Efficient synthesis of α- and β-chacotriosyl glycosides using appropriate donors, and their cytotoxic activity

Article abstract of DOI:10.1016/j.carres.2008.02.012

Natural steroidal glycosides containing α-l-rhamnopyranosyl-(1→4)-[α-l-rhamnopyranosyl-(1→2)]-β-d-glucopyranose (chacotriose) at the oligosaccharide moiety exhibit anti-cancer and anti-herpes activities. To investigate the structure-activity relationships of the aglycone parts of chacotriosides, we developed a synthesis method for chacotriosyl glycosides having various aglycones. In the process, it was revealed that α-chacotriosyl glycosides could be obtained mainly by using a trichloroacetimidate donor, while β-chacotriosyl glycosides were afforded by using phosphite and phosphate donors. In cytotoxicity tests using the A549 and HepG2 cell lines, naturally occurring β-chacotriosyl diosgenin and cholestanol exhibited higher activities than the corresponding α-chacotriosyl glycosides.

Full text of DOI:10.1016/j.carres.2008.02.012

Available online at www.sciencedirect.com
Carbohydrate Research 343 (2008) 1309–1315
Efficient synthesis of a- and b-chacotriosyl glycosides
using appropriate donors, and their cytotoxic activity
Hiroyuki Miyashita,a Yuuki Kai,a Toshihiro Noharab and Tsuyoshi Ikedaa,*
aFaculty of Medical and Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan
bFaculty of Pharmaceutical Sciences, Sojo University, 4-22-1 Ikeda, Kumamoto 860-0082, Japan
Received 15 January 2008; received in revised form 12 February 2008; accepted 14 February 2008
Available online 20 February 2008
Abstract—Natural steroidal glycosides containing a-L-rhamnopyranosyl-(1?4)-[a-L-rhamnopyranosyl-(1?2)]-b-D-glucopyranose
(chacotriose) at the oligosaccharide moiety exhibit anti-cancer and anti-herpes activities. To investigate the structure–activity
relationships of the aglycone parts of chacotriosides, we developed a synthesis method for chacotriosyl glycosides having various
aglycones. In the process, it was revealed that a-chacotriosyl glycosides could be obtained mainly by using a trichloroacetimidate
donor, while b-chacotriosyl glycosides were afforded by using phosphite and phosphate donors. In cytotoxicity tests using the A549
and HepG2 cell lines, naturally occurring b-chacotriosyl diosgenin and cholestanol exhibited higher activities than the correspond-
ing a-chacotriosyl glycosides.
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords: Chacotriose; Glycoside; Transglycosylation; Cytotoxicity
1. Introduction
part (chacotriose), and the a-anomeric glycoside of dios-
cin exhibit little or no activity. These results obtained by
our group indicate that the naturally occurring b-glyco-
sidic linkage in glycosides plays an important role in
determining biological activity. Although facile synthetic
methods for chacotriosyl derivatives have been demon-
strated,7–14 our method6 may be useful for directly trans-
ferring the chacotriose moiety to various aglycones.
However, a-chacotriosyl glycosides with weak biological
activities were predominantly afforded in our previous
study.6 Thus, as it is generally known that the reactivity
and stereoselectivity of glycosylations are considerably
influenced by the properties of the donor, catalyst, and
solvent, we describe the facile synthetic route for b-chac-
otriosyl glycosides predominantly by investigating a
variety of chacotriosyl donors. Furthermore, the cyto-
toxicities of the obtained neosaponins have been
determined.
We have studied the steroidal oligosaccharides in many
solanaceous plants and have clarified their chemical
structures and certain biological activities.1,2 Our
detailed studies on the relationship between the struc-
tures and bioactivities of the steroidal glycosides from
Solanum plants have suggested that spirostanol-type ste-
roidal glycosides containing the chacotriose moiety
exhibited potent anti-cancer3,4 and anti-herpes5 activi-
ties. Therefore, we have developed a facile synthetic
method for chacotriosyl donors from D-glucose and
L-rhamnose that can be used to transfer the moiety to a
variety of aglycones to afford a/b neosaponins, including
a natural steroidal glycoside dioscin (b-chacotriosyl dios-
genin).6 In cytotoxicity tests using the PC-12 (lung can-
cer) and HCT116 (colon cancer) cell lines, dioscin
exhibits moderate cytotoxicity [GI50: 2.29 lM (PC-12);
3.07 lM (HCT116)] equivalent to that of cisplatin. On
the other hand, the aglycone part (diosgenin), the sugar
2. Results and discussion
4-Tolyl (2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl)-(1?4)-
[(2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl)-(1?2)]-3,6-
*
Corresponding author. Tel.: +81 96 371 4738; fax: +81 96 362
7799; e-mail: tikeda@gpo.kumamoto-u.ac.jp
0008-6215/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2008.02.012

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H. Miyashita et al. / Carbohydrate Research 343 (2008) 1309–1315
di-O-pivaloyl-1-thio-b-D-glucopyranoside
(1)
was
The chacotriosyl donors 1–5 that were obtained were
transferred to aglycones (diosgenin and cholestanol)
using an acid-washed molecular sieve (MSAW 300)
and a promoter (Table 1). First, the glycosylation of 1
with diosgenin was carried out in the presence of NIS
with boron trifluoride etherate (BF3ꢀEt2O) or trimethyl-
silyl trifluoromethanesulfonate (TMSOTf) as the pro-
moter in anhydrous dichloromethane (CH2Cl2). As a
result, the a-form of protected glycoside 6 was predom-
inantly obtained in 48% yield (entry 1, a/b = 71/29).
