Structural analogues of diosgenyl saponins: Synthesis and anticancer activity

Article abstract of DOI:10.1016/j.bmc.2009.09.046

Saponins display various biological activities including anti-tumor activity. Recently intensive research has been focused on developing saponins for tumor therapies. The diosgenyl saponin dioscin is one of the most common steroidal saponins and exhibits

Full text of DOI:10.1016/j.bmc.2009.09.046

Bioorganic & Medicinal Chemistry 17 (2009) 7670–7679
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structural analogues of diosgenyl saponins: Synthesis and anticancer activity
Matthew J. Kaskiw a, Mary Lynn Tassotto b, Mac Mok a, Stacey L. Tokar a, Roxanne Pycko a,b,c
,
John Th’ng a,b,c, Zi-Hua Jiang a,
*
a Department of Chemistry, Lakehead University, 955 Oliver Road, Thunder Bay, ON, Canada P7B 5E1
b Regional Cancer Care, Thunder Bay Regional Health Sciences Centre, 980 Oliver Road, Thunder Bay, ON, Canada P7B 6V4
c Medical Sciences Division, Northern Ontario School of Medicine, 955 Oliver Road, Thunder Bay, ON, Canada P7B 5E1
a r t i c l e i n f o
a b s t r a c t
Article history:
Saponins display various biological activities including anti-tumor activity. Recently intensive research
has been focused on developing saponins for tumor therapies. The diosgenyl saponin dioscin is one of
the most common steroidal saponins and exhibits potent anticancer activity in several human cancer
cells through apoptosis-inducing pathways. In this paper, we describe the synthesis of several diosgenyl
Received 19 August 2009
Revised 18 September 2009
Accepted 22 September 2009
Available online 26 September 2009
saponin analogues containing either a 2-amino-2-deoxy-b-
D-glucopyranosyl residue or an a-L-rhamno-
pyranosyl-(1?4)-2-amino-2-deoxy-b- -glucopyranosyl residue with different acyl substituents on the
D
Keywords:
Saponin
Diosgenin
Dioscin
Aromatic nitro functionality
Cytotoxicity
Anticancer
amino group. The cytotoxic activity of these compounds was evaluated in MCF-7 breast cancer cells
and HeLa cervical cancer cells. Structure–activity relationship studies show that the disaccharide saponin
analogues are in general less active than their corresponding monosaccharide analogues. The incorpora-
tion of an aromatic nitro functionality into these saponin analogues does not exhibit significant effect on
their cytotoxic activity.
Ó 2009 Elsevier Ltd. All rights reserved.
Synthesis
1. Introduction
cancer cells is considered to be the most suitable method of anti-
cancer therapy.9 Because of their unique apoptosis-inducing prop-
Saponins are glycosides of steroids and triterpenoids that are
widely distributed in terrestrial plants and in some marine organ-
isms. In recent years, saponins have received considerable atten-
tion because of their various biological activities. In particular,
their favorable anti-tumorigenic properties have attracted inten-
sive research in developing saponins for tumor therapies. Diosge-
nyl saponins are the most abundant steroidal saponins and many
of them display potent anticancer activity. The diosgenyl saponin
dioscin (Chart 1) is one of the most common steroidal saponins
found in plants and exhibits cytotoxicity in several cancer cells
erties in cancer cells, dioscin and other diosgenyl saponins are
currently being explored for tumor therapy.10–12
In an effort to study the structure–activity relationship and de-
velop more potent anticancer agents, a number of dioscin deriva-
tives and analogues have been synthesized.2,13–16 Previously, we
reported a group of diosgenyl saponin analogues containing a 2-
amino-2-deoxy-b-D-glucopyranose residue with different acyl sub-
stituents at the amino group (structure I, Chart 1).17 Moderate
cytotoxic activity was found for most analogues against neuroblas-
toma (SK-N-SH) cells, breast cancer (MCF-7) cells, and cervical can-
cer (HeLa) cells. In the present paper, we describe the synthesis and
anticancer property of new saponin analogues containing either a
monosaccharide residue (structure I, Chart 1) or a disaccharide res-
idue (structure II, Chart 1) with an acyl substituent at the amino
group. In particular, some analogues contain an aromatic nitro
functionality which is known to be a common functionality per-
forming electron transfer. Electron transfer and oxidative stress
have been increasingly implicated in the actions of anticancer
drugs.18,19 Electron transfer pathways can endogenously lead to
the formation of reactive oxygen species, which cause oxidative
stress in cells and lead to the induction of apoptosis. We hypothe-
size that the incorporated aromatic nitro group will exert oxidative
stress in cancer cells and thus improve the cytotoxic activity of
these saponin analogues.
with an IC50 value typically in l
M.1–6 Dioscin has also been shown
to induce apoptosis in HL-60 (human leukemia) cells7 and HeLa
(cervical cancer) cells6 through activation of caspase-9 and -3, to-
gether with down-regulation of anti-apoptotic Bcl-2 protein. Re-
cently, Wang et al.8 reported that mitochondria was the major
cellular target of dioscin in its induction of apoptosis in HL-60 cells
and that dioscin exerted its cytotoxicity through multiple apopto-
sis-inducing pathways. Apoptosis permits the removal of damaged,
senescent or unwanted cells in multi-cellular organisms without
damaging the cellular microenvironment. Since cancer cells have
gained the ability to evade apoptosis, re-introducing apoptosis into
* Corresponding author. Tel.: +1 807 766 7171; fax: +1 807 346 7775.
E-mail address: zjiang@lakeheadu.ca (Z.-H. Jiang).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.09.046

M. J. Kaskiw et al. / Bioorg. Med. Chem. 17 (2009) 7670–7679
7671
26
O
O
20
27
22
11
O
O
H
H
16
OH
O
OH
O
H
H
H
H
4
3
O
R
O
O
HO
O
HO
HO
6
NH
4
O
R
O
I R = H
HO
Dioscin
O
HO
HO
R = acyl
O
OH
HO
4
OH
II R
=
HO
OH
Chart 1. Diosgenyl saponin dioscin and structural analogues I and II.
2. Results and discussion
2.1. Chemical synthesis
OBz
O
OH
O
HO
BzO
HO
HO
7
OH
OBz
6
NH
NH
Troc
Troc
Two diosgenyl saponin analogues incorporating an aromatic
nitro group at the 2-amino group of the -glucosamine residue
D
a
(4 and 5, Scheme 1) were synthesized. Starting from the previously
reported amine 117, 3-nitrobenzoic acid and 3,5-dinitrobenzoic acid
werecoupledto theamineinthepresenceof 2-(1H-benzotriazole-1-
yl)-1,1,3,3-tetramethylaminium hexafluorophosphate (HBTU) and
diisopropylethylamine (DIPEA) to give 2 and 3, respectively, in good
yield. Compounds 2 and 3 were then treated with sodium methoxide
to provide saponin analogues 4 and 5.
For the synthesis of saponin analogues containing a disaccha-
ride residue, we first tried the convergent strategy by preparing
the disaccharide donor, which will then be coupled to the diosge-
OBz
O
RO
BzO
8 R = H
9 R = Bz
OH
NH
Troc
Scheme 2. Reagents and conditions: (a) BzCl (2.1 equiv), DMAP, pyridine–CH2Cl2,
ꢀ40 °C, 65% for 8 and 14% for 9.
nyl aglycone. Since the 4-OH group in the D-glucosamine deriva-
at ꢀ40 °C, compound 8 was obtained in 65% yield, together with
14% of 9. The structure of 8 was confirmed through extensive 2D
NMR spectral analysis (including 1H–1H COSY and 1H–13C HSQC).
tives is known to be substantially less reactive than the 3-OH
group,20 we attempted to prepare 1,3,6-tri-O-benzoylated 7 as
the potential glycosylation acceptor to build the disaccharide from
the readily available trichloroethoxycarbonyl (Troc) protected glu-
cosamine derivative 6 (Scheme 2). When 6 was treated with
3.1 equiv of benzoyl chloride in the presence of 4-dimethylamino-
pyridine (DMAP) at ꢀ40 °C, compound 7 was not formed in any
detectable amount. Instead, the 3,6-di-O-benzoylated 8 and the
3,4,6-tri-O-benzoylated product 9 were obtained in 23% and 27%,
respectively, together with an 8% of a less polar compound which
was structurally not confirmed but likely to be the 1,3,4,6-tetra-
O-benzoylated product according to its 1H NMR spectral data. This
result indicated that the anomeric hydroxyl group was the least
reactive among the four hydroxyl groups present in 6. Thus, we
Glycosylation of 8 with L-rhamnopyranosyl imidate 10 in the
presence of trimethylsilyl trifluoromethanesulfonate (TMSOTf) as
the catalyst at ꢀ20 °C afforded disaccharide 11 in 71% yield
(Scheme 3). The formation of the di-rhamnopyranosylated trisac-
charide in ꢁ15% yield was observed from its crude 1H NMR spec-
trum but an analytically pure product was not obtained. The
glycosidic linkage at 4-O-position in 11 was confirmed by convert-
ing 11 into its acetylated derivative 12. The 1H NMR spectrum of 12
showed an additional acetyl group signal at d 2.28 ppm and a new
signal at d 6.24 ppm (d, J = 3.5 Hz, H-1I), indicating the presence of
an acetyl group at the anomeric position, which in turn confirmed
that compound 11 had a free anomeric hydroxyl group. Compound
11 was then treated with trichloroacetonitrile and 1,8-diazabicy-
clo[5,4,0]undec-7-ene (DBU) to give imidate 13 in 80% yield. Glyco-
sylation of donor 13 with diosgenin in the presence of TMSOTf as
the catalyst provided product 14 in 80% yield. The newly formed
decided to prepare 3,-6-di-O-benzoylated derivative
8 which
would allow regio-selective glycosylation at 4-OH over the ano-
meric-OH if 8 were used as a glycosylation acceptor (Scheme 2).
When compound 6 was treated with 2.1 equiv of benzoyl chloride
O
O
O
O
O
O
H
H
H
OAc
O
OAc
O
OH
O
H
H
H
H
H
H
HO
HO
AcO
AcO
AcO
AcO
O
O
O
NH2
NH
NH
b
a
R
R
O
O
2
3
R = H
R = NO2
4
5
R = H
1
R = NO2
NO2
NO2
Scheme 1. Reagents and conditions: (a) for 2, 3-nitrobenzoic acid, HBTU, DIPEA, DMF, rt, 75%; for 3, 3,5-dinitrobenzoic acid, HBTU, DIPEA, DMF, rt, 87%; (b) NaOCH3, CH3OH–
CH2Cl2 (1:1), rt, 93% for 4 and 78% for 5.

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M. J. Kaskiw et al. / Bioorg. Med. Chem. 17 (2009) 7670–7679
b-glycosidic linkage and the previously formed
a
-rhamnopyrano-
logues were confirmed by 1H NMR, 13C NMR, and mass spectro-
scopic data.
syl linkage in 14 were confirmed by the anomeric signals in its
1H and 13C NMR spectra: d 4.77 ppm (d, J = 8.0 Hz, H-1I) and
4.87 ppm (br s, H-1II); d 98.89 and 99.88 ppm (C-1I and C-1II).
