Synthesis and cytotoxic activity of diosgenyl saponin analogues

Article abstract of DOI:10.1016/j.bmc.2007.12.022

Diosgenyl saponins are steroidal glycosides that are often found as major components in many traditional oriental medicines. Recently, a number of naturally occurring diosgenyl saponins have been shown to exert cytotoxic activity against several strains o

Full text of DOI:10.1016/j.bmc.2007.12.022

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3209–3217
Synthesis and cytotoxic activity of diosgenyl saponin analogues
Matthew J. Kaskiw,a Mary Lynn Tassotto,b John Th’nga,b,c and Zi-Hua Jianga,*
aDepartment of Chemistry, Lakehead University, 955 Oliver Road, Thunder Bay, Ont., Canada P7B 5E1
bRegional Cancer Care, Thunder Bay Regional Health Sciences Centre, 980 Oliver Road, Thunder Bay, Ont., Canada P7B 6V4
cMedical Sciences Division, Northern Ontario School of Medicine, 955 Oliver Road, Thunder Bay, Ont., Canada P7B 5E1
Received 6 October 2007; revised 7 December 2007; accepted 11 December 2007
Available online 27 December 2007
Abstract—Diosgenyl saponins are steroidal glycosides that are often found as major components in many traditional oriental med-
icines. Recently, a number of naturally occurring diosgenyl saponins have been shown to exert cytotoxic activity against several
strains of human cancer cells. Use of these saponin compounds for cancer treatment is hampered due to the lack of understanding
of their action mechanism as well as limited access to such structurally complicated molecules. In the present paper, we have pre-
pared a group of diosgenyl saponin analogues which contain a b-D-2-amino-2-deoxy-glucopyranose residue having different substit-
uents at the amino group. Moderate cytotoxic activity is found for most analogues against neuroblastoma (SK-N-SH) cells, breast
cancer (MCF-7) cells, and cervical cancer (HeLa) cells. The analogue 13 that contains an a-lipoic acid residue exhibits the highest
potency against all three cancer cell lines with IC50 ranging from 4.8 lM in SK-N-SH cells to 7.3 lM in HeLa cells. Preliminary
mechanistic investigation with one saponin analogue (10) shows that the compound induces cell cycle arrest at G1 phase in
SK-N-SH cells, but the same compound induces cell cycle arrest at G2 phase in MCF-7 cells. This result suggests that the cytotoxic
activity of these saponin analogues may involve different action mechanisms in cell lines derived from different cancer sites.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
attached to the 3-hydroxyl group of the aglycone is usu-
ally b-D-glucopyranose which in turn often has an a-L-
rhamnopyranose residue attached to the 2-O-position
and another sugar or sugar chain to the 3-O- or 4-O-po-
sition.1 Diosgenyl saponins have great potential to be
explored for cancer treatment. However, due to the
complexity of their structures and thus limited accessi-
bility, as well as a poor understanding of the underlying
mechanism of their antitumor activity, no diosgenyl
saponin molecule has yet been clinically used as an anti-
cancer agent. To date, limited information on the struc-
ture activity relationship (SAR) of diosgenyl saponins
and their cytotoxicity is available.8 Although both the
aglycone and the carbohydrate moieties are required
for the exhibition of their antitumor activity, the exact
role of the carbohydrate moiety remains unclear. Re-
cently, Myszka et al.14 reported the apoptosis-inducing
property in B cell chronic leukemia cells by synthetic
diosgenyl 2-amino-2-deoxy-b-D-glucopyranoside hydro-
chloride and one urea derivative in combination with
cladribine (2-CdA). Their finding indicates that diosge-
nyl saponin analogues carrying only one sugar residue
might show good antitumor potential. We initiated
our research on diosgenyl saponins in order to discover
simpler structural analogues with potent anticancer
activity. Toward that end, we have designed and
Saponins are steroid or triterpenoid glycosides that are
widely distributed in plants and some marine organisms.
Saponins exhibit a broad spectrum of biological activi-
ties and have attracted much attention in recent years.1
Diosgenyl saponins bear the diosgenin as the aglycone,
and they are often found as major components in many
traditional Chinese medicines. Various biological activi-
ties have been observed for this group of saponins,
including antibacterial,2 antiviral,3 antifungal,4 and anti-
inflammatory activities.5 Recently, a number of diosge-
nyl saponins were found to exhibit cytotoxic properties
against several strains of human cancer cells,6–8 and
these saponins exerted their antitumor effect by inducing
apoptosis in cancer cells.9–12 Among a great number of
naturally occurring diosgenyl saponins, dioscin9,12,13 is
best known to have these properties (Fig. 1).
The structural diversity of naturally occurring diosgenyl
saponins lies mainly in the sugar moiety. The first sugar
Keywords: Saponin; Diosgenin; Cytotoxicity; Anticancer; Synthesis.
*
Corresponding author. Tel.: +1 807 766 7171; fax: +1 807 346
7775; e-mail: zjiang@lakeheadu.ca
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.022

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M. J. Kaskiw et al. / Bioorg. Med. Chem. 16 (2008) 3209–3217
26
O
20
O
27
O22
11
H
O
H
16
OH
O
H
H
OH
3
H
H
O
O
HO
HO
O
HO
HO
O
6
O
O
NH
HO
R
O
HO
HO
Dioscin
Analogues
OH
OH
Figure 1. The structures of diosgenyl saponin dioscin and designed analogues.
synthesized a group of analogues having a b-D-glucosa-
mine residue attached to the aglycone (Fig. 1). The pres-
ence of the NH2 group allows the straightforward
preparation of structural analogues with different sub-
stituents at the amino group. In this paper, we are
describing the synthesis and cytotoxic activity of these
saponin analogues against a number of human cancer
cells. Also presented are the structural features in rela-
tion to their activity and the preliminary result of the
saponin effects on cell cycle arrest.
which can be cleaved under reductive b-elimination.20
Due to its neighboring group participating capacity,
the Troc group has been widely employed in the synthe-
ses of b-glycosides of glucosamine derivatives.21,22 For
our present targets, we choose the readily available gly-
cosyl imidate 122 as the glycosylation donor (Scheme 1)
to construct the b-linked glycosides. Thus, the glycosyl-
ation reaction between diosgenin and 1 in the presence
of trimethylsilyl trifluoromethanesulfonate (TMSOTf)
as the catalyst affords the expected b-glycoside 2 in
98% yield. The b-linkage in 2 is confirmed by the cou-
1
pling constant of the anomeric proton of its H NMR
2. Results and discussion
2.1. Chemical synthesis
spectral data (d 4.80, d, J = 8.5 Hz).22 Compound 2 is then
treated with zinc dust in acetic acid to provide free amine 3
in good yield. Treatment of 3 with various acylating re-
agents gives compounds 4–8 in good yield (73–92%).
