Synthesis of a cholestane glycoside OSW-1 with potent cytostatic activity.

Article abstract of DOI:10.1016/S0008-6215(02)00126-X

The potent antitumor agent OSW-1 was synthesized from the protected aglycone in different ways. It was proven that direct glycosylation of the aglycone in its hemiketal form could be achieved, affording the protected OSW-1 in a moderate yield. Alternatively, regioselective protection of the triol obtained by reduction of the aglycone, followed by glycosylation, deprotection and oxidation yielded the same OSW-1 derivative. The third approach to this compound consisted of glycosylation of the previously described lactol [Morzycki, J. W.; Gryszkiewicz, A. Polish J. Chem. 2001, 75, 983-989], reaction of the resulting aldehyde with a Grignard reagent, and oxidation. OSW-1 obtained on removal of the protective groups was identical with the natural product.

Full text of DOI:10.1016/S0008-6215(02)00126-X

Carbohydrate Research 337 (2002) 1269–1274
www.elsevier.com/locate/carres
Synthesis of a cholestane glycoside OSW-1 with potent cytostatic
activity
Jacek W. Morzycki,* Agnieszka Wojtkielewicz
Institute of Chemistry, Uni6ersity of Bialystok, al. Pilsudskiego 11/4, 15-443 Bialystok, Poland
Received 11 January 2002; received in revised form 7 May 2002; accepted 22 May 2002
Abstract
The potent antitumor agent OSW-1 was synthesized from the protected aglycone in different ways. It was proven that direct
glycosylation of the aglycone in its hemiketal form could be achieved, affording the protected OSW-1 in a moderate yield.
Alternatively, regioselective protection of the triol obtained by reduction of the aglycone, followed by glycosylation, deprotection
and oxidation yielded the same OSW-1 derivative. The third approach to this compound consisted of glycosylation of the
previously described lactol [Morzycki, J. W.; Gryszkiewicz, A. Polish J. Chem. 2001, 75, 983–989], reaction of the resulting
aldehyde with a Grignard reagent, and oxidation. OSW-1 obtained on removal of the protective groups was identical with the
natural product. © 2002 Elsevier Science Ltd. All rights reserved.
Keywords: OSW-1; Glycosylation; Saponins; Steroids; Antitumor activity
1. Introduction
first performed the coupling of the protected OSW-1
aglycone with a sugar imidate.5 In a new synthetic route
to the saponin OSW-1, a different approach to the
synthesis of the steroid and the sugar parts of the
molecule was recently reported, but the same coupling
procedure was employed.6
The steroidal saponin OSW-1 (5) belongs to a family
of similar compounds isolated from the bulbs of Or-
nithogalum saundersiae, a perennial grown in southern
Africa.1 The saponins feature common cholestane agly-
cone (3b,16b,17a-trihydroxycholest-5-en-22-one) and
vary at the 2¦ O-acyl group of the sugar moiety. In
vitro assays showed that the saponins were extremely
toxic against a broad spectrum of malignant tumor
cells, including drug-resistant leukemia and various
lung cancer cell lines. The anticancer activities (IC50
values) of OSW-1 are from 10 to 100 times more potent
than those of the clinically used anticancer agents (e.g.,
mitomycin C, adriamycin, cisplatin, camptothecin, and
taxol).2
The steroid aglycone and the sugar moiety are both
important for biological activity of OSW-1. Removal of
the acetyl and the 4-methoxybenzoyl groups from the
sugar part diminished the cytotoxicity by 1000 times.2
The steroidal glycosides bearing the disaccharide moi-
ety of OSW-1 at different positions were proved at least
10,000 times less active than OSW-1.7 The mechanism
of action of OSW-1 is still unknown but its cytotoxicity
profile is strikingly similar2,8 to that of cephalostatins, a
group of the dimeric steroid–pyrazine marine alka-
loids.9 It was hypothesized that these two groups of
compounds might form a similar active intermediate,
presumably a C-22 oxocarbenium ion.4,9b Therefore,
the presence of both a 16b,17a-diol and a C-22 car-
bonyl group is essential for the biological activity of
OSW-1. The recently performed structure–activity rela-
tionship studies confirmed this conclusion.10 However,
synthesis of further analogues is necessary for determi-
nation of the minimal pharmacophor requirements.
We have recently reported a synthesis of the saponin
OSW-1 aglycone.3 The first synthesis of the aglycone
was described by Fuchs et al.4 The Chinese chemists
* Corresponding author. Tel.: +48-85-7457604; fax: +48-
85-7457581
E-mail address: morzycki@uwb.edu.pl (J.W. Morzycki).