Although the yield was good for the convergent synthesis of 14
according to Schemes 2 and 3, both compounds 8 and 11 existed as
2.2. Biological studies
Previously, we reported the synthesis of several monosaccha-
ride diosgenyl saponin analogues and their moderate cytotoxic
activity against neuroblastoma (SK-N-SH) cells, breast cancer
(MCF-7) cells, and cervical cancer (HeLa) cells.17 The analogue con-
a
/b mixtures and therefore presented challenges in chromato-
graphic separation from unwanted products which also existed
as /b mixtures. Thus, we investigated an alternative route to ac-
a
cess 14 starting from the previously reported intermediate 1517
(Scheme 4). Compound 15 was treated with guanidinium ni-
trate–sodium methoxide at room temperature to give the O-
deacetylated product 16 in 90% yield leaving the N-Troc group in-
tact.21 Selective benzoylation of 16 with 2.1 equiv of benzoyl chlo-
ride in the presence of DMAP at ꢀ40 °C gave 3,6-di-O-benzoylated
product 17 in 69% yield. The structure of 17 was confirmed by
taining an
against all three cancer cell lines with IC50 ranging from 4.8
in SK-N-SH cells to 7.3 M in HeLa cells. The role of -lipoic acid
a-lipoic acid residue exhibits the highest potency
l
M
l
a
in biological systems is very complex. Its disulfide bond allows
the molecule to act as an oxidant as well as a reductant; however,
based on its redox potential, its oxidizing behavior is more pro-
nounced in biological systems.22 Wenzel et al.23 showed that
a-li-
1H–1H COSY 2D NMR spectroscopy. Reaction of 17 with
L-rhamno-
poic acid induced apoptosis in human colon cancer cells by
Å
pyranosyl donor 10 using TMSOTf as the catalyst afforded 14 in
87% yield, which was identical, as indicated by 1H and 13C NMR
spectra and TLC, with compound 14 prepared through Scheme 3.
The final steps for the preparation of disaccharide saponin ana-
logues (23–26, Scheme 5) followed the same approach as that used
for the synthesis of monosaccharide analogues (4 and 5, Scheme 1).
Thus, the N-Troc protecting group in 14 was removed by treating
with zinc dust in acetic acid, affording amine 18 in 95%. Amine
increasing superoxide free radical (O2ꢀ )-generation in mitochon-
drial. In recent years, extensive evidence supports the involvement
of reactive oxygen species (ROS) and oxidative stress (OS) in the
mechanism of many anticancer drugs.18 Several saponin analogues
synthesized here (4, 5, 25, and 26) contain an aromatic nitro func-
tionality which is anticipated to exert oxidative stress in cells and
thus improve their anticancer potency. In addition, the preparation
of disaccharide saponin analogues (23–26) would allow us to study
18 was then treated with benzoyl chloride, ( )-a-lipoic acid, 3-
the effect of the a-L-rhamnosyl moiety at 4-O-position of the inner
nitrobenzoic acid, and 3,5-dinitrobenzoic acid to give N-acylated
products 19–22, respectively, in 76–83% yield. Finally the O-acetyl
and O-benzoyl groups in 19–22 were removed by treating with so-
dium methoxide to provide saponin analogues 23–26, respectively,
in very good yield. The structures of the synthesized saponin ana-
sugar residue of these saponin analogues.
The cytotoxic activity of the synthesized saponin analogues was
measured through MTT assay24 using breast cancer (MCF-7) and
cervical cancer (HeLa) cells. The anticancer drug Doxorubicin was
also included in the study as a comparison. The data presented in
OBz
O
OBz
O
O
OC(NH)CCl
3
BzO
O
OH
b
a
BzO
NH
O
8
O
NH
+
AcO
AcO
Troc
O
AcO
AcO
OAc
AcO
AcO
Troc
OAc
OAc
11
OAc
12
10
c
O
OBz
O
O
O
H
Diosgenin
BzO
Troc
NH
O
OBz
O
AcO
AcO
H
H
OC(NH)CCl
3
d
O
O
OAc
BzO
Troc
O
NH
13
AcO
AcO
14
OAc
Scheme 3. Reagents and conditions: (a) TMSOTf, CH2Cl2, ꢀ20 °C, 71%; (b) Ac2O, pyridine, rt, 86%; (c) CCl3CN, DBU, rt, 80%; (d) TMSOTf, CH2Cl2, rt, 80%.
O
O
O
O
H
H
OBz
O
OR
O
H
H
H
H
b
c
HO
BzO
RO
RO
O
14
O
NH
NH
10
Troc
Troc
17
15 R = Ac
16 R = H
a
Scheme 4. Reagents and conditions: (a) guanidinium nitrate, NaOCH3, CH3OH–CH2Cl2 (9:1), rt, 90%; (b) BzCl (2.1 equiv), DMAP, pyridine–CH2Cl2, ꢀ40 °C, 69%; (c) TMSOTf,
CH2Cl2, rt, 87%.

M. J. Kaskiw et al. / Bioorg. Med. Chem. 17 (2009) 7670–7679
7673
O
O
O
H
O
H
c
OBz
O
OH
O
H
H
H
H
O
O
O
BzO
O
HO
NH
O
NH
O
AcO
AcO
R
HO
R
14 R = Troc
18 R = H
a
HO
OAc
OH
b
CO
CO
CO
CO
CO
20 R =
22 R =
23 R =
25 R =
24 R =
26 R =
19 R =
21 R =
S
S
S
S
CO
O2N
CO
O2N
CO
NO2
NO2
NO2
NO2
Scheme 5. Reagents and conditions: (a) Zn dust, HOAc, rt, 95%; (b) for 19, BzCl, pyridine, rt, 80%; for 20, ( )-
a-lipoic acid, HBTU, DIPEA, DMF, rt, 83%; for 21, 3-nitrobenzoic
acid, HBTU, DIPEA, DMF, rt, 76%; for 22, 3,5-dinitrobenzoic acid, HBTU, DIPEA, DMF, rt, 83%; (c) NaOCH3, CH3OH–CH2Cl2, rt, 91–96%.
Table 1 show that these analogues are significantly less active than
Doxorubicin. Comparing compounds with the same N-substitution,
all disaccharide saponin analogues (23–26) show lower cytotoxic-
ity by a magnitude of 2–6-fold in both MCF-7 cells and HeLa cells
than their corresponding monosaccharide analogues. The largest
decrease in potency is observed for analogues containing the
the aromatic ring as indicated by the similar IC50 values between
N-(3,5-dinitrobenzoyl)-substituted analogues and N-benzoyl-
substituted analogues (5 vs I when R = Bz, and 26 vs 23). These
data suggest that the aromatic nitro functionality exhibits only
marginal effect on the cytotoxicity of these saponin analogues
when compared to simple N-benzoyl substitution.
a
-lipoic acid residue (24 vs monosaccharide analogue I when
R = -lipoyl). For example, compound 24 has the IC50 value of
M against HeLa cells while the monosaccharide analogue I
-lipoyl) has the IC50 of 7.3 M against HeLa cells. With re-
a
3. Conclusion
39.9
l
(R =
a
l
We have synthesized several diosgenyl saponin analogues con-
spect to the effect of aromatic nitro functionality on the cytotoxic-
ity, the N-(3-nitrobenzoyl)-substituted analogues are more active
than the N-benzoyl-substituted analogues in both MCF-7 and HeLa
cells (4 vs structure I when R = Bz, and 25 vs 23), which indicates
that the addition of the nitro group on the aromatic ring increases
the cytotoxicity of these compounds. However, this enhancement
seems to be diminished when a second nitro group is present on
taining either a 2-amino-2-deoxy-b-
D
-glucopyranosyl residue or
-glucopyr-
an -rhamnopyranosyl-(1?4)-2-amino-2-deoxy-b-D
a-
L
anosyl residue with different acyl substituents on the amino group.
The cytotoxic activity of these compounds was evaluated in MCF-7
breast cancer cells and HeLa cervical cancer cells. Structure–activ-
ity relationship analysis indicated that the addition of an
a-L-rhamnopyranosyl residue at 4-O-position of the inner b-D-glu-
cosamine residue resulted in decrease in the cytotoxicity of these
saponin analogues. The incorporation of an aromatic nitro func-
tionality onto the amino group seems to exert only marginal effect
on their anticancer activity.
Table 1
The in vitro cytotoxic activity (IC50
,
l
M) of diosgenyl saponin analoguesa,b
Compound
Rc
Cancer cell line
4. Experimental
MCF-7
HeLa
Monosaccharide analogue I
Id
Id
4
Bz
-Lipoyl
19.4
6.9
13.6
18.7
18.0
7.3
16.1
21.0
4.1. General methods
a
3-NO2-Bz
All air and moisture sensitive reactions were performed under
nitrogen atmosphere. All commercial reagents were used as sup-
plied. Anhydrous pyridine was distilled over potassium hydroxide,
dichloromethane over calcium hydride, and methanol from mag-
nesium turnings and a catalytic amount of iodine. Anhydrous
N,N-dimethylformamide (DMF) was purchased from Aldrich. ACS
grade solvents were purchased from Fisher Scientific and used
for chromatography without distillation. TLC plates (Silica Gel 60
5
3,5-(NO2)2-Bz
Disaccharide analogue II
f
23
Bz
>50e
18.8
22.8
39.5
0.73
24
a
-Lipoyl
39.9
25
3-NO2-Bz
39.4
f
26
3,5-(NO2)2-Bz
Doxorubicin
1.03
a
The inhibitory concentration (IC50) is the concentration in
l
M that inhibits cell
F254, thickness 0.25 mm) and Silica Gel 60 (40–63 lm) for flash
growth by 50% in 3 days compared to cells that remained untreated.
b
IC50 results are based on the average absorbance readings from eight replicate
column chromatography were purchased from SILICYCLE INC.,
Canada. 1H and 13C NMR spectra were recorded on a Varian Unity
Inova 500 MHz spectrometer. Tetramethylsilane (TMS, d 0.00 ppm)
or solvent peaks were used as internal standards for 1H and 13C
NMR spectra. The chemical shifts were given in ppm and coupling
constants in Hz indicated to a resolution of 0.5 Hz. Multiplicity of
wells per concentration of compound tested.
c
R group refers to the substituent at the amino group; see general structure in
Chart 1.
d
Data from Ref. 17.
e
Tested to 50
Tested to 50
l
l
M and the absorbance decreased to 68% at 50
M, no change of absorbance.
lM.
f

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M. J. Kaskiw et al. / Bioorg. Med. Chem. 17 (2009) 7670–7679
proton signals is indicated as follows: s (singlet), d (doublet), dd
(double doublet), t (triplet), q (quartet), m (multiplet), br (broad),
and approx. (approximate). For spectroscopic assignments, sugar
carbon atoms are designated with 1, 2, 3, . . ., and diosgenyl carbon
atoms with 10, 20, 30 . . ., etc. In the case of disaccharide residue, su-
gar units are designated as I, II, beginning at the reducing end of
the molecule, according to the Whelan system (Rule 2-Carb-
37.2).25 MALDI mass spectra were obtained from Bruker Daltonics
Apex-Qe FTICR MS mass spectrometer and ESI mass spectra were
measured on the Applied Biosystems Mariner Biospectrometry
Workstation, both at the University of Alberta, Canada. Optical
rotations were measured with Perkin–Elmer 343 Polarimeter at
22 °C. Elemental analyses were carried out on a CEC (SCP) 240-
XA Analyzer instrument by Lakehead University Instrumentation
Laboratory (LUIL).
