was deprotected as
described in Section 3.7 to give 14 (27 mg, 97%) as a
white solid: [a]D ꢀ102.0 (c 0.11, MeOH); HRESIMS:
calcd for C H NaO 891.4718, found m/z 891.4785
d 5.85 (1H, s, H-12), 5.49 (1H, d, J 3.7 Hz, Glc H-1),
0
4.79 (1H, s, Rha H-1), 4.70 (1H, s, Rha H-1), 2.33
(1H, s, H-9), 1.31, 1.30, 1.24, 1.13, 1.08, 0.98, 0.71 (each
1
3
3H, s, tert-CH · 7); C NMR (in C D N): d 38.9, 26.4,
4
1
5
72
16
3
5
5
+
[
M+Na] ; H NMR (in C D N): d 6.39 (1H, s, Rha
83.2, 42.6, 55.1, 17.4, 32.6, 44.0, 61.7, 37.0, 199.2, 128.1,
169.3, 45.2, 26.5, 26.4, 31.8, 48.5, 41.9, 43.2, 31.6, 38.2,
28.3, 16.3, 16.7, 18.5, 23.1, 28.7, 28.4, 181.3 (C-1–30),
95.5, 79.4, 72.3, 78.7, 71.7, 61.2 (Glc C-1–6), 102.6,
72.5, 72.7, 73.4, 70.1, 18.5 (Rha [1!2] C-1–6), 103.1,
72.0, 72.7, 73.4, 69.6, 18.5 (Rha [1!4] C-1–6).
5
5
0
H-1), 5.85 (1H, s, Rha H-1), 5.52 (1H, d, J 7.3 Hz,
Glc H-1), 1.77 (3H, d, J 6.1 Hz, Rha H-6), 1.63 (3H,
d, J 6.1 Hz, Rha H-6), 1.14 (3H, d, J 7.3 Hz, H-21),
0
1
.06 (3H, s, H-19), 0.84 (3H, s, H-18), 0.70 (3H, d, J
5
.5 Hz, H-27).
3
.7.3. Cholesterol-3b-yl a-L-rhamnopyranosyl-(1!4)-
3.7.6. Glycyrrhetic acid-3b-yl a-L-rhamnopyranosyl-
(1!4)-[a-L-rhamnopyranosyl-(1!2)]-b-D-glucopyran-
oside (18). Compound 12 (38 mg, 0.028 mmol) was
deprotected as described in Section 3.7 to give 18
(25 mg, 96%) as a white solid: [a]D +27.8 (c 0.10,
MeOH); HRESIMS: calcd for C H NaO 947.4980,
[
a-L-rhamnopyranosyl-(1!2)]-a-D-glucopyranoside (15).
Compound 9 (40 mg, 0.032 mmol) was deprotected as
described in Section 3.7 to give 15 (27 mg, 99%) as a
white solid: [a]D +5.49 (c 0.11, MeOH); HRESIMS:
calcd for C H NaO 863.5132, found m/z 863.5180
4
1
5
76
14
48 76
+ 1
17
+
[
M+Na] ; H NMR (in C D N): d 5.97 (1H, s, Rha
found m/z 947.5123 [M+Na] ; H NMR (in C D N):
5 5
5
5
0
H-1), 5.87 (1H, s, Rha H-1), 5.53 (1H, d, J 3.7 Hz,
Glc H-1), 1.71 (3H, d, J 6.7 Hz, Rha H-6), 1.69 (3H,
d, J 6.7 Hz, Rha H-6), 0.98 (3H, d, J 6.1 Hz, H-21),
d 5.86 (1H, s, H-12), 5.49 (1H, d, J 7.3 Hz, Glc H-1),
0
4.86 (1H, s, Rha H-1), 4.70 (1H, s, Rha H-1), 2.47
0
(1H, s, H-9), 1.73 (3H, d, J 6.1 Hz, Rha H-6), 1.59
0
0
.91 (3H, s, H-19), 0.90 (3H, d, J 6.7 Hz, H-27), 0.89
(3H, d, J 6.1 Hz, Rha H-6), 1.30, 1.28, 1.27, 1.25,
1
3
13
(
(
3H, d, J 6.7 Hz, H-26), 0.66 (3H, s, H-18); C NMR
in C D N): d 37.4, 28.7, 79.6, 40.9, 141.2, 121.0, 32.2,