0008-6215/02/$ - see front matter © 2002 Elsevier Science Ltd. All rights reserved.
PII: S0008-6215(02)00126-X

1270
J.W. Morzycki, A. Wojtkielewicz / Carbohydrate Research 337 (2002) 1269–1274
2. Results and discussion
TES groups on the sugar moiety) were simultaneously
removed with a soft Lewis acid – Pd(MeCN)2Cl2 at
room temperature. The yield of OSW-1 (5) was satisfac-
tory, but the high cost of the reagent and the formation
of a minor 3b-chloro product 6, due to the presence of
chlorides in the reaction mixture, encouraged the inves-
tigation of deprotection under different conditions. A
high yield of OSW-1 (5) was also achieved using a
catalytic amount of p-TsOH in dioxane/water at 75 °C
(no glycoside hydrolysis was observed).
In this paper the results of glycosylation of the
protected OSW-1 aglycone (1) and related compounds
are described. The disaccharide of OSW-1, namely 2-O-
(4-methoxybenzoyl)-b-
D
-xylopyranosyl-(1“3)-2-O-ace-
tyl-a- -arabinopyranose, was obtained according to the
L
known procedure.5 The corresponding disaccharide
trichloroacetimidate was also prepared by the method
described in the literature5 and used as a glycosyl
donor. In the previous paper, an improved synthesis of
the OSW-1 aglycone (1) in its hemiketal form, with the
ring B double bond and the 3b-OH group protected as
an i-steroid ether, was reported.3 Compound 1 proved
to be stereochemically pure, but its configuration at
C-22 could not be elucidated directly from the spectra.
Presumably, the configuration is 22R since this com-
pound was calculated, using the MM+ force field, to be
over 4 kcal/mol more stable than its 22S epimer.
LiAlH4 reduction of 1 afforded the triol 2a as a C-22
diastereoisomeric mixture with one epimer prevailing
(Scheme 1). The triol 2a was regioselectively protected
at O-22 as the benzyl ether 2b. Only one epimer of 2b
could be easily isolated in its pure state from the
reaction mixture. This predominant epimer (its configu-
ration was not established, but it was not important for
further synthesis) was taken for the coupling reaction
with a disaccharide donor. The glycosylation proceeded
smoothly under the promotion of TMSOTf, yielding
3b. The hydrogenolysis of the benzyl ether was followed
by oxidation of the 22-OH group to the ketone 4 with
pyridinium dichromate. Finally, all protective groups
(3a,5a-cyclo-6b-methoxy protection of aglycone and
The direct glycosylation of the protected aglycone (1)
with the disaccharide trichloroimidate was also studied.
The conversion was 20% and two products were formed
in the ratio of about 1:1. In addition to the desired
protected OSW-1 (4), the glycoside 8 of the hemiketal
was obtained (Scheme 2). Similar glycosylation of the
hemiacetal 9 was also performed. Compounds 1 and 9
were obtained from the readily available hydroxy-lac-
tone 7 by reaction with isoamyl lithium or diisobutyl
aluminum hydride, respectively. In the case of the
glycosylation of 9, the conversion was slightly above
50% and three products were formed. The major one,
aldehyde 11, was accompanied by the hemiacetal gly-
coside 10 and the orthoester 12. The latter compound
was formed by the well known mechanism involving
participation of acetate at the 2% position.11 The alde-
hyde 11 seems to be a versatile starting material for
synthesis of OSW-1 analogues with different side
chains. The reaction 11 with isoamyl magnesium bro-
mide was carried out at low temperature (−70 °C).
The ester groups remained intact under these condi-
tions, but an undesired retroaldol reaction accompanied
the Grignard reaction. The alkylation product was
Scheme 1.

J.W. Morzycki, A. Wojtkielewicz / Carbohydrate Research 337 (2002) 1269–1274
1271
Scheme 2.
formed as a single stereoisomer, which occurred to be
identical with compound 3a obtained by the method
described above. The 16-O-glycoside devoid side chain,
17-ketone 13, was the retroaldol reaction product. The
retroaldol reaction was promoted by a strongly basic
Grignard reagent.
It can be concluded from this study that the previ-
ously described hydroxy-lactone 7 is a valuable inter-
mediate in the synthesis of the highly potent cytostatic
saponin OSW-1. Synthesis of the side chain analogues
of OSW-1 from the hydroxy-lactone 7 and the aldehyde
11 using the reactions with less basic organometallic
reagents, and evaluation of the antitumor activities of
the analogues will be published in due course.
matograms were developed on aluminum TLC sheets
precoated with silica gel 60 F254 (E. Merck) and visual-
ized with 50% H2SO4 after heating. All solvents were
dried and freshly distilled prior to use.