Next, 2 was used with diphenyl sulfoxide and 2,4,6-tri-
tert-butylpyrimidine (TTBP) as promoters,17 and then
transferred to diosgenin to afford 6 (a/b = 80/20) in
22% yield. Since the yield and b-selectivity of this glyc-
osylation method were not satisfactory, we performed
the glycosylation of 3 with diosgenin using BF3ꢀEt2O
as a promoter to afford 6 (a/b = 69/29) in 64% yield.
However, because the b-selectivity was not sufficiently
high, even though the yield was improved over that
using 1 and 2, glycosylation was attempted in polar
solvents (entries 5–7). Glycosylation in all of the polar
solvents predominantly afforded b-glycoside, but the
total yield was sharply decreased because of the fact that
neither 3 nor diosgenin easily dissolve in polar solvents.
On the other hand, when 418 was reacted with diosgenin
under BF3ꢀEt2O in CH2Cl2, a naturally occurring b-gly-
coside was mainly afforded (a/b = 26/74) in 48% yield
prepared from 4-tolyl 3,6-di-O-pivaloyl-1-thio-b-D-
glucopyranoside15 and a-L-rhamnopyranosyl trichloro-
acetimidate according to the method of Cheng et al.16
The thiotolyl moiety of chacotrioside 1 was deprotected
with N-iodosuccinimide (NIS) to yield chacotriosyl
derivative 2, and the 1-hydroxy group at the reducing
end of 2 (Scheme 1) was converted into various leaving
groups for transglycosylation. First, trichloroacetimi-
date 36 (a-form, 86%) was easily produced from 2 in
the presence of trichloroacetonitrile and 1, 8-diazabi-
cyclo[5.4.0]undec-7-ene. Accordingly, diethyl phosphite
4 (a-form, 93%) and diphenyl phosphate 5 (a-form,
86%) were synthesized from 2, which correspond
to diethyl chlorophosphite and diphenyl phosphoryl
chloride under triethylamine, respectively.
OPiv
R1
O
O
PivO
1 : R1 = STol, R2 = H
O
R2
a
AcO
O
2 : R1 = H, R2 = OH
AcO
b
3 : R1 = H, R2 = OC(NH)CCl3
4 : R1 = H, R2 = OP(OEt)2
5 : R1 = H, R2 = OPO(OPh)2
c
OAc
AcO
d
O
AcO
OAc
Scheme 1. Reagents and conditions: (a) TMSOTf, CH2Cl2, H2O, 62%;
(b) CCl3CN, DBU, CH2Cl2, 86%; (c) ClP(OEt)2, Et3N, CH2Cl2, 93%;
(d) ClPO(OPh)2, Et3N, CH2Cl2, 86%.
Table 1. Glycosylation of donors 1–5 with diosgenin or cholestanol
OPiv
O
Diosgenin
or
O
PivO
OR
O
Cholestanol
AcO
O
1-5
Promoter
MSAW 300
AcO
6 : R = Diosgenyl
7 : R = Cholestanyl
OAc
AcO
O
AcO
OAc
Entries
Donor
Acceptor
Promoter
Solvent
a/b
Yield (%)
1
2
1
D
D
NIS/BF3ꢀEt2O
CH2Cl2
CH2Cl2
71/29
76/24
48
43
NIS/TMSOTf
3
2
3
D
Ph2SO/TTBP/Tf2O
CH2Cl2
80/20
22
4
5
6
7
8
D
D
D
D
C
BF3ꢀEt2O
BF3ꢀEt2O
BF3ꢀEt2O
BF3ꢀEt2O
BF3ꢀEt2O
CH2Cl2
CH3CN
CH3CH2CN
CH3CN/Et2O
CH2Cl2
69/31
24/76
26/74
25/75
81/19
64
9
14
29
42
9
10
11
12
13
4
5
D
D
D
D
C
BF3ꢀEt2O
TMSOTf
TMSOTf
TMSOTf
BF3ꢀEt2O
CH2Cl2
CH2Cl2
Et2O
CH3CN/Et2O
CH2Cl2
26/74
24/76
33/67
31/69
28/72
48
53
34
31
44
14
15
D
C
TMSOTf
TMSOTf
CH2Cl2
CH2Cl2
27/73
30/70
43
46
D: diosgenin, C: cholestanol.

