Synthesis of neosaponins having an α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]- D-glucopyranosyl glyco-linkage

Article abstract of DOI:10.1016/S0040-4039(01)00173-3

To verify the role of the α-L-rhamnopyranosyl-(1→4)-[α-L-rhamnopyranosyl-(1→2)]- β-D-glucopyranosyl (chacotriosyl) moiety of steroidal glycosides from Solaum plants in their antitumor and antivirus activities, chacotriosides of diosgenin, cholesterol, and glycrrhetic acid were synthesized by developing our trans-oligoglycosidation. The chacotriosyl trichloroacetimidate was linked to the aglycones to afford neoglycosides as a mixture of α and β anomers, that was easily separated with ODS chromatography.

Full text of DOI:10.1016/S0040-4039(01)00173-3

TETRAHEDRON
LETTERS
Pergamon
Tetrahedron Letters 42 (2001) 2353–2356
Synthesis of neosaponins having an a-
(1“4)-[a- -rhamnopyranosyl-(1“2)]-
glyco-linkage
L
-rhamnopyranosyl-
L
D
-glucopyranosyl
Tsuyoshi Ikeda,a Hiroyuki Miyashita,a Tetsuya Kajimotob and Toshihiro Noharaa,*
aFaculty of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Kumamoto 862-0973, Japan
bDepartment of Biotechnology, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei,
Tokyo 184-8588, Japan
Received 25 December 2000; revised 22 January 2001; accepted 26 January 2001
Abstract—To verify the role of the a-
L
-rhamnopyranosyl-(1“4)-[a-
L
-rhamnopyranosyl-(1“2)]-b-D-glucopyranosyl (chacotriosyl)
moiety of steroidal glycosides from Solaum plants in their antitumor and antivirus activities, chacotriosides of diosgenin,
cholesterol, and glycrrhetic acid were synthesized by developing our trans-oligoglycosidation. The chacotriosyl trichloroacetimi-
date was linked to the aglycones to afford neoglycosides as a mixture of a and b anomers, that was easily separated with ODS
chromatography. © 2001 Elsevier Science Ltd. All rights reserved.
Recent developments in glycobiology have revealed the
important roles of many glycoconjugates in immune
responses, viral and bacterial infections, inflammation
and many other inter- and intracellular signal transduc-
tions. Among the huge number of glycoconjugates, we
have been interested in the steroidal glycosides, which
are often found as the major components in traditional
Chinese medicines, because of their remarkable phar-
macological and biological activities.1 Especially, the
steroidal glycosides isolated from Solanum plants, e.g.
S. dulcamara, S. lyratum, and S. nigrum,2 exhibited
cytotoxicity toward human cancer cells3 and herpes
simplex virus type 1 (HSV-1)4 in good accordance with
the fact that these medicinal plants have been utilized
to treat patients suffering from cancer or herpes infec-
tion. Our detailed studies on the relationship between
the structure and bioactivities of the glycosides from
Solanum plants showed a potent anti-tumor activity in
a-
L
-rhamnopyranosyl-(1“4)-[a-
L-rhamnopyranosyl-
-glucopyranosyl (chacotriosyl) steroids,
(1“2)]-b-
D
while the compounds having other sugar chains were
almost inactive.3 In addition, Kuo reported that the
chacotriosyl moiety of solamar-gine (chacotriosyl sola-
sodine), a major steroidal alkaloid from Solanum
plants, caused triggering cell death by apoptosis.5
Therefore, we embarked on a synthetic study of several
chacotriosyl glycoconjugates to examine their biological
activities.