1.23, 1.09, 0.74 (each 3H, s, tert-CH · 7); C NMR
(in C D N): d 39.5, 26.5, 88.4, 39.5, 55.3, 17.3, 32.6,
3
5
5
5
5
3
1
2.1, 50.4, 36.9, 21.3, 28.5, 42.5, 56.9, 24.5, 40.0, 56.4,
2.0, 19.4, 36.1, 18.9, 36.5, 24.2, 39.8, 20.8, 23.0, 22.7
43.9, 61.7, 37.0, 199.1, 128.2, 169.3, 45.2, 26.5, 26.3,
31.7, 48.4, 41.9, 43.2, 31.5, 38.2, 28.3, 16.3, 16.7, 18.5,
23.2, 28.6, 28.3, 181.2 (C-1–30), 101.2, 79.1, 76.3, 77.8,
77.6, 61.2 (Glc C-1–6), 102.6, 71.9, 72.1, 73.7, 69.1,
18.5 (Rha [1!2] C-1–6), 104.1, 72.0, 72.3, 73.4, 70.0,
(
C-1–27), 98.3, 79.4, 72.8, 78.7, 72.3, 61.5 (Glc C-1–6),
1
1
04.2, 72.8, 72.9, 74.0, 70.5, 18.7 (Rha [1!2] C-1–6),
03.2, 72.6, 72.8, 74.0, 70.4, 18.6 (Rha [1!4] C-1–6).
1
8.4 (Rha [1!4] C-1–6).
3
.7.4. Cholesterol-3b-yl a-L-rhamnopyranosyl-(1!4)-
[
a-L-rhamnopyranosyl-(1!2)]-b-D-glucopyranoside (16).
3.8. Allyl a-L-rhamnopyranosyl-(1!4)-[a-L-rhamno-
pyranosyl-(1!2)]-b-D-glucopyranoside (19)
Compound 10 (30 mg, 0.024 mmol) was deprotected as
described in Section 3.7 to give 16 (22 mg, 98%) as a
white solid: [a]D ꢀ42.2 (c 0.10, MeOH); HRESIMS:
calcd for C H NaO 863.5132, found m/z 863.5165
Fully protected trisaccharide 4 (500 mg, 0.54 mmol) was
dissolved in 2 M NaOH–dioxane (1:1, 4.0 mL) at room
temperature. After stirring for 14 h, the mixture was
4
1
5
76
14
+
[
M+Na] ; H NMR (in C D N): d 6.78 (1H, d, J
5 5
+
6
.1 Hz, Glc H-1), 6.40 (1H, s, Rha H-1), 5.86 (1H, s,
subjected to Amberlite IR-120B (H form), and the
0
Rha H-1), 1.78 (3H, d, J 6.1 Hz, Rha H-6), 1.64 (3H,
d, J 6.1 Hz, Rha H-6), 1.08 (3H, s, H-19), 0.97 (3H,
d, J 6.7 Hz, H-21), 0.90 (3H, d, J 6.7 Hz, H-27), 0.89
products were extracted using MeOH and concentrated
under diminished pressure to yield 19 (280 mg) as a col-
orless oil. [a]D +20.1 (c 0.10, MeOH); FABMS (m/z):
0
1
3
+
1
(
(
3H, d, J 6.7 Hz, H-26), 0.66 (3H, s, H-18); C NMR
in C D N): d 37.6, 30.2, 78.7, 39.0, 141.0, 122.0, 32.3,
513 [M+H] ; H NMR (in C D N): d 6.33 (1H, s,
5 5
0
Rha H-1), 6.07 (1H, m, H-2), 5.82 (1H, s, Rha H-1),
5
5

H. Miyashita et al. / Carbohydrate Research 342 (2007) 2182–2191
2189
5
.37 (1H, br d, J 17.7 Hz, H-3a), 5.15 (1H, d, J 10.4 Hz,
(–NHCH C H ), 32.1, 30.0 · 2, 29.9 · 2 29.8, 29.7,
2
13 27
H-3b), 4.42 (1H, d, J 7.3 Hz, Glc H-1), 4.34 (1H, m, H-
1
Rha H-6, Rha H-6).