H. Miyashita et al. / Carbohydrate Research 343 (2008) 1309–1315
1311
O
O
c
α
α
α-form)
(
6
8
OH
O
O
O
a
HO
6
O
O
HO
HO
OH
HO
c
c
O
β
6
8β
(β-form)
HO
OH
OH
α
7
α
α
9
( -form)
O
O
O
HO
O
b
O
HO
7
HO
OH
HO
O
c
HO
β
9
(
β
7
β-form)
OH
Scheme 2. Reagents and conditions: (a) ODS column chromatography (90% MeOH); (b) ODS column chromatography (95% MeOH); (c) 3% KOH/
MeOH, 65 °C.
Table 2. Cytotoxic activity (IC50
)
(entry 9). Subsequently, to further increase the b-selec-
tivity, we attempted glycosylation using 4 in polar
solvents (entries 11 and 12). Unfortunately, the b-selec-
tivity did not increase over that in entry 10. Finally, 5
was transferred to diosgenin, mainly affording a natu-
rally occurring b-glycoside (entry 14), as in the case of
4. To obtain the other neosaponin, donors 3–5 were gly-
cosylated with cholestanol (entries 8, 13, and 15). The
BF3ꢀEt2O-promoted glycosylation of 3 with cholestanol
predominantly afforded the a-form of protected glyco-
side 7 in 42% yield (entry 8; a/b = 81/19); on the other
hand, the TMSTOf-promoted glycosylation of 5 with
cholestanol mainly afforded the b-form of 7 in 44% yield
(entry 13; a/b = 28/72). Only by changing the leaving
group into a donor could a-glycoside and b-glycoside
be easily synthesized separately. The technique of trans-
ferring distinct types of chacotriosyl donors to the agly-
cone after building chacotriose is a useful procedure to
construct a library of a- and b-chacotriosyl glycosides
having various aglycones.
The a/b anomeric mixtures of protected neoglycosides
6 and 7 thus obtained were separated on an ODS col-
umn to afford pure a- and b-protected neoglycosides
6a/b and 7a/b. Each protected neoglycoside (6a/b,
7a/b) was deprotected in the usual manner to afford
the corresponding neosaponins (8a/b, 9a/b) (Scheme 2).
The cytotoxicities of neosaponins 8a/b and 9a/b, dios-
genin, cholestanol, and chacotriose6 toward human lung
cancer (A549) and human hepatoblastoma (HepG2) cell
lines were determined19 (Table 2). Dioscin (8b) exhibited
moderate cytotoxicity against A549, which was the same
as that of 5-fluorouracil (5FU). However, compound 8a,
which is an anomeric isomer of dioscin, showed weak
activity. Interestingly, the same tendency that the
b-chacotriosyl cholestanol 9b had higher activity than
9a was observed even though these had weaker activities
compared with those of 8a and 8b. In addition, 8a and
A549
2.4
HepG2
0.57
5-Fluorouracil
8a
12.2
2.5
15.1
7.3
>50
>50
>50
8.1
4.1
8b (dioscin)
9a
9b
Chacotriose
Cholestanol
Diosgenin
>50
>50
>50
>50
>50
8b exhibited moderate cytotoxicity against HepG2,
while 9a and 9b did not show activity. Furthermore,
only diosgenin, cholestanol, and chacotriose did not
exhibit high cytotoxicity against A549 and HepG2. As
a consequence, it is suggested that the aglycone moiety
and the b-glycosidic linkage of the chacotriosyl glyco-
side are essential for cytotoxicity. It is thought that
our method, in which an oligosaccharide moiety is trans-
ferred to various aglycones to obtain the a- and b-linked
neosaponins efficiently, is useful to investigate the struc-
ture–activity relationships of bioactive glycoconjugate
compounds such as natural products.