4.10–4.20 (m, 2H, H-2, H-6a), 4.32 (dd, 1H, J 12.0, 5.0 Hz, H-6b),
4.40 (approx. q, 1H, J ꢃ 7.5 Hz, H-160), 4.93 (d, 1H, J 8.5 Hz, H-
1), 5.19 (dd, 1H, J 9.5, 9.5 Hz, H-4), 5.23 (m, 1H, H-60), 5.52 (dd,
1H, J 10.0, 10.0 Hz, H-3), 7.04 (d, 1H, J 8.5 Hz, NH), 8.94 (d, 2H,
J 2.0 Hz, Ar-H), 9.14 (t, 1H, J 2.0 Hz, Ar-H); 13C NMR (125 MHz,
CDCl3): d 14.72, 16.46, 17.34, 19.53, 20.84, 21.02, 28.97, 29.59,
39.91, 30.47, 31.51, 31.55, 32.00, 32.20, 37.01, 37.29, 39.09,
39.89, 40.42, 41.77, 50.17, 56.02, 56.62, 62.23, 62.53, 67.04,
68.90, 72.05, 72.82, 79.82, 80.95, 99.32 (C-1), 109.51 (C-220),
121.51 (C-60), 122.27, 127.45, 137.59, 140.25 (C-50), 148.87,
162.97 (CO), 169.52 (CO), 171.01 (CO), 171.99 (CO); LRESIMS
(m/z): 918 (M+Na+); HRMALDIMS calcd for C46H62N3O15 (M+H+):
896.41754, found: 896.41724.
4.4. Diosgenyl 2-deoxy-2-(3-nitrobenzamido)-b-D-glucopyrano-
side (4)
4.2. Diosgenyl 3,4,6-tri-O-acetyl-2-deoxy-2-(3-nitrobenzamido)-
b-
D
-glucopyranoside (2)
Compound 2 (220 mg, 0.258 mmol) was dissolved in dry dichlo-
romethane–methanol (1:1, 8 mL) and treated with catalytical
amount of sodium methoxide at room temperature for 16 h. The
solution was then neutralized with weakly acidic resin (IRC-50,
H+ form) and concentrated in vacuo. The residue was purified
through Sephadex LH-20 gel filtration chromatography (dichloro-
methane–methanol, 1:1) to give 4 (173 mg, 93%). Rf 0.34 (hex-
A mixture of compound 117 (380 mg, 0.54 mmol), 3-nitroben-
zoic acid (135 mg, 0.81 mmol), 2-(1H-benzotriazole-1-yl)-1,
1,3,3-tetramethylaminium hexafluorophosphate (HBTU, 307 mg,
0.81 mmol), diisopropylethylamine (DIPEA, 209 mg, 0.28 mL,
1.62 mmol) in dry dimethyl formamide (5 mL) was stirred at room
temperature for 16 h. The solvent was then removed under high
vacum and the residue dissolved in dichloromethane (100 mL).
The organic layer was washed with sodium bicarbonate solution
(20 mL) and dried with sodium sulfate and concentrated. The res-
idue was purified by repeated flash chromatography (hexane–
ethyl acetate, 1:1; then dichloromethane–methanol, 100:2) to give
ane–ethyl acetate–methanol, 1:1:0.2);
½
a 2D2
ꢂ
ꢀ36.9 (c 1.0,
chloroform–methanol, 1:1); 1H NMR (500 MHz, CDCl3–CD3OD,
1:1): d 0.77 (s, 3H, CH3), 0.79 (d, 3H, J 6.5 Hz, CH3), 0.95 (s, 3H,
CH3), 0.96 (d, 3H, J 6.5 Hz, CH3), 2.08 (m, 1H), 2.28 (m, 1H), 3.35
(m, 2H, H-260a, H-260b), 3.46 (m, 2H), 3.54 (m, 1H, H-30), 3.71
(dd, 1H, J 10.0, 9.0 Hz), 3.77 (dd, 1H, J 12.0, 5.0 Hz, H-6a), 3.85
(dd, 1H, J 10.0, 8.5 Hz, H-2), 3.90 (dd, 1H, J 12.0, 2.5 Hz, H-6b),
4.40 (approx. q, 1H, J 8.5 Hz, H-160), 4.75 (d, 1H, J 8.5 Hz, H-1),
5.27 (m, 1H, H-60), 7.71 (dd, 1H, J 8.0, 8.0 Hz, Ar-H), 8.23 (br d,
1H, J 8.0 Hz, Ar-H), 8.38 (br d, 1H, J 8.0 Hz, Ar-H), 8.74 (dd, 1H, J
2.0, 2.0 Hz, Ar-H); 13C NMR (125 MHz, CDCl3–CD3OD, 1:1): d
14.88, 16.80, 17.51, 19.84, 21.55, 29.39, 30.21, 30.94, 31.99,
32.19, 32.37, 32.77, 37.54, 37.97, 39.75, 40.46, 41.03, 42.41,
50.92, 57.27, 57.92, 62.42, 62.86, 67.58, 71.71, 75.02, 77.06,
80.12, 81.80, 100.59 (C-1), 110.39 (C-220), 122.41 (C-60), 123.09,
126.71, 130.58, 134.19, 137.36, 141.15 (C-50), 149.04, 167.79
(CO); LRESIMS (m/z): 759.4 (M+Clꢀ); HRESIMS calcd for
C40H56N2O10Cl (M+Clꢀ): 759.36290, found (negative mode):
759.36311.
2 (345 mg, 75%). Rf 0.35 (hexane–ethyl acetate, 1:1); ½a D22
ꢀ20.8 (c
ꢂ
1.0, chloroform–methanol, 1:1); 1H NMR (500 MHz, CDCl3): d 0.76
(s, 3H, CH3), 0.79 (d, 3H, J 6.0 Hz, CH3), 0.93 (s, 3H, CH3), 0.96 (d, 3H,
J 7.0 Hz, CH3), 2.04 (s, 3H, CH3CO), 2.06 (s, 3H, CH3CO), 2.10 (s, 3H,
CH3CO), 3.36 (dd, 1H, J 11.0, 11.0 Hz, H-260a), 3.47 (dd, 1H, J 11.0,
4.5 Hz, H-260b), 3.50 (m, 1H, H-30), 3.85 (ddd, 1H, J 10.0, 5.0,
2.5 Hz, H-5), 4.15–4.20 (m, 2H, H-2, H-6a), 4.33 (dd, 1H, J 12.0,
5.0 Hz, H-6b), 4.39 (approx. q, 1H, J ꢃ 8.0 Hz, H-160), 4.96 (d, 1H, J
8.5 Hz, H-1), 5.19 (dd, 1H, J 10.0, 9.5 Hz, H-4), 5.21 (m, 1H, H-60),
5.56 (dd, 1H, J 10.0, 10.0 Hz, H-3), 7.01 (d, 1H, J 8.5 Hz, NH), 7.61
(t, 1H, J 8.0 Hz, Ar-H), 8.16 (d, 1H, J 8.0 Hz, Ar-H), 8.29 (br d, 1H, J
8.0 Hz, Ar-H), 8.49 (t, 1H, J 1.5 Hz, Ar-H); 13C NMR (125 MHz,
CDCl3): d 14.67, 16.40, 17.30, 19.48, 20.79, 20.97, 28.93, 29.54,
29.84, 30.42, 31.49, 31.50, 31.94, 32.18, 36.96, 37.28, 38.97,
40.37, 41.72, 50.12, 55.61, 56.58, 62.20, 62.59, 66.98, 69.06,
71.93, 72.88, 79.77, 80.90, 99.44 (C-1), 109.44 (C-220), 121.01 (C-
60), 126.38, 130.12, 133.73, 135.77, 140.41 (C-50), 148.12, 165.26
(CO), 169.49 (CO), 170.92 (CO), 171.77 (CO); LRESIMS (m/z): 873
(M+Na+); HRMALDIMS calcd for C46H63N2O13 (M+H+): 851.43247,
found: 851.43213.
4.5. Diosgenyl 2-deoxy-2-(3,5-dinitrobenzamido)-b-D-glucopyra-
noside (5)
In a similar way as described for the synthesis of 4, compound 3
(120 mg, 0.134 mmol) was treated with sodium methoxide to pro-
vide 5 (80 mg, 78%). Rf 0.30 (dichloromethane–methanol, 100:8);
½
a 2D2
ꢂ
ꢀ2.2 (c 0.3, chloroform–methanol, 1:1); 1H NMR (500 MHz,
4.3. Diosgenyl 3,4,6-tri-O-acetyl-2-deoxy-2-(3,5-dinitrobenzam-
ido)-b-D-glucopyranoside (3)
CDCl3–CD3OD, 1:1): d 0.77 (s, 3H, CH3), 0.80 (d, 3H, J 6.0 Hz,
CH3), 0.95 (s, 3H, CH3), 0.97 (d, 3H, J 6.5 Hz, CH3), 2.10 (m, 1H),
2.27 (m, 1H), 3.37 (m, 2H), 3.45–3.57 (m, 3H), 3.73 (dd, 1H, J
10.0, 9.0 Hz), 3.80 (dd, 1H, J 12.0, 4.5 Hz, H-6a), 3.85 (dd, 1H, J
10.5, 8.5 Hz, H-2), 3.90 (dd, 1H, J 12.0, 2.5 Hz, H-6b), 4.41 (m, 1H,
H-160), 4.78 (d, 1H, J 8.5 Hz, H-1), 5.28 (m, 1H, H-60), 9.13 (d, 2H,
J 2.0 Hz, Ar-H), 9.16 (t, 1H, J 2.0 Hz, Ar-H); 13C NMR (125 MHz,
CDCl3–CD3OD, 1:1): d 13.70, 15.62, 16.34, 18.67, 20.38, 28.20,
29.04, 29.77, 30.81, 31.00, 31.19, 31.59, 36.37, 36.77, 38.61,
39.28, 39.85, 41.23, 49.73, 56.09, 56.92, 61.25, 61.67, 66.42,
70.49, 73.78, 75.93, 78.85, 80.63, 99.25 (C-1), 109.22 (C-220),
120.48 (C-60), 121.32, 127.24, 137.96, 139.89 (C-50), 148.29,
164.04 (CO); LRESIMS (m/z): 804.4 (M+Clꢀ); HRESIMS calcd for
C40H55N3O12Cl (M+Clꢀ): 804.34798, found (negative mode):
804.34718.