OPiv
O
OH
OPiv
O
O
HO
HO
a
O
b
HO
PivO
O
O
R2
R1
PivO
Me
AcO
OH
O
OH
O
1
2
AcO
OAc
NH
Me
O
C
4 : R1= H, R2= OAllyl
5 : R1= R2= H or OH
CCl3
AcO
O
c
AcO
OAc
Me
AcO
O
3
d
6 : R1= OC(NH)CCl3, R2= H
AcO
OAc
Scheme 1. Reagents and conditions: (a) pivaloyl chloride, pyridine, 0°C for 3 h, 68%; (b) 3, MS4A, CH2Cl2, N2, −78°C, 1 h, 89%;
(c) Pd[P(Ph)3]4, AcOH, 80°C, 71%; (d) CCl3CN, DBU, CH2Cl2, 0°C, 2 h, 75%.
Keywords: neosaponin; glycoconjugate; trans-oligoglycosidation; Solanum plant; chacotriose.
* Corresponding author. Tel.: +81-96-371-4380; fax: +81-96-362-7799; e-mail: none@gpo.kumamoto-u.ac.jp
0040-4039/01/$ - see front matter © 2001 Elsevier Science Ltd. All rights reserved.
PII: S0040-4039(01)00173-3

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T. Ikeda et al. / Tetrahedron Letters 42 (2001) 2353–2356
We have reported the trans-oligoglycosylation of bioac-
tive glycolinkages from natural glycosides to various
aglycones.6 This method is advantageous in the case
where the oligoglycosyl moiety transferred is naturally
abundant and/or easily prepared on a large scale.
Applying this method to the synthesis of a series of
neo-chacotriosides (one of neosaponins), we prepared
allyl chacotrioside as the original glycoside and trans-
formed to chacotriosyl trichloroacetimidate as the gly-
cosyl donor in the first step. In the next step, the
trichloroacetimidate is activated with a Lewis acid to
form the glycosyl linkage with several aglycones. In
order to investigate the bioactivity of the synthesized
neosaponins in comparison with the library of steroidal
glycosides isolated from Solanum plants in our labora-
tory, we chose diosgenin, cholesterol, and glycyrrhetinic
acid as the aglycone moieties.
However, stereoselectivity of the reaction was not satis-
factory to afford the b anomer, so that several condi-
tions were examined (Table 1, entries 2–8). In the
beginning, the reaction temperature and Lewis acid
were, respectively changed to room temperature (entry
2) and to BF3·Et2O (entry 5); however, the ratio of the
b anomer was not increased. This result suggested that
the glycosylation reaction was controlled thermody-
namically via an Sn1-type reaction; therefore, the glyco-
sylation was attempted under a ‘normal procedure’, i.e.
adding the Lewis acid to a solution of diosgenin and 6
in CH2Cl2 (entries 3, 4, 6–8). A catalytic amount of
TMSOTf was used in entries 3 and 4. Entry 4 reacted at
22°C showed an increase of both b selectivity and the
chemical yield, while entry 3 was not changed selectivity
as well as in the ‘inverse procedure’. When an excess
amount of BF3·Et2O was employed (entries 6 and 7),
the ratio of b anomer was the most increased in all the
cases. On the other hand, when a catalytic amount of
BF3·Et2O was employed (entry 8), the yield of glycosy-
lation was best, but the b selectivity was similar to that
in entry 4. These results suggested that relatively high
reaction temperature is effective to give the high b
selectivity and the chemical yield (entries 3, 4, 6, 7).
Probably, kinetically controlled glycosylation was
slightly increased owing to the higher reaction tempera-
ture. Furthermore, the excess amounts of BF3·Et2O
under the ‘normal procedure’ increased b stereo selec-
tivity. The glycosyl donor 6 might be converted to a
highly reactive carbenium-ion intermediate, and then
the excess amounts of BF3·Et2O could be coordinated
at the thermodynamically stable a face of the interme-
diate, and SN2 reaction took place to increase b
selectivity.
Allyl b-
with pivaloyl chloride in pyridine at 0°C to give an allyl
3,6-di-O-pivaloyl-b-
-glucopyranoside 28 (Scheme 1).
