Synthesis of novel spirostanic saponins and their cytotoxic activity

Article abstract of DOI:10.1016/j.bmc.2007.10.089

This study was carried out to assess the cytotoxicity of several new synthetic steroidal saponins against the human myeloid leukemia cell lines (HL-60 and U937) and against human melanoma cells (SK-MEL-1). Several diosgenyl glycosides analyzed showed strong cell growth inhibition which was associated with alterations in cell cycle progression and induction of apoptosis. Studies of cytochrome c release and caspase-9 activation suggest a main role of the intrinsic pathway of apoptosis in the mechanism of cytotoxicity caused by this kind of compounds.

Full text of DOI:10.1016/j.bmc.2007.10.089

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 2063–2076
Synthesis of novel spirostanic saponins and their cytotoxic activity
a,b
Juan C. Hernandez, Francisco Leon, Ignacio Brouard,a Fernando Torres,b,c
a,b
´
´
b,c
b,c
Sara Rubio,b,c Jose Quintana, Francisco Estevez and Jaime Bermejoa,
*
´
´
aInstituto de Productos Naturales y Agrobiolog´ıa-C.S.I.C., Instituto Universitario de Bio-Orga´nica, ‘‘Antonio Gonza´lez’’,
Av. Astrofı´sico F. Sa´nchez 3, 38206 La Laguna, Tenerife, Spain
bInstituto Canario de Investigacio´n del Ca´ncer (ICIC), Av. Astrofı´sico F. Sa´nchez 2, 38206 La Laguna, Tenerife, Spain
cDepartment of Biochemistry, Molecular Biology and Physiology, University of Las Palmas de Gran Canaria,
Plaza Dr. Pasteur s/n, 35016 Las Palmas de Gran Canaria, Spain
Received 2 July 2007; revised 23 October 2007; accepted 30 October 2007
Available online 4 November 2007
´
Dedicated to the memory of Professor Antonio G. Gonzalez.
Abstract—This study was carried out to assess the cytotoxicity of several new synthetic steroidal saponins against the human mye-
loid leukemia cell lines (HL-60 and U937) and against human melanoma cells (SK-MEL-1). Several diosgenyl glycosides analyzed
showed strong cell growth inhibition which was associated with alterations in cell cycle progression and induction of apoptosis.
Studies of cytochrome c release and caspase-9 activation suggest a main role of the intrinsic pathway of apoptosis in the mechanism
of cytotoxicity caused by this kind of compounds.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
Consequently, various syntheses of steroidal saponins
have been successfully developed.12–15 As a part of
our research focused on the cytotoxicity of several ste-
roidal saponins isolated from Dracaena draco,5,16 we de-
signed the synthesis of ten analogs containing a
diosgenyl a-L-rhamnopyranosyl-(1 ! 2)-b-D-glucopy-
ranoside skeleton. Mimaki et al.,17 employing molecular
models and molecular dynamic studies, suggested that
the three-dimensional structure of the diglycoside moi-
ety contributed to the cytotoxic activity of the molecule.
In that study, they revealed that in the diosgenyl a-L-
rhamnopyranosyl-(1 ! 2)-b-D-glucopyranoside deriva-
tive, which is cytotoxic, the diglycoside exists in a
vertically oriented conformation against the steroid
plane. However, in the diosgenyl a-L-rhamnopyrano-
Saponins are a very large group of glycosylated second-
ary metabolites isolated primarily from plants, but also
found in lower marine organisms.1 Saponins are be-
lieved to be some of the principal constituents of many
plant drugs and traditional medicines worldwide, and
are described as being responsible for most of the ob-
served biological effects. Although the biological role
of saponins in plants is not yet fully understood, they
are considered to be a part of the natural chemical de-
fense mechanism of plants against attack by pathogens.2
Saponins consist of a hydrophobic aglycone, which may
have a triterpenoid or steroid structure, linked by a gly-
cosidic bond to a sugar moiety normally including glu-
cose, rhamnose, galactose, or xylose, among others.
Steroidal saponins are amphipathic molecules of special
interest due to the biological activities they exhibit. In
recent years several research groups have evaluated their
cytotoxic activities.3–11
syl-(1 ! 4)-b-D-glucopyranoside
derivative,
which
presents a conformation with the diglycoside and the
steroid in the same plane, the activity was not relevant.
Starting from this hypothesis, we proposed the synthesis
of several saponins analogous to those obtained from
Dracaena draco,5,16 always having the diosgenyl b-D-
glucopyranoside basic structure and including different
monosaccharides (Glc, Gal, Man, Ara, Xyl, and Fuc)
as well as the same derivative with rhamnose, linked
through the C-20 of the glucose. Then, the most potent
Keywords: Synthesis; Spirostanic saponins; Cytotoxicity; Apoptosis;
Leukemia; Human melanoma cells; Cytochrome c release; Caspase.