In a similar way as described for the preparation of 2, com-
pound 1 (200 mg, 0.28 mmol) was treated with 3,5-dinitrobenzoic
acid (91 mg, 0.43 mmol) in the presence of HBTU (162 mg,
0.43 mmol), DIPEA (110 mg, 0.15 mL, 0.85 mmol) in dry dimethyl-
formamide (5 mL) to give 3 (223 mg, 87%) after chromatographic
purification (hexane–ethyl acetate, 1.75:1). Rf 0.50 (hexane–ethyl
acetate, 1:1); ½a D22
ꢂ
ꢀ14.3 (c 1.0, chloroform–methanol, 1:1); 1H
NMR (500 MHz, CDCl3): d 0.76 (s, 3H, CH3), 0.78 (d, 3H, J
6.5 Hz, CH3), 0.94 (s, 3H, CH3), 0.96 (d, 3H, J 7.0 Hz, CH3), 2.02
(s, 3H, CH3CO), 2.06 (s, 3H, CH3CO), 2.10 (s, 3H, CH3CO), 3.36
(dd, 1H, J 11.0, 11.0 Hz, H-260a), 3.46 (dd, 1H, J 11.0, 4.5 Hz, H-
260b), 3.52 (m, 1H, H-30), 3.83 (dd, 1H, J 9.5, 5.0, 2.5 Hz, H-5),

M. J. Kaskiw et al. / Bioorg. Med. Chem. 17 (2009) 7670–7679
7675
4.6. 3,6-Di-O-benzoyl-2-deoxy-2-(2,2,2-trichloroethoxycarbon-
ylamino)- /b- -glucopyranose (8) and 3,4,6-tri-O-benzoyl-2-de-
3H, J 6.0 Hz, CH3), 1.89 (s, 3H, CH3CO), 1.96 (s, 3H, CH3CO), 2.02 (s,
3H, CH3CO), 3.70–3.74 (m, 2H, OH, H-5II), 4.15 (dd, 1H, J 9.5, 9.5 Hz,
H-4I), 4.19 (ddd, 1H, J 10.0, 9.5, 3.0 Hz, H-2I), 4.40 (d, 1H, J 12.0 Hz,
Troc-Ha), 4.41 (m, 1H), 4.48 (dd, 1H, J 12.5, 3.0 Hz, H-6Ia), 4.65 (d,
1H, J 12.0 Hz, Troc-Hb), 4.87–4.94 (m, 3H), 5.13–5.18 (m, 2H), 5.36
(br s, 1H, H-1I), 5.74 (d, 1H, J 10.0 Hz, NH), 5.77 (dd, 1H, J 11.0,
10.5 Hz, H-3I), 7.40–7.62 (m, 6H, Ar-H), 8.08 (m, 4H, Ar-H); 13C
a
D
oxy-2-(2,2,2-trichloroethoxycarbonylamino)-a/b-D-glucopyra-
nose (9)
To a solution of 6 (3.0 g, 8.46 mmol) and 4-dimethylamino-
pyridine (DMAP, 103 mg, 0.10 mmol) in anhydrous pyridine
(9 mL) and dry CH2Cl2 (6 mL), benzoyl chloride (2.06 mL,
17.77 mmol) in dry CH2Cl2 (8 mL) was added drop-wise under
N2 atmosphere at ꢀ40 °C. The reaction was carefully monitored
by TLC every 20 min. After being stirred at room temperature
for 2 h, the reaction was quenched with methanol (1.0 mL) and
the mixture further stirred for 30 min. The solution was then di-
luted with CH2Cl2 (200 mL) and washed with cold HCl solution
(4 N, 50 mL) and saturated NaHCO3 solution (30 mL). The organic
layer was dried over anhydrous Na2SO4 and concentrated under
vacuum. The residue was subjected to flash chromatography
(hexane–ethyl acetate, 1.5:1) to yield 8 (3.10 g, 65%) and 9
NMR (125 MHz, CHCl3) for the
a-isomer: d 17.04, 20.99, 54.91,
62.74, 67.60, 68.95, 69.41, 70.16, 70.52, 72.12, 74.47, 75.91, 92.10
(C-1I), 95.31 (Troc-CCl3), 98.36 (C-1II), 128.54, 128.70, 129.58,
130.03, 130.35, 133.46, 133.63, 154.70 (Troc-CO), 166.29 (CO),
166.59 (CO), 170.05 (CO), 170.32 (CO), 170.58 (CO); Anal. Calcd for
C35H38Cl3NO16: C, 50.34; H, 4.59; N, 1.68. Found: C, 50.31; H, 4.55;
N, 1.52.
For structure confirmation that the glycosylation occurred at 4-
OH in 8, disaccharide 11 was converted into its acetylated deriva-
tive 12. To a cooled solution (ice water bath) of 11 (78 mg,
0.09 mmol) in pyridine (2.1 mL), acetic anhydride (1.7 mL) was
added. The mixture was stirred at room temperature for 4 h. The
solution was diluted with CH2Cl2 (150 mL) and washed succes-
sively with cold HCl solution (4 N, 20 mL), and saturated NaHCO3
solution (20 mL ꢄ 2). The organic layer was dried over anhydrous
Na2SO4 and concentrated under vacuum. The residue was subjected
to flash column chromatography (hexane–ethyl acetate, 1.5:1) to
give 12 (70 mg, 86%). Rf 0.73 (CH2Cl2–MeOH, 25:1); 1H NMR
(500 MHz, CDCl3): d 0.67 (d, 3H, J 6.0 Hz, CH3), 1.91 (s, 3H, CH3CO),
1.97 (s, 3H, CH3CO), 2.01 (s, 3H, CH3CO), 2.28 (s, 3H, CH3CO), 3.70
(m, 1H, H-5II), 4.17 (m, 2H), 4.33 (ddd, 1H, J 10.5, 10.0, 3.5 Hz, H-
2I), 4.39 (d, 1H, J 12.0 Hz, Troc-Ha), 4.51 (dd, 1H, J 12.5, 2.5 Hz, H-
6Ia), 4.67 (d, 1H, J 12.0 Hz, Troc-Hb), 4.75 (m, 1H, H-6Ib), 4.89 (dd,
1H, J 10.0, 9.5 Hz, H-4II), 4.90 (d, 1H, J 1.5 Hz, H-1II), 5.15 (m, 3H,
NH, H-2II, H-3II) 5.65 (dd, 1H, J 11.0, 10.5 Hz, H-3I), 6.24 (d, 1H, J
3.5 Hz, H-1I), 7.42–7.50 (m, 4H, Ar-H), 7.59 (m, 2H, Ar-H), 8.05
(m, 4H, Ar-H).
(666 mg, 14%), both as anomeric mixtures (
a
/b, 8:1). For 8: Rf
0.26 (hexane–ethyl acetate, 1.5:1); ½a D22
ꢂ
+0.05 (c 0.5, CHCl3) for
the anomeric mixture (a
/b = 8:1); 1H NMR (500 MHz, CDCl3)
for the
a-isomer: d 3.40 (br s, 1H, OH), 3.69 (br s, 1H, OH),
3.87 (dd, 1H, J 10.0, 9.5 Hz, H-4), 4.21 (ddd, 1H, J 10.5, 9.0,
3.5 Hz, H-2), 4.34 (m, 1H, H-5), 4.41 (d, 1H, J 12.0 Hz, Troc-
Ha), 4.66 (dd, 1H, J 12.0, 2.0 Hz, H-6a), 4.74 (dd, 1H, J 12.0,
4.0 Hz, H-6b), 4.76 (d, 1H, J 12.0 Hz, Troc-Hb), 5.40 (d, 1H, J
3.5 Hz, H-1), 5.65 (dd, 1H, J 10.5, 9.5 Hz, H-3), 5.81 (d, 1H, J
9.0 Hz, NH), 7.44 (m, 4H, Ar-H), 7.57 (m, 2H, Ar-H), 8.04 (m,
4H, Ar-H); 13C NMR (125 MHz, CDCl3) for the
a-isomer: d
54.06 (C-2), 63.58 (C-6), 69.63 (C-4), 70.80 (C-5), 74.44 (Troc-
CH2), 74.68 (C-3), 92.44 (Troc-CCl3), 95.45(C-1), 128.68, 129.13,
129.80, 130.08, 130.31, 133.57, 133.91, 154.43 (Troc-CO),
167.23 (CO), 168.07 (CO); Anal. Calcd for C23H22Cl3NO9: C,
49.09; H, 3.94; N, 2.49. Found: C, 49.08; H, 3.84; N, 2.23. For
9: Rf 0.48 (hexane–ethyl acetate, 1.5:1); ½a D22
ꢂ
+26.4 (c 0.5, CHCl3)
for the anomeric mixture (a
/b = 8:1); 1H NMR (500 MHz, CDCl3)
4.8. 2,3,4-Tri-O-acetyl-a-L-rhamnopyranosyl-(1?4)-3,6-di-O-
for the
a-isomer: d 3.92 (br s, 1H, OH), 4.33 (ddd, 1H, J 10.0,
benzoyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-
a-D-
3.5 Hz, H-2), 4.41 (dd, 1H, J 12.5, 4.0 Hz, H-6a), 4.47 (d, 1H, J
12.0 Hz, Troc-Ha), 4.63–4.69 (m, 2H, H-5, H-6b), 4.70 (d, 1H, J
12.0 Hz, Troc-Hb), 5.47 (br s, 1H, H-1), 5.70 (d, 1H, J 10.0 Hz,
NH), 5.72 (dd, 1H, J 9.5, 10.0 Hz), 5.90 (dd, 1H, J 10.5, 10.0 Hz),
7.26–7.57 (m, 10H, Ar-H), 7.92 (m, 3H, Ar-H), 8.05 (m, 2H, Ar-
glucopyranosyl trichloroacetimidate (13)
To a solution of 11 (500 mg, 0.58 mmol) in anhydrous CH2Cl2
(3 mL), trichloroacetonitrile (CCl3CN, 1.0 mL, 9.97 mmol) and
H); 13C NMR (125 MHz, CDCl3) for the
a-isomer: d 54.73 (C-2),
1,8-diazabicyclo[5,4,0]undec-7-ene
(DBU,
88 mg,
87 lL,
0.58 mmol) were added and stirred at room temperature for
20 min. The solid was filtered through celite and the filtrate
was concentrated in vacuo. The residue was purified with flash
chromatography (hexane–ethyl acetate, 1.5:1 with 0.5% triethyl-
amine) to afford 13 (466 mg, 80%) as a syrup. Rf 0.29 (hexane–
63.00 (C-6), 68.40, 69.49, 71.41, 74.54, 92.31 (C-1), 95.37
(Troc-CCl3), 128.60, 128.63, 128.97, 129.03, 129.71, 130.01,
130.15, 133.43, 133.64, 154.43 (Troc-CO), 165.42 (CO), 166.63
(CO), 166.87 (CO); Anal. Calcd for C30H26Cl3NO10: C, 54.03; H,
3.93; N, 2.10. Found: C, 54.03; H, 3.92; N, 1.89.
