Synthesis of cholestane glycosides bearing OSW-1 disaccharide or its 1→4-linked analogue and their antitumor activities

Article abstract of DOI:10.1016/j.bmcl.2010.07.085

For further structure-activity relationship (SAR) research of OSW saponins, a cholestane glycoside, namely 3β, 16β, 26-trihydroxycholest-5-en-22- one 16-O-(2-O-4-methoxybenzoyl-β-d-xylopyranosyl)-(1→3)-2-O-acetyl- α-l-arabinopyranoside (1) together with t

Full text of DOI:10.1016/j.bmcl.2010.07.085

Bioorganic & Medicinal Chemistry Letters 20 (2010) 5439–5442
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of cholestane glycosides bearing OSW-1 disaccharide or its
1?4-linked analogue and their antitumor activities
Dan Zheng a, , Liang Zhou a, , Yuyao Guan a, Xiaozhuo Chen a, Wanqi Zhou b, Xiaoguang Chen b,
Pingsheng Lei a,
*
a Key Laboratory of Bioactivity Substance and Resources Utilization of Chinese Herbal Medicine, Ministry of Education, Institute of Materia Medica, Peking Union Medical College
& Chinese Academy of Medical Sciences, Peking 100050, PR China
b Department of Pharmacology, Institute of Materia Medica, Peking Union Medical College & Chinese Academy of Medical Sciences, Peking 100050, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
For further structure–activity relationship (SAR) research of OSW saponins, a cholestane glycoside, namely
Received 9 June 2010
Revised 20 July 2010
Accepted 22 July 2010
Available online 29 July 2010
3b, 16b, 26-trihydroxycholest-5-en-22-one 16-O-(2-O-4-methoxybenzoyl-b-
D-xylopyranosyl)-(1?3)-2-
O-acetyl- -arabinopyranoside (1) together with two 1?4-linked disaccharide analogues (2 and 3) were
a-L
synthesized. Their cytotoxic activities were evaluated by the standard MTT assay. Compound 1 showed
potent cytotoxicity against five types of human tumor cells, with IC50 ranging between 1.3 and 73 nM.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
OSW-1 analogue
1?4-Linked disaccharide analogue
Cholestane glycosides
Synthesis
Cytotoxicity
OSW-1 belongs to a family of saponins isolated from the bulbs
of Ornitnogalum saundersiae by Sashida et al.1 It was found that
OSW-1 exhibited exceptionally potent cytotoxic activities on vari-
ous malignant tumor cells in vitro,2 with IC50 values ranging be-
tween 0.1 and 0.7 nM, which are around 10–100 times more
potent than clinically applied anticancer agents, such as mitomycin
C, adriamycin, cisplatin, camptothecin, and taxol. OSW-1 was also
found to be cytostatic in the US National Cancer Institute 60-cell
in vitro screen, with a mean IC50 of 0.78 nM and a mean IC100 of
58 nM. Because of its high potentiality, approaches toward the syn-
thesis of the aglycone3 and the whole structure4–8 of OSW-1 have
been developed by several groups. Various analogues9–19 have also
been prepared and tested for cytotoxicity in recent years. Accord-
ing to the previous SAR studies, it is known that: (1) the steroidal
C17-side chain could tolerate certain modifications without signif-
icant loss of activity; (2) the disaccharide moiety is of importance
to cytotoxicity, removal of the acetyl (Ac) and the 4-methoxyben-
zoyl (MBz) groups on the disaccharide moiety decreased the activ-
ity about 1000 times; (3) it is also necessary to have the
oligosaccharide chain attached to 16-b-OH, other substitute posi-
remains to be discovered about the structural elements of OSW-1
that are associated with its high activity.
In the synthesis of aglycone part of OSW-1, the introduction of
17-OH involves a series of tedious processes and expensive, high
toxic reagents such as OsO4. But no study has reported the neces-
sity of 17-OH to its activity. In modification of disaccharide, Ma
et al.9 synthesized OSW-1 analogues bearing 1?3 and 1?4 linked
OSW-1 disaccharide attached at 3-OH for the first time, but both of
them were inactive. Their work could not demonstrate which kind
of linkage would contribute to the activity as the substitute posi-
tion of disaccharide at 3-OH might be inappropriate. Therefore,
for further SAR research of OSW saponins and simplifying the syn-
thesis procedure, we designed and synthesized 3b, 16b, 26-trihydr-
oxycholest-5-en-22-one (17-deoxy-26-hydroxyl OSW-1 aglycone)
from diosgenin, which was then attached with OSW-1 disaccharide
and its 1?4 linked analogue at 16-b-OH, affording three OSW-1
analogues 1, 2, 3, whose antitumor activities were tested (Fig. 1).
The sapogenin 9 was prepared from diosgenin according to the
method reported by several groups previously.20,21 Diosgenin was
protected with t-butyldimethylsilyl group at 3-OH to provide 4,
and then oxyfunctionalized of C-16 and 5(6)-double bond with Ox-
one in the presence of NaHCO3 to afford the mixture of epoxide
diastereomers 5a and 5b, which could be distinguished by their
1H NMR spectra.21 The mixture of 5a and 5b was treated with
Zn/KI in Ac2O/HOAc to give 16, 22-dione 6. Removal of the 26-O-
acetyl group in 6 by treating with CH3ONa/CH3OH provided 7a
tions (e.g., 3-OH, 16-a-OH) led to deactivation. However, much
* Corresponding author. Tel.: +86 10 63162006; fax: +86 10 63017757.
E-mail addresses: lei@imm.ac.cn, wangdizhu007@163.com (P. Lei).
Authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.07.085