Selecti6e benzylation of triol 2a.—To the stirred solu-
tion of 2a (100 mg, 0.22 mmol) in THF (6 mL), NaH
(14 mg of 60% dispersion in mineral oil, 0.33 mmol)
was added at 0 °C. The reaction mixture was stirred 15
min at 0 °C, then BnBr (0.04 mL, 0.33 mmol) and
Bu+4 I− (10 mg, 0.04 mmol) were added. The reaction
mixture was stirred 1.5 h at reflux. The reaction was
carefully quenched with water and extracted with ether.
The extract was dried over MgSO4 and solvent was
evaporated in vacuo.
22 - Benzyloxy - 6i - methoxy - 3h,5h - cyclocholestane-
16i,17h-diol (2b). Pure 2b (48 mg, 40%) was eluted
from a silica gel column with EtOAc (12.5%)–hexane,
mp 130–132 °C (rectangular plates); Rf 0.35 (7:3 hex-
ane–EtOAc); [h]2D1 +11.1° (c 0.5, CHCl3). IR (CHCl3):
3. Experimental
General methods.—Melting points were determined
on a Kofler apparatus of the Boe¨tius type. Optical
rotations were measured on a Perkin–Elmer 141 polar-
imeter in CHCl3 or in MeOH. NMR spectra were
recorded with the Bruker AC 200F (200 MHz) and
Avance 500 (500 MHz) spectrometers using CDCl3 or
C5D5N solutions with TMS as the internal standard
(only selected signals in the NMR spectra are reported).
Infrared spectra were recorded on a Nicolet Series II
Magna-IR 550 FT-IR spectrometer as chloroform solu-
tions unless otherwise stated. Mass spectra were ob-
tained at 70 eV with an AMD-604 spectrometer.
Compounds 1, 2a and 9 were obtained according to the
earlier described procedures.3a,3c The reaction products
were isolated by column chromatography performed on
70–230 mesh silica gel (J. T. Baker). Thin-layer chro-
1
w 3621, 3406, 1468, 1455, 1384, 1090 cm−1. H NMR
(200 MHz, CDCl3): l 7.33 (m, 5 H), 4.69 (s, 1 H), 4.68
(d, 1 H, J 11.2 Hz), 4.44 (d, 1 H, J 11.2 Hz), 4.11 (dd,
1 H, J 8.8, 4.2 Hz), 3.96 (m, 1 H), 3.34 (s, 3 H), 2.78
(m, 1 H), 1.05 (d, 3 H, J 7.5 Hz), 1.03 (s, 3 H), 0.94 (s,
3 H), 0.93 (d, 6 H, J 6.4 Hz), 0.65 (m, 1 H), 0.43 (dd,
1 H, J 8.0, 5.1 Hz). 13C NMR (50 MHz, CDCl3): l
137.9 (C), 128.5 (CH×3), 127.8 (CH), 127.7 (CH), 87.2
(C), 82.5 (CH), 82.4 (CH), 81.3 (CH), 70.3 (CH2), 56.6
(CH3), 47.8 (C), 47.5 (CH), 47.1 (CH), 43.3 (C), 37.1
(CH2), 35.11 (C), 35.06 (CH2), 35.00 (CH2×2), 34.97
(CH), 33.4 (CH2), 30.5 (CH), 28.4 (CH), 28.3 (CH2),
24.9 (CH2), 22.8 (CH3), 22.4 (CH3), 22.3 (CH2), 21.4
(CH), 19.2 (CH3), 13.7 (CH3), 13.1 (CH2), 7.5 (CH3).

1272
J.W. Morzycki, A. Wojtkielewicz / Carbohydrate Research 337 (2002) 1269–1274
Glycosylation of compound 2b.—A solution of 2-O-
acetyl-3-O-[3,4-di-O-triethylsilyl-2-O-(4-methoxybenzo-
-arabino-
pyranosyl trichloroacetimidate (106 mg, 0.11 mmol)
and compound 2b (54 mg, 0.1 mmol) in dry CH2Cl2 (15
113.3 (CH), 100.8 (CH), 88.7 (C). ESIMS (m/z): 1254.8
(M+Na+).
yl)-b-
D
-xylopyranosyl]-4-O-(triethylsilyl)-b-
L
Oxidation of alcohol 3a.—To a solution of 3a (10
mg, 0.008 mmol) in CH2Cl2 (3 mL) pyridinium dichro-
mate (6 mg, 0.016 mmol) was added, and the reaction
mixture was stirred for 1 h. The solvent was evaporated
in vacuo at rt. The crude product was purified by silica
gel column chromatography to afford 4 in quantitative
yield. The protected OSW-1 (4) proved identical in all
respects with the compound 4 obtained by direct glyco-
sylation of the aglycone 1 as described below.