29.6, 29.4, 27.2, 26.3, 23.0 (–CH – · 12), 14.2 (–CH ),
2
3
a), 4.05 (1H, m, H-1b), 1.69, 1.61 (each 3H, d, J 6.1 Hz,
102.2, 78.8, 77.0, 78.2, 77.1, 61.3 (Glc C-1–6), 102.6,
0
72.6, 72.4, 74.1, 70.5, 18.7 (Rha [1!2] C-1–6), 102.9,
72.6, 72.2, 73.8, 69.8, 18.5 (Rha [1!4] C-1–6).
3
2
.9. General procedure for the preparation of compounds
0 and 21
3.10. General procedure for the preparation of compounds
2–25
2
O gas was added to a soln of allyl chacotrioside 19
3
(
ꢀ
0.4 mmol) in MeOH (7.0 mL) at a temperature below
70 ꢁC. After the color of the mixture changed to blue,
Carboxylic acid chloride (0.3 mmol) was added to a
soln of chacotriosyl derivative (0.1 mmol) in 2 M
NaOAc/THF (2:3, 5.0 mL) at room temperature. After
stirring for 2 h, the mixture was concentrated under
diminished pressure, and the residue was purified by
silica gel column chromatography.
O gas was substituted with nitrogen gas, and dimethyl
3
sulfide (10.0 mmol) was added to the mixture. The mix-
ture was stirred for 18 h and concentrated under dimin-
ished pressure to yield an aldehyde. The aldehyde
(
0.4 mmol) was dissolved in MeOH (3.0 mL) containing
alkyl amine (2.0 mmol) adjusted to pH 6–7, and
3.10.1. N-Octanoyl-octylaminoethyl a-L-rhamnopyran-
osyl-(1!4)-[a-L-rhamnopyranosyl-(1!2)]-b-D-glucopyran-
oside (22). This was synthesized as described in Section
3.10 using compound 20 (60 mg, 0.10 mmol) and octa-
noyl chloride (0.04 mL, 0.25 mmol). The product was
purified by column chromatography (7:3:0.3:0.2 CHCl3–
MeOH–H O–NH OH) to give compound 22 (70 mg,
NaBH CN (0.5 mmol) was added. After stirring for
3
4
0 h, the mixture was concentrated under diminished
pressure, and the residue was purified by silica gel col-
umn chromatography.
3
.9.1. Octylaminoethyl a-L-rhamnopyranosyl-(1!4)-
2
4
[
a-L-rhamnopyranosyl-(1!2)]-b-D-glucopyranoside (20).
97%) as a colorless oil: [a] +67.1 (c 0.10, MeOH); HRE-
D
This was synthesized as described in Section 3.9 using
SIMS: calcd for C H NNaO 776.4408, found m/z
3
6
67
15
+
1
compound 19 (200 mg, 0.39 mmol) and octylamine
776.4398 [M+Na] ; H NMR (in C D N): d 6.29 (1H,
5 5
s, Rha H-1), 5.78 (1H, s, Rha H-1), 4.76 (1H, d, J
0
(
0.3 mL, 2.3 mmol). The product was purified by
column chromatography (6:4:0.8:0.2 CHCl –MeOH–
7.3 Hz, Glc H-1), 1.36–1.22 (22H, m, –CH – · 11), 0.83
3
2
1
3
H O–NH OH) to give compound 20 (180 mg, 73%) as
(6H, t, J 6.6 Hz, –CH · 2); C NMR (in C D N): d
2
4
3
5
5
a colorless oil: [a] +27.8 (c 0.10, MeOH); HRFABMS:
67.8 (–OCH CH NH–), 49.1 (–OCH - CH NH–), 47.8
2 2 2 2
D
calcd for C H NO 628.3544, found m/z 628.3438
(–NHCH C H ), 172.8 (–COCH –), 46.1 (–COCH –),
2 7 15 2 2
2
8
54
14
+
1
[
1
M+H] ; H NMR (in C D N): d 6.11 (1H, s, Rha H-
), 5.66 (1H, s, Rha H-1), 4.73 (1H, d, J 7.3 Hz, Glc
22.9–33.5 (–CH – · 11), 14.3 · 2 (–CH · 2), 102.0,
5
5
2
3
0
78.6, 77.5, 78.0, 77.0, 61.2 (Glc C-1–6), 102.9, 72.8,
72.4, 74.1, 70.4, 18.7 (Rha [1!2] C-1–6), 102.9, 72.7,
72.3, 73.8, 69.8, 18.4 (Rha [1!4] C-1–6).