*
Corresponding author. Tel.: +34 922318583; fax: +34 922318571;
e-mail: jbermejo@ull.es
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.10.089

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J. C. Hernandez et al. / Bioorg. Med. Chem. 16 (2008) 2063–2076
2064
cytotoxic compound was submitted to additional
modifications.
The use of boron trifluoride-diethyl etherate complex
as Lewis acid18 and trichloroacetonitrile as leaving
group allowed the coupling of the monosaccharides
in a stereoselective manner and with acceptable yields
affording the corresponding coupling products 12–18
(Scheme 2).
Some chemotherapeutic drugs have been shown to use
apoptotic pathways to mediate their cytotoxic effects.
An important goal in chemotherapy is to find new cyto-
toxic agents that are able to increase or restore the abil-
ity of tumor cells to undergo apoptosis. Apoptotic cell
death is characterized by morphological and biochemi-
cal changes, which largely result from the activation of
a family of cysteine proteases known as caspases. There
are two primary caspase activation pathways, involving
either stimulation of cell surface death receptors (the
extrinsic pathway) or perturbation of mitochondria
(the intrinsic pathway). Many anti-cancer drugs induce
apoptosis by activating the intrinsic pathway. This cell
death pathway involves the release of cytochrome c
and the activation of the apoptosome-catalyzed caspase
cascade. The apoptosome is a protein complex consist-
ing of cytochrome c, apoptotic protease-activating fac-
tor 1, and caspase-9. Apoptosome assembly processes
and activates the initiator caspase-9. Active caspase-9
then triggers a caspase cascade to induce apoptosis.
Finally, a two-step deprotection protocol employing
AcOH (80%), followed by basic deacetylation with
NaOMe in MeOH, gave the desired new spirostane-type
steroidal saponins 20, 21, 23, and 24, and the known
natural ones 19, 22, and 25. The physical data of the
known compounds were in accordance with those re-
ported in the literature.21–23 Analysis of the 1H and
13C NMR spectra of 20, 21, 23 and 24 together with
homonuclear correlation (COSY) and heteronuclear
multiple coherence (HMQC and HMBC) and TROESY
experiments allowed the structure of these new com-
pounds to be unambiguously deduced. Correlations be-
tween C-20 of the glucopyranoside unit and H-100 of the
second monosaccharide unit as well as the ROE effect
between H-20 and H-100 confirmed the interglycosidic
linkage. The connection between the aglycone moiety
and the saccharide portion was confirmed by a correla-
tion between the C-10 of glucose and H-3 of the aglycone
and the ROE effect between H-10 and H-3.
This study was undertaken to investigate the effects of
synthetic diosgenyl glycosides on cell growth and death
in human tumor cells, and associated biological events
such as induction of apoptosis and impairment of cell
cycle progression.
Preliminary results on cytotoxicity bioassays showed
that compound 25 was the most potent cytotoxic agent
against the three tumor cell lines tested (Table 2), and
thus subsequent modifications were developed over this
structure. It has been reported that the presence of acet-
yl groups usually increases cytotoxic potency.17
2. Results and discussion
Chemistry: In order to develop the synthesis of a set of
new steroidal saponins, we initially decided to prepare
the common precursor diosgenyl b-D-glucopyranoside,
known as trillin (3), suitably protected as 4,6-O-benzyl-
idene-3-O-pivaloyl-b-D-glucopyranoside 4a. Compound
3, as shown in Scheme 1, was prepared starting from
Thus, following the methodology described by Yu
et al.,24 the selective introduction of acetyl groups
employing the lipase Novozyme 435 was performed
(Scheme 3). This procedure afforded a mono- and a
diacetyl derivative named diosgenyl a-L-rhamnopyrano-
syl-(1 ! 2)-6-O-acetyl-b-D-glucopyranoside (27) and
diosgenyl 4-O-acetyl-a-L-rhamnopyranosyl-(1 ! 2)-6-
O-acetyl-b-D-glucopyranoside (26), respectively.
2,3,4,6-tetra-O-benzoyl-a-D-glucopyranosyl
bromide
(1) and commercially available diosgenin, using AgOTf
as coupling promoter with high stereoselectivity and
was prepared in good yield (67%). Hydrolysis of the es-
ter groups of 2 under basic conditions yielded 3, which
was protected as the O-benzylidene derivative 4. At-
tempts at selective monoprotection of the hydroxyl
group located at C-30 under conditions described by Li
et al.18 using TBDMSCl and imidazole in DMF were
unrewarded in our case because starting material was
recovered.