ethyl acetate, 1.5:1);
½
a 2D2 +26.2 (c 0.5, CHCl3); 1H NMR
ꢂ
(500 MHz, CDCl3): d 0.68 (d, 3H, J 6.0 Hz, CH3), 1.90 (s, 3H,
CH3CO), 1.96 (s, 3H, CH3CO), 2.01 (s, 3H, CH3CO), 3.71 (m, 1H,
H-5II), 4.22 (dd, 1H, J 9.5, 9.5 Hz, H-4I), 4.30 (m, 1H, H-5I), 4.42
(ddd, 1H, J 10.5, 10.0, 3.5 Hz, H-2I), 4.48 (d, 1H, J 12.0 Hz, Troc-
Ha), 4.53 (dd, 1H, J 12.5, 3.5 Hz, H-6Ia), 4.63 (d, 1H, J 12.0 Hz,
Troc-Hb), 4.79 (dd, 1H, J 12.5, 1.5 Hz, H-6Ib), 4.88 (dd, 1H, J
10.0, 10.0 Hz, H-4II), 4.91 (br s, 1H, H-1II), 5.15 (m, 1H, H-2II),
5.17 (dd, 1H, J 10.0, 3.5 Hz, H-3II), 5.22 (d, 1H, J 10.0 Hz, NH),
5.71 (dd, 1H, J 10.5, 9.5 Hz, H-3I), 6.42 (d, 1H, J 3.5 Hz, H-1I),
7.47 (m, 4H, Ar-H), 7.60 (m, 2H, Ar-H), 8.06 (m, 4H, Ar-H), 8.81
(s, 1H, NH); 13C NMR (125 MHz, CDCl3): d 17.06, 20.88, 20.92,
20.96, 54.46, 62.20, 67.68, 68.58, 70.11, 70.74, 71.66, 71.85,
74.61, 75.89, 90.83, 94.87 (C-1I), 95.09 (Troc-CCl3), 99.03 (C-1II),
128.65, 128.69, 129.19, 129.81, 129.99, 130.25, 133.43, 133.92,
154.44 (Troc-CO), 160.70 (CO), 165.94 (CO), 166.73 (CO), 170.01
(CO), 170.20 (CO); Anal. Calcd for C37H38Cl6N2O16: C, 45.37; H,
3.91; N, 2.86. Found: C, 45.12; H, 3.62; N, 2.63.
4.7. 2,3,4-Tri-O-acetyl-
benzoyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonylamino)-
-glucopyranose (11)
a
-
L
-rhamnopyranosyl-(1?4)-3,6-di-O-
a
/b-
D
A suspension of 8 (5.0 g, 8.88 mmol), 10 (3.88 g, 8.88 mmol), and
activated molecular sieves (4 Å, 3.0 g) in anhydrous CH2Cl2 (15 mL)
was stirred under N2 atmosphere at room temperature for 30 min
and then cooled to ꢀ20 °C. A solution of trimethylsilyl trifluoro-
methanesulfonate (TMSOTf, 5.5 mL, 0.02 M in dry CH2Cl2) was
added drop-wise. The mixture was stirred for 30 min, quenched by
saturated NaHCO3 solution (10 mL), extracted with CH2Cl2
(3 ꢄ 20 mL) and dried over Na2SO4. The filtrate was concentrated
invacuowith theresultingresiduepurifiedbyflashchromatography
(hexane–ethyl acetate, 1.5:1) to give 11 (5.26 g, 71%) as an anomeric
mixture (
+19.8 (c
a
/b = 10:1). Rf 0.29 (hexane–ethyl acetate, 1.5:1); ½a D22
ꢂ
0.5,
CHCl3)
for
the
anomeric
mixture
(a a-isomer: d 0.68 (d,
/b = 10:1); 1H NMR (500 MHz, CDCl3) for the

7676
M. J. Kaskiw et al. / Bioorg. Med. Chem. 17 (2009) 7670–7679
4.9. Diosgenyl 2,3,4-tri-O-acetyl-
a
-
L
-rhamnopyranosyl-(1?4)-
residual methanol which interferes with the subsequent reaction.
3,6-di-O-benzoyl-2-deoxy-2-(2,2,2-trichloroethoxycarbonyla-
mino)-b- -glucopyranoside (14)
Rf 0.19 (CH2Cl2–MeOH, 14.3:1); ½a D22
ꢂ
ꢀ45.0 (c 0.5, MeOH); 1H
D
NMR (500 MHz, C5D5N): d 0.70 (d, 3H, J 5.5 Hz, CH3), 0.84 (s, 3H,
CH3), 0.92 (s, 3H, CH3), 1.15 (d, 3H, J 7.0 Hz, CH3), 2.49 (m, 1H),
2.62 (m, 1H), 3.51 (dd, 1H, J 11.0, 10.0 Hz, H-260a), 3.60 (dd, 1H, J
10.0, 3.5 Hz, H-260b), 3.85 (m, 1H, H-30), 4.05 (m, 1H, H-5), 4.30
(dd, 1H, J 9.0, 9.0 Hz), 4.33 (dd, 1H, J 10.0, 9.0 Hz), 4.42–4.49 (m,
2H, H-2, H-6a), 4.55 (approx. q, 1H, J ꢃ 7.5 Hz, H-160), 4.61 (br d,
1H, J 11.5 Hz, H-6b), 4.95 (d, 1H, J 12.0 Hz, Troc-Ha), 5.17 (d, 1H,
J 12.0 Hz, Troc-Hb), 5.18 (m, 1H, H-60), 5.22 (d, 1H, J 8.0 Hz, H-1),
6.61 (br s, 1H, OH), 7.52 (br s, 1H, OH), 7.59 (br s, 1H, OH), 9.26
(d, 1H, J 9.0 Hz, NH); 13C NMR (125 MHz, C5D5N): d 15.39, 16.69,
17.68, 19.79, 21.42, 29.60, 30.44, 30.94, 31.94, 32.14, 32.53,
37.35, 37.70, 39.79, 40.18, 40.78, 42.30, 50.54, 56.95, 60.17,
63.10, 63.21, 67.20, 67.54, 72.64, 74.94, 76.60, 79.04, 81.42, 97.50
(Troc-CCl3), 101.47 (C-1), 109.61 (C-220), 121.98 (C-60), 141.22 (C-
50), 156.20 (CO); Anal. Calcd for C36H54Cl3NO9: C, 57.56; H, 7.25;
N, 1.86. Found: C, 57.61; H, 7.41; N, 1.67.
4.9.1. Method A
A suspension of 13 (230 mg, 0.23 mmol), diosgenin (95 mg,
0.23 mmol), and activated molecular sieves (4 Å, 2.0 g) in anhy-
drous CH2Cl2 (3 mL) was stirred at room temperature for 15 min
under N2 atmosphere. A solution of trimethylsilyl trifluorometh-
anesulfonate (TMSOTf, 0.45 mL, 0.01 M in dry CH2Cl2) was added
drop-wise. The mixture was stirred for 15 min, quenched by satu-
rated NaHCO3 solution (10 mL), extracted with CH2Cl2 (3 ꢄ 10 mL)
and dried over Na2SO4. The filtrate was concentrated in vacuo with
the resulting residue purified by flash chromatography (hexane–
ethyl acetate–methanol, 5:2:0.05) to give compound 14 (221 mg,
80%) as white foam.
4.9.2. Method B
A
suspension of 17 (230 mg, 0.24 mmol), 10 (191 mg,
4.11. Diosgenyl 3,6-di-O-benzoyl-2-deoxy-2-(2,2,2-trichloroeth-
oxycarbonylamino)-b-D-glucopyranoside (17)
0.44 mmol), and activated molecular sieves (4 Å, 2.0 g) in anhy-
drous CH2Cl2 (5 mL) was stirred at room temperature for 15 min
under N2 atmosphere. A solution of trimethylsilyl trifluorometh-
anesulfonate (TMSOTf, 0.44 mL, 0.01 M in dry CH2Cl2) was added
drop-wise. The mixture was stirred for 30 min, quenched by satu-
rated NaHCO3 solution (5 mL), extracted with CH2Cl2 (3 ꢄ 20 mL)
and dried over Na2SO4. The filtrate was concentrated in vacuo with
the resulting residue purified by flash chromatography (hexane–
ethyl acetate–methanol, 5:2:0.05) to give compound 14 (262 mg,
87%) as white foam.
To a solution of 16 (386 mg, 0.51 mmol) and 4-dimethylamino-
pyridine (DMAP, 6 mg, 0.05 mmol) in anhydrous pyridine (4 mL)
and CH2Cl2 (2 mL), benzoyl chloride (0.12 mL, 1.03 mmol) in dry
CH2Cl2 (2 mL) was added drop-wise under N2 atmosphere at
ꢀ40 °C. After being stirred at room temperature for 2 h, the reac-
tion was quenched with MeOH (1.0 mL) and the mixture stirred
for 30 min. The solution was then diluted with CH2Cl2 (150 mL)
and washed with cold HCl solution (4 N, 30 mL) and saturated
NaHCO3 solution (30 mL). The organic layer was dried over anhy-
drous Na2SO4 and concentrated under vacuum. The residue was
subjected to flash chromatography (toluene–acetone, 11:1) to give
Rf 0.17 (hexane–ethyl acetate–methanol, 6:2:0.05); ½a D22
ꢀ52.5
ꢂ
(c 0.2, CHCl3); 1H NMR (500 MHz, CDCl3): d 0.67 (d, 3H, J 6.0 Hz,
CH3), 0.77 (s, 3H, CH3), 0.79 (d, 3H, J 6.0 Hz, CH3), 0.95 (s, 3H,
CH3), 0.98 (d, 3H, J 6.5 Hz, CH3), 1.91 (s, 3H, CH3CO), 1.95 (s, 3H,
CH3CO), 2.00 (s, 3H, CH3CO), 3.38 (dd, 1H, J 11.5, 10.5 Hz, H-
260a), 3.48 (m, 2H, H-260b, H-30), 3.69 (m, 1H, H-5II), 3.75 (m, 1H,
H-2I), 3.86 (m, 1H, H-5I), 4.04 (dd, 1H, J 9.0, 9.5 Hz, H-4I)), 4.41 (ap-
prox. q, 1H, J ꢃ 7.5 Hz, H-160), 4.52 (dd, 1H, J 12.0, 5.0 Hz, H-6Ia),
4.54 (d, 1H, J 12.0 Hz, Troc-Ha), 4.71 (d, 1H, J 12.0 Hz, Troc-Hb),
4.77 (d, 1H, J 8.0 Hz, H-1I), 4.80 (dd, 1H, J 12.5, 2.5 Hz, H-6Ib),
4.87 (br s, 1H, H-1II), 4.88 (dd, 1H, J 10.0, 9.5 Hz, H-4II), 5.12 (m,
1H, H-2II), 5.15 (dd, 1H, J 10.0, 3.5 Hz, H-3II), 5.29 (m, 1H, H-60),
5.60 (dd, 1H, J 10.5, 9.0 Hz, H-3I), 7.45 (m, 4H, Ar-H), 7.58 (m, 2H,
Ar-H), 8.05 (m, 4H, Ar-H); 13C NMR (125 MHz, CDCl3): d 14.74,
16.48, 17.02, 17.35, 19.53, 20.91, 28.98, 29.71, 30.49, 31.56,
32.03, 32.25, 36.96, 37.18, 39.01, 39.95, 40.45, 41.78, 50.16,
56.67, 57.17, 62.25, 62.99, 67.05, 67.66, 68.65, 70.19, 70.73,
73.25, 73.45, 74.49, 80.17, 81.01, 95.50 (Troc-CCl3), 98.89 (C-1II),
99.88 (C-1I), 109.51 (C-220), 121.99 (C-60), 128.60, 128.67, 129.52,
130.02, 130.22, 133.28, 133.72, 140.49 (C-50), 154.35 (CO), 166.06
(CO), 166.39 (CO), 170.05 (CO), 170.16 (CO), 170.26 (CO); Anal.