H-1), 1.25–1.11 (12H, m, –CH – · 6), 0.81 (3H, t,
2
1
3
J 7.3 Hz, –CH3);
C NMR (in C D N): d 65.5
5 5
(
–OCH CH NH–), 48.3 (–OCH CH NH–), 48.1
2 2 2 2
(
–NHCH C H ), 31.9, 29.3 · 2, 27.1, 26.3, 22.8
3.10.2. N-Tetradecanoyl-octylaminoethyl a-L-rhamnopyr-
anosyl-(1!4)-[a-L-rhamnopyranosyl-(1!2)]-b-D-gluco-
pyranoside (23). This was synthesized as described in
Section 3.10 using compound 20 (57 mg, 0.09 mmol)
and tetradecanoyl chloride (0.05 mL, 0.18 mmol). The
product was purified by column chromatography
(8:2:0.2 CHCl –MeOH–NH OH) to give compound 23
2
7
15
(
6
–CH – · 6), 14.2 (–CH ), 102.2, 78.8, 77.0, 78.1, 77.1,
2
3
1.3 (Glc C-1–6), 102.6, 72.6, 72.4, 74.1, 70.5, 18.7
(
(
Rha [1!2] C-1–6), 102.9, 72.6, 72.2, 73.8, 69.8, 18.5
Rha [1!4] C-1–6).
3
.9.2. Tetradecylaminoethyl a-L-rhamnopyranosyl-(1!4)-
3
4
[
a-L-rhamnopyranosyl-(1!2)]-b-D-glucopyranoside (21).
(56 mg, 73%) as a colorless oil: [a]D +18.2 (c 0.10,
This was synthesized as described in Section 3.9 using
MeOH); HRESIMS: calcd for C H NNaO
15
4
2
79
+
1
compound 19 (100 mg, 0.20 mmol) and tetradecylamine
860.5347, found m/z 860.5364 [M+Na] ; H NMR (in
0
(
150 mg, 0.70 mmol). The product was purified by
C D N): d 6.28 (1H, s, Rha H-1), 5.76 (1H, s, Rha
5
5
column chromatography (7:3:0.4:0.1 CHCl –MeOH–
H-1), 4.74 (1H, d, J 6.7 Hz, Glc H-1), 1.39–1.24 (34H,
3
1
3
H O–NH OH) to give compound 21 (98 mg, 71%) as
m, –CH – · 17), 0.84 (6H, t, J 6.7 Hz, –CH · 2);
C
2
4
2
3
a colorless oil: [a] +27.8 (c 0.10, MeOH); HRFABMS:
NMR (in C D N): d 67.8 (–OCH CH NH–), 49.1
5 5 2 2
D
calcd for C H NO 712.4483, found m/z 712.4595
(–OCH2- CH NH–), 47.8 (–NHCH C H ), 172.8
2 2 7 15
3
4
66
14
+
1
[
M+H] ; H NMR (in C D N): d 6.12 (1H, s, Rha
(–COCH –), 46.2 (–COCH –), 22.9–33.5 (–CH – · 17),
5
5
2
2
2
0
H-1), 5.67 (1H, s, Rha H-1), 4.73 (1H, d, J 7.6 Hz,
14.3 · 2 (–CH · 2), 102.8, 78.6, 77.5, 78.0, 77.0, 61.2
3
Glc H-1), 1.28–1.17 (24H, m, –CH – · 12), 0.87 (3H, t,
(Glc C-1–6), 102.9, 72.8, 72.5, 74.1, 70.4, 18.7 (Rha
[1!2] C-1–6), 102.9, 72.7, 72.4, 73.9, 69.7, 18.5 (Rha
[1!4] C-1–6).