It is noteworthy that trisaccharides with pharmacologi-
cal activity are reported in the literature.3,5 We therefore
synthesized, starting from 4a, a novel derivative 30, con-
taining a trisaccharide core attached to diosgenin, taking
advantage of a ‘one-pot’ procedure.25 In this method,
trichloroacetamidate glycosyl and thioglycoside were
used as sequential glycosyl donors and two glycosidic
linkages (Scheme 4).26
A report was found in the literature on the difficulty of
accomplishing the selective monoprotection of the hy-
droxyl groups at C-20 and C-30 of a glucopyranosyl
unit.18,19 As an alternative, treatment of the above-men-
tioned O-benzylidene derivative 4 with pivaloyl chlo-
ride19 provided a mixture of protected alcohols at the
desired C-30 position (45%, 4a), at C-20 (9.5%, 4b), and
at both C-20 and C-30 (5%, 4c). Compound 4a, which
has the free –OH group at C-20, was submitted to glyco-
sylation reaction with the trichloroacetamidate partners
(5-11, Table 1) obtained from their corresponding natu-
ral monosaccharides.20
3. Biological evaluation
3.1. Diosgenyl glycosides inhibit growth of human tumor
cell lines
Initial studies on the cytotoxicity of diosgenyl a-L-
rhamnopyranosyl-(1 ! 2)-b-D-glucopyranoside
(25)
showed that this compound was a potent cytotoxic
agent against HL-60 cells.17 In the present report, we de-
scribe the chemical modifications that were developed

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J. C. Hernandez et al. / Bioorg. Med. Chem. 16 (2008) 2063–2076
2065
O
O
HO
Diosgenin
O
HO
HO
PhCOO
O
O
c
OH
a, b
O
PhCOO
PhCOO
HO
RO
RO
RO
OH
PhCOO
O
Br
D-glucose
O
2. R = PhCO
3. R = H
1
RO
d
O
O
e
Ph
Ph
O
O
f
O
O
O
Diosgenin
4a. R1 = H; R2 = Piv
4b. R1 = Piv; R2 = H
4c. R1 = Piv; R2 = Piv
O
Diosgenin
R2O
HO
R1O
HO
4
˚
Scheme 1. Reagents and conditions: (a) PhCOCl, DMAP, Py; (b) HBr/AcOH (30:70), CH2Cl2; (c) diosgenin, AgOTf, CH2Cl2, ꢁ20 ꢁC–rt, 4 A MS;
(d) NaOMe in MeOH 0.5 M; (e) PhCH(OMe)2, CSA, DMF, 50 ꢁC; (f) PivCl, Py, rt.
over this basic structure to obtain new semi-synthetic
diosgenyl glycosides and the effect of these chemical
changes on the growth of human myeloid (HL-60 and
U937) and melanoma (SK-MEL-1) cell lines (Table 2).
linked to C-400 on the rhamnose unit is relevant for anti-
proliferative activity. In consonance with these results,
compound 30, which contains the hydroxyl group at
C-400 attached to a second rhamnose, was also signifi-
cantly less cytotoxic than 25 but similar to 26, with no
noteworthy differences among cell lines. Next, we
decided to determine the influence of a pentose instead
of rhamnose on the cytotoxicity of the diosgenyl glyco-
sides. Therefore, compounds 22 and 23, containing
arabinose and xylose, respectively, were synthesized.
The results indicated that the presence of these pentoses
did not improve cytotoxicity. Contrarily, the antiprolif-
erative capability of both compounds was decreased, as
compared to compound 25, in all human cell lines. As
occurs with other substitutions, these chemical changes
affected the cell lines under study to different degrees.
A dramatic decrease in growth inhibitory activity was
specifically observed on SK-MEL-1 in response to the
above compounds (IC50 > 100 lM). These results sup-
port the role of rhamnose as a necessary structural
requirement for diosgenyl glycosides’ cytotoxicity.
Growth inhibition of human tumor cells in culture was
determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyl-2H-tetrazolium bromide (MTT) dye-reduction
assay. Antiproliferative studies of compound 25 indi-
cated that these diosgenyl glycosides displayed strong
cytotoxic properties in all assayed cell lines, with an
IC50 of about 5–7 lM. Interestingly this compound
was more potent than etoposide, a chemotherapy drug
for the treatment of some types of cancer, against SK-
MEL-1 melanoma cells (results not shown). Since an
important structural component of compound 25 is
rhamnose, we next decided to evaluate the role of this
monosaccharide. Therefore, saponin glycosides contain-
ing different hexoses were synthesized. The results indi-
cated that those compounds containing glucose (19),
galactose (20), mannose (21) or fucose (24) instead of
rhamnose were, in general, less cytotoxic in all cell lines
tested. However, a certain cell-type dependence was also
observed with some chemical changes. For example,
compounds 21 and 24 presented IC50s similar to those
of compound 25 (IC50 ꢀ 5 to 11 lM) on HL-60 cells.