The coupling reaction between 3 and N-acetyl-L-alanine
results in almost complete racemization of the alanine res-
idue when N,N0-diisopropylcarbodiimide (DIC) is used as
the condensation agent. The so-formed diastereomeric
A number of diosgenyl saponins and derivatives have
been synthesized in recent years,15,16 most notably by
the group of Yu.17–19 The 2,2,2-trichloroethoxycarbonyl
(Troc) group is an efficient amine-protection group
O
O
O
O
H
H
OAc
OAc
O
OAc
O
Diosgenin
H
H
H
c
H
O
b
AcO
AcO
AcO
AcO
AcO
AcO
O
O
3
a
TrocHN
OC(NH)CCl3
TrocHN
NH2
2
1
O
O
O
O
H
H
d
OAc
O
OH
H
H
H
H
O
HO
HO
AcO
AcO
O
O
NHR
NHR
9 R = Ac
4 R = Ac
5 R = Bz
10 R = Bz
11 R = HO2CCH2CH2CO
6 R = HO2CCH2CH2CO
CH3
CH3
12 R =
7 R =
AcHN
CO
AcHN
CO
CO
CO
13 R =
8 R =
S
S
S
S
Scheme 1. Reagents and conditions: (a) TMSOTf, CH2Cl2, 0 ꢁC, 30 min, 98%; (b) Zn dust, HOAc, rt, 16 h, 96%; (c) for 4, Ac2O, pyridine, rt, 4 h,
92%; for 5, BzCl, pyridine, rt, 4 h, 84%; for 6, succinic anhydride, pyridine, rt, 4 h, 73%; for 7, AcHN-L-Ala-OH, HBTU, DIPEA, DMF, rt, 16 h,
78%; for 8, ( )-a-lipoic acid, HBTU, DIPEA, DMF, rt, 16 h, 83%; (d) NaOCH3, CH3OH–CH2Cl2, rt, 4 h, 95–100%.

M. J. Kaskiw et al. / Bioorg. Med. Chem. 16 (2008) 3209–3217
3211
O
O
O
O
H
H
OR
O
H
H
OR
O
RO
RO
H
H
O
RO
RO
O
NH
O
a
NH
+
HO
O
+
3
OH
O
OH
O
O
O
O
O
H
HN
H
H
14 R = Ac
16 R = H
b
RO
RO
O
O
15 R = Ac
17 R = H
b
RO
Scheme 2. Reagents and conditions: (a) HBTU, DIPEA, DMF, rt, 16 h, 21% for 14 and 56% for 15; (b) CH3ONa, CH3OH/CH2Cl2 (1:1), rt, 4 h, 98–
100%.
Table 1. The in vitro cytotoxic activity (IC50, lM) of synthetic saponin
analoguesa,b,c
mixture of 7 is chromatographically inseparable, but
readily recognized on its H NMR spectrum (500 MHz
1
Compound
Cancer cell line
MCF-7
in CDCl3: d 4.84, d, J = 8.5 Hz for the anomeric proton
of the sugar residue in L-alanine-bearing isomer; d 4.90,
d, J = 8.5 Hz for the anomeric proton of the sugar residue
in D-alanine-bearing isomer). This racemization can be
largely suppressed (<5%) when 2-(1H-benzotriazole-
1-yl)-1,1,3,3,-tetramethylaminium hexafluorophosphate
(HBTU)23 is employed as the activating agent. The final
removal of the acetyl groups in 4–8 by treatment with so-
dium methoxide in dichloromethane–methanol affords
the corresponding diosgenyl saponin analogues 9–13,
respectively. The structures of compounds 9–13 have been
confirmed by 1H NMR, 13C NMR, and MS spectral data.
SK-N-SH
HeLa
21.5 1.06
9
10
11.6 0.88
14.9 1.62
14.1 1.37
15.3 0.42
4.8 0.28
18.3 0.63
19.4 0.35
22.0 1.41
11.0 0.35
6.9 0.60
11.3 0.42
19.2 0.56
0.06
18.0 1.41
13.9 0.88
20.0 1.27
7.3 0.56
11
12
13
16
16.3 0.56
19.5 0.98
>20.0
>20.0
17
Doxorubicin
a The inhibitory concentration (IC50) is the concentration in lM that
inhibits cell growth by 50% in three days compared to cells that
remained untreated.
b IC50 results are based on the average absorbance readings from eight
replicate wells per concentration of compound tested, and represent
the average IC50 SD value obtained from a minimum of n = 2
independent MTT assays for each compound.
Many biological systems interact through multiple
simultaneous molecular contacts, and in recent years
the design of multivalent ligands and inhibitors has be-
come a focus of inquiry in molecular biochemistry and
drug discovery.24 In the present study, we have prepared
one dimeric diosgenyl saponin analogue (Scheme 2),
hoping to improve the antitumor activity of the mono-
meric analogue. Thus, the reaction of two equivalents
of amine 3 with one equivalent of dicarboxylic acid, adi-
pic acid, in the presence of HBTU as the activating
agent, gives the desired dimer 15 in 56% yield, together
with 21% of the monomeric product 14. Compounds 14
and 15 are treated with sodium methoxide to give sapo-
nin analogues 16 and 17, respectively, in nearly quanti-
tative yield. Their structures have been confirmed by
spectroscopic methods.
c MCF-7 cells were treated with the chemotherapeutic agent Doxoru-
bicin as a positive control for the MTT assay.
the method described by Carmichael et al.,25 and the re-
sults are listed in Table 1. The results are presented as
the inhibitory concentration (IC50) that inhibits the
growth of cells by 50% when compared with untreated
cells. Moderate inhibitory activity was observed for
most compounds against all three cancer cell lines, with
IC50 values in the lM range, varying from 4.8 lM (for
compound 13 in SK-N-SH cells) to greater than
20 lM (for compounds 16 and 17 in HeLa cells). When
comparing SK-N-SH and HeLa cells, the cervical cancer
cell line (HeLa) is slightly more resistant to the inhibi-
tory effects of all compounds, with the exception of com-
pound 11. At concentrations greater than the IC50
values, the MTT absorbance readings for each of com-
pounds 9–13 against all three cancer cell lines ap-
proached zero, indicating that the compounds were
cytotoxic at those concentrations and that cell death
had occurred. These observations, in addition to the loss
of cell viability shown by the MTT assay, indicate that
the saponin analogues tested have both growth inhibi-
tory and cytotoxic properties.
2.2. Biological evaluation
Each of these saponin analogues is new compound, with
the exception of compound 9. The synthesis of com-
pound 9 was reported earlier,14 but no biological activity
has yet been described for this compound. We investi-
gated the cytotoxic activity of each of these compounds
against several human cancer cell lines including SK-N-
SH (neuroblastoma), MCF-7 (breast cancer), and HeLa
(cervical cancer). The rapid assay employed was the
MTT method that measures cell viability, according to

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M. J. Kaskiw et al. / Bioorg. Med. Chem. 16 (2008) 3209–3217
In regard to the structure–activity relationship (SAR) of
these saponin compounds, the structural modification at
the 2-position of the sugar residue appears to affect the
potency and selectivity of their anticancer activity. Most
notably, compound 13 which has an a-lipoic acid resi-
due is the most active one against all three cancer cell
lines. a-Lipoic acid is a biogenic antioxidant that phys-
iologically acts as a coenzyme in the process of oxidative
decarboxylation of a-keto acids. In cells, the disulfide
bond of a-lipoic acid can readily be reduced to produce
its open chain form as dihydrolipoic acid.26 a-Lipoic
acid has been reported to inhibit the proliferation of
WM35 and A350 melanoma cells at different concentra-
tions,27 although the exact mechanism is unclear. Other
disulfide organic sulfur compounds (OSC) are also
known for their antiproliferative actions on human can-
cers.28 Generally, disulfides can act as oxidants as well as
reductants in biological systems, but their oxidizing
behavior is more pronounced.29 Whether or not the in-
creased cytotoxicity of compound 13 might be related
to the redox property of the a-lipoic acid residue at-
tached at the 2-N-position of the sugar is worth further
investigation. Among all the analogues, compounds 11
and 16 are perhaps structurally most close: compound
11 bears a succinic acid residue, while 16 an adipic acid
residue which has two more CH2 units. Compound 11
(IC50 22.0 lM) is less active than 16 (IC50 11.3 lM)
against MCF-7 cells, but their relative activity is re-
versed against HeLa cells (IC50 13.9 lM for 11 and
IC50 > 20.0 lM for 16). Selective activity against differ-
ent cancer cells is also observed for compounds 12 (with
the L-alanine residue) and 16. Both compounds are more
active against MCF-7 cells than against the other two
cell types. The dimeric analogue 17 shows, however,
lower activity than the monomeric compound 16 against
all three cancer cell lines tested. This may indicate that
divalent structures do not result in increased anticancer
activity for diosgenyl saponins.
at either G1 or G2 phase in different cancer cells suggests
that the cytotoxic effect of diosgenyl saponins may in-
volve different action mechanisms in different cancer
cells.