3. Experimental
3.1. General methods
Optical rotations were measured using a JASCO DIP-
1000 KUY digital polarmeter (l = 0.5 cm). 1H NMR
and 13C NMR spectra were recorded on JNM-ECX
400 MHz and JEOL a-500 MHz NMR spectrometers.
Elucidation of the chemical structures was based on
1H, 13C, COSY, HMQC, and HMBC NMR experi-
ments. The chemical shifts are reported in parts per mil-
lion (ppm) relative to the residual solvent peaks, and the

1312
H. Miyashita et al. / Carbohydrate Research 343 (2008) 1309–1315
coupling constants (J) are reported in terms of Hertz.
FABMS and HRFABMS spectra were obtained using
a glycerol matrix in the positive-ion mode by using a
JEOL JMS-DX-303HF spectrometer. HRESIMS analy-
sis was performed using a JMS-T100LC. Column chro-
matography was carried out using Silica Gel 60 (Kanto
Chemical, Co., Inc., 70–230 mesh and 230–400 mesh);
Amberlite IR-120B; MB-3 (Organo Co., Ltd); and
Chromatorex ODS (Fuji Silysia Chemical Co., Ltd) col-
umns. TLC was performed on precoated Silica Gel 60
F254 plates (E. Merck).
a colorless oil. Physicochemical data were identical to
those described in Ref. 6.
3.4. (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-a-D-glucopyranosyl diethyl phosphite (4)
Diethyl chlorophosphite (20 lL, 138 lmol) was added
to a stirred solution of trisaccharide 2 (55 mg, 55 lmol)
and Et3N (25 lL, 181 lmol) in CH2Cl2 (1.5 mL) at 0 °C
under nitrogen. After stirring for 30 min, the reaction
was quenched with MeOH, followed by stirring at room
temperature for 30 min. The mixture was concentrated
under diminished pressure, and the residue was purified
by silica gel column chromatography (2:1 hexane–
3.2. 4-Tolyl (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-1-thio-b-D-glucopyranoside (1)
1
EtOAc) to yield 4 (52 mg, 93%) as a colorless oil: H
BF3ꢀEt2O (2.0 mL, 16.0 mmol) was added to a suspen-
sion of 4-tolyl 3,6-di-O-pivaloyl-1-thio-b-D-glucopyran-
oside (2.6 g, 5.8 mmol) and MSAW 300 (4.0 g) in
CH2Cl2 (10 mL) at ꢁ78 °C under nitrogen. After stir-
ring for 1 h, a-L-rhamnopyranosyl trichloroacetimidate
(8.6 g, 19.8 mmol) in CH2Cl2 (13 mL) was added to
the mixture, followed by stirring for 5 h at room temper-
ature. The mixture was diluted with CHCl3 and filtered
using Celite. The filtrate was washed with satd aq NaH-
CO3 and satd aq NaCl, dried over MgSO4, and concen-
trated under diminished pressure. The residue was
purified by silica gel column chromatography (3:1
hexane–EtOAc) to yield 1 (3.4 g, 55%) as a colorless
NMR (in CDCl3): d 1.20, 1.23 (each 9H, s,
C(CH3)3 ꢂ 2), 1.31 (3H, t, J 7.3 Hz, OCH2CH3), 1.33
(3H, t, J 7.0 Hz, OCH2CH3), 1.94, 1.98, 2.02, 2.04,
2.11, 2.12 (each 3H, s, COCH3 ꢂ 6), 4.76 (1H, s, Rha
H-1), 4.89 (1H, s, Rha0 H-1), 5.20 (1H, dd, J 3.7 Hz,
Glc H-1), 5.55 (1H, dd, J 9.1, 9.2 Hz, Glc H-3).
3.5. (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-a-D-glucopyranosyl diphenyl phosphate (5)
Diphenyl phosphoryl chloride (40 mL, 193 lmol) was
added to a stirred solution of trisaccharide 2 (20 mg,
20 lmol), DMAP (3 mg, 25 lmol), and Et3N (30 lL,
217 lmol) in CH2Cl2 (1.2 mL) at 0 °C under nitrogen.
After stirring for 1 h, the reaction was quenched with
MeOH, followed by stirring at room temperature for
30 min. The mixture was concentrated under diminished
pressure, and the residue was purified by silica gel col-
umn chromatography (3:2 hexane–EtOAc) to yield 5
1
oil: [a]D +9.2 (c 0.13, CHCl3); H NMR (in CDCl3): d
1.15 (3H, d, J 6.1 Hz, Rha H-6), 1.17, 1.19 (each 9H,
s, C(CH3)3 ꢂ 2), 1.23 (3H, d, J 6.1 Hz, Rha0 H-6),
1.96 ꢂ 2, 2.03, 2.05, 2.11, 2.12 (each 3H, s, COCH3 ꢂ 6),
2.34 (1H, s, phCH3), 4.72 (1H, d, J 8.5 Hz, Glc H-1),
4.87 (1H, s, Rha H-1), 4.92 (1H, s, Rha0 H-1); 13C
NMR (in CDCl3): d 99.5, 77.7, 72.0, 77.4, 75.4, 62.7
(Glc C-1–6), 98.0, 69.9, 69.2, 70.5, 67.9, 17.1 (Rha
[1?2] C-1–6), 97.3, 69.5, 68.7, 70.2, 66.7, 17.1 (Rha
[1?4] C-1–6); HRESIMS: calcd for C47H66NaO21S:
1021.3715; found: m/z 1021.3798 [M+Na]+.