However, these substances were completely ineffective
on SK-MEL-1 cells if compared to 25 (IC50 > 100 lM
vs IC50 = 7 lM). Selective acetylation was also per-
formed on compound 25 to yield the mono- and diacet-
ylated derivatives 27 and 26, respectively. Compound 27
was highly cytotoxic (Fig. 1A and B) and no impact on
the IC50s was observed when compared with its deacet-
ylated counterpart 25. This result indicated that the hy-
droxyl group at C-60 of glucose may not play a
significant role in the cytotoxicity of this compound.
Contrarily, a significant decrease in cytotoxic potency
was observed for the diacetylated derivative 26 in all cell
lines tested, which suggests that the free hydroxyl group
3.2. Effect of diosgenyl glycosides on cell cycle
progression
A detailed inspection of Table 2 reveals that of the three
human cell lines used in this study, the growth of promy-
elocytic HL-60 cells was, in general, highly susceptible to
the cytotoxicity induced by diosgenyl glycosides and,
therefore, further studies were performed in this cell line.
Thus, only those compounds with low IC50 (615 lM)
were selected to explore the mechanism through which
diosgenyl glycosides decrease cell viability. Therefore,
we first analyzed whether cell growth inhibition induced
by compounds 20, 21, 24, 25, and 27 is mediated via
alteration in cell cycle progression.
Consistent with growth inhibitory effects, the cytometric
flow studies (Table 3) reveal that compounds 21 and 24 in-

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J. C. Hernandez et al. / Bioorg. Med. Chem. 16 (2008) 2063–2076
2066
Table 1. Synthesis of disaccharide saponins by coupling reaction
Donor
Acceptor
Coupling product
Yield Deprotection product
Yield
OAc
O
Ph
O
HO
Ph
O
O
O
HO
O
AcO
AcO
O
O
O-diosgenin
12
O-diosgenin
PivO
O
HO
O
O-diosgenin
HO
HO
AcO
PivO
77
50
52
64
56
58
49
58
O
O
AcO
HO
AcO
OC(=NH)CCl3
AcO
AcO
HO
19
4a
HO
5
AcO
HO
HO
HO
OH
Ph
O
OAc
O
O
O
O
O-diosgenin
O-diosgenin
PivO
OAc
HO
AcO
AcO
OAc
O
O
4a
4a
4a
4a
4a
4a
38
41
49
47
40
41
O
O
13
OC(=NH)CCl3
AcO
AcO
HO
20
6
HO
AcO
HO
HO
OH
O
AcO
AcO
OAc
OAc
O
O
AcO
14
AcO
Ph
HO
21
AcO
O
HO
HO
HO
O
O
O
O
OC(=NH)CCl3
O-diosgenin
O-diosgenin
PivO
7
O
O
Ph
O
HO
O
AcO
AcO
O
O
HO
O
O-diosgenin
O-diosgenin
PivO
O
HO
O
AcO
O
OC(=NH)CCl3
HO
HO
AcO
AcO
AcO
15
22
8
HO
Ph
O
OAc
HO
HO
HO
O
O
O
O
O-diosgenin
O-diosgenin
PivO
AcO
OH
AcO
AcO
O
O
O
O
OC(=NH)CCl3
16
23
HO
AcO
AcO
HO
9
Ph
O
HO
HO
HO
OC(=NH)CCl3
OAc
O
PivO
O
O
O
O-diosgenin
O-diosgenin
O
O
O
O
O
OAc
OAc
OH
17
24
AcO
AcO
OH
OAc
10
OH
OAc
Ph
O
HO
HO
OC(=NH)CCl3
O
O
O-diosgenin
O-diosgenin
PivO
HO
O
O
O
18
AcO
O
O
OAc
11
25
HO
AcO
HO
AcO
OH
OAc
duce significant G1 arrest at the expense of S- and G2/M-
phase cell population following treatment over 16 h with
concentrations from 10 to 30 lM. Concentrations of
30 lM were used to demonstrate that the effects on cell cy-
cle progression were dose-dependent. Doses higher than
the antiproliferative IC50 were used to identify the pri-
mary targets and early mechanism of action of diosgenyl
glycosides.
30 lM of compounds. Taken together, the results indi-
cated that compounds 21 and 24 versus 25 and 27 dis-
played antiproliferative activities through different
mechanisms that involve cell cycle alteration. Selective
modulation of different cell cycle-regulatory proteins
could explain the differences among them, although
other possibilities cannot be ruled out. No evidence
of change in the phases of the cell cycle was observed
in response to compound 20. In this context it is
important to note that galactose present in 20 is a dia-
stereomer of mannose, which is the monosaccharide
present in 21. The results suggest that not only the
configuration of the hydroxyl group in compound 21
plays an important role in the cell cycle arrest but also
the configuration of the glycosidic bond between the
monosaccharides.