Calcd for C62H78Cl3NO18: C, 60.46; H, 6.38; N, 1.14. Found: C,
60.14; H, 6.48; N, 0.89.
compound 17 (340 mg, 69%). Rf 0.24 (toluene–acetone, 11:1); ½a D22
ꢂ
ꢀ33.5 (c 0.2, CHCl3); 1H NMR (500 MHz, CDCl3): d 0.78 (s, 3H, CH3),
0.79 (d, 3H, J 6.5 Hz, CH3), 0.97 (s, 3H, CH3), 0.98 (d, 3H, J 8.0 Hz,
CH3), 3.38 (dd, 1H, J 11.0, 11.0 Hz, H-260a), 3.40 (br s, 1H, OH),
3.48 (dd, 1H, J 11.5, 3.5 Hz, H-260b), 3.53 (m, 1H, H-30), 3.75–3.81
(m, 3H, H-2, H-4, H-5), 4.41 (approx. q, 1H, J ꢃ 7.5 Hz, H-160),
4.57 (d, 1H, J 12.0 Hz, Troc-Ha), 4.63 (d, 1H, J 12.5, 1.5 Hz, H-6a),
4.71 (d, 1H, J 12.0 Hz, Troc-Hb), 4.72 (m, 1H, H-6b), 4.83 (d, 1H, J
8.5 Hz, H-1), 5.27 (m, 1H, H-60), 5.40 (d, 1H, J 9.0 Hz, NH), 5.50
(dd, 1H, J 10.0, 8.5 Hz, H-3), 7.38–7.45 (m, 4H, Ar-H), 7.56 (m,
2H, Ar-H), 8.02 (m, 4H, Ar-H); 13C NMR (125 MHz, CDCl3): d
14.75, 16.49, 17.35, 19.56, 21.00, 28.98, 29.72, 30.49, 31.56,
32.03, 32.25, 36.99, 37.25, 39.03, 39.97, 40.46, 41.78, 50.21,
56.56, 56.68, 62.26, 63.99, 67.05, 70.02, 74.23, 74.48, 75.81,
80.10, 81.00, 95.61 (Troc-CCl3), 99.90 (C-1), 109.53 (C-220),
121.99 (C-60), 128.62, 128.68, 129.17, 129.80, 130.10, 130.25,
133.50, 133.85, 140.51 (C-50), 154.46 (CO), 167.21 (CO), 167.51
(CO); Anal. Calcd for C50H62Cl3NO11: C, 62.60; H, 6.51; N, 1.46.
Found: C, 63.00; H, 6.32; N, 1.10.
4.12. Diosgenyl 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl-(1?4)-
2-benzamido-3,6-di-O-benzoyl-2-deoxy-b-D-glucopyranoside
(19)
4.10. Diosgenyl 2-deoxy-2-(2,2,2-trichloroethoxycarbonylami-
no)-b-D-glucopyranoside (16)
Guanidinium nitrate (3.72 g, 30.4 mmol) was dissolved in
MeOH–CH2Cl2 (9:1, 300 mL) and sodium methoxide in methanol
solution (1.0 M, 6 mL) was added. To the above solution, com-
pound 1517 (2.88 g, 3.29 mmol) was added and the mixture was
stirred at room temperature for 20 h. The mixture was then neu-
tralized by adding weak acidic ion-exchange resin (Amberlite
IRC-50, H+ form). The resin was filtered and the filtrate concen-
trated in vacuo. The residue was subjected to flash chromatogra-
phy (CH2Cl2–MeOH, 12.5:1) to give 16 (2.21 g, 90%). The
compound was freeze-dried from dioxane to ensure removal of
4.12.1. Preparation of amine 18
To a solution of 14 (217 mg, 0.17 mmol) in acetic acid (10 mL),
zinc dust (2.0 g) was added and the mixture was stirred at room tem-
perature for 20 h. The mixture was filtered and the solid was thor-
oughly washed with CH2Cl2 (100 mL). The filtrate was
concentrated in vacuo and the residue was re-dissolved in CH2Cl2
(200 mL). The solution was washed with saturated NaHCO3
solution (20 mL), dried with Na2SO4, and concentrated under vac-
uum to give a white solid 18 (180 mg, 96%) which was used without
further purification in the next step.

M. J. Kaskiw et al. / Bioorg. Med. Chem. 17 (2009) 7670–7679
7677
To a cooled solution (ice bath) of 18 (85 mg, 0.08 mmol) in pyr-
idine (2.0 mL), benzoyl chloride (14.0 L, 0.12 mmol) was added.
4.14. Diosgenyl 2,3,4-tri-O-acetyl-
3,6-di-O-benzoyl-2-deoxy-2-(3-nitrobenzamido)-b-
noside (21)
a
-
L
-rhamnopyranosyl-(1?4)-
l
D-glucopyra-
The mixture was stirred at room temperature for 4 h. The solution
was diluted with CH2Cl2 (150 mL), washed with cold HCl solution
(4 N, 20 mL) and saturated NaHCO3 solution (20 mL). The organic
layer was dried over anhydrous Na2SO4 and concentrated under
vacuum. The residue was subjected to flash chromatography (tolu-
ene–acetone, 9:1) to give 19 (92 mg, 80%). Rf 0.25 (toluene–ace-
In a similar way as described for the preparation of 2, com-
pound 18 (150 mg, 0.14 mmol) was coupled with 3-nitrobenzoic
acid (28 mg, 0.16 mmol) in the presence of HBTU (53 mg,
0.14 mmol) and DIPEA (0.05 mL, 0.28 mmol) in dry dimethylform-
amide (5.0 mL) to give 21 (143 mg, 83%). Rf 0.28 (toluene–acetone,
tone, 9:1); ½a 2D2
ꢂ
ꢀ33.9 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3):
d 0.69 (d, 3H, J 6.0 Hz, CH3), 0.76 (s, 3H, CH3), 0.79 (d, 3H, J
6.5 Hz, CH3), 0.92 (s, 3H, CH3), 0.97 (d, 3H, J 7.0 Hz, CH3), 1.90 (s,
3H, CH3CO), 1.95 (s, 3H, CH3CO), 2.01 (s, 3H, CH3CO), 3.37 (dd,
1H, J 10.5, 11.5 Hz, H-260a), 3.49 (m, 2H, H-260b, H-30), 3.72 (m,
1H, H-5II), 3.93 (m, 1H, H-5I), 4.14 (dd, 1H, J 9.0, 9.5 Hz, H-4I),
4.27 (m, 1H, H-260), 4.40 (approx. q, 1H, J ꢃ 7.0 Hz, H-160), 4.55
(dd, 1H, J 12.0, 5.0 Hz, H-6Ia), 4.83 (dd, 1H, J 12.0, 2.0 Hz, H-6Ib),
4.88 (dd, 1H, J 10.0, 10.0 Hz, H-4II), 4.91 (d, 1H, J 8.5 Hz, H-1I),
4.92 (br s, 1H, H-1II), 5.16 (m, 1H, H-2II), 5.17 (m, 1H, H-3II), 5.21
(m, 1H, H-60), 5.71 (dd, 1H, J 9.0, 9.0 Hz, H-3I), 6.24 (d, 1H, J
9.0 Hz, NH), 7.28–7.60 (m, 11H, Ar-H), 8.00 (m, 2H, Ar-H), 8.08
(m, 2H, Ar-H); 13C NMR (125 MHz, CDCl3): d 14.73, 16.46, 17.02,
17.34, 19.50, 20.91, 28.98, 29.71, 30.48, 31.53, 32.02, 32.22,
36.93, 37.21, 39.14, 39.93, 40.43, 41.77, 50.16, 55.51, 56.65,
62.24, 63.11, 67.03, 67.61, 68.73, 70.17, 70.76, 73.27, 74.02,
76.81, 79.94, 80.99, 98.67 (C-1II), 100.22 (C-1I), 109.49 (C-220),
121.61 (C-60), 126.96, 128.61, 128.73, 129.53, 130.04, 130.15,
131.65, 133.26, 133.60, 134.65, 140.64 (C-50), 166.12 (CO), 166.92
(CO), 168.06 (CO), 170.04 (CO), 170.18 (CO), 170.23 (CO); LRESIMS
(m/z): 1182 (M+Na+); HRESIMS calcd for C66H81NO17Na (M+Na+):
1182.5396, found: 1182.5394.
11:1); ½a 2D2
ꢂ
ꢀ29.8 (c 1.0, CHCl3); 1H NMR (500 MHz, CDCl3): d 0.72
(d, 3H, J 6.5 Hz, CH3), 0.76 (s, 3H, CH3), 0.79 (d, 3H, J 6.5 Hz, CH3),
0.91 (s, 3H, CH3), 0.97 (d, 3H, J 7.0 Hz, CH3), 1.90 (s, 3H, CH3CO),
1.95 (s, 3H, CH3CO), 2.02 (s, 3H, CH3CO), 3.37 (dd, 1H, J 11.0,
11.0 Hz, H-260a), 3.47 (m, 1H, H-260b), 3.53 (m, 1H, H-30), 3.73
(m, 1H, H-5II), 3.98 (m, 1H, H-5I), 4.19 (dd, 1H, J 9.0, 9.0 Hz, H-4I),
4.36 (dd, 1H, J 10.0, 8.0 Hz, H-2I), 4.40 (approx. q, 1H, J ꢃ 7.0 Hz,
H-160), 4.55 (dd, 1H, J 12.5, 5.0 Hz, H-6Ia), 4.88 (m, 1H, H-6Ib),
4.90 (dd, 1H, J 9.5, 10.0 Hz, H-4II), 4.95 (br s, 1H, H-1II), 4.96 (d,
1H, J 8.0 Hz, H-1I), 5.15 (m, 2H, H-2II, H-3II), 5.19 (m, 1H, H-60),
5.71 (dd, 1H, J = 9.0, 9.0 Hz, H-3I), 6.69 (d, 1H, J 9.0 Hz, NH), 7.39
(m, 1H, Ar-H), 7.46–7.61 (m, 6H, Ar-H), 7.94 (m, 1H, Ar-H), 8.02
(m, 2H, Ar-H), 8.08 (m, 2H, Ar-H), 8.25 (m, 1H, Ar-H), 8.42 (m,
1H, Ar-H); 13C NMR (125 MHz, CDCl3): d 14.72, 16.46, 17.05,
17.34, 19.49, 20.92, 20.97, 28.98, 29.75, 30.48, 31.51, 31.56,
32.03, 32.22, 36.90, 37.19, 39.34, 39.93, 40.42, 41.77, 50.16,
55.56, 56.64, 62.23, 63.00, 67.02, 67.64, 68.82, 70.09, 70.66,
73.24, 74.18, 76.32, 79.71, 80.97, 98.53 (C-1II), 99.99 (C-1I),
109.47 (C-220), 121.92 (C-60), 122.04, 126.24, 128.64, 128.69,
129.28, 129.98, 130.06, 130.18, 133.15, 133.35, 133.82, 136.13,
140.44 (C-50), 148.27, 165.63 (CO), 166.19 (CO), 167.13 (CO),
170.01 (CO), 170.26 (CO), 170.35 (CO); LRESIMS (m/z): 1228
(M+Na+); HRESIMS calcd for C66H80N2O19Na (M+Na+): 1227.5252,
found: 1227.5240.