1
(19 mg, 86%) as a colorless oil: H NMR (in CDCl3):
d 1.19, 1.22 (each 9H, s, C(CH3)3 ꢂ 2), 1.87, 1.93,
1.98, 2.05, 2.11 ꢂ 2 (each 3H, s, COCH3 ꢂ 2), 4.79
(1H, s, Rha H-1), 4.83 (1H, s, Rha0 H-1), 5.52 (1H,
dd, J 9.1, 9.8 Hz, Glc H-3), 6.00 (1H, d, J 3.7 Hz, Glc
H-1).
3.3. Preparation of (2,3,4-tri-O-acetyl-a-L-rhamnopyr-
anosyl)-(1?4)-[(2,3,4-tri-O-acetyl-a-L-rhamnopyran-
osyl)-(1?2)]-3,6-di-O-pivaloyl-a-D-glucopyranoside (2)6
3.6. General procedure for the glycosylation of (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-glucopyranosyl diosgenin (6a, 6b)
TMSOTf (50 lL, 276 mmol) was added to a suspension
of 1 (200 mg, 200 lmol) and NIS (100 mg, 444 lmol) in
CH2Cl2 (4.0 mL) at ꢁ78 °C under nitrogen. After stir-
ring for 10 min, H2O (1.0 mL, 56 mmol) was added to
the mixture, followed by stirring for 3 h at room temper-
ature. Next, Na2S2O3 (20 mg, 127 mmol) and NaHCO3
(20 mg, 238 mmol) were added to the mixture, followed
by stirring for 1 h. The precipitate was filtered, and the
filtrate was concentrated under diminished pressure.
The residue was purified by silica gel column chromato-
graphy (2:1 hexane–acetone) to yield 2 (111 mg, 62%) as
3.6.1. Procedure
oside. 4-Tolyl
A
using 4-tolyl thio-b-chacotori-
thio-b-chacotorioside (50 mg,
1
50 lmol), diosgenin (60 mg, 144 lmol), NIS (25 mg,
111 lmol), and MSAW 300 (200 mg) were suspended
in CH2Cl2 (2.0 mL) at room temperature. After stirring
for 30 min, TMSTOf (1 M solution in Et2O, 10 lL) was
added to the mixture dropwise. The mixture was stirred
for 30 min at room temperature, diluted with CHCl3,

H. Miyashita et al. / Carbohydrate Research 343 (2008) 1309–1315
1313
washed with satd aq NaHCO3 and satd aq NaCl, and
concentrated under diminished pressure. The obtained
residue was purified by column chromatography (2:1
hexane–EtOAc) to yield a mixture of 6a and 6b as a
colorless oil. The mixture was separated by ODS
column chromatography (9:1 MeOH–H2O) to yield 6a
(22 mg, 34%) and 6b (9 mg, 14%), both as white solids.
Physicochemical data were identical to those described
in Ref. 6.
and then purified by column chromatography (2:1
hexane–EtOAc) to yield a mixture of 6a and 6b as a
colorless oil. The mixture was separated by ODS column
chromatography (9:1 MeOH–H2O) to yield 6a (4 mg,
12%) and 6b (13 mg, 41%), both as white solids.
Physicochemical data were identical to the products of
Section 3.6.1.
3.6.5. Procedure E using chacotriosyl diphenyl phos-
phate. Chacotriosyl diphenyl phosphate 5 (30 mg,
27 lmol), diosgenin (20 mg, 48 lmol), and MSAW 300
(150 mg) were suspended in CH2Cl2 (1.5 mL) at room
temperature. After stirring for 30 min, TMSTOf (1 M
solution in Et2O, 20 lL) was added to the mixture in a
dropwise fashion. After stirring for 2 h at room
temperature, the reaction was quenched with MeOH,
followed by stirring at room temperature for 30 min.
The residue was concentrated under diminished pressure
and then purified by column chromatography (2:1
hexane–EtOAc) to yield a mixture of 6a and 6b as a col-
orless oil. The mixture was separated by ODS column
chromatography (9:1 MeOH–H2O) to yield 6a (4 mg,
11%) and 6b (11 mg, 32%), both as white solids.