5440
D. Zheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5439–5442
O
O
OAc
OAc
O
STol
OR
c
O
a
OH
OH
O
AcO
RO
b
OAc
OR
O
O
O
O
O
AcO
AcO
11 R=Ac
12 R=H
OH
10
O
OH
O
HO
HO
HO
HO
OMBz
HO
HO
OMBz
O
STol
OAc
O
O
STol
OR
e
2
1
O
HO
OH
O
O
HO
O
HO
OMBz
HO
O
O
13 R=H
14 R=Ac
15
OH
O
OAc
d
O
OH
OH
AcO
O
O
OH
OMBz
O
Scheme 2. Reagents and conditions: (a) p-Thiocresol, BF3ÁEt2O, CH2Cl2, 0 °C to rt.,
8 h, 85%; (b) CH3ONa, CH3OH, rt, 2 h, 90%; (c) (CH3)2C(OCH3)2, CSA, DMF, under
vacuum, 5 h, 95%; (d) Ac2O, DMAP, Et3N, CH2Cl2, 1.5 h, 90%; (e) Amberlite IR-120
(H+), CH3OH, 50 °C, 4 h, 81%.
O
O
HO
AcO
HO
HO
O
HO
HO
HO
OMBz
OSW-1
3
Figure 1. Structures of OSW-1 and synthesized OSW-1 analogues.
Glycosylation of diol acceptor 15 with xylosyl donor 16 in the
presence of BF3ÁEt2O afforded the desired 1?4-linked disaccharide
17 as the major product in 61% yield, which was separated from its
1?3-linked isomer 18 (in 18% yield) through silica gel column
chromatography. Their structures were determined by 2D NMR.
The classification of the glycosylation position was also confirmed
by comparison of the ‘acylation shift’4 of 17 with its acetylation
product 19, or 18 with its acetylation product 20. The chemical
shift of ara-H-3 was found to be downshifted to 5.05 ppm from
3.70 to 3.66 ppm after acetylation; the chemical shift of ara-H-4
for 18 was 3.87–3.86 ppm, while in 20 it was 5.18–5.14 ppm. After
the silylation of 17 and 18 separately, 21 and 22 were afforded,
which were subsequently converted to the corresponding trichlo-
roacetimidate 23 and 24 in satisfactory yield (Scheme 3). The ara-
binosyl moiety of 21 is conformationally preferred the 1C4 form
and the xylosyl unit was still 4C1, which was deduced from the cou-
pling constants between trans H-1 and H-2 of both sugars, that is,
ara-H-1 was broad single, and J1,2 (xyl) = 6.0 Hz.
Under the promotion of trimethylsilyl triflate (TMSOTf), cou-
pling of 16b, 22b-diol aglycone 9 with disaccharide imidate 24
gave 16-glycosidation saponin 25 in 60% yield after chromatogra-
phy on silica gel which indicated the 16-OH in 9 was more active
than 22-OH under the condition of Schmidt glycosylation. The
saponin 25 was exposed to oxidation with pyridinium dichromate
(PDC) to afford 22-one 26. Removal of all protecting groups by
sequential treatment with 2,3-dichloro-5,6-dicyanobenzoquinone
and 7b in 64% yield, which then underwent ring-chain tautomer-
ization (Scheme 1). When the mixture of 7a and 7b were exposed
to silylation using TBDMSCl in pyridine, only 7a could be silylated
to afford 8, and 7b was still untouched. Dione 8 was then reduced
under NaBH4/CeCl3 to provide 16b, 22b-dihydroxyl aglycone 9 in a
yield of 73%. The absolute configuration of 22(S) was determined
by Mosher’s method in our lab.
The synthesis of saccharide moiety was referred to Yu and Jin’s
route.4,5,9 Analyzingtheirmethods, itis foundthattheanomericsub-
stitute of acceptor plays an important role in the regioselective gly-
cosylation with the xylose donor: when 1-OH was
a-form and
substituted with thioether, due to the stereoelectronic effect, 4-OH
is more active than 3-OH. So for regioselective synthesis of 1?4-
linked disaccharide, we synthesized p-methylphenyl 2-O-acetyl-1-
thio-
readily prepared from
Scheme 2. Tetraacetyl-
followed by deacetylation to give 12 in excellent yield. Compound
12 was then isopropylidenated to form 3, 4-O-isopropylidene-
a
-
L
-arabinopyranoside 15 as an acceptor. The acceptor 15 was
-arabinose in six steps as illustrated in
-arabinose 10 underwent thioglycosidation
L
L
a-L-
arabinopyranoside 13 in the yield of 95%. After acetylation to 2-OH
of 13, isopropylidene group was removed to give diol acceptor 15
in 81% yield. The xylosyl donor 16 was smoothly prepared by the ap-
proach of Jin5 with modified workup procedures.
O
O
O
c
b
O
O
OH
O
OH
RO
Diosgenin R = H
TBSO
O
TBSO
O
5b
5a
a
4
R = TBS
O
O
O
OAc
OH
d
O
OH
O
O
TBSO
TBSO
TBSO
7a
7b
6
O
OH
OTBS
OTBS
e
f
O
OH
7a
TBSO
TBSO
8
9
Scheme 1. Reagents and conditions: (a) TBDMSCl, DMAP, imidazole, DMF, 50 °C, 30 min, 98%; (b) Oxone, NaHCO3, acetone/H2O/CH2Cl2, rt, 48 h, 85%; (c) Zn powder, KI, HOAc/
Ac2O, rt, 24 h, 86%; (d) CH3ONa, CH3OH/CH2Cl2, 64%; (e) TBDMSCl, DMAP, pyridine, 79%; (f) NaBH4/CeCl3Á7H2O, THF, 73%.

D. Zheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5439–5442
5441
O
STol
OAc
O
O
STol
OAc
O
STol
OAc
HO
OH
RO
O
O
O
OR
15
a
PMBO
b
OMBz
PMBO
OMBz
OPMB
OPMB
O
OC(=NH)CCl3
OMBz
18 R = H
20 R = Ac
22 R = TES
17 R = H
19 R = Ac
21 R = TES
b
c
c
PMBO
OPMB
16
Scheme 3. Reagents and conditions: (a) BF3ÁEt2O, À60 to À40 °C, 4 Å MS, CH2Cl2, 61% for 17, 18% for 18; (b) Ac2O/pyridine, DMAP, 60% for 19, 55% for 20; (c) TESCl, pyridine,
DMAP, 70% for 21, 80% for 22.
Table 1
(DDQ) in CH2Cl2/H2O and bis (acetonitrile) palladium (II) chloride
in acetone/H2O in one operation5 furnished the analogue 1 in the
yield of 65% (Scheme 4).
Cytotoxic activities of compounds 1–3 against tumor cellsa
Tumor cells
IC50 (M)
Coupling of 9 with disaccharide imidate 23 using the same
method, 16-glycosidation saponin 27 was formed in 42% yield,
and, unexpectedly, 16, 22-bidesmosidic steroid glycoside 28 was
also achieved in a yield of 25%.Their structures were determined
by 2D NMR, for example, in HMBC spectrum of 28, two overlapped
ara-H-1 (d 4.36 br s, 2H) show long range correlations with C-16 (d
72.49) and C-22 (d 77.00), respectively. The saponin 27 was ex-
posed to oxidation with PDC to afford 22-one 29. Removal of all
protecting groups of 28 and 29 by the same method used in the
synthesis of 1, furnished the analogues 2, 3 in the yield of 75%
and 59%, respectively (Scheme 4). The arabinosyl residues of 27
and 28 were still in 1C4 form, but after deprotection, changed to
the 4C1 form according to their 1H NMR spectra.
The in vitro antitumor activities of the synthetic glycosides 1, 2,
and 3 against HCT-8, BEL-7402, BGC-823, A2780 and A-549 were
evaluated by the standard MTT assay using taxol as positive con-
trol. The results are listed in Table 1. It was shown that compound
1, 17-deoxy-26-hydroxyl OSW-1, exhibited potent cytotoxic activ-
ity on various cancer cell lines especially BGC-832 (IC50 1.3 nM),
which is comparable to that of OSW-1. This result indicated the
17-OH could be truncated without great loss of activity. On the
1
2
3
Taxol
HCT-8
2.3 Â 10À8
3.9 Â 10À8
1.3 Â 10À9
7.3 Â 10À8
4.6 Â 10À8
>10À5
>10À5
>10À5
>10À5
>10À5
>10À5
>10À5
>10À5
>10À5
>10À5
5.1 Â 10À8
6.0 Â 10À9
<1.0 Â 10À9
<1.0 Â 10À9
1.6 Â 10À8
BEL-7402
BGC-823
A2780
A-549
a
The in vitro cytotoxicities against HCT-8 (colon carcinoma), BEL-7402 (liver
cancer), BGC-823 (stomach carcinoma), A2780 (ovarian cancer) and A-549 (lung
carcinoma) cell lines were evaluated by the standard MTT assay.