The 2 and 4 positions of the glucose donor 2 were
D
-glucopyranoside 17 was selectively protected
D
glycosylated with 2,3,4-tri-O-acetyl-a-L-rhamnosyl
trichloroacetimidete 39a using boron trifluoride etherate
(BF3·Et2O)9 as the promoter to give fully protected
chacotrioside 4. The trisaccharide 5 was converted to
an a-trichloroacetimidate 610 by the deallylation with
Pd[P(Ph)3]4.11
The trisaccharide donor 6 was transferred to diosgenin
by Schmidt’s ‘inverse procedure’,12 in which the
hydroxyl group of the acceptor was activated with
trimethylsilyl trifluoromethanesulfonate (TMSOTf) due
to the steric hindrance of 3-OH in steroids and triter-
penoids, and the fully protected glycoside 7 was
obtained as a mixture of a and b anomers (1:0.24,
a-anomer; l 4.93, d, J=3.7 Hz, b-anomer; l 4.47, d,
J=7.3 Hz) in 59% yield (Table 1, entry 1).
Therefore, we chose the conditions shown in entry 7 to
synthesize other neosaponins. Cholesterol and gly-
cyrrhetinic acid methyl ester were glycosylated with 6 in
Table 1. Results of glycosylation of 6 and diosgenin
Entries
Lewis acid (equiv.)
Temp (°C)
[a:b]
Yield (%)
1
2
3
4
5
6
7
8
I.P.a
TMSOTf (3.2)
TMSOTf (2.2)
TMSOTf (0.01)
TMSOTf (0.01)
BF3·Et2O (12)
BF3·Et2O (12)
BF3·Et2O (11)
BF3·Et2O (0.05)
0
1:0.24
1:0.28
1:0.26
1:0.35
1:0.25
1:0.43
1:0.44
1:0.36
58.3
63.4
68.4
72.1
53.5
60.2
64.3
81.5
I.P.a
−78“r.t.
−78“r.t.
N.P.b
N.P.b
I.P.a
22
0
0
20
20
N.P.b
N.P.b
N.P.b
a I.P.=inverse procedure.
b N.P.=normal procedure.

T. Ikeda et al. / Tetrahedron Letters 42 (2001) 2353–2356
2355
O
COOH
7α
8α
O
O
Diosgenin
c
7
a
b
HO
HO
HO
7β
9α
8β
c
c
13 : Diosgenin 14 : Cholesterol 15 : Glycyrrhetinic
Acid
10α
OH
O
OH
Cholesterol
O
O
O
6
OR
9
HO
HO
a
b
b
O
O
10β
12α
9β
HO
O
O
HO
c
OR
HO
HO
OH
HO
OH
HO
O
O
11α
a
d
HO
HO
11
OH
OH
Glycyrrhetinic Acid
30-methylester
R = 13
R = 14
R = 15
8α
10α
12α
8β
10β
12β
R = 13
R = 14
R = 15
12β
11β
d
Scheme 2. Reagents and conditions: (a) BF3·Et2O, CH2Cl2, rt; (b) ODS (90% MeOH); (c) 3% KOH/MeOH, reflux, 92–97%; (d)
3% LiOH/MeOH, reflux, 80–85%.
the same manner described above (entry 7) to afford an
a and b anomer mixture of chacotriosyl cholesterol 9
(55.8%, a:b=1:0.73) and chacotriosyl glycyrrehetic acid
11 (52.6%, a:b=1:0.56), respectively. The anomeric
mixtures of a- and b-chacotriosides were separated by
octadodecyl silica gel (ODS) column chromatography
using 90% MeOH to give a and b chacotriosides (7a,
7b, 9a, 9b, 11a, and 11b), respectively. Each glycoside
separated by ODS was deprotected in the usual manner
to give 8a, 8b, 10a, 10b, 12a, and 12b13 (Scheme 2).