Physicochemical data were identical to the products of
Section 3.6.1.
3.6.2. Procedure
B
using 1-hydroxy-a-chacotori-
oside. Trifluoromethanesulfonic anhydride (15 lL,
9 lmol) was added to a solution of 2 (22 mg, 22 lmol),
diphenyl sulfoxide (40 mg, 198 lmol), and TTBP
(50 mg, 202 mmol) in CH2Cl2 at ꢁ40 °C. After stirring
for 1.5 h, diosgenin (35 mg, 84 lmol) was added to the
mixture at ꢁ40 °C. The reaction mixture was stirred
for 1 h before the addition of excess Et3N. The mixture
was diluted with CH2Cl2, washed with satd aq NaHCO3
and satd aq NaCl, and concentrated under diminished
pressure. The residue was purified by silica gel column
chromatography (2:1 hexane–EtOAc) to yield a mixture
of 6a and 6b as a colorless oil. The mixture was sepa-
rated by ODS column chromatography (9:1 MeOH–
H2O) to yield 6a (5 mg, 18%) and 6b (1 mg, 4%), both
as white solids. Physicochemical data were identical to
the products of Section 3.6.1.
3.7. General procedure for the glycosylation of (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-
glucopyranosyl cholestanol (7a, 7b)
3.6.3. Procedure C using chacotriosyl trichloroacet-
imidate. Chacotriosyl imidate 3 (48 mg, 64 lmol),
diosgenin (400 mg, 0.97 mmol), and MSAW 300
(800 mg) were suspended in CH2Cl2 (8.0 mL) at 20 °C.
After stirring for 30 min, BF3ꢀEt2O (10% solution in
CH2Cl2, 2.0 lmol) was added dropwise to the mixture.
The mixture was stirred for 2 h at room temperature,
diluted with CHCl3, and filtered using Celite. The filtrate
was washed with satd aq NaHCO3 and satd aq NaCl,
dried over MgSO4, and concentrated under diminished
pressure. The residue was purified by column chromato-
graphy (2:1 hexane–EtOAc) to yield a mixture of 6a and
6b as a colorless oil. The mixture was separated by ODS
column chromatography (9:1 MeOH–H2O) to yield 6a
(26 mg, 44%) and 6b (12 mg, 20%), both as white solids.
Physicochemical data were identical to the products of
Section 3.6.1.
3.7.1. Procedure A using chacotriosyl trichloroacet-
imidate. Glycosylation of 3 (40 mg, 39 lmol) with
cholestanol (60 mg, 178 lmol) was accomplished as
described in Section 3.6.3, followed by purification by
column chromatography (3:1 hexane–EtOAc) to yield
a mixture of 7a and 7b as a colorless oil. The mixture
was separated by ODS column chromatography (19:1
MeOH–H2O) to afford 7a (17 mg, 34%) and 7b (4 mg,
8%), both as white solids. Data for a-glycoside (7a):
15
1
½aꢃD ꢁ22.1 (c 0.21, CHCl3); H NMR (in CDCl3): d
0.66 (3H, s, H-19), 0.86 (3H, d, J 6.1 Hz, H-27), 0.87
(3H, s, H-18), 0.89 (3H, d, J 6.1 Hz, H-21), 1.16 (3H,
d, J 6.7 Hz, Rha H-6), 1.21 (3H, d, J 6.1 Hz, Rha0
H-6), 1.20, 1.23 (each 9H, s, C(CH3)3 ꢂ 2), 1.95, 1.98,
2.02, 2.03, 2.11, 2.12 (each 3H, s, COCH3 ꢂ 6), 4.72
(1H, s, Rha H-1), 4.88 (1H, s, Rha0 H-1), 5.03 (1H, d,
J 3.1 Hz, Glc H-1); 13C NMR (in CDCl3): d 37.1,
27.9, 78.8, 29.7, 45.2, 29.3, 31.9, 35.6, 54.5, 35.6, 21.3,
28.3, 39.0, 56.4, 24.2, 28.8, 56.6, 12.1, 12.4, 35.8, 20.6,
36.2, 23.9, 39.6, 29.3, 22.8, 22.6 (C-1–27), 97.6, 80.0,
71.3, 77.5, 71.2, 62.6 (Glc C-1–6), 99.9, 69.6, 68.8,
71.0, 67.9, 17.6 (Rha [1?2] C-1–6), 96.3, 68.9, 68.0,
70.3, 66.9, 17.2 (Rha0 [1?4] C-1–6); HRESIMS: calcd
for C67H106O22Na: 1286.7151; found: m/z 1286.6833
3.6.4. Procedure D using chacotriosyl diethyl phos-
phite. Chacotriosyl diethyl phosphite
4
(25 mg,
25 lmol), diosgenin (15 mg, 36 lmol), and MSAW 300
(140 mg) were suspended in CH2Cl2 (1.5 mL) at room
temperature. After stirring for 30 min, TMSTOf (1 M
solution in Et2O, 20 lL) was added to the mixture in a
dropwise fashion. After stirring for 1.5 h at room
temperature, the reaction was quenched with MeOH,
followed by stirring at room temperature for 30 min.