other hand, compound 2, the 1?4-linked disaccharide isomer of
1, was inactive in a concentration of 10À5
lM, neither of compound
3. The results implied that the 1?3 glycosidic linkage in disaccha-
ride is critical to the cytotoxicities.
Structural superimposition analysis was also implemented in
our study with Accelrys Discovery Studio program package. The
minimum energy conformations of compounds 1, 2 and OSW-1
were calculated and aligned as shown in Figure 2. The result
showed no obvious difference between compounds 1 and 2 in
topological structure of molecules. In consideration of the great
R1
R2
O
O
OC(=NH)CCl3
OTBS
O
e
TESO
O
OAc
c
a, b
O
1
O
22
AcO
9
O
OTES
TBSO
PMBO
OMBz
OPMB
PMBO
PMBO
OMBz
25 R1 = OH R2 = H
24
d
26 R1,R2 = O
R1
R2
OTBS
OR
e
2
3
TBSO
27 R1 = OH R2 = H
29 R1,R2 = O
O
OC(=NH)CCl3
d
c
O
OAc
OTES
a, b
PMBO
O
21
9
OR
OMBz
OPMB
OTBS
23
OR
e
28
TESO
TBSO
O
O
O
PMBO
R =
PMBO
OMBz
OAc
Scheme 4. Reagents and conditions: (a) NIS, CH2Cl2/H2O, rt, 3 h, (b) DBU, CCl3CN, CH2Cl2, À15 °C, 10 h, 86% for 23, 70% for 24 for two steps; (c) TMSOTf, CH2Cl2, 4 Å MS,
À25 °C, 60% for 25, 42% for 27, 25% for 28; (d) PDC, CH2Cl2, rt, 84% for 26, 80% for 29, (e) DDQ, CH2Cl2/H2O, then Pd(CH3CN)2Cl2, acetone/H2O, 96% for 1, 75% for 2, 59% for 3.

5442
D. Zheng et al. / Bioorg. Med. Chem. Lett. 20 (2010) 5439–5442
Acknowledgements
Financial support of this research by National Natural Science
Foundation of China (NNSFC 30772632) and National Program of
New Drug Innovation and Production (2009ZX09301-003-9-1) is
gratefully acknowledged by authors.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2010.07.085.
References and notes
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Figure 2. Superimposition of compounds 1 (red), 2 (blue) and OSW-1 (green).
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activity difference of these two isomers, it is indicated that the ara-
4-OH may play an important role for the biological activity, which
is in accord with Tschamber’s19 research findings.
Insummary, basedonthepreviousSARdataonthecytotoxicityof
OSW saponins, three glycosides bearing the OSW-1 disaccharide or
its 1?4-linked analogue were synthesized in a facile way and their
cytotoxicities were determined. These results suggested that the
17-OH might not be of importance for antitumor activity, and the
1?3 glycosidic linkage in disaccharide is critical to the
cytotoxicities.
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