,
mL) was stirred with 4 A molecular sieves (225 mg) at
rt for 15 min, then the reaction mixture was cooled to
−68 °C (EtOH–dry ice bath) and a 0.14 M solution of
TMSOTf (0.24 mL) in CH2Cl2 was slowly added. The
reaction mixture was stirred for further 30 min,
quenched with Et3N and filtered. The filtrate was evap-
orated in vacuo and the crude product 3b was purified
by silica gel column chromatography with EtOAc
(2.5%)–hexane.
Glycosylation of hemiketal 1.—A solution of 2-O-
acetyl-3-O-[3,4-di-O-triethylsilyl-2-O-(4-methoxybenzo-
yl)-b-
D
-xylopyranosyl]-4-O-(triethylsilyl)-b-L-arabino-
22 - Benzyloxy - 6i - methoxy - 3h,5h - cyclocholestane-
pyranosyl trichloroacetimidate (600 mg, 0.64 mmol)
16i,17h-diol
methoxybenzoyl)-i-
yl-4-O-triethylsilyl-h-
16-O-{O-[3,4-di-O-triethylsilyl-2-O-(4-
-xylopyranosyl]-(1“3)-2-O-acet-
-arabinopyranoside} (3b). Com-
and aglycone 1 (250 mg, 0.5 mmol) in dry CH2Cl2 (15
,
D
mL) was stirred with 4 A molecular sieves (1.5 g) at rt
L
for 15 min, then the reaction mixture was cooled to
−68 °C (EtOH–dry ice bath) and a 0.14 M solution of
TMSOTf (1.3 mL) in CH2Cl2 was slowly added. The
reaction mixture was stirred for further 30 min,
quenched with Et3N and filtered. The filtrate was evap-
orated in vacuo and the products were separated by
silica gel column chromatography. Elution with EtOAc
(2.5%)–hexane afforded compound 4 (68 mg, 10%),
further elution with EtOAc (4%)–hexane yielded 8 (64
mg, 9%). 1:2 EtOAc–hexane eluted the unreacted agly-
cone 1 (200 mg, 80%).
pound 3b was obtained in 59% (78 mg) as an amor-
phous white solid; Rf 0.39 (19:1 benzene–EtOAc). IR
(CHCl3): w 3399, 1721, 1607, 1458, 1255, 1103, 1019
1
cm−1. H NMR (200 MHz, CDCl3): l 8.04 (d, 2 H, J
8.9 Hz), 7.29 (m, 5 H), 6.89 (d, 2 H, J 8.9 Hz), 5.11 (s,
1 H), 4.90 (m, 3 H), 4.66 (d, 1 H, J 11.5 Hz), 4.47 (m,
1 H), 4.36 (d, 1 H, J 11.5 Hz), 4.31 (bs, 1 H), 3.90–4.10
(m, 3 H), 3.87 (s, 3 H), 3.70–3.85 (m, 2 H), 3.53 (m, 2
H), 3.34 (m, 2 H), 3.30 (s, 3 H), 2.75 (m, 1 H), 2.05 (s,
3 H). 13C NMR (50 MHz, CDCl3): l 169.0 (C), 164.5
(C), 164.4 (C), 163.3 (C), 137.8 (CH), 132.1 (CH), 129.0
(C), 128.4 (CH), 128.2 (CH), 128.1 (CH), 127.7 (CH),
122.7 (C), 113.0 (CH). ESIMS (m/z): 1344.8 (M+Na+).
Remo6al of the benzyl protecti6e group in 3b.—A
suspension of 3b (20 mg, 0.015 mmol), 10% Pd/C (30
mg) and Et3N (0.007 mL) in EtOAc (1 mL) and EtOH
(1 mL) was stirred under H2 atmosphere (50 atm) at
50 °C overnight. The catalyst was then filtered off and
the solvent was evaporated in vacuo from the filtrate
affording crude product, which was purified by silica
gel column chromatography. Elution with EtOAc
(25%)–hexane yielded 3a.
Protected OSW-1 4. Compound 4 was obtained as an
oil; Rf 0.33 (19:1 benzene–EtOAc). IR (CHCl3): w 3476,
1738, 1716, 1690, 1607, 1461, 1256, 1101, 1018 cm−1
.