4.13. Diosgenyl 2,3,4-tri-O-acetyl-
3,6-di-O-benzoyl-2-deoxy-2-{5-[(R/S)-1,2-dithiolan-3-yl]-
pentanamido}-b- -glucopyranoside (20)
a-L-rhamnopyranosyl-(1?4)-
D
4.15. Diosgenyl 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl-(1?4)-
Toasolutionof 18(95 mg, 0.09 mmol)indryDMF(5.0 mL), ( )-
a-
3,6-di-O-benzoyl-2-deoxy-2-(3,5-dinitrobenzamido)-b-D-gluco-
lipoic acid (18 mg, 0.09 mmol), DIPEA (0.03 mL, 0.17 mmol), and
HBTU (33 mg, 0.09 mmol) were added and the mixture was stirred
at room temperature for 16 h. The solvent was removed in vacuo
and the residue was dissolved in CH2Cl2 (150 mL) and washed with
H2O (10 mL ꢄ 2). The organic layer was dried over anhydrous
Na2SO4 and concentrated under vacuum. The residue was subjected
to flash chromatography (toluene–acetone, 9:1) to give 20 (92 mg,
83%) as an inseparable diastereomeric mixture (in 1:1 ratio). Rf
pyranoside (22)
In a similar way as described for the preparation of 2, com-
pound 18 (150 mg, 0.14 mmol) was treated with 3,5-dinitrobenzo-
ic acid (34 mg, 0.16 mmol) in the presence of HBTU (53 mg,
0.14 mmol), DIPEA (0.05 mL, 0.28 mmol) in dry DMF (5.0 mL) to
give 22 (143 mg, 83%). Rf 0.32 (toluene–acetone, 11:1); ½a D22
ꢂ
ꢀ25.9 (c 0.5, CHCl3); 1H NMR (500 MHz, CDCl3): d 0.73 (d, 3H, J
6.0 Hz, CH3), 0.76 (s, 3H, CH3), 0.78 (d, 3H, J 6.0 Hz, CH3), 0.91 (s,
3H, CH3), 0.96 (d, 3H, J 6.5 Hz, CH3), 1.91 (s, 3H, CH3CO), 1.97 (s,
3H, CH3CO), 2.03 (s, 3H, CH3CO), 3.37 (dd, 1H, J 11.0, 11.0 Hz, H-
260a), 3.47 (m, 1H, H-260b), 3.54 (m, 1H, H-30), 3.73 (m, 1H, H-
5II), 4.01 (m, 1H, H-5I), 4.20 (dd, 1H, J 9.5, 8.5 Hz, H-4I), 4.40 (ap-
prox. q, 1H, J ꢃ 7.0 Hz, H-160), 4.41 (m, 1H, H-2I), 4.55 (dd, 1H, J
12.5, 5.0 Hz, H-6Ia), 4.90 (dd, 1H, J 9.5, 10.0 Hz, H-4II), 4.92 (m,
1H, H-6Ib), 4.96 (br s, 1H, H-1II), 4.97 (d, 1H, J 8.0 Hz, H-1I), 5.13
(m, 1H, H-2II), 5.14 (dd, 1H, J 10.0 Hz, 3.0 Hz, H-3II), 5.19 (m, 1H,
H-60), 5.67 (dd, 1H, J 9.0, 8.5 Hz, H-3I), 7.01 (d, 1H, J 9.0 Hz, NH),
7.38–7.62 (m, 6H, Ar-H), 8.01–8.10 (m, 4H, Ar-H), 8.80 (d, 2H, J
2.0 Hz, Ar-H), 9.08 (t, 1H, J 2.0 Hz, Ar-H); 13C NMR (125 MHz,
CDCl3): d 14.73, 16.48, 17.08, 17.36, 19.49, 20.90, 20.95, 28.99,
29.79, 30.49, 31.51, 31.57, 32.05, 32.23, 36.90, 37.18, 39.45,
39.93, 40.44, 41.78, 50.17, 55.66, 56.65, 62.25, 62.92, 67.04,
67.71, 68.86, 70.05, 70.60, 73.22, 74.19, 75.99, 79.60, 80.98, 98.44
(C-1II), 99.74 (C-1I), 109.50 (C-220), 121.32 (C-60), 122.10, 127.43,
128.70, 128.80, 129.03, 129.88, 130.09, 130.20, 133.46, 134.07,
137.89, 140.27 (C-50), 148.78, 163.40 (CO), 166.29 (CO), 167.27
(CO), 170.01 (CO), 170.35 (CO), 170.55 (CO); LRESIMS (m/z): 1273
(M+Na+); HRESIMS calcd for C66H79N3O21Na (M+Na+): 1272.5103,
found: 1272.5095.
0.29(toluene–acetone, 9:1); ½a D22
ꢂ ꢀ50.7(c0.33, CHCl3)for thediaste-
reomeric mixture (1:1); 1H NMR (500 MHz, CDCl3): d 0.68 (d, 3H, J
6.0 Hz, CH3), 0.78 (s, 3H, CH3), 0.79 (d, 3H, J 6.5 Hz, CH3), 0.95 (s,
3H, CH3), 0.98 (d, 3H, J 7.0 Hz, CH3), 1.91 (s, 3H, CH3CO), 1.95 (s,
3H, CH3CO), 2.00 (s, 3H, CH3CO), 3.03–3.15 (m, 2H, CH2S), 3.31 (m,
1H), 3.37 (dd, 1H, J 11.0, 11.0 Hz, H-260a), 3.46 (m, 2H, H-30, H-
260b), 3.69 (m, 1H, H-5II), 3.86 (m, 1H, H-5I), 4.04 (m, 1H, H-2I),
4.05 (dd, 1H, J 9.0, 9.5 Hz, H-4I), 4.42 (approx. q, 1H, J ꢃ 7.5 Hz, H-
160), 4.51 (dd, 1H, J 12.0, 5.0 Hz, H-6Ia), 4.78 (m, 2H, H-1I, H-6Ib),
4.87 (br s, 1H, H-1II), 4.88 (dd, 1H, J 9.5, 9.5 Hz, H-4II), 5.14 (m, 1H,
H-2II), 5.15 (m, 1H, H-3II), 5.28 (m, 1H, H-60), 5.54 (d, 1H, J 9.0 Hz,
NH), 5.56 (m, 1H, H-3I), 7.45 (m, 4H, Ar-H), 7.58 (m, 2H, Ar-H), 8.05
(m, 4H, Ar-H); 13C NMR (125 MHz, CDCl3): d 14.72, 16.47, 17.01,
17.33, 19.53, 20.90, 20.95, 25.35, 28.76, 28.98, 29.71, 30.48, 31.56,
32.03, 32.26, 34.66, 36.64, 36.95, 37.20, 38.57, 39.22, 39.94, 40.29,
40.44, 41.78, 50.17, 54.98, 56.27, 56.67, 62.26, 63.07, 67.04, 67.59,
68.72, 70.15, 70.74, 73.17, 73.89, 76.80, 79.72, 80.99, 98.62 (C-1II),
99.90 (C-1I), 109.49 (C-220), 121.92 (C-60), 128.58, 128.72, 129.61,
130.01, 130.18, 133.25, 133.67, 140.58 (C-50), 166.08 (CO), 166.68
(CO), 170.02 (CO), 170.16 (CO), 170.22 (CO), 172.96 (CO); LRESIMS
(m/z): 1267 (M+Na+); HRESIMS calcd for C67H89NO17S2Na (M+Na+):
1266.5469, found: 1266.5463.

7678
M. J. Kaskiw et al. / Bioorg. Med. Chem. 17 (2009) 7670–7679
4.16. Diosgenyl
a-
L-rhamnopyranosyl-(1?4)-2-benzamido-2-
32.86, 33.25, 38.07, 38.57, 40.35, 41.02, 41.54, 43.02, 51.74,
deoxy-b-
D
-glucopyranoside (23)
57.90, 58.80, 62.12, 63.86, 67.99, 70.77, 72.36, 72.58, 73.87,
74.46, 77.15, 80.13, 80.63, 82.32, 101.26 (C-1II), 103.13 (C-1I),
110.71 (C-220), 122.85 (C-60), 123.42, 127.18, 131.26, 134.56,
138.14, 141.84 (C-50), 149.79, 168.49 (CO); LRESIMS (m/z): 893
(M+Na+); HRESIMS calcd for C46H66N2O14Na (M+Na+): 893.4411,
found: 893.4403.
In a similar way as described for the preparation of 4, com-
pound 19 (50 mg, 0.04 mmol) was treated with sodium methoxide
to yield 23 (33 mg, 95%). Rf 0.47 (CH2Cl2–MeOH, 5:1); ½a D22
ꢀ55.2 (c
ꢂ
0.33, MeOH); 1H NMR (500 MHz, C5D5N plus two drops of D2O): d
0.68 (d, 3H, J 5.0 Hz, CH3), 0.80 (s, 3H, CH3), 0.81 (s, 3H, CH3), 1.13
(d, 3H, J 7.0 Hz, CH3), 1.68 (d, 3H, J 5.5 Hz, CH3), 2.39 (m, 1H), 2.68
(m, 1H), 3.49 (dd, 1H, J 10.5, 10.5 Hz, H-260a), 3.58 (m, 1H, H-260b),
3.78 (m, 1H, H-5I), 3.83 (m, 1H, H-30), 4.13 (dd, 1H, J 12.0, 4.5 Hz, H-
6Ia), 4.26 (br d, 1H, J 11.0 Hz, H-6Ib), 4.37 (dd, 1H, J 9.5, 9.5 Hz),
4.52 (m, 2H), 4.57 (dd, 1H, J 9.0, 3.0 Hz, H-3II), 4.63 (dd, 1H, J
9.0 Hz, 9.5 Hz), 4.72 (br s, 1H, H-2II), 4.76 (dd, 1H, J 9.5 Hz,
9.5 Hz), 5.02 (m, 1H), 5.54 (d, 1H, J 8.5 Hz, H-1I), 5.71 (m, 1H, H-
60), 5.93 (br s, 1H, H-1II), 7.40 (m, 3H, Ar-H), 8.40 (m, 2H, Ar-H),
9.57 (d, 1H, J 8.0 Hz, NH); 13C NMR (125 MHz, C5D5N): d 13.38,
16.67, 17.66, 18.83, 19.66, 21.39, 29.57, 30.47, 30.92, 31.90,
32.12, 32.54, 37.28, 37.71, 39.87, 40.14, 40.75, 42.27, 50.52,
55.42, 56.92, 61.75, 63.17, 67.17, 70.74, 72.97, 73.17, 74.07,
74.35, 77.67, 79.05, 79.49, 81.41, 100.65 (C-1II), 103.24 (C-1I),
109.59 (C-220), 122.11 (C-60), 128.44, 129.00, 131.64, 137.14,
141.03 (C-50), 168.84 (CO); LRESIMS (m/z): 848 (M+Na+); HRESIMS
calcd for C46H67NO12Na (M+Na+): 848.4560, found: 848.4552.