3. Conclusion
We have prepared a group of diosgenyl saponin ana-
logues bearing a b-D-2-amino-2-deoxy-glucopyranose
residue with different substituents at the amino group.
Moderate cytotoxic activity is found for most analogues
against SK-N-SH neuroblastoma cells, MCF-7 breast
cancer cells, and HeLa cervical cancer cells. The ana-
logue 13 that contains an a-lipoic acid residue exhibits
the highest activity against all three cell lines with IC50
value in the lM range. Further research is focused on
the design and synthesis of more structural analogues
with increased cytotoxicity and selectivity, as well as
more detailed investigation of their action mechanisms.
4. Experimental
4.1. General method
All air and moisture sensitive reactions were performed
under nitrogen atmosphere. Anhydrous N,N-dimethyl-
formamide (DMF) was purchased from Aldrich, and
other dry solvents were prepared in usual way. ACS
grade solvents were purchased from Fisher and used
for chromatography without distillation. TLC plates
(silica gel 60F254, thickness 0.25 mm) and silica gel 60
(40–63 lm) for flash column chromatography were pur-
chased from SILICYCLE Inc., Canada. 1H and 13C
NMR spectra were recorded on Varian Unity Inova
500 MHz spectrometer. Tetramethylsilane (TMS, d
0.00 ppm) or solvent peaks were used as internal stan-
dards for 1H and 13C spectra. Multiplicity of proton sig-
nals is indicated as follows: s (singlet), d (doublet), t
(triplet), q (quartet), m (multiplet), br (broad), and ap-
prox (approximate). For spectroscopic assignments, su-
gar carbon atoms are designated with 1, 2, 3. . ., and
diosgenyl carbon atoms are designated with 10, 20,
30 . . ., etc. Optical rotations were measured on a Per-
kin-Elmer 241 Polarimeter at room temperature (20–
22 ꢁC). Low resolution electrospray ionization (ESI)
mass spectra were obtained from a Biflex-IV MALDI
linear/reflector instrument at the University of Mani-
toba, Canada, and high resolution (HR) ESI mass spec-
tra were measured on the Applied Biosystems Mariner
Biospectrometry Workstation at the University of
Alberta, Canada.
The action mechanism of the anticancer activity of dios-
genyl saponins is still poorly understood. Recently, the
cytotoxicity of these saponins has been shown to corre-
late with their propensity for cellular internalization.30
These compounds have also been reported to induce
apoptosis in cancer cells.10–12,14 Dioscin9 and its struc-
tural analogue, methyl protodioscin,31,32 were shown
to induce G2/M cell cycle arrest in HepG2 liver cancer
cells and human leukemia K562 cells. Since the MTT as-
say of the present study measures the activity of reduc-
tases in the mitochondria of viable cells, it is a relative
measure of the number of cells that continue to undergo
active metabolism, and a reduction in the MTT absor-
bance reading relative to untreated cells does not di-
rectly measure cell death. We have since tested the
synthesized saponin compounds against these and other
cancer cell lines from different tissue origins and found
variations in cellular responses, including the mecha-
nisms of cell death (manuscript in preparation). Our pre-
liminary investigation shows that in neuroblastoma
(SK-N-SH) cells, compound 10 induces cell cycle arrest
at G1 phase while in breast cancer (MCF-7) cells, it in-
duces cell cycle arrest at G2 phase (data not shown).
The finding that compound 10 induces cell cycle arrest
4.2. Diosgenyl 3,4,6-tri-O-acetyl-2-deoxy-2-(2,2,2-trichlo-
roethoxycarbonylamino)-b-D-glucopyranoside (2)
A suspension of 1 (5.48 g, 8.77 mmol), diosgenin (3.63 g,
˚
8.64 mmol), and activated molecular sieves (4 A, 2.0 g)
in anhydrous CH2Cl2 (50 mL) was stirred at room tem-
perature for 15 min and then cooled to 0 ꢁC under N2
atmosphere. A solution of trimethylsilyl trifluorometh-
anesulfonate (TMSOTf, 8.0 mL, 0.02 M in CH2Cl2)

M. J. Kaskiw et al. / Bioorg. Med. Chem. 16 (2008) 3209–3217
3213
was added dropwise. The mixture was stirred for 30 min,
quenched by Et3N (0.1 mL), and then filtered through
Celite. The filtrate was concentrated in vacuo with the
resulting residue purified by flash column chromatogra-
phy (CH2Cl2/MeOH, 100:1) to give compound 2 (7.21 g,
q, 1H, J ꢂ 8.0 Hz, H-160), 4.85 (d, 1H, J = 8.0 Hz, H-1),
5.05 (dd, 1H, J = 9.5, 9.5 Hz, H-4), 5.34 (m, 1H, H-60),
5.38 (dd, 1H, J = 9.5. 9.5 Hz, H-3), 5.46 (d, 1H,
J = 8.5 Hz, NH); 13C NMR (125 MHz, CDCl3): d
14.34, 14.74, 16.49, 17.35, 19.59, 21.02, 22.87, 23.64,
29.00, 29.64, 29.92, 30.50, 31.59, 31.80, 32.04, 32.23,
37.06, 37.35, 39.04, 39.94, 40.47, 41.81, 50.23, 55.71,
56.68, 62.27, 62.48, 67.07, 69.02, 71.84, 72.35, 79.81,
81.02, 99.45, 109.53, 122.10, 140.57, 169.10, 170.50,
170.80, 171.10; HRESI-MS Calcd for C41H61NO11Na
[M+Na]+: 766.4137, found (positive mode): 766.4140.