2. (a) Encyclopedia of Contemporary Chinese Medical
Plants; Chi, S. Ed. (translation editor, Sugi, M.); Kogyo
Chosakai: Tokyo, 1980; pp. 79–87; (b) Chinese Drug
Dictionary; Koso New Medical College, Ed., Shanghai
Science and Technology Publishing Co.: Shanghai, 1978;
Vol. 1, pp. 630–631.
3. Nakamura, T.; Komori, C.; Lee, Y.-Y.; Hashimoto, F.;
Yahara, S.; Nohara, T.; Ejima, A. Biol. Pharm. Bull.
1996, 19, 564–566.
4. Ikeda, T.; Ando, J.; Miyazono, A.; Zhu, X.-H.; Tsuma-
gari, H.; Nohara, T.; Yokomizo, K.; Uyeda, M. Biol.
Pharm. Bull. 2000, 23, 364–365.
5. (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.
The cytotoxicity of the obtained neosaponins (8a, 8b,
10a, 10b, 12a, and 12b) was tested with HCT116 and
PC-12 cell lines. Compound 8b showed cytotoxicity
{IC50: 2.66 mg/mL (HCT116); 1.99 mg/mL (PC-12)},
while the other neosaponins showed no cytotoxicity (>5
mg/mL).
6. (a) Ikeda, T.; Kajimoto, T.; Kinjo, J.; Nakayama, K.;
Nohara, T. Tetrahedron Lett. 1998, 39, 3513–3516; (b)
Ikeda, T.; Kinjo, J.; Kajimoto, T.; Nohara, T. Heterocy-
cles 2000, 52, 775–798.
In conclusion, facile preparation of an a-
L
-rhamnopy-
-glu-
ranosyl-(1“4)-[a- -rhamnopyranosyl-(1“2)]-a-
L
D
copyranosyl (chacotriosyl) trichloroacetimidate (6) has
been achieved (from the allyl glucoside to 6 by 4 steps
in 32% overall yield) in spite of the fact that the a
glycosides were more predominant than the b glyco-
sides in the oligoglycosylation. Since only the 8b
showed cytotoxicity, both the b stereochemistry of
chacotrioside and the steroidal aglycone moiety are
crucial for the cytotoxicity. To investigate futher struc-
ture-activity relationship, synthesis of more b-chacor-
tiosyl derivatives and bioassays are now in progress.
7. Compound 1 was readily prepared from 1,2,3,4,6-penta-
O-acetyl-D-glucose and allylalchohol in the presence of
BF3·Et2O. (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.
8. (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. 1 1999, 465–473.
9. (a) Schmidt, R. R. Advan. Carbohydr. Chem. Biochem.
1994, 50, 21–123; (b) Schmidt, R. R.; Michel, J. Angew.
Chem., Int. Ed. Engl. 1980, 39, 731–732.
Acknowledgements
10. Compound 6: [h]D25 +16.0° (c 0.43, CHCl3); lH (CDCl3,
500 MHz): 1.15 (3H, d, J=6.7 Hz, rha-6), 1.17 (3H, d,
J=6.1 Hz, rha-6), 1.21, 1.23 (18H, s, Piv), 1.97, 1.98,
2.04×2, 2.12×2 (18H, s, acetyl), 4.81 (1H, br s, rha-1),
4.91 (1H, d, J=1.8 Hz, rha-1), 5.65 (1H, dd, J=9.2, 9.8
Hz, glc-3), 6.42 (1H, d, J=3.7 Hz, glc-1a), 8.77 (1H, s,
-NH); FABMS (positive) m/z 1058 (M+Na)+.
This work was supported in part by a Grant-in-Aid for
Scientific Research (No. 11771387 to T.I.) from the
Ministry of Education, Science and Culture, Japan.
11. Nakayama, K.; Uoto, K.; Higashi, K.; Soga, T.;
Kusama, T. Chem. Pharm. Bull. 1992, 40, 1718–1720.
12. Schmidt, R. R.; Toepfer, A. Tetrahedron. Lett. 1991, 32,
3353–3356.