4.19. Diosgenyl
a-
L-rhamnopyranosyl-(1?4)-2-deoxy-2-(3,5-
dinitrobenzamido)-b-
D
-glucopyranoside (26)
In a similar way as described for the preparation of 4, com-
pound 22 (62 mg, 0.05 mmol) was converted to 26 (42 mg, 91%).
Rf 0.55 (CH2Cl2–MeOH, 5:1); ½a D22
ꢂ
ꢀ58.0 (c 0.1, MeOH); 1H NMR
(500 MHz, CD3OD): d 0.74 (s, 3H, CH3), 0.75 (d, 3H, J 6.0 Hz, CH3),
0.90 (d, 3H, J 7.0 Hz, CH3), 0.92 (s, 3H, CH3), 1.20 (d, 3H, J 6.0 Hz,
CH3), 2.03 (m, 1H), 2.26 (m, 1H), 3.27 (m, 2H, 2 H-260), 3.37 (dd,
1H, J 10.0, 9.5 Hz), 3.38 (m, 1H), 3.50 (m, 1H, H-30), 3.59 (dd, 1H,
J 9.5, 3.5 Hz, H-3II), 3.61 (dd, 1H, J 9.5, 9.5 Hz), 3.68 (dd, 1H, J
12.0, 4.0 Hz), 3.77 (dd, 1H, J 10.5, 9.0 Hz), 3.82 (m, 2H), 3.85 (dd,
1H, J 10.5, 8.5 Hz), 3.92 (m, 1H, H-5II), 4.33 (approx. q, 1H,
J ꢃ 7.5 Hz, H-160), 4.70 (d, 1H, J 8.0 Hz, H-1I), 5.25 (m, 1H, H-60),
9.04 (d, J 2.0 Hz, Ar-H), 9.11 (t, 1H, J 2.0 Hz, Ar-H); The signal for
the L
-rhamnopyranosyl anomeric proton (H-1II) was not observed;
it was likely buried under the large solvent peak at d 4.87 ppm. 13C
NMR (125 MHz, CD3OD): d 15.03, 16.89, 17.65, 17.96, 19.91, 22.09,
30.01, 30.80, 31.58, 32.54, 32.86, 33.25, 38.07, 38.56, 40.34, 41.02,
41.53, 43.02, 51.74, 57.89, 59.02, 62.09, 63.85, 67.98, 70.77, 72.36,
72.57, 73.85, 74.47, 77.20, 80.03, 80.51, 82.32, 101.04 (C-1II),
103.17 (C-1I), 110.71 (C-220), 122.19 (C-60), 122.91, 128.59,
139.53, 141.79 (C-50), 150.25, 166.09 (CO); LRESIMS (m/z): 938
(M+Na+); HRESIMS calcd for C46H65N3O16Na (M+Na+): 938.4262,
found: 938.4256.
4.17. Diosgenyl
S)-1,2-dithiolan-3-yl]-pentanamido}-b-
a
-
L
-rhamnopyranosyl-(1?4)-2-deoxy-2-{5-[(R/
D-glucopyranoside (24)
In a similar way as described for the preparation of 4, com-
pound 20 (50 mg, 0.04 mmol) was converted to 24 (46 mg, 96%)
as a diastereomeric mixture in 1:1 ratio. Rf 0.53 (CH2Cl2–MeOH,
5:1); ½a 2D2
ꢀ78.0 (c 0.1, MeOH) for the diastereomeric mixture
ꢂ
(1:1); 1H NMR (500 MHz, CD3OD): d 0.76 (d, 3H, J 6.5 Hz, CH3),
0.77 (s, 3H, CH3), 0.92 (d, 3H, J 7.0 Hz, CH3), 0.99 (s, 3H, CH3),
1.20 (d, 3H, J 6.5 Hz, CH3), 2.21 (m, 2H), 2.30 (m, 1H), 2.41 (m,
1H), 3.05 (m, 1H), 3.13 (m, 1H), 3.25–3.31 (m, 2H), 3.36 (dd, 1H, J
10.5, 10.5 Hz, 1H, H-260), 3.41 (m, 1H), 3.46 (m, 1H), 3.49–3.64
(m, 5H), 3.75–3.79 (m, 2H), 3.90 (m, 1H), 4.35 (approx. q, 1H,
J ꢃ 7.5 Hz, H-160), 4.53 (d, 0.5H, J 8.0 Hz, H-1I), 4.54 (d, 0.5H, J
8.0 Hz, H-1I), 4.82 (d, 1H, J 1.5 Hz, H-1II), 5.33 (m, 1H, H-60); 13C
NMR (125 MHz, CD3OD): d 15.03, 16.92, 17.65, 17.99, 20.09,
22.11, 27.02, 30.02, 30.13, 30.78, 31.58, 32.54, 32.88, 33.32,
40.39, 41.04, 41.46, 41.56, 43.04, 51.74, 57.69, 57.91, 70.76,
72.34, 72.57, 73.85, 74.58, 76.99, 80.21, 82.35, 101.02 ((C-1II),
103.10 (C-1I), 110.73 (C-220), 122.91 (C-60), 141.98 (C-50), 176.47
(CO); LRESIMS (m/z): 932 (M+Na+); HRESIMS calcd for
C47H75NO19S2Na (M+Na+): 932.4628, found: 932.4619.
4.20. Cell lines, cell culture, and medium
Cell lines used in this study include MCF-7 (breast cancer)
and HeLa (cervical cancer). MCF-7 cell line was supplied by the
American Type Culture Collection (Manassas, Virginia), and HeLa
cell line was obtained from the Lady Davis Institute for Medical
Research (Montreal, Quebec). All cell lines were grown in Dul-
becco’s Modified Eagle’s Medium (Sigma) containing 10% fetal
bovine serum (PAA Laboratories) at 37 °C in a 5% CO2 humidified
atmosphere.
4.21. Cell proliferation assay
Measurement of cell proliferation was determined using the 3-
4.18. Diosgenyl
nitrobenzamido)-b-
a
-
L
-rhamnopyranosyl-(1?4)-2-deoxy-2-(3-
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
D
-glucopyranoside (25)
(MTT) assay as described by Carmichael et al.24 Briefly, 2000 cells
were plated out into each well of a 96-well plate and allowed to
adhere. Saponin analogues, dissolved in dimethylsulfoxide (DMSO,
Sigma) and diluted with tissue culture medium, were then added
In a similar way as described for the preparation of 4, com-
pound 21 (50 mg, 0.04 mmol) was converted to 25 (32 mg,
92%). Rf 0.50 (CH2Cl2–MeOH, 5:1); ½a D22
ꢂ
ꢀ41.8 (c 0.5, MeOH);
at increasing concentrations (0–50 lM, eight wells per concentra-
1H NMR (500 MHz, CD3OD): d 0.75 (m, 6H, 2CH3), 0.91 (d, 3H,
J 7.0 Hz, CH3), 0.92 (s, 3H, CH3), 1.21 (d, 3H, J 6.5 Hz, CH3),
3.27 (dd, 1H, J 10.5, 11.5 Hz, H-260a), 3.28 (m, 1H, H-260b)),
3.38 (dd, 1H, J 10.0, 9.5 Hz), 3.39 (m, 1H), 3.49 (m, 1H, H-30),
3.57–3.60 (m, 2H), 3.68 (dd, 1H, J 12.0, 4.0 Hz, H-6Ib), 3.75–
3.87 (m, 4H), 3.93 (m, 1H, H-5II), 4.34 (approx. q, 1H, J ꢃ 7.5 Hz,
H-160), 4.70 (d, 1H, J 8.5 Hz, H-1I), 5.24 (m, 1H, H-60), 7.72 (t, 1H,
J 8.0 Hz, Ar-H), 8.20 (m, 1H, Ar-H), 8.38 (m, 1H, Ar-H), 8.69 (m,
tion). The cells were incubated in the presence of each saponin
analogue for 72 h. MTT reagent (Sigma) dissolved in phosphate
buffered saline was added to each well at a concentration of
0.5 mg/mL, and the cells were incubated for four additional hours.
Following this time, the medium containing the MTT reagent was
aspirated, and DMSO (100 lL) was added to each well. The absor-
bance of each well was measured in a microplate reader (Power-
Wave XS, Bio-Tek) at a wavelength of 490 nm. The IC50 value for
each compound tested was determined by plotting concentration
versus percent absorbance obtained in the MTT assay. The IC50 re-
sults listed in Table 1 represent the average IC50 value obtained
from multiple MTT assays for each compound.
1H, Ar-H); The signal for the L-rhamnopyranosyl anomeric pro-
ton (H-1II) was not observed; it was likely buried under the large
solvent peak at d 4.87 ppm. 13C NMR (125 MHz, CD3OD): d 15.04,
16.91, 17.65, 17.97, 19.92, 22.10, 30.02, 30.80, 31.58, 32.54,

M. J. Kaskiw et al. / Bioorg. Med. Chem. 17 (2009) 7670–7679
7679
10. Bachran, C.; Bachran, S.; Sutherland, M.; Bachran, D.; Fuchs, H. Mini-Rev. Med.
Chem. 2008, 8, 575.
Acknowledgments
11. Gnoula, C.; Mégalizzi, V.; Nève, N. D.; Sauvage, S.; Ribaucour, F.; Guissou, P.;
Duez, P.; Dubois, J.; Ingrassia, L.; Lefranc, F.; Kiss, R.; Mijatovic, T. Int. J. Oncol.
2008, 32, 5.
12. Lee, M.-S.; Yuet-Wa, J. C.; Kong, S.-K.; Yu, B.; Eng-Choon, V. O.; Nai-Ching, H.
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This work was supported by Natural Sciences and Engineering
Research Council of Canada (NSERC, Grant 312630) and Lakehead
University.
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