The residue was concentrated under diminished pressure
15
[M+Na]+. Data for b-glycoside (7b): ½aꢃD ꢁ52.1 (c

1314
H. Miyashita et al. / Carbohydrate Research 343 (2008) 1309–1315
0.22, CHCl3); 1H NMR (in CDCl3): d 0.66 (3H, s, H-19),
0.86 (3H, d, J 6.7 Hz, H-27), 0.89 (3H, d, J 6.7 Hz,
H-21), 1.08 (3H, s, H-18), 1.15 (3H, d, J 6.1 Hz, Rha
H-6), 1.17 (9H, s, C(CH3)3), 1.19 (3H, d, J 6.1 Hz, Rha0
H-6), 1.21 (each 9H, s, C(CH3)3), 1.95, 1.98, 2.02, 2.04,
2.10, 2.12 (each 3H, s, COCH3 ꢂ 6), 4.60 (1H, d, J
7.4 Hz, Glc H-1), 4.82 (1H, s, Rha H-1), 4.88 (1H, s,
Rha0 H-1); 13C NMR (in CDCl3): d 37.1, 27.1, 79.2,
29.1, 45.1, 29.4, 31.9, 35.6, 54.5, 35.5, 21.3, 28.0, 39.1,
56.4, 24.1, 28.6, 56.4, 12.3, 12.4, 35.9, 20.6, 36.1, 23.9,
39.6, 29.3, 22.7, 22.5 (C-1–27), 97.5, 80.2, 71.1, 77.5,
71.1, 62.6 (Glc C-1–6), 99.8, 69.5, 68.8, 71.2, 67.9, 17.8
(Rha [1?2] C-1–6), 96.6, 68.8, 68.0, 70.3, 66.8, 17.5
(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); HRESIMS: calcd for C45H78O14Na: 865.5289;
found: m/z 865.5183 [M+Na]+.
3.9. Cholestanol-3b-yl a-L-rhamnopyranosyl-(1?4)-[a-L-
rhamnopyranosyl-(1?2)]-b-D-glucopyranoside (9b)
Compound 7b (18 mg, 14 lmol) was dissolved in 3%
KOH/MeOH (3.0 mL) and heated at 65 °C. After stir-
ring for 20 h, the mixture was purified by column
chromatography (MCI gel, CHP20P; water, 100 mL;
MeOH, 80 mL) and then concentrated under diminished
(Rha0
[1?4] C-1–6); HRESIMS: calcd for
15
C67H106O22Na: 1286.7151; found: m/z 1286.6945
pressure to afford 9a (11 mg, 92%) as a white solid: ½aꢃD
[M+Na]+.
ꢁ46.3 (c 0.23, CHCl3); 1H NMR (in CDCl3) d: 0.65 (3H,
s, H-18), 0.90 (3H, d, J 6.7 Hz, H-26), 0.91 (3H, d, J
6.7 Hz, H-27), 0.98 (3H, d, J 6.7 Hz, H-21), 1.09 (3H,
s, H-19), 1.63 (3H, d, J 6.1 Hz, Rha H-6), 1.78 (3H, d,
J 6.1 Hz, Rha0 H-6), 4.37 (1H, d, J 7.3 Hz, Glc H-1),
5.87 (1H, s, Rha H-1), 6.41 (1H, s, Rha0 H-1); 13C
NMR (in C5D5N): d 37.6, 29.9, 79.8, 39.1, 45.3, 29.3,
32.1, 32.0, 50.4, 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, 20.7, 23.0,
22.6 (C-1–27), 97.2, 78.1, 76.8, 78.0, 77.9, 61.3 (Glc
C-1–6), 102.1, 72.6, 72.7, 74.2, 69.5, 18.6 (Rha [1?2]
C-1–6), 102.9, 72.6, 72.9, 73.9, 70.4, 18.5 (Rha [1?4]
C-1–6); HRESIMS: calcd for C45H78O14Na: 865.5289;
found: m/z 865.5112 [M+Na]+.