Moreover, the IC50 values were determined at 72 h of
treatment, while the experiments of flow cytometry
were analyzed after a short incubation time. Interest-
ingly, compounds 25 and 27 also produced alterations
in cell cycle progression although they induced G2–M
arrest at the expense of G1 phase cell number. A sig-
nificant increase in the S-phase was also observed at

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J. C. Hernandez et al. / Bioorg. Med. Chem. 16 (2008) 2063–2076
2067
O
HO
Ph
O
O
Ac
O
HO
O
Diosgenin
O Diosgenin
a
b, c
PivO
HO
O
OC(=NH)CCl
3
HO
AcO
O
O
4a
O
O
Glycosyl donors
5-11
Coupling products 12-18
Deprotection products 19-25
Scheme 2. Reagents: (a) BF3ÆEt2O; (b) AcOH (80%); (c) NaOMe, MeOH.
3.3. Diosgenyl glycosides induce apoptosis in human
myeloid leukemia cells
Table 2. Effects of synthetic steroidal saponins on the growth of
human tumor cell linesa
Compound
IC50 (lM)
To determine whether diosgenyl glycosides decrease
HL-60 cell viability through apoptosis activation, quan-
tification of the number of hypodiploid cells (i.e., apop-
totic cells) by flow cytometry was performed.
HL-60
U937
SK-MEL-1
19
20
21
22
23
24
25
26
27
30
88 12
>100
43 12
>100
>100
>100
>100
>100
>100
15
11
3
4
12
56
1
4
The results indicate that the percentage of apoptotic cells
increased from 5 1% (control) to 12.5 0.5% (2.5-fold
increase) and 18 0.5% (3.6-fold increase) after 16 h of
treatment with 10 lM of diosgenyl glycosides 25 and
27, respectively (Fig. 2A). Using a higher concentration
(30 lM) of compounds 21 and 24 the percentage of
apoptotic cells increased from 4.5 1% to 22 1% (5-
fold) and to 18 2% (fourfold), respectively (Fig. 2B
and C). Doses of 10–30 lM were used to demonstrate
that the induction of apoptosis was dose-dependent.
38 10
41
10
5
4
4
1
8
2
3
80 21
28
6
8
3
2
1
1
7
2
33
7
18
5
17
4
4
8
2
22
21
37
a Cells were cultured for 72 h and the IC50 values were calculated as
described in Section 5. The data shown represent means SEM of 3–
5-independent experiments with three determinations in each.
Compound 20 did not induce apoptosis at either con-
centration tested (Fig. 2A and B). Etoposide, which in-
duces morphological changes and internucleosomal
DNA fragmentation characteristic of apoptotic cell
death in human leukemia cells, was used as a positive
These data indicate a considerable effect on the extent as
well as the nature of the cell cycle arrest in HL-60 cells
when the diosgenyl b-D-glucopyranoside skeleton is
modified by substitution at C-20 with different glycosyl
moieties.
HO
HO
AcO
HO
AcO
HO
HO
O
O
O
O
O
O
O Diosgenin
O Diosgenin
O Diosgenin
HO
HO
a
O
O
O
HO
AcO
HO
HO
HO
HO
OH
25
OH
26
OH
27
Scheme 3. Reagents: (a) Novozyme 435, vinyl acetate, THF.
O
Diosgenin
O
Ph
O
O
PivO
SPh
O
a
O
OC(=NH)CCl
O
3
AcO
HO
4a
O
O
AcO
O
29
OAc
O
O
O
O
AcO
11
AcO
28
OAc
O
O
b, c
HO
O
HO
HO
O
O
O
O
30
HO
OH
O
HO
HO
OH
˚
Scheme 4. Reagents and conditions: (a) 4 A MS, TMSOTf, NIS, CH2Cl2, ꢁ20 ꢁC; (b) AcOH (80%), 70 ꢁC; (c) NaOH, MeOH/THF/H2O (1:1:1).

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J. C. Hernandez et al. / Bioorg. Med. Chem. 16 (2008) 2063–2076
2068
A
A
B
20
Control
125
100
75
50
25
0
15
10
5
Comp-27
0
0
1
10
100
C
20 21 24 25 27
Compound 27 (µM)
Compound
Figure 1. (A) Effect of diosgenyl glycoside 27 on human HL-60 cell
viability. Cells were cultured in the presence of the indicated doses of
compound 27 for 72 h, and thereafter cell viability was determined by
the MTT assay as described in Section 5. The results of a represen-
tative experiment are shown. Each point represents the average of
triplicate experiments. (B) Effects of diosgenyl glycoside 27 on the
morphology of HL-60 cells as visualized by phase contrast microscopy.
The cells were untreated (control) or treated with 10 lM of compound
27 (lower panel) for 16 h.