2
1
3
J 6.7 Hz, –CH3);
C NMR (in C D N): d 65.4
5 5
(
–OCH CH NH–), 48.3 (–OCH2- CH NH–), 48.1
2 2 2

2190
H. Miyashita et al. / Carbohydrate Research 342 (2007) 2182–2191
0
13
1.59 (3H, d, J 6.1 Hz, Rha H-6); C NMR (in
C D N): d 96.5, 79.6, 75.5, 79.5, 78.9, 61.5 (Glc C-1–
6), 103.1, 72.3, 72.8, 73.3, 70.4, 18.5 (Rha [1!2] C-1–
6), 103.0, 72.4, 72.9, 74.0, 70.4, 18.6 (Rha [1!4] C-1–6).
3
.10.3. N-Octanoyl-tetradecylaminoethyl a-L-rhamnopyr-
anosyl-(1!4)-[a-L-rhamnopyranosyl-(1!2)]-b-D-glucopy-
ranoside (24). This was synthesized as described in Sec-
tion 3.10 using compound 21 (80 mg, 0.11 mmol) and
octanoyl chloride (0.06 mL, 0.37 mmol). The product
was purified by column chromatography (4:1:0.1
CHCl –MeOH–NH OH) to give compound 24 (74 mg,
5
5
3
a
3.12. Cytotoxicity bioassays
3
4
7
9%) as a colorless oil: [a]D +11.8 (c 0.07, MeOH);
Growth inhibition experiments were carried out in qua-
druplicate in 96-well microplates, and the number of via-
ble cells at the end of incubation period was determined
HRESIMS: calcd for C H NNaO 860.5347, found
m/z 860.5336 [M+Na] ; H NMR (in C D N): d 6.32
4
2
1
79
15
+
5
5
0
22
(
1H, s, Rha H-1), 5.80 (1H, s, Rha H-1), 4.77 (1H, d,
using an MTT assay. Each sample was dissolved in
J 6.7 Hz, Glc H-1), 1.36–1.18 (34H, m, –CH - · 17),
Me SO (2.0 mg/mL) and diluted with an experimental
2
2
1
3
0
.85 (6H, t, J 6.7 Hz, –CH · 2);
C NMR (in
growth medium such that the final Me SO concentra-
3
2
C D N): d 68.0 (–OCH CH NH–), 49.1 (–OCH -
tion was less than 0.5%. PC-12 and HCT116 cells were
seeded in a 96-well microplate at a concentration of
1000 cells/well. They were continuously cultured with-
out or with five concentrations (5000, 1000, 200, 40,
and 8 ng/mL) of test compounds for 72 h starting from
the next day. After adding MTT (20 lL, 5 mg/mL in
phosphate-buffered saline; Sigma), the medium was re-
moved, and the blue dye that formed was dissolved in
5
5
2
2
2
CH NH–), 47.8 (–NHCH C H ), 176.0 (–COCH –),
2
2
13 27
2
4
6.3 (–COCH –), 23.0–33.5 (–CH – · 17), 14.3 · 2
2
2
(
1
–CH · 2), 102.0, 78.7, 77.5, 78.0, 77.0, 61.3 (Glc C-
3
–6), 103.0, 72.8, 72.5, 74.1, 70.5, 18.8 (Rha [1!2]
C-1–6), 103.0, 72.7, 72.4, 73.9, 69.8, 18.5 (Rha [1!4]
C-1–6).
3
.10.4. N-Tetradecanoyl-tetradecylaminoethyl a-L-rhamno-
150 lL of Me SO. The absorbance was measured, and
2
pyranosyl-(1!4)-[a-L-rhamnopyranosyl-(1!2)]-b-D-gluco-
pyranoside (25). This was synthesized as described in
Section 3.10 using compound 21 (80 mg, 0.11 mmol)
and tetradecanoyl chloride (0.09 mL, 0.37 mmol). The
product was purified by column chromatography
the T/C (%) score was calculated by the formulae de-
scribed below. A graph of the concentration of samples
against T/C (%) was plotted. The concentration value
that corresponded with 50% T/C was designated as the
GI50 value
(
(
9:1:0.1 CHCl –MeOH–NH OH) to give compound 25
3 4
85 mg, 83%) as a colorless oil: [a]D +33.4 (c 0.10,
T =C ð%Þ ¼ ðT ꢀ SÞ=ðC ꢀ SÞ ꢁ 100
MeOH); HRESIMS: calcd for C H NNaO
15
where T is the OD550 value of the cell with the samples
after a 3-d incubation, C the OD550 value of the cell
without the samples, and S the OD550 value of the cell
before the samples were added.