1H NMR (500 MHz, CDCl3): l 8.05 (d, 2 H, J 8.9 Hz),
6.91 (d, 2 H, J 8.9 Hz), 4.93 (dd, 1 H, J 4.5, 3.6 Hz),
4.82 (brs, 1 H), 4.75 (dd, 1 H, J 5.0, 2.8 Hz), 4.35 (s, 1
H), 4.29 (m, 1 H), 4.23 (d, 1 H, J 2.7 Hz), 3.98–3.89
(m, 2 H), 3.86 (s, 3 H), 3.82 (brs, 1 H), 3.77 (t, 1 H, J
5.1 Hz), 3.70 (dd, 1 H, J 7.7, 5.4 Hz), 3.61 (dd, 1 H, J
8.3, 5.1 Hz), 3.31 (s, 3 H), 3.25–3.29 (m, 1 H), 3.15 (d,
1 H, J 7.4 Hz), 2.75 (m, 1 H), 2.49 (m, 1 H), 2.26 (m,
2 H), 1.95 (s, 3 H), 1.15 (d, 3 H, J 7.4 Hz, H-21), 1.02
(s, 3 H), 0.9–1.0 (m, 34 H), 0.73 (m, 6 H), 0.55–0.65
(m, 18 H), 0.41 (dd, 1 H J 8.0, 5.1 Hz). 13C NMR (125
MHz, CDCl3): l 219.6 (C), 168.7 (C), 164.7 (C), 163.4
(C), 132.0 (CH), 128.3 (CH), 122 (C), 113.4 (CH).
ESIMS (m/z): 1252.8 (M+Na+).
6i-Methoxy-3h,5h-cyclocholestane-16i,17h,22-triol
16-O-{O-[3,4-di-O-triethylsilyl-2-O-(4-methoxybenzoyl)-
i-
h-
D
-xylopyranosyl]-(1“3)-2-O-acetyl-4-O-triethylsilyl-
L
-arabinopyranoside} (3a). Compound 3a (16 mg,
86%) was obtained as an oil; Rf 0.31 (7:3 hexane–
EtOAc); [h]2D1 −21.4° (c 0.3, CHCl3). IR (CHCl3): w
(20S)-6i-Methoxy-3h,5h-cyclofurostane-17h,22-diol
22-O-{O-[3,4-di-O-triethylsilyl-2-O-(4-methoxybenzoyl)
3592, 3435, 1720, 1607, 1463, 1255, 1101, 1018 cm−1
.
1H NMR (500 MHz, CDCl3): l 8.01 (d, 2 H, J 8.9 Hz),
6.88 (d, 2 H, J 8.9 Hz), 4.88 (m, 2 H), 4.84 (m, 1 H),
4.46 (bs, 1 H), 4.29 (m, 1 H), 3.92–4.07 (m, 3 H), 3.86
(s, 3 H), 3.78 (m, 2 H), 3.57 (m, 1 H), 3.30 (s, 3 H and
m, 1 H), 2.75 (m, 1 H), 2.30 (m, 1 H), 1.96 (s, 3 H), 1.02
(s, 3 H), 0.80–0.99 (m, 36 H), 0.77 (d, 3 H, J 6.6 Hz),
0.73 (d, 3 H, J 6.5 Hz), 0.50–0.65 (m, 20 H), 0.40 (dd,
1 H, J 7.8, 5.1 Hz). 13C NMR (50 MHz, CDCl3): l
168.8 (C), 164.5 (C), 163.3 (C), 131.9 (CH), 122.7 (C),
-i-
yl-h-
D
-xylopyranosyl]-(1“3)-2-O-acetyl-4-O-triethylsil-
L
-arabinopyranoside} (8). Compound 8 was ob-
tained as an oil; Rf 0.12 (19:1 benzene–EtOAc); [h]D21
+9.8° (c 0.5, CHCl3). IR (CHCl3): w 3510, 1727, 1607,
1460, 1256, 1098, 1011 cm−1 1H NMR (500 MHz,
.
CDCl3): l 7.96 (d, 2 H, J 8.9 Hz), 6.89 (d, 2 H, J 8.9
Hz), 5.35 (d, 1 H, J 3.2 Hz), 5.01 (dd, 1 H, J 9.3, 3.3
Hz), 4.96 (t, 1 H, J 7.3 Hz), 4.69 (d, 1 H, J 6.6 Hz), 4.23
(t, 1 H, J 7.6), 4.08 (bs, 1 H), 4.01 (dd, 1 H, J 11.5, 4.4

J.W. Morzycki, A. Wojtkielewicz / Carbohydrate Research 337 (2002) 1269–1274
1273
(20S)-6i-Methoxy-16i,17h-dihydroxy-3h,5h-cyclo-
bisnorcholanaldehyde 22,16-hemiacetal 22-O-{O-[3,4-di-
Hz), 3.86 (s, 3 H), 3.75 (t, 1 H, J 7.7), 3.69 (m, 1 H),
3.55 (dd, 1 H, J 11.8, 3.6 Hz), 3.31 (s, 3 H), 3.24 (dd, 1
H, J 11.6, 8.6 Hz), 2.76 (m, 1 H), 2.36 (m, 1 H), 1.75 (s,
3 H), 0.81–1.05 (m, 43 H), 0.50–0.65 (m, 19 H), 0.43
(dd, 1 H, J 7.9, 5.1 Hz). 13C NMR (50 MHz, CDCl3):
l 169.8 (C), 164.5 (C), 163.2 (C), 131.7 (CH), 128.3
(CH), 122.8 (C), 115.1 (C), 113.4 (CH). ESIMS (m/z):
1252.8 (M+Na+).