B
C
25
20
15
10
Control
Comp-24
5
0
control and induced 48.6 2.6% of apoptotic cells (data
not shown). Morphological changes characteristic of
apoptotic cells (fragmented and condensed chromatin)
were visualized by fluorescent microscopy. Figure 2D
shows a representative field of cells exposed to 10 lM
of compound 25 for 6 h displaying such morphological
changes and etoposide was also included as a positive
control.
SubG1
C
20 21 24
DNA content
Compound
D
Comp-25
Etoposide
Control
3.4. Involvement of caspase-3 activation in diosgenyl
glycoside induced cell death
Figure 2. Induction of apoptosis in HL-60 cells by diosgenyl glycosides.
(A) Cells were incubated with 10 lM of the indicated compounds for
16 h, and the percentages of apoptotic cells were determined by flow
cytometry. The results of a representative experiment are shown, and
each point represents the average SE of triplicate determinations. (B)
Cells were treated with 30 lM of the indicated compounds and the
percentages of apoptotic cells were determined by flow cytometry as
above. (C) HL-60 cells were treated with 30 lM of 24, harvested at 16 h,
and subjected to DNA flow cytometry. (D) Cells were cultured in the
absence (control) or presence of 10 lM of compound 25 for 6 h; then
they were stained with Hoechst 33258 and nuclei were visualized using
fluorescence microscopy. Etoposide was used as a positive control.
Next, we examined whether these diosgenyl glycosides
(compounds 21, 24, 25, and 27) induce pro-caspase-3
cleavage since caspase-3 is the most active effector cas-
pase to be involved in apoptosis induced by cytotoxic
agents. Cleavage of procaspase-3 by diosgenyl glyco-
sides was determined by immunoblotting using a poly-
clonal antihuman caspase-3 antibody that recognized
the Mr 32,000 proenzyme (procaspase-3). The results
indicate that these compounds at 10 lM promote the
cleavage of procaspase-3 (Fig. 3, upper panel). An
Table 3. Effect of synthetic steroidal saponins on cell cycle distribution of HL-60 cells
%G0/G1
%S
%G2-M
Control
20
49.1 1.2
50.8 0.1
49.9 1.2
52.0 0.2
61.5 1.0
52.2 0.7
58.9 4.5
46.7 0.2
41.7 3.1
44.9 0.4
33.8 4.5
22.4 1.0
21.7 0.8
22.1 0.8
20.8 0.2
17.4 2.0
21.6 0.5
17.4 0.1
22.7 1.1
23.1 0.1
22.8 0.1
25.2 3.5
28.5 1.8
27.5 0.5
28.0 0.7
27.1 0.7
21.1 3.5
26.2 0.1
23.7 0.9
30.5 0.1
35.2 0.4
32.3 0.4
41.0 6.4
10 lM
30 lM
10 lM
30 lM
10 lM
30 lM
10 lM
30 lM
10 lM
30 lM
21
24
25
27
The cells were cultured with the indicated compounds for 16 h and the cell cycle distribution was determined by flow cytometry. The results of a
representative experiment are expressed as means SE of duplicate determinations

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J. C. Hernandez et al. / Bioorg. Med. Chem. 16 (2008) 2063–2076
2069
important processing of procaspase-3 was observed at
30 lM concentration, indicating that this effect was
dose-dependent (results not shown).
A
Cyt c
Cytosol
β-Actin
Since poly(ADP-ribose) polymerase (PARP) is a typical
substrate for caspase-3, we also examined whether these
compounds induce PARP cleavage. Western blot analy-
sis using a polyclonal antibody which recognizes the Mr
85,000 cleaved form of PARP shows the generation of
the 85 kDa fragment in diosgenyl glycoside-treated cells
after 6 h of treatment (Fig. 3, middle panel). Membranes
were stripped and reprobed with b-actin antibody as
loading control (Fig. 3, lower panel).
C
21 24 25 27
Compound
B
Cyt c
Cytosol
β-Actin
Cyt c
Mitochondria
β-Actin
3.5. Diosgenyl glycoside-induced apoptosis involves mito-
chondrial cytochrome c release, but does not affect the
expression of Bcl-2
C
21 24 25
27
Compound
Figure 4. Analysis of cytochrome c release from mitochondria induced
by diosgenyl glycosides. (A) HL-60 cells were treated with 10 lM of
the indicated diosgenyl glycosides and harvested at 6 h. After
treatment, cytosolic extracts were prepared, separated by sodium
dodecyl sulfate–polyacrylamide gel electrophoresis, and cytochrome c
was detected by immunoblotting. b-Actin was used as loading control.
(B) U937 cells were treated as above and cytosolic or mitochondrial
lysates were analyzed by immunoblotting with an anti-cytochrome c
antibody.