The GI50 value was defined as the concentration of
sample necessary to inhibit the growth to 50% of the
control.
4
8
91
+
1
9
44.6286, found m/z 944.6263 [M+Na] ; H NMR (in
0
C D N): d 6.30 (1H, s, Rha H-1), 5.80 (1H, s, Rha H-
5
5
1
–
), 4.78 (1H, d, J 6.7 Hz, Glc H-1), 1.39–1.25 (46H, m,
1
3
CH – · 23), 0.86 (6H, t, J 7.1 Hz, –CH · 2);
C
2
3
NMR (in C D N): d 67.8 (–OCH CH NH–), 49.1
5
5
2
2
(
–OCH CH NH–), 47.8 (–NHCH C H ), 172.8
2 2 2 13 27
(
1
–COCH –), 46.1 (–COCH –), 22.8–33.5 (–CH – · 23),
2
2
2
4.3 · 2 (–CH · 2), 102.0, 78.6, 77.5, 78.0, 77.1, 61.2
3
Acknowledgement
(
Glc C-1–6), 102.9, 72.8, 72.5, 74.1, 70.5, 18.8 (Rha
[
1!2] C-1–6), 103.0, 72.7, 72.4, 73.9, 69.7, 18.5 (Rha
We thank Mr. Akira Ejima of Daiichi Pharmaceutical
Co., Ltd, for measuring the cytotoxicity against PC-12
and HCT116 cells.
[
1!4] C-1–6).
3
.11. a-L-Rhamnopyranosyl-(1!4)-[a-L-rhamnopyran-
osyl-(1!2)]-a-D-glucopyranose (26)
Supplementary data
Compound 5 (102 mg, 0.079 mmol) was dissolved in 3%
KOH/MeOH (7.0 mL) and heated at 65 ꢁC. After
stirring for 20 h, the mixture was purified by column
chromatography (MCI gel, CHP P; water, 250.0 mL;
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2
007.06.008.
2
0
MeOH, 150.0 mL), and the organic solvent was concen-
trated under diminished pressure to yield 26 (51 mg,
References
. Hostettmann, K.; Marston, A. Saponins; Cambridge
University Press: Cambridge, UK, 1995.
. (a) Nohara, T.; Yahara, S.; Kinjo, J. Nat. Prod. Sci. 1998,
9
4%) as a colorless oil. [a]D ꢀ45.1 (c 0.05, MeOH);
1
HRESIMS: calcd for C H NaO 495.1690, found
m/z 495.1485 [M+Na] ; H NMR (in C D N): d 5.91
1
8
32
14
+
1
5
5
2
(
(
1H, d, J 3.7 Hz, Glc H-1), 5.83 (1H, s, Rha H-1) 5.76
1H, s, Rha H-1), 1.73 (3H, d, J 6.1 Hz, Rha H-6),
4, 203–214; (b) Nohara, T. Yakugaku Zasshi 2004, 124,
0
183–205.

H. Miyashita et al. / Carbohydrate Research 342 (2007) 2182–2191
2191
3
4
. (a) Nakamura, T.; Komori, C.; Lee, Y.; Hashimoto, Y.;
Yahara, S.; Nohara, T.; Ejima, A. Biol. Pharm. Bull. 1996,
10. (a) Ogawa, T.; Beppu, K.; Nakabayashi, S. Carbohydr.
Res. 1981, 93, C6; (b) Ferrier, R. J.; Furneaux, R. H.
Methods Carbohydr. Chem. 1980, 8, 251.
11. (a) Jiang, L.; Chan, T. H. J. Org. Chem. 1998, 63, 6035–
6038; (b) Kurahashi, T.; Mizutani, T.; Yoshida, J. J.
Chem. Soc., Perkin Trans. 4 1999, 465–474.
12. Liu, L. D.; Liu, H. W. Tetrahedron Lett. 1989, 30, 35–
38.
13. Schmidt, R. R.; Toepfer, A. Tetrahedron Lett. 1991, 32,
3353–3356.