O-triethylsilyl-2-O-(4-methoxybenzoyl)-i-
D-xylopyra-
nosyl]-(1“3)-4-O-triethylsilyl-h- -arabinopyranosyl 1,2-
L
orthoacetate} (12). Compound 12 was obtained as an
amorphous solid; Rf 0.25 (23:2 benzene–EtOAc); [h]D21
−4.0° (c 0.5, CHCl3). IR (CHCl3): w 3566, 1723, 1606,
1458, 1255, 1099, 1010 cm−1 1H NMR (500 MHz,
.
CDCl3): l 8.01 (d, 2 H, J 8.9 Hz), 6.92 (d, 2 H, J 8.9
Hz), 5.34 (d, 1 H, J 3.9), 5.29 (d, 1 H, J 4.8), 5.01 (t, 1
H, J 7.7 Hz), 4.77 (d, 1 H, J 7.2 Hz), 4.15 (m, 2 H),
4.02 (m, 1 H), 3.97 (dd, 1 H, J 11.6, 4.4 Hz), 3.87 (s, 3
H), 3.82–3.86 (m, 2 H), 3.70–3.75 (m, 2 H), 3.63 (dd,
1 H, J 11.5, 3.4 Hz), 3.33 (s, 3 H), 3.23 (dd, 1 H, J 11.6,
8.9 Hz), 2.77 (m, 1 H), 2.32 (dd, 1 H, J 7.0, 5.0 Hz),
1.67 (s, 3 H), 1.05 (s, 3 H), 0.80–1.00 (m, 40 H),
0.50–0.67 (m, 20 H), 0.45 (dd, 1 H, J 8.0, 5.1 Hz). 13C
NMR (50 MHz, CDCl3): l 164.7 (C), 163.2 (C), 131.6
(CH), 128.3 (CH), 122.9 (C), 121.6 (C), 113.5 (CH),
103.9 (CH), 100.6 (CH), 97.4 (CH). ESIMS (m/z):
1182.6 (M+Na+).
Glycosylation of hemiacetal 9.—The coupling reac-
tion of 9 with the disaccharide donor (Sugar*ꢀ
OꢀC(ꢁNH)CCl3) was performed according to the
procedure described above. The products obtained (10,
11, and 12) were separated and purified by silica gel
column chromatography. Pure aldehyde 11 (yield 30%)
was eluted with EtOAc (5%)–hexane, further elution
with EtOAc (7.5%)–hexane yielded consecutively com-
pounds 10 (39%) and 12 (9%).
(20S)-6i-Methoxy-16i,17h-dihydroxy-3h,5h-cyclo-
bisnorcholanaldehyde 16-O-{O-[3,4-di-O-triethylsilyl-2-
O-(4-methoxybenzoyl)-i-
D
-xylopyranosyl]-(1“3)-2-O-
acetyl-4-O-triethylsilyl-h-
L
-arabinopyranoside} (11).
Compound 11 was obtained as a white foam; Rf 0.34
(23:2 benzene–EtOAc); [h]2D1 −13.3° (c 0.5, CHCl3). IR
(CHCl3): w 3517, 2735, 1725, 1607, 1255, 1097, 1033
Alkylation of the aldehyde 11.—A solution of
isoamyl magnesium bromide in anhyd ether was pre-
pared from magnesium (6 mg, 0.26 mmol) and isoamyl
bromide (0.032 mL, 0.26 mmol). The Grignard reagent
was added dropwise to a stirred solution of compound
11 (150 mg, 0.13 mmol) in anhyd ether (10 mL) under
argon at −70 °C. The reaction mixture was stirred 4 h
at gradually increasing temperature to 0 °C. The reac-
tion mixture was quenched with satd aq NH4Cl and the
product was extracted with ether. The extract was dried
over MgSO4 and solvent was evaporated in vacuo. The
products were separated by silica gel column chro-
matography. Elution with EtOAc (15%)–hexane
yielded ketone 13 (71 mg, 50%). Further elution with
EtOAc (25%)–hexane afforded 3a (31 mg, 20%) identi-
cal (Rf, optical rotation, NMR spectra) with the com-
pound 3a described above.