The vast majority of conventional anti-cancer agents
indirectly exploit mitochondria to exert their cytotoxic
action. In order to evaluate whether apoptosis induced
by diosgenyl glycosides 21, 24, 25, and 27 in HL-60
and U937 cells involves cytochrome c release from mito-
chondria to cytosol, we subjected cytosolic preparations
to immunoblot analysis. The exposure of HL-60 cells to
these diosgenyl glycosides resulted in increased cytosolic
cytochrome c and this response was observed at 6 h of
treatment (Fig. 4A). Similar results were obtained with
compounds 21 and 27 on U937 cells (Fig. 4B). Since
the Bcl-2 family proteins are the central regulator of
cytochrome c release and caspase activation,27 we tested
whether Bcl-2 is involved in the apoptosis induction by
diosgenyl glycosides. The results indicate that the
expression of Bcl-2 was unchanged after exposure to
compounds 21, 24, 25, and 27 (results not shown).
Pro-Caspase-9
Caspase-9
C
21 24 25 27
Compound
Figure 5. Western blot analysis for the cleavage of procaspase-9. HL-
60 cells were cultured in the presence of 10 lM of the indicated
diosgenyl glycosides and harvested at 6 h. Total cell lysates were
subjected to sodium dodecyl sulfate–polyacrylamide gel electrophore-
sis followed by immunoblotting with an anti-procaspase-9 antibody
that also recognizes the proteolytic fragments generated.
3.6. Diosgenyl glycoside-induced cell death involves
caspase-9 activation
Once cytochrome c is in the cytoplasm, it binds to
apoptotic protease-activating factor-1 (Apaf-1), which
then permits recruitment of procaspase-9. Oligomeri-
zation results in autoactivation of procaspase-9. In
the mitochondrial pathway, the complex of cyto-
chrome c, Apaf-1, and caspase-9, called the ‘apopto-
some’, is a critical activator of effector caspases.
Incubation of HL-60 cells with 10 lM diosgenyl glyco-
sides 21, 24, 25, and 27 for 6 h promoted cleavage of
the inactive pro-caspase-9 to the active 37 kDa frag-
ment (Fig. 5).
Pro-Caspase-3
PARP (fragment)
β-Actin
C
21 24 25 27
Compound
Figure 3. Western blot analysis for the cleavage of procaspase-3 and
for the cleavage of poly (ADP-ribose) polymerase. Cells were treated
with 10 lM of the indicated diosgenyl glycosides for 6 h and whole cell
lysates were subjected to sodium dodecyl sulfate–polyacrylamide gel
electrophoresis followed by blotting with an anti-procaspase-3 anti-
body (upper panel) or with an anti-poly(ADP-ribose) polymerase
(PARP) antibody which recognizes the 85 kDa fragment (middle
panel). The migration positions of full-length procaspase-3, as well as
of PARP cleavage product p85, are indicated. Data shown are
representative of two experiments performed. b-Actin was used as
loading control (lower panel). Control lane (C) refers to untreated
cells.
Taken together, these results allow us to conclude that
the mitochondria play an important role in the activa-
tion of caspase-3, proteolytic cleavage of poly(ADP-ri-
bose) polymerase, and induction of apoptosis triggered
by diosgenyl glycosides in human myeloid cells.
4. Conclusions
In conclusion, we have synthesized a series of diosgenyl
glycosides binding different monosaccharide units to

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J. C. Hernandez et al. / Bioorg. Med. Chem. 16 (2008) 2063–2076
2070
diosgenyl b-glucopyranoside (trillin) and evaluated the
cytotoxicity against three different human cell lines
(HL-60, U937, and SK-MEL-1). Not all diosgenyl glyco-
sides were equally cytotoxic to the three cell lines. SK-
MEL-1 cells were resistant to compounds 19–24, while
compounds 25 and 27 were the most cytotoxic toward
all cell lines assayed. Induction of cell death by diosgenyl
glycosides 21 and 24 in human leukemia cells was associ-
ated with cell cycle delays at the G1 (and S) phase. How-
ever, compounds 25 and 27 induced G2/M arrest like most
DNA-damaging agents that arrest cells at the G2/M tran-
sition. Cell death induced by diosgenyl glycosides 21, 24,
25, and 27 was preceded by a rapid release of cytochrome
c from mitochondria into the cytosol and subsequent cas-
pase activation involving caspase-9 and -3 to cleave poly
(ADP-ribose) polymerase. These findings suggest that
these diosgenyl glycosides present anti-tumor activity,
which may be mediated by apoptosis caused by cyto-
chrome c release and caspase activation in human leuke-
mia cells. Our data should contribute to the development
of diosgenyl glycosides or related drugs as potential can-
cer chemotherapeutic or chemopreventive agents.