94%) as a white foam. Rf 0.36 (hexane/acetone, 1.5:1);
22
D
½aꢀ ꢁ34.72 (c 0.71, CHCl3); 1H NMR (500 MHz,
CDCl3): d 0.78 (s, 3H, CH3), 0.80 (d, 3H, J = 6.0 Hz,
CH3), 0.98 (d, 3H, J = 7.0 Hz, CH3), 0.99 (s, 3H,
CH3), 2.03 (s, 3H, CH3CO), 2.04 (s, 3H, CH3CO),
2.08 (s, 3 H, CH3CO), 3.37 (dd, 1H, J = 11.0, 11.0 Hz,
H-260a), 3.46–3.55 (m, 3H, H-260b, H-30, H-2), 3.72
(m, 1H, H-5), 4.09–4.15 (m, 1H, H-6b), 4.28 (dd, 1H,
J = 12.0, 4.5 Hz, H-6a), 4.41 (approx q, 1H, J ꢂ 7.5 Hz,
H-160), 4.68 (d, 1H, J = 11.5 Hz, Troc-H), 4.79 (d, 1H,
J = 11.5 Hz, Troc-H), 4.82 (d, 1H, J = 8.5 Hz, H-1),
5.05 (dd, 1H, J = 9.5, 10.0 Hz, H-4), 5.31 (m, 1H, H-
60), 5.38 (dd, 1H, J = 9.5, 10.5 Hz, H-3), 5.48 (d, 1H,
J = 9.0 Hz, NH); 13C NMR (125 MHz, CDCl3): d
14.35, 14.68, 16.42, 17.29, 19.51, 20.81, 20.87, 20.95,
28.91, 29.54, 30.41, 31.49, 31.96, 32.18, 36.96, 37.24,
38.86, 39.86, 40.38, 41.72, 50.14, 56.60, 60.60, 62.18,
62.38, 66.96, 69.05, 71.70, 71.92, 74.50, 79.96, 80.93,
95.65, 99.35, 109.45, 122.02, 140.39, 154.17, 169.71,
170.84, 170.94; HRESI-MS Calcd for C42H60NO12Cl3-
Na [M+Na]+: 898.3073, found (positive mode):
898.3072.
4.4. Diosgenyl 3,4,6-tri-O-acetyl-2-benzamido-2-deoxy-b-
D-glucopyranoside (5)
In a similar way as described for the preparation of 4,
compound 3 (160 mg, 0.228 mmol) was treated with
benzoyl chloride (0.03 mL, 0.26 mmol) in pyridine
(2.0 mL) to give 5 (155 mg, 84%) as a white solid. Rf
22
0.56 (CH2Cl2/MeOH, 25:1); ½aꢀ ꢁ34.7 (c 0.36, CHCl3);
D
1H NMR (500 MHz, CDCl3): d 0.76 (s, 3H, CH3), 0.78
(d, 3H, J = 5.5 Hz, CH3), 0.95 (s, 3H, CH3), 0.96 (d, 3H,
J = 7.5 Hz, CH3), 1.99 (s, 3H, CH3CO), 2.03 (s, 3H,
CH3CO), 2.09 (s, 3 H, CH3CO), 3.37 (dd, 1H,
J = 11.0, 11.0 Hz, H-260a), 3.46 (dd, 1H, J = 11.0,
3.5 Hz, H-260b), 3.50 (m, 1H, H-2), 3.76–3.78 (m, 1H,
H-5), 3.98 (m, 1H, H-30), 4.11 (d, 1H, J = 11.5 Hz, H-
6b), 4.30 (dd, 1H, J = 12.0, 4.7 Hz, H-6a), 4.39 (approx
q, 1H, J ꢂ 7.5 Hz, H-160), 4.95 (d, 1H, J = 8.0 Hz, H-1),
5.12 (dd, 1H, J = 9.5, 9.5 Hz, H-4), 5.25 (m, 1H, H-60),
5.52 (dd, 1H, J = 9.5, 9.5 Hz, H-3), 6.23 (d, 1H,
J = 9.0 Hz, NH); 13C NMR (125 MHz, CDCl3): d
14.71, 16.45, 17.33, 19.53, 20.91, 21.01, 28.97, 29.62,
30.47, 31.55, 32.00, 32.22, 37.00, 37.34, 38.99, 39.91,
40.43, 41.77, 50.19, 55.84, 56.64, 62.24, 62.53, 67.03,
68.69, 71.94, 72.43, 79.95, 80.98, 99.75, 109.49, 121.96,
127.02, 128.90, 131.90, 134.54, 140.56, 167.91, 169.69,
170.98, 171.16; HRESI-MS Calcd for C46H63NO11Na
[M+Na]+: 828.4293, found (positive mode): 828.4289.
4.3. Diosgenyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-b-
D-glucopyranoside (4)
To a solution of 2 (1.65 g, 1.88 mmol) in acetic acid
(40 ml), zinc dust (4.0 g) was added and the mixture
was stirred at room temperature for 16 h. The mixture
was filtered and the solid was thoroughly washed with
CH2Cl2 (100 mL). The filtrate was concentrated in va-
cuo and the residue was re-dissolved with CH2Cl2
(400 mL). The solution was washed with saturated
NaHCO3 solution (50 mL), dried with Na2SO4, and
concentrated under vacuum to give a white solid 3
(1.27 g, 96%) which was used without further purifica-
tion in the next step.
4.5. Diosgenyl 3,4,6-tri-O-acetyl-2-(3-carboxylpropanami-
do)-2-deoxy-b-D-glucopyranoside (6)
To a cooled solution (ice water bath) of 3 (97 mg,
0.14 mmol) in pyridine (3.2 mL), acetic anhydride
(2.5 mL) was added. The mixture was stirred at room
temperature for 4 h. The solution was diluted with
CH2Cl2 (100 mL) and washed successively with cold
4 N HCl solution (100 mL) and saturated NaHCO3
solution (50 mL · 2). The organic layer was dried over
anhydrous NaSO4 and concentrated under vacuum.
The residue was subjected to flash column chromatogra-
phy (hexane/CH2Cl2/ethyl acetate, 3:3:7) to give the pure
compound 4 (94 mg, 92%) as a white solid. Rf 0.40
In a similar way as described for the preparation of 4,
compound 3 (193 mg, 0.27 mmol) was treated with suc-
cinic anhydride (33 mg, 0.33 mmol) in pyridine (3.0 mL)
to give compound 6 (141 mg, 73%) as a white solid. Rf
22
0.43 (CH2Cl2/MeOH, 12:1); ½aꢀ ꢁ58.8 (c 0.11, CHCl3);
D
1H NMR (500 MHz, CDCl3): d 0.78 (s, 3H, CH3), 0.79
(d, 3H, J = 7.0 Hz, CH3), 0.95 (d, 3H, J = 7.5 Hz, CH3),
1.00 (s, 3H, CH3), 2.01 (s, 3H, CH3CO), 2.02 (s, 3H,
CH3CO), 2.07 (s,
3 H, CH3CO), 2.46 (t, 2H,
J = 7.5 Hz, CH2CO), 2.70 (m, 2H, CH2CO), 3.37 (dd,
1H, J = 11.0, 11.0 Hz, H-260a), 3.45 (dd, 1H, J = 11.0,
5.0 Hz, H-260b), 3.49 (m, 1H, H-2), 3.70–3.77 (m, 2H,
H-5, H-30), 4.10 (dd, 1H, J = 12.5, 2.5 Hz H-6a), 4.26
(dd, 1H, J = 12.5, 5.5 Hz, H-6b), 4.39 (approx q, 1H,
J ꢂ 8.0 Hz, H-160), 4.85 (d, 1H, J = 8.0 Hz, H-1), 5.02
(dd, 1 H, J = 10.0, 10.0 Hz, H-4), 5.32 (m, 1H, H-60),
5.37 (dd, 1H, J = 10.0, 10.0 Hz, H-3), 6.03 (d, 1H,
J = 8.5 Hz, NH);HRESI-MS Calcd for C43H63NO13Na
[M+Na]+: 824.4192, found (positive mode): 824.4193.