1
cm−1. H NMR (500 MHz, CDCl3): l 9.54 (d, 1 H, J
0.8 Hz), 8.00 (d, 2 H, J 8.9 Hz), 6.92 (d, 2 H, J 8.9 Hz),
4.92 (dd, 1 H, J 6.7, 5.3 Hz), 4.86 (m, 1 H), 4.67 (d, 1
H, J 5.2 Hz), 4.14 (d, 1 H, J 4.5 Hz), 4.11 (m, 1 H),
3.98 (m, 1 H), 3.87 (s, 3 H), 3.65–3.86 (m, 4 H), 3.31 (s,
3 H), 3.22 (dd, 1 H, J 11.5, 7.4 Hz), 3.12 (m, 1 H), 3.07
(s, 1 H), 2.74 (m, 1 H), 1.90 (s, 3 H), 0.85–1.11 (m, 36
H), 0.50–0.65 (m, 20 H), 0.41 (dd, 1 H, J 7.9, 5.1 Hz).
13C NMR (50 MHz, CDCl3): l 207.6 (CH), 168.9 (C),
164.6 (C), 163.3 (C), 131.8 (CH), 128.3 (CH), 122.7 (C),
113.4 (CH). ESIMS (m/z): 1182.7 (M+Na+).
(20S)-6i-Methoxy-16i,17h-dihydroxy-3h,5h-cyclo-
bisnorcholanaldehyde 22,16-hemiacetal 22-O-{O-[3,4-di-
O-triethylsilyl-2-O-(4-methoxybenzoyl)-i-
nosyl]-(1“3)-2-O-acetyl-4-O-triethylsilyl-h-
D
-xylopyra-
-arabino-
L
6i-Methoxy-3h,5h-cycloandrostan-16i-ol-17-one 16-
pyranoside} (10). Compound 10: mp 126–130 °C
(prisms from hexane–CH2Cl2); Rf 0.22 (23:2 benzene–
EtOAc); [h]2D1 −12.0° (c 0.5, CHCl3). IR (CHCl3): w
O-{O-[3,4-di-O-triethylsilyl-2-O-(4-methoxybenzoyl)-
i-D
-xylopyranosyl]-(1“3)-2-O-acetyl-4-O-triethylsilyl-
h-
L
-arabinopyranoside} (13). Compound 12 was ob-
3518, 1731, 1606, 1458, 1255, 1097, 1004 cm−1
.
1H
tained as an oil; Rf 0.25 (4:1 hexane–EtOAc); [h]D21
NMR (500 MHz, CDCl3): l 7.98 (d, 2 H, J 8.9 Hz),
6.90 (d, 2 H, J 8.9 Hz), 5.34 (d, 1 H, J 4.8 Hz), 4.99 (t,
1 H, J 7.5 Hz), 4.90 (m, 1 H), 4.63 (d, 1 H, J 7.0 Hz),
4.55 (d, 1 H, J 5.7 Hz), 4.00 (m, 1 H), 3.97 (m, 1 H),
3.87 (s, 3 H), 3.83 (m, 1 H), 3.65–3.75 (m, 3 H), 3.39
(dd, 1 H, J 11.8, 1.8 Hz), 3.33 (s, 3 H), 3.19 (dd, 1 H,
J 11.5, 8.9 Hz), 2.76 (m, 1 H), 2.41 (m, 1 H), 1.86 (s, 3
H), 1.04 (s, 3 H), 0.92–1.00 (m, 20 H), 0.82–0.87 (m, 14
H), 0.45–0.67 (m, 20 H). 13C NMR (50 MHz, CDCl3):
l 169.6 (C), 164.8 (C), 163.6 (C), 163.3 (C), 131.8 (CH),
128.2 (CH), 122.4 (C), 113.4 (CH), 105.2 (CH). ESIMS
(m/z): 1182.7 (M+Na+).
−0.1° (c 1.5, CHCl3). IR (CHCl3): w 1739, 1726, 1607,
1
1255, 1096 cm−1. H NMR (200 MHz, CDCl3): l 7.95
(d, 2 H, J 8.8 Hz), 6.89 (d, 2 H, J 8.8 Hz), 4.98 (m, 2
H), 4.78 (d, 1 H, J 6.5 Hz), 4.68 (d, 1 H, J 6.9 Hz), 3.95
(m, 2 H), 3.85 (s, 3 H), 3.68–3.84 (m, 4 H), 3.32 (s, 3
H), 2.78 (m, 1 H). 13C NMR (50 MHz, CDCl3): l 217.8
(C), 168.8 (C), 164.5 (C), 163.2 (C), 131.8 (CH), 128.3
(CH), 122.9 (C), 113.3 (CH), 101.7 (CH), 100.4 (CH),
81.9 (CH). ESIMS (m/z): 1123.7 (M+Na+).