3.7.2. Procedure B using chacotriosyl diethyl phos-
phite. Glycosylation of 4 (52 mg, 51 lmol) with choles-
tanol (80 mg, 206 lmol) was accomplished as described
in Section 3.6.4, followed by purification by column
chromatography (3:1 hexane–EtOAc) to yield a mixture
of 7a and 7b as a colorless oil. The mixture was sepa-
rated by ODS column chromatography (19:1 MeOH–
H2O) to afford 7a (8 mg, 12%) and 7b (21 mg, 32%),
both as white solids. Physicochemical data were identi-
cal to the products of Section 3.7.1.
3.7.3. Procedure E using chacotriosyl diphenyl phos-
phate. Glycosylation of 4 (19 mg, 17 lmol) with
cholestanol (30 mg, 77 lmol) was accomplished as
described in Section 3.6.5, followed by purification by
column chromatography (3:1 hexane–EtOAc) to yield
a mixture of 7a and 7b as a colorless oil. The mixture
was separated by ODS column chromatography (19:1
MeOH–H2O) to afford 7a (3 mg, 14%) and 7b (7 mg,
32%), both as white solids. Physicochemical data were
identical to the products of Section 3.7.1.
3.10. Cancer cell growth inhibitory bioassay
The in vitro inhibitory effects of compounds 8a–9b on
the proliferation of human lung carcinoma (A549) and
hepatoblastoma (HepG2) cell lines were measured with
the MTT method.20 Human tumor cells were main-
tained as adherent cell cultures in the MEM medium
supplemented with 1 mM sodium pyruvate and 10%
fetal bovine serum (FBS) at 37 °C in a humidified incu-
bator containing 5% CO2. The cells were counted, trans-
ferred into 96-well microtiter plates at a density of
5 ꢂ 103 cells per well in 100 lL of culture medium,
and then incubated with 10% FBS for 24 h prior to
the addition of the test compounds. The test compounds
were dissolved in DMSO (100 lL each) and diluted with
the medium (without FBS) to the desired concentration.
To microtiter wells emptied of their original media, the
test compounds were then added, followed by incu-
bation for 24 h. At the final concentration of 0.5%,
DMSO exhibited no interference with the growth of
the cell lines. A MTT solution (10 lL/well) was added
to cells, which were then incubated for 2 h at 37 °C.
The absorbances of the resulting solutions were mea-
sured at 450 nm on an automated microplate reader
(Labsystems Multiskan MS). Each experiment was
repeated twice in triplicate.
3.8. Cholestanol-3b-yl a-L-rhamnopyranosyl-(1?4)-[a-L-
rhamnopyranosyl-(1?2)]-a-D-glucopyranoside (9a)
Compound 7a (41 mg, 32 lmol) was dissolved in 3%
KOH/MeOH (5.0 mL) and heated at 65 °C. After stir-
ring for 20 h, the mixture was purified by column
chromatography (MCI gel, CHP20P; water, 250 mL;
MeOH, 150 mL) and then concentrated under dimin-
ished pressure to afford 9a (27 mg, 98%) as a white solid:
15
½aꢃD +35.1 (c 0.16, CHCl3); 1H NMR (in CDCl3): d 0.65
(6H, s, H-18, 19), 0.90 (3H, d, J 6.7 Hz, H-26, 27), 0.96
(3H, d, J 6.7 Hz, H-21), 1.67 (3H, d, J 6.1 Hz, Rha H-6),
1.72 (3H, d, J 6.1 Hz, Rha0 H-6), 5.52 (1H, s, Glc H-1),
5.85 (1H, s, Rha H-1), 5.97 (1H, s, Rha0 H-1); 13C NMR
(in C5D5N): d 37.6, 30.2, 78.7, 39.0, 45.3, 29.4, 32.3,
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

H. Miyashita et al. / Carbohydrate Research 343 (2008) 1309–1315
1315
8. Wang, Y.; Zhang, Y.; Zhu, Z.; Zhu, S.; Li, Y.; Li, M.; Yu,
B. Bioorg. Med. Chem. 2007, 15, 2528–2532.
Supplementary data
9. Li, W.; Qiu, Z.; Wang, Y.; Zhang, Y.; Li, M.; Yu, J.;
Zhang, L.; Zhu, Z.; Yu, B. Carbohydr. Res. 2007, 342,
2705–2715.
10. Zhu, S.; Zhang, Y.; Li, M.; Yu, J.; Zhang, L.; Li, Y.; Yu,
B. Bioorg. Med. Chem. Lett. 2006, 15, 5629–5632.
11. Zhang, Y.; Li, Y.; Guo, T.; Guan, H.; Shi, J.; Yu, Q.; Yu,
B. Carbohydr. Res. 2005, 340, 1453–1459.
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2008.02.012.
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