22
1
(CH2Cl2/MeOH, 16:1); ½aꢀ ꢁ61.0 (c 0.36, CHCl3); H
D
NMR (500 MHz, CDCl3): d 0.79 (s, 3H, CH3), 0.79
(d, 3H, J = 6.5 Hz, CH3), 0.87 (d, 3H, J = 7.0 Hz,
CH3), 0.89 (s, 3H, CH3), 1.96 (s, 3H, CH3CO), 2.02 (s,
3H, CH3CO), 2.04 (s, 3H, CH3CO), 2.08 (s, 3H,
CH3CO), 3.38 (dd, 1H, J = 11.0, 11.0 Hz, H-260a),
3.48 (dd, 1H, J = 11.0, 4.5 Hz, H-260b), 3.50 (m, 1H),
3.69–3.78 (m, 2 H), 4.10 (dd, 1 H, J = 12.0, 2.0 Hz, H-
6b), 4.26 (dd, 1H, J = 12.0, 5.0 Hz, H-6a), 4.40 (approx

3214
M. J. Kaskiw et al. / Bioorg. Med. Chem. 16 (2008) 3209–3217
4.6. Diosgenyl 3,4,6-tri-O-acetyl-2-[(S)-2-acetamido-propa-
namido]-2-deoxy-b-D-glucopyranoside (7)
50.21, 55.73, 56.07, 56.58, 56.66, 62.26, 62.49, 67.04,
69.08, 71.81, 72.27, 79.72, 79.95, 80.99, 99.32, 109.50,
122.11, 140.54, 169.71, 170.98, 170.99, 172.98; HRESI-
MS Calcd for C47H71NO11S2Na [M+Na]+: 912.4360,
found (positive mode): 912.4358.
To a solution of 3 (300 mg, 0.43 mmol) in dry DMF
(5.0 mL), N-acetyl-L-alanine (56 mg, 0.43 mmol), diiso-
propylethylamine (DIPEA, 0.15 mL, 0.86 mmol), and 2-
(1H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexa-
fluorophosphate (HBTU, 163 mg, 0.43 mmol) were
added and the mixture was stirred for 16 h. The solvent
was removed in vacuo and the residue was dissolved with
CH2Cl2 (150 mL) and washed with H2O (10 mL · 2). The
organic layer was dried over anhydrous Na2SO4 and con-
centrated under vacuum. The residue was subjected to
flash column chromatography (CH2Cl2/MeOH, 25:1) to
give compound 7 (272 mg, 78%) as a white solid. Rf 0.36
4.8. Diosgenyl 2-acetamido-2-deoxy-b-D-glucopyrano-
side (9)
Compound 4 (100 mg, 0.13 mmol) was dissolved in
MeOH/CH2Cl2 (2:1, 9 mL) and then NaOMe solution
in methanol (0.6 mL) was added until pH 10. After stir-
ring for 4 h at room temperature, the mixture was neu-
tralized by adding weak acidic ion-exchange resin
(Amberlite IRC-50, H+ form). The resin was filtered
and the filtrate concentrated in vacuo. The residue was
applied to a Sephadex LH-20 gel filtration column
(MeOH/CH2Cl2, 2:1) to yield compound 9 (81 mg,
98%) as a white solid. Rf 0.21 (CH2Cl2/MeOH, 2:1);
22
D
1
(CH2Cl2/MeOH, 25:1); ½aꢀ ꢁ53.2 (c 0.64, CHCl3); H
NMR (500 MHz, CDCl3): d 0.81 (s, 3H, CH3), 0.82 (d,
3H, J = 6.0 Hz, CH3), 0.98 (d, 3H, J = 7.0 Hz, CH3),
1.02 (s, 3H, CH3), 1.99 (s, 3H, CH3CO), 2.01 (s, 3H,
CH3CO), 2.04 (s, 3H, CH3CO), 2.10 (s, 3H, CH3CO),
3.37 (m, 1H, H-260a), 3.48–3.51 (m, 2H, H-260b, H-2),
3.74–3.83 (m, 2H, H-30, H-5), 4.12 (dd, 1H, J = 12.0,
2.0 Hz H-6b), 4.29 (dd, 1H, J = 12.0, 4.5 Hz, H-6a), 4.29
(m, 1H, CHNHAc), 4.43 (approx q, 1H, J ꢂ 7.5 Hz, H-
160), 4.80 (d, 1H, J = 8.5 Hz, H-1), 4.91 (dd, 1H, J = 9.5,
9.5 Hz, H-4), 5.20 (dd, 1H, J = 10.5, 10.5 Hz, H-3), 5.26
(m, 1H, H-60), 7.62 (d, 1H, J = 7.5 Hz, NH), 7.87 (d,
1H, J = 8.5 Hz, NH); 13C NMR (125 MHz, CDCl3): d
14.74, 16.50, 17.35, 18.27, 19.59, 20.90, 21.01, 23.40,
29.01, 29.67, 30.51, 31.60, 32.05, 32.27, 37.04, 37.34,
39.03, 39.95, 40.47, 41.82, 49.15, 50.26, 55.32, 56.69,
62.30, 62.52, 67.07, 69.10, 71.81, 72.31, 79.88, 81.00,
98.15, 108.16, 120.68, 139.30, 168.34, 169.00, 169.60,
169.61, 171.54; HRESI-MS Calcd for C44H66N2O12Na
[M+Na]+: 837.4508, found (positive mode): 837.4506.
22
D
22
D
½aꢀ ꢁ66.6 (c 0.26, CH3OH/CHCl3, 1:1); lit.14 ½aꢀ
1
ꢁ75 (c 0.5, CH3OH/CHCl3, 1:1); H NMR (500 MHz,
C5D5N plus two drops of CD3OD): d 0.73 (d, 3H,
J = 6.0 Hz, CH3), 0.84 (s, 3H, CH3), 0.96 (s, 3H,
CH3), 1.18 (d, 3H, J = 7.0 Hz, CH3), 2.18 (s, 3H,
CH3CO), 3.50 (dd, 1H, J = 10.5, 10.5 Hz, H-260a),
3.60 (dd, 1H, J = 10.5, 3.5 Hz, H-260b), 3.83–3.88 (m,
1H, H-30), 3.91–3.94 (m, 1H, H-5), 4.14 (m, 1 H, H-2),
4.30 (dd, 1H, J = 12.0, 6.0 Hz, H-6a), 4.37–4.44 (m,
2H, H-3, H-4), 4.50 (dd, 1H, J = 10.0, 2.0 Hz, H-6b),
4.55 (approx q, 1H, J = 7.0 Hz, H-160), 5.21 (d, 1H,
J = 7.5 Hz, H-1), 5.35–5.36 (m, 1H, H-60), 8.89 (d, 1H,
J = 8.0 Hz, NH); 13C NMR (125 MHz, C5D5N plus
two drops of CD3OD): d 15.54, 16.86, 17.82, 19.92,
21.57, 24.08, 29.73, 30.64, 31.07, 32.12, 32.28, 32.66,
32.74, 37.50, 37.91, 40.00, 40.34, 40.93, 42.43, 50.71,
57.13, 58.49, 63.13, 63.34, 67.33, 72.81, 76.76, 78.98,
78.99, 81.57, 101.27, 109.74, 122.20, 141.44, 171.01;
ESI-MS Calcd for C35H55NO8 [M]+: 617.39, found (po-
sitive mode): 618.64 [M+H]+. The NMR spectral data
are in general agreement with the reported data14 which
were measured in DMSO-d6.