Deprotection of the functional groups.—Compound 4
(29 mg, 0.024 mmol) was dissolved in the solution of
PdCl2 (1.6 mg) in MeCN (2 mL), acetone (1.6 mL) and

1274
J.W. Morzycki, A. Wojtkielewicz / Carbohydrate Research 337 (2002) 1269–1274
water (0.06 mL). The reaction mixture was stirred 60 h
at rt, evaporated in vacuo and chromatographed on
silica gel column. Elution with MeOH (5%)–CH2Cl2
afforded compound 6 (5 mg, 26%), further elution with
MeOH (7%)–CH2Cl2 yielded OSW-1 (5, 15 mg, 74%)
as an amorphous solid.
85.6 (C). ESIMS (m/z): 913.4 (M+Na+). Mass calcd
for C47H67ClNaO14: 913.4112; found: 913.4104.
Acknowledgements
We thank Dr. L. Siergiejczyk for assistance in
recording some NMR spectra and Mrs. J. Maj for help
in preparation of the manuscript. Financial support
from the State Committee for Scientific Research,
Poland (Grant No. 7 T09A 045 20) is gratefully
acknowledged.
Saponin OSW-1 (5). Compound 5: Rf 0.18 (93:7
CH2Cl2–MeOH); [h]2D1 −44.0° (c 0.5, MeOH); lit.
−43.2°, −45.2°.1,5 IR (CHCl3): w 3594, 3458, 1728,
1
1692, 1606, 1512, 1259, 1170, 1034 cm−1. H NMR
(500 MHz, C5D5N): l 8.30 (d, 2 H, J 8.9 Hz), 7.07 (d,
2 H, J 8.9 Hz), 5.64 (m, 1 H), 5.51 (dd, 1 H, J 8.0, 6.1
Hz), 5.37 (d, 1 H, J 4.0 Hz), 5.09 (d, 1 H, J 7.6 Hz),
4.76 (s, 1 H), 4.56 (d, 1 H, J 6.0 Hz), 4.37 (m, 1 H), 4.31
(dd, 1 H, J 11.4, 5.1 Hz), 4.19–4.25 (m, 2 H), 4.10–4.18
(m, 3 H), 3.81 (m, 1 H), 3.76 (s, 3 H), 3.65–3.75 (m, 2
H), 3.18 (q, 1 H, J 7.4 Hz), 1.96 (s. 3 H), 1.28 (d, 3 H,
J 7.4 Hz), 1.07 (s, 3 H), 0.99 (s, 3 H), 0.89 (d, 3 H, J 6.3
Hz), 0.86 (d, 3 H, J 6.3 Hz). 13C NMR (125 MHz,
C5D5N): l 219.8 (C), 170.1 (C), 166.3 (C), 164.8 (C),
142.8 (C), 133.3 (CH), 122.0 (CH), 115.0 (CH), 104.5
(CH), 101.7 (CH), 97.3 (C), 89.2 (CH), 86.6 (C), 81.7
(CH), 77.1 (CH), 75.9 (CH), 72.9 (CH), 72.2 (CH), 71.6
(CH), 68.6 (CH), 67.8 (CH2), 66.3 (CH2), 56.4 (CH3),
51.0 (CH), 49.4 (CH), 47.4 (C), 47.2 (CH), 44.3 (CH2),
40.1 (CH2), 38.7 (CH2), 37.7 (C), 35.5 (CH2), 34.5 (C),
33.6 (CH2), 33.5 (CH2), 33.4 (CH2), 33.1 (CH2), 32.9
(CH), 28.6 (CH), 23.7 (CH3), 23.4 (CH3), 21.8 (CH2),
21.7 (CH3), 26.5 (CH3), 14.5 (CH3), 12.8 (CH3). ESIMS
(m/z): 895.4 (M+Na+). Mass calcd for C47H68NaO15:
895.4450; found: 895.4472.
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–
1
1693, 1606, 1512, 1259, 1170, 1033 cm−1. H NMR
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4.71 (m, 2 H), 4.15 (m, 3 H), 3.88 (s, 3 H), 3.69–3.87
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(C), 140.7 (C), 132.2 (CH), 121.4 (CH), 114.0 (CH),
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