References
1. Studies in Plant Science, 6; Yang, C.-R.; Tanaka, O.,
Eds.; Advances in plant glycosides, chemistry and biol-
ogy. Elsevier: Amsterdam, 1999.
13. Selected spectroscopic data: 8a: [h]3D0−26.1° (c 0.11,
MeOH); lH (C5D5N): 0.70 (3H, d, J=5.5 Hz, H-27),

2356
T. Ikeda et al. / Tetrahedron Letters 42 (2001) 2353–2356
0.84, 0.88 (each 3H, s, H-18, 19), 1.15 (3H, d, J=6.7 Hz,
0.10, MeOH); lH (C5D5N) 0.66 (3H, s, H-18), 0.90, 0.91
(each 3H, d, J=6.7 Hz, H-26, 27), 0.97 (3H, d, J=6.7
Hz, H-21), 1.08 (3H, s, H-19), 1.64, 1.78 (each 3H, d,
J=6.1 Hz, rha-6×2), 4.96 (1H, d, J=7.9 Hz, glc-1), 5.37
(1H, br s, H-6), 5.86 (1H, s, rha-1), 6.40 (1H, s, rha-1);
FABMS (positive) m/z 863 (M+Na)+. 12a: [h]D30 +56.5° (c
0.13, MeOH); lH (C5D5N): 0.98, 1.08, 1.13, 1.24, 1.30,
1.33, 1.38 (each 3H, s, H-23–H-29), 1.63 (6H, d, J=6.1
Hz, rha-6×2), 5.49 (1H, br s, glc-1), 5.85 (1H, s, rha-1),
6.01 (1H, s, rha-1), 6.44 (1H, br s, H-12); FABMS
(positive) m/z 947 (M+Na)+. 12b: [h]3D0 +27.8 ° (c 0.10,
MeOH); lH (C5D5N): 1.09, 1.23, 1.25, 1.28, 1.30, 1.34,
1.43 (each 3H, s, H-23ꢀ29), 1.59, 1.70 (each 3H, d,
J=6.1 Hz, rha-6×2), 4.91 (1H, d, J=7.9 Hz, glc-1), 5.86
(1H, s, rha-1), 6.00 (1H, s, rha-1), 6.61 (1H, br s, H-12);
FABMS (positive) m/z 1058 (M+Na)+.
H-21), 1.68, 1.70 (each 3H, d, J=6.1 Hz, rha-6×2), 5.33
(1H, br s, H-6), 5.52 (1H, d, J=3.7 Hz, glc-1), 5.86 (1H,
s, rha-1), 5.95 (1H, s, rha-1); FABMS (positive) m/z 891
(M+Na)+. 8b: [h]D30−102.0° (c 0.43, MeOH); lH (C5D5N):
0.71 (3H, d, J=5.5 Hz, H-27), 0.84, 1.06 (each 3H, s,
H-18, 19), 1.14 (3H, d, J=7.3 Hz, H-21), 1.63, 1.77 (each
3H, d, J=6.1 Hz, rha-6×2), 4.95 (1H, d, J=7.9 Hz,
glc-1), 5.33 (1H, br s, H-6), 5.85 (1H, s, rha-1), 6.40 (1H,
s, rha-1); FABMS (positive) m/z 891 (M+Na)+. 10a: [h]D30
+5.5° (c 0.11, MeOH); lH (C5D5N): 0.66 (3H, s, H-18),
0.91 (6H, d, J=6.1 Hz, H-26, 27), 0.91 (3H, s, H-19),
0.98 (3H, d, J=6.1 Hz, H-21), 1.69, 1.71 (each 3H, d,
J=6.1 Hz, rha-6×2), 5.36 (1H, br s, H-6), 5.53 (1H, d,
J=3.7 Hz, glc-1), 5.87 (1H, s, rha-1), 5.97 (1H, s, rha-1);
FABMS (positive) m/z 863 (M+Na)+. 10b: [h]D30 −42.2° (c
.
.