4.7. Diosgenyl 3,4,6-tri-O-acetyl-2-{5-[(R/S)-1,2-dithiolan-
3-yl]-pentanamido}-2-deoxy-b-D-glucopyranoside (8)
In a similar way as described for the preparation of 7,
compound 3 (150 mg, 0.21 mmol) was treated with
( )-a-lipoic acid (44 mg, 0.21 mmol), DIPEA
(0.075 mL, 0.43 mmol), and HBTU (82 mg, 0.21 mmol)
4.9. Diosgenyl 2-benzamido-2-deoxy-b-D-glucopyrano-
side (10)
in dry DMF (5.0 mL) to give 8 (158 mg, 83%) as a white
22
D
solid. Rf 0.32 (hexane/EtOAc/MeOH, 1:1:0.025); ½aꢀ
1
ꢁ175.0 (c 0.03, CHCl3); H NMR (500 MHz, CDCl3):
d 0.78 (s, 3H, CH3), 0.79 (d, 3H, J = 6.5 Hz, CH3),
0.96 (d, 3H, J = 7.0 Hz, CH3), 0.99 (s, 3H, CH3), 2.01
(s, 3H, CH3CO), 2.02 (s, 3H, CH3CO), 2.07 (s, 3H,
CH3CO), 2.23 (m, 1H, CHHCO), 2.45 (m, 1H,
CHHCO), 3.08–3.20 (m, 2H, CH2S), 3.17 (m, 1 H),
3.37 (dd, 1H, J = 10.5, 10.5 Hz, H-260a), 3.45–3.56 (m,
3H, H-260b, H-2, CHS), 3.65 (m, 1H, H-30), 3.70 (m,
1H, H-5), 4.09 (dd, 1H, J = 12.0, 2.5 Hz, H-6b), 4.26
(dd, 1H, J = 12.0, 5.0 Hz, H-6a), 4.40 (approx q, 1H,
J ꢂ 7.5 Hz, H-160), 4.89 (two sets of d, each 0.5H,
J = 8.0 Hz, H-1), 5.03 (dd, 1H, J = 9.5, 9.5 Hz, H-4),
5.33 (m, 1H, H-60), 5.41 (two sets of dd, each 0.5H,
J = 10.0, 10.0 Hz, H-3), 5.48 (two sets of d, each 0.5H,
J = 8.5 Hz, NH); 13C NMR (125 MHz, CDCl3): d
14.72, 16.49, 17.34, 19.59, 20.88, 20.96, 25.42, 25.47,
28.98, 29.64, 30.48, 31.59, 32.04, 32.28, 34.78, 34.83,
36.67, 37.04, 37.32, 38.65, 39.11, 39.92, 40.45, 41.79,
In a similar way as described for the preparation of 9,
compound 5 (71 mg, 0.09 mmol) was treated with so-
dium methoxide to give 10 (55 mg, 92%) as a white solid.
22
Rf 0.18 (CH2Cl2/MeOH, 12:1); ½aꢀ ꢁ28.0 (c 0.16,
D
1
CHCl3/CH3OH, 1:1); H NMR (500 MHz, C5D5N): d
0.69 (d, 3H, J = 5.5 Hz, CH3), 0.82 (s, 3H, CH3), 0.83
(s, 3H, CH3), 1.14 (d, 3H, J = 6.0 Hz, CH3), 2.43 (m, 1
H), 2.69 (m, 1H), 3.50 (dd, 1H, J = 10.5, 10.5 Hz, H-
260a), 3.59 (dd, 1H, J = 10.5, 3.0 Hz, H-260b), 3.90 (m,
1H, H-30), 4.06 (m, 1H, H-5), 4.32 (m, 1H, H-6a), 4.43
(m, 1H, H-2), 4.54 (approx q, 1H, J = 7.5 Hz, H-160),
4.60 (br d, 1H, J = 11.0 Hz, H-6b), 4.68–4.80 (m, 2H,
H-3, H-4), 5.22 (m, 1H, H-60), 5.56 (d, 1H, J = 8.5 Hz,
H-1), 6.58 (s, 1H, OH), 7.37–7.44 (m, 5H, 3 Ar–H, 2
OH), 8.39 (d, 2 H, J = 6.5 Hz, 2 Ar–H), 9.44 (d, 1H,
J = 8.0 Hz, NH); 13C NMR (125 MHz, C5D5N): d
15.51, 16.80, 17.80, 19.80, 21.51, 29.71, 30.47, 30.64,
31.05, 32.04, 32.25, 32.68, 37.43, 37.87, 40.09, 40.28,

M. J. Kaskiw et al. / Bioorg. Med. Chem. 16 (2008) 3209–3217
3215
40.89, 42.40, 50.64, 57.07, 59.47, 63.31, 67.31, 73.10,
76.30, 79.10, 79.17, 81.54, 101.16, 109.71, 122.19,
128.51, 129.06, 131.67, 137.32, 141.26, 141.28, 168.94;
ESI-MS Calcd for C40H57NO8 [M]+: 679.41, found (po-
sitive mode): 680.69 [M+H]+.
CH3), 1.15 (d, 3H, J = 7.0 Hz, CH3), 2.22 (m, 1 H),
2.48 (t, 2H, J = 7.0 Hz), 2.54 (m, 1 H), 2.72 (m, 1 H),
2.97–3.08 (m, 2H, CH2S), 3.47–3.53 (m, 2H, H-260a,
CHS), 3.60 (dd, 1H, J = 12.0, 3.5 Hz, H-260b), 3.87–
3.94 (m, 1H, H-30), 4.00 (m, 1H, H-5), 4.25 (dd, 1H,
J = 9.0, 9.0 Hz, H-4), 4.40 (dd, 1H, J = 12.0, 5.5 Hz,
H-6a), 4.50–4.59 (m, 4H, H-2, H-3, H-6b, H-160), 5.32
(two sets of d, each 0.5H, J = 8.0 Hz, H-1), 5.36 (m,
1H, H-60), 8.82 (d, 1H, J = 7.5 Hz, NH); 13C NMR
(125 MHz, C5D5N): d 15.51, 16.83, 17.79, 19.99, 21.57,
26.54, 29.74, 30.68, 31.06, 32.14, 32.26, 32.66, 32.76,
35.46, 37.28, 37.52, 37.89, 39.12, 40.14, 40.32, 40.86,
40.92, 42.42, 50.70, 57.12, 57.28, 58.45, 63.20, 63.33,
67.31, 72.99, 76.79, 78.89, 79.02, 81.54, 101.22, 109.72,
122.25, 141.45, 173.74; ESI-MS Calcd for C41H65NO8S2
[M]+: 763.42, found (positive mode): 764.70 [M+H]+.
4.10. Diosgenyl 2-(3-carboxylpropanamido)-2-deoxy-b-D-
glucopyranoside (11)
In a similar way as described for the preparation of 9,
compound 6 (66 mg, 0.08 mmol) was treated with so-
dium methoxide to give 11 (55 mg, quantitatively) as a
22
D
white solid. Rf 0.28 (CHCl3/MeOH/H2O, 6:4:0.5); ½aꢀ
ꢁ29.4 (c 0.14, CHCl3/CH3OH, 1:1); 1H NMR
(500 MHz, CD3OD): d 0.69 (s, 3H, CH3), 0.70 (d, 3H,
J = 5.5 Hz, CH3), 0.86 (d, 3H, J = 7.0 Hz, CH3), 0.93
(s, 3H, CH3), 2.40 (m, 4 H), 3.14–3.26 (m, 3 H), 3.32–
3.50 (m, 3 H), 3.54–3.60 (m, 1 H), 3.74–3.82 (m, 1 H),
4.29 (approx q, 1H, J = 7.5 Hz, H-160), 4.46 (d, 1H,
J = 8.0 Hz, H-1), 4.63–4.68 (m, 1 H), 5.27 (m, 1H, H-
60), 8.89 (d, 1H, J = 9.5, NH); 13C NMR (125 MHz,
CD3OD): d 15.90, 16.96, 17.69, 20.04, 22.16, 30.07,
30.83, 31.63, 32.61, 32.96, 32.97, 33.10, 33.36, 33.58,
38.15, 38.68, 40.18, 41.10, 41.60, 43.08, 51.84, 57.97,
57.98, 63.00, 63.92, 68.03, 72.15, 76.20, 78.05, 80.42,
82.38, 101.28, 110.74, 122.80, 142.09, 175.93, 178.86;
ESI-MS Calcd for C37H57NO10 [M]+: 675.40, found (po-
sitive mode): 676.38 [M+H]+, 698.86 [M+Na]+.