14. Nakayama, K.; Uoto, K.; Higashi, K.; Soga, T.; Kusama,
T. Chem. Pharm. Bull. 1992, 40, 1718–1720.
15. Numata, M.; Sugimoto, M.; Ito, Y.; Ogawa, T. Carbo-
hydr. Res. 1990, 203, 205–217.
16. (a) Ikeda, T.; Kinjo, J.; Kajimoto, T.; Nohara, T.
Heterocycles 2000, 52, 775–798; (b) Lee, Y. C.; Lee, R.
T. In Neoglycoconjugates; Lee, Y. C., Lee, R. T., Eds.;
Academic Press: California, 1994.
17. Borch, R. F.; Bernstein, M. D.; Durst, H. D. J. Am. Chem.
Soc. 1971, 93, 2897–2904.
1
9, 564–566; (b) Ikeda, T.; Tsumagari, H.; Honbu, T.;
Nohara, T. Biol. Pharm. Bull. 2003, 26, 1198–1201.
. Ikeda, T.; Ando, J.; Miyazono, A.; Zhu, X. H.; Tsuma-
gari, H.; Nohara, T.; Yokomizo, K.; Uyeda, M. Biol.
Pharm. Bull. 2000, 23, 363–364.
5
6
. Kawasaki, T.; Yamauchi, T. Chem. Pharm. Bull. 1962, 10,
7
03–708.
. (a) Hsu, S. H.; Tsai, T. R.; Lin, C. N.; Yen, M. H.; Kuo,
K. W. Biochem. Biophys. Res. Commun. 1996, 229, 1–5; (b)
Chang, L. C.; Tsai, T. R.; Wang, J. J.; Lin, C. N.; Kuo, K.
W. Biochem. Biophys. Res. Commun. 1998, 242, 21–25; (c)
Liang, C. H.; Liu, L. F.; Shiu, L. Y.; Huang, Y. S.; Chang,
L. C.; Kuo, K. W. Biochem. Biophys. Res. Commun. 2004,
3
22, 751–758.
7
. (a) Zhu, S.; Zhang, Y.; Li, M.; Yu, J.; Zhang, L.; Li, Y.;
Yu, B. Bioorg. Med. Chem. Lett. 2006, 16, 5629–5632; (b)
Zhang, Y.; Li, Y.; Guo, T.; Guan, H.; Shi, J.; Yu, Q.; Yu,
B. Carbohydr. Res. 2005, 340, 1453–1459; (c) Yang, Z.;
Wong, E. L.; Shum, T. Y.; Che, C. M.; Hui, Y. Org. Lett.
18. Schmidt, R. R.; Zimmermann, P. Angew. Chem., Int. Ed.
Engl. 1986, 25, 725.
2
005, 7, 669–672; (d) Zhang, Y.; Li, Y.; Zhu, S.; Guan, H.;
Lin, F.; Yu, B. Carbohydr. Res. 2004, 339, 1753–1759; (e)
Li, M.; Han, X.; Yu, B. Carbohydr. Res. 2003, 338, 117–
19. Kinjo, M.; Oka, K.; Naito, S.; Kohga, S.; Tanaka, K.;
Oboshi, S.; Hayata, Y.; Yasumoto, K. Br. J. Cancer 1979,
39, 15–23.
20. Brattain, M. G.; Fine, W. D.; Khaled, F. M.; Thompson,
J.; Brattain, D. E. Cancer Res. 1981, 41, 1751–1756.
21. Mahato, S. B.; Sahu, N. P.; Ganguly, A. N. Indian J.
Chem. 1980, 19B, 817–819.
1
21; (f) Lahmann, M.; Gyback, H.; Garegg, P. J.;
Oscarson, S.; Suhr, R.; Thiem, J. Carbohydr. Res. 2002,
37, 2153–2159.
3
8
. Ikeda, T.; Miyashita, H.; Kajimoto, T.; Nohara, T.
Tetrahedron Lett. 2001, 42, 2353–2356.
9
. Vic, G.; Crout, D. H. G. Carbohydr. Res. 1995, 279, 315–
22. Carmichael, J.; Degrraff, W. G.; Gazdar, A. F.; Minna, J.
D.; Mitchell, J. B. Cancer Res. 1987, 47, 936–942.
3
19.