4.13. Diosgenyl 3,4,6-tri-O-acetyl-2-(5-carboxylpentami-
do)-2-deoxy-b-D-glucopyranoside (14) and N,N0-bis(dios-
genyl 3,4,6-tri-O-acetyl-2-deoxy-b-D-glucopyranosid-2-
yl) hexandiamide (15)
In a similar way as described for the preparation of 7,
compound 3 (130 mg, 0.19 mmol) was treated with adipic
acid (12 mg, 0.085 mmol), DIPEA (0.064 mL,
0.37 mmol), and HBTU (70 mg, 0.19 mmol) in dry
DMF (5.0 mL) to give the monomeric product 14
(15 mg, 21%) and the dimeric product 15 (71 mg, 56%).
22
D
For 14: Rf 0.24 (hexane/EtOAc/MeOH, 1:1:0.1); ½aꢀ
1
4.11. Diosgenyl 2-[(S)-2-acetamido-propanamido]-2-
deoxy-b-D-glucopyranoside (12)
ꢁ36.8 (c 0.25, CHCl3); H NMR (500 MHz, CDCl3): d
0.78 (s, 3H, CH3), 0.79 (d, 3H, J = 7.0 Hz, CH3), 0.96
(d, 3H, J = 7.0 Hz, CH3), 0.99 (s, 3H, CH3), 2.01 (s, 6H,
2 CH3CO), 2.07 (s, 3H, CH3CO), 2.29 (m, 2 H), 2.34 (m,
2 H), 3.37 (dd, 1H, J = 11.0, 11.0 Hz, H-260a), 3.46–3.52
(m, 2H, H-260b, H-2), 3.72–3.77 (m, 2H, H-5, H-30),
4.10 (dd, 1H, J = 12.0, 1.5 Hz, H-6b), 4.28 (dd, 1H,
J = 12.0, 4.5 Hz, H-6a), 4.41 (approx q, 1H, J ꢂ 7.5 Hz,
H-160), 4.85 (d, 1H, J = 8.0 Hz, H-1), 5.03 (dd, 1H,
J = 9.5, 10.0 Hz, H-4), 5.31 (m, 1H, H-60), 5.38 (dd, 1H,
J = 9.5, 10.5 Hz, H-3), 5.86 (d, 1H, J = 9.0 Hz, NH);
13C NMR (125 MHz, CDCl3): d 14.73, 16.50, 17.35,
19.58, 20.88, 20.96, 21.02, 21.03, 24.30, 24.99, 28.98,
29.63, 29.91, 30.47, 31.56, 31.57, 32.03, 32.26, 36.36,
37.03, 37.33, 39.05, 39.93, 40.45, 41.79, 50.21, 55.37,
56.67, 62.24, 62.53, 67.05, 69.14, 71.75, 72.50, 79.76,
81.00, 99.44, 109.54, 122.03, 140.57, 169.73, 171.00,
171.22, 173.12, 178.16; ESI-MS Calcd for C45H67NO13
In a similar way as described for the preparation of 9,
compound 7 (168 mg, 0.21 mmol) was treated with so-
dium methoxide to give 12 (141 mg, quantitatively) as a
22
white solid. Rf 0.21 (CH2Cl2/MeOH, 8:1); ½aꢀ ꢁ44.8 (c
D
0.36, CHCl3/CH3OH, 1:1); 1H NMR (500 MHz, CDCl3/
CD3OD, 1:1): d 0.71 (s, 3H, CH3), 0.72 (d, 3H,
J = 5.0 Hz, CH3), 0.88 (d, 3H, J = 7.0 Hz, CH3), 0.92 (s,
3H, CH3), 1.29 (d, 3H, J = 7.0 Hz, CH3), 1.91 (s, 3H,
CH3CO), 3.21 (m, 1 H), 3.26 (m, 1 H), 3.30 (m, 1 H),
3.38 (m, 1 H), 3.40–3.51 (m, 3 H), 3.64 (dd, 1H,
J = 12.0, 5.0 Hz, H-6a), 3.78 (dd, 1H, J = 12.0, 2.5 Hz,
H-6b), 4.24 (q, 1H, J = 7.0 Hz, CHNHAc), 4.33 (approx
q, 1H, J = 7.5 Hz, H-160), 4.47 (d, 1H, J = 8.0 Hz, H-1),
5.25 (m, 1H, H-60); 13C NMR (125 MHz, CDCl3/
CD3OD, 1:1): d 15.63, 17.56, 18.27, 18.95, 20.59, 22.29,
23.66, 30.10, 30.89, 31.65, 32.71, 32.92, 33.10, 33.50,
38.27, 38.65, 40.21, 41.17, 41.75, 43.11, 50.73, 51.63,
57.98, 58.58, 63.22, 63.53, 68.33, 72.33, 75.40, 77.57,
80.53, 82.53, 100.65, 111.14, 123.11, 141.95, 173.20,
175.20. ESI-MS Calcd for C38H60N2O9 [M]+: 688.43,
found (positive mode): 689.61 [M+H]+, 711.57 [M+Na]+.
[M]+: 829.46, found (positive mode): 830.71 [M+H]+.
22
D
For 15: Rf 0.29 (hexane/EtOAc/MeOH, 1:1:0.1); ½aꢀ
1
ꢁ61.7 (c 0.29, CHCl3); H NMR (500 MHz, CDCl3): d
0.78 (s, 3H, CH3), 0.80 (d, 3H, J = 7.0 Hz, CH3), 0.97
(d, 3H, J = 7.0 Hz, CH3), 0.99 (s, 3H, CH3), 2.02 (s, 3H,
CH3CO), 2.03 (s, 3H, CH3CO), 2.08 (s, 3H, CH3CO),
2.19 (m, 2 H), 3.37 (dd, 1H, J = 11.0, 11.0 Hz, H-260a),
3.48 (dd, 1H, J = 11.0, 4.0 Hz, H-260b), 3.50 (m, 1H, H-
2), 3.73–3.81 (m, 2H, H-5, H-30), 4.09–4.12 (dd, 1H,
J = 12.0, 2.5 Hz H-6b), 4.28 (dd, 1H, J = 12.0, 5.0 Hz,
H-6a), 4.41 (approx q, 1H, J ꢂ 7.5 Hz, H-160), 4.90 (d,
1H, J = 8.5 Hz, H-1), 5.06 (dd, 1H, J = 9.5, 10.0 Hz, H-
4), 5.29 (m, 1H, H-60), 5.45 (dd, 1H, J = 9.5, 10.5 Hz, H-
3), 6.13 (d, 1H, J = 8.5 Hz, NH); 13C NMR (125 MHz,
CDCl3): d 14.74, 16.51, 17.36, 19.61, 20.91, 21.03, 21.10,
24.31, 28.99, 29.61, 29.91, 30.50, 31.58, 31.59, 32.04,
4.12. Diosgenyl 2-deoxy-2-{5-[(R/S)-(1,2-dithiolan-3-yl)]-
pentamido}-b-D-glucopyranoside (13)
In a similar way as described for the preparation of 9,
compound 8 (139 mg, 0.16 mmol) was converted to 13
(119 mg, quantitatively) as a white solid. Rf 0.24
22
(CH2Cl2/MeOH, 12:1); ½aꢀ ꢁ44.8 (c 0.21, CHCl3/
D
1
CH3OH, 1:1); H NMR (500 MHz, C5D5N): d 0.70 (d,
3H, J = 5.5 Hz, CH3), 0.85 (s, 3H, CH3), 0.99 (s, 3H,

3216
M. J. Kaskiw et al. / Bioorg. Med. Chem. 16 (2008) 3209–3217
32.28, 36.06, 37.06, 37.35, 39.05, 39.92, 40.46, 41.80,
50.23, 55.34, 56.67, 62.26, 62.49, 67.06, 69.25, 71.72,
72.76, 79.59, 80.98, 99.33, 109.52, 121.97, 140.69,
169.70, 170.98, 171.17, 173.01; HRESI-MS Calcd for
C84H124N2O22Na [M+Na]+: 1535.8538; found (positive
mode): 1535.8533.
by the American Type Culture Collection (Manassas,
Virginia), and HeLa cells were obtained from the Lady
Davis Institute for Medical Research (Montreal, Que-
bec). All cell lines were grown in Dulbecco’s modified
Eagle’s medium (Sigma) containing 10% fetal bovine
serum (PAA Laboratories) at 37 ꢁC in a 5% CO2 humid-
ified atmosphere.
4.14. Diosgenyl 2-(5-carboxylpentamido)-2-deoxy-b-D-
glucopyranoside (16)
4.17. Cell proliferation assay
In a similar way as described for the preparation of 9,
compound 14 (26 mg, 0.03 mmol) was converted to 16
(22 mg, quantitatively) as a white solid. Rf 0.20
Measurement of cell proliferation was performed using
the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazoli-
um bromide (MTT) assay as described by Carmichael
et al.25 Briefly, 2000 cells were plated out into each well
of a 96-wellplate and allowed to adhere. Saponin ana-
logues (dissolved in DMSO (Sigma) and diluted with tis-
sue culture medium) were added at increasing
concentrations (0–30 lM, 8 wells per concentration).
The cells were incubated in the presence of each saponin
analogue for 72 h. MTTreagent (Sigma) dissolved in
phosphate-buffered saline was added to each well at a
concentrationof 0.5 mg/mL, and the cells were incu-
bated 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
(PowerWave XS, Bio-Tek) at a wavelength of 490 nm.
The IC50 value for each compound tested was deter-
mined by plotting concentration vs. percent absorbance
obtained in the MTT assay. The IC50 results listed in Ta-
ble 1 represent the average IC50 value obtained from
multiple MTT assays for each compound.
22
(CH2Cl2/MeOH, 12:1); ½aꢀ ꢁ14.0 (c 0.14, CHCl3/
D
1
CH3OH, 1:1); H NMR (500 MHz, C5D5N): d 0.68 (d,
3 H, J = 5.0 Hz, CH3), 0.81 (s, 3H, CH3), 0.95 (s, 3H,
CH3), 1.13 (d, 3H, J = 7.0 Hz, CH3), 2.52 (m, 5 H),
2.70 (m, 1 H), 3.50 (dd, 1H, J = 10.5, 10.5 Hz, H-
260a), 3.58 (dd, 1H, J = 10.5, 3.5 Hz, H-260b), 3.88 (m,
1H, H-30), 3.97 (m, 1H, H-5), 4.21 (dd, 1H, J = 9.0,
9.0 Hz, H-4), 4.36 (dd, 1H, J = 12.0, 5.5 Hz, H-6a),
4.43–4.58 (m, 4H, H-2, H-3, H-6b, H-160), 5.25 (d,
1H, J = 7.5 Hz, H-1), 5.33 (m, 1H, H-60), 8.82 (m, 1H,
J = 8.0 Hz, NH); 13C NMR (125 MHz, C5D5N): d
15.30, 16.62, 17.59, 19.73, 21.34, 25.60, 26.39, 29.51,
30.49, 30.85, 31.87, 32.05, 32.43, 32.50, 34.92, 37.18,
37.28, 37.69, 39.80, 40.11, 40.70, 42.31, 50.49, 56.89,
58.28, 63.11, 64.74, 67.11, 72.83, 76.70, 78.74, 78.83,
81.34, 101.04, 109.52, 121.94, 141.29, 173.64, 176.06;
ESI-MS calcd for C39H61NO10 [M]+: 703.43, found (po-
sitive mode): 704.71 [M+H]+.
4.15. N,N0-Bis(diosgenyl 2-deoxy-b-D-glucopyranosid-2-
yl) hexandiamide (17)
Acknowledgments
In a similar way as described for the preparation of 9,
compound 15 (27 mg, 0.02 mmol) was converted to 17
(22.5 mg, quantitatively) as a white solid. Rf 0.35
This work was supported by Natural Sciences and Engi-
neering Research Council of Canada (NSERC, Grant
312630) and Lakehead University.
22
(CH2Cl2/MeOH, 5:1); ½aꢀ ꢁ39.2 (c 0.14, CHCl3/
D
1
CH3OH, 1:1); H NMR (500 MHz, C5D5N): d 0.68 (d,
3H, J = 5.5 Hz, CH3), 0.86 (s, 3 H, CH3), 0.95 (s, 3H,
CH3), 1.15 (d, 3 H, J = 7.0 Hz, CH3), 2.49 (m, 1 H),
2.55 (m, 2 H), 2.68 (m, 1 H), 3.50 (dd, 1H, J = 10.5,
10.5 Hz, H-260a), 3.59 (dd, 1H, J = 10.5, 2.5 Hz, H-
260b), 3.89 (m, 1H, H-30), 3.95 (m, 1H, H-5), 4.21 (dd,
1H, J = 9.0, 9.0 Hz, H-4), 4.36 (dd, 1H, J = 12.0,
5.5 Hz, H-6a), 4.45 (dd, 1H, J = 10.0, 9.0 Hz, H-3),
4.52–4.58 (m, 3H, H-2, H-6b, H-160), 5.25 (d, 1H,
J = 8.5 Hz, H-1), 5.32 (m, 1H, H-60), 8.75 (m, 1H,
J = 9.0 Hz, NH); 13C NMR (125 MHz, C5D5N): d
15.52, 16.89, 17.80, 19.96, 21.58, 26.20, 29.73, 30.69,
31.06, 32.12, 32.28, 32.69, 32.76, 37.20, 37.51, 37.89,
39.92, 40.33, 40.94, 42.44, 50.71, 57.12, 58.36, 63.28,
63.34, 67.32, 72.93, 76.95, 78.80, 79.01, 81.56, 101.16,
109.73, 122.21, 141.41, 174.09; HRESI-MS Calcd for
C72H112N2O16Na [M+Na]+: 1283.7904, found (positive
mode): 1283.7903.
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