2.05 (s, 3H, –OAc), 3.38 (t, 1H, J = 10.8 Hz, H-26),
3.45 (m, 3H, H-26, H-3, H-50), 3.57 (t, 1H, J = 9.4 Hz,
H-40), 3.67 (m, 2H, H-500, H-20), 3.71 (m, 1H, H-60),
4.09 (m, 1H, H-600), 4.29–4.42 (m, 3H, H-60, H-600, H-
16), 4.61 (d, 1H, J = 8.0 Hz, H-10), 4.67 (d, 1H,
J = 7.9 Hz, H-100), 4.97 (t, 1H, J = 7.2 Hz, H-200), 5.08
(m, 2H, H-300, H-400), 5.26 (t, 1H, J = 7.9 Hz, H-30),
5.35 (br d, 1H, H-6), 5.43 (s, 1H, Ph-CH), 7.29–7.35
(m, 5H, Ph). 13C NMR (d, CDCl3, 75 MHz): 177.0
(CO-Piv), 170.7 (CO-Ac), 170.2 (CO-Ac), 169.8 (CO-
Ac), 169.4 (CO-Ac), 140.4 (C-5), 128.2 (Ph), 125.8
(Ph), 121.8 (C-6), 109.3 (C-22), 101.1 (PhCH), 100.7
(C-100), 100.2 (C-10), 80.80 (C-16), 80.20 (C-3), 78.9 (C-
40), 78.7 (C-20), 73.6 (C-30), 73.0 (C-300), 71.0 (C-500),
71.0 (C-200), 68.8 (C-60), 68.4 (C-400), 66.85 (C-26), 65.4
(C-50), 62.19 (C-17), 62.0 (C-600), 56.51 (C-14), 50.07
(C-9), 41.62 (C-20), 40.27 (C-13), 39.78 (C-12), 38.69
(C-4), 37.12 (C-1), 36.81 (C-10), 32.05 (C-7), 31.83 (C-
2), 31.42 (C-15), 31.40 (C-8), 30.30 (C-25), 29.56 (C-
23), 28.81 (C-24), 27.16 (CH3-Piv), 20.86 (C-11), 20.76
(CH3-Ac), 20.55 (CH3-Ac) and 19.36 (C-19), 17.12 (C-
27), 16.28 (C-18), 14.51 (C-21). HRFABMS: 1079.5514
(M+H)+ (calcd for C59H83O18, 1079.5579) (5), 665
(12), 398 (14), 397 (56), 331 (36). Anal. Calcd for
C59H82O18: C, 65.66; H, 7.66. Found: C, 65.66; H, 7.28.
5. Experimental
5.1. General experimental procedures
5.2.2. Diosgenyl 2,3,4,6-tetra-O-acetyl-b-D-galactopyran-
osyl-(1 ! 2)-4,6-O-benzylidene-3-O-pivaloyl-b-D-glucopy-
20
D
Optical rotations were recorded in a Perkin-Elmer 343
ranoside (13). White amorphous solid, ½aꢃ ꢁ38ꢁ (c
1
1
polarimeter. H NMR and 13C NMR spectra were ob-
0.102, CHCl3). H NMR (d, CDCl3, 400 MHz): 0.76
(m, 6H, CH3-18, CH3-27), 0.95 (d, 3H, J = 6.7 Hz,
CH3-21), 1.00 (s, 3H, CH3-19), 1.20 (s, 9H, CH3-Piv),
1.95 (s, 3H, –OAc), 2.02 (s, 3H, –OAc), 2.04 (s, 3H, –
OAc), 2.11 (s, 3H, –OAc), 3.35 (t, 1H, J = 10.9 Hz, H-
26), 3.48 (m, 3H, H-26, H-3, H-50), 3.59 (t, 1H,
J = 9.5 Hz, H-40), 3.68–3.73 (m, 2H, H-20, H-60), 3.86
(t, 1H, J = 7.0 Hz, H-500), 4.0 (dd, 1H, J = 10.5; 5.0 Hz,
H-60), 4.12 (m, 2H, H-600), 4.39 (dd, 1H, J = 14.2;
6.6 Hz, H-16), 4.63 (m, 2H, H-10, H-100), 4.89 (dd, 1H,
J = 10.5; 3.3 Hz, H-300), 5.14 (dd, 1H, J = 10.4; 8.0 Hz,
H-200), 5.26 (t, 1H, J = 8.5 Hz, H-30), 5.35 (m, 2H, H-6,
H-400), 5.43 (s, 1H, Ph-CH), 7.29–7.35 (m, 5H, Ph). 13C
NMR (d, CDCl3, 75 MHz): 177.0 (CO-Piv), 170.4,
170.2 and 169.9 (CO-Ac), 140.6 (C-5), 128.2 (Ph),
125.8 (Ph), 121.7 (C-6), 109.3 (C-22), 101.1 (PhCH),
100.9 (C-100), 100.3 (C-10), 80.82 (C-16), 80.16 (C-3),
78.80 (C-40), 78.23 (C-20), 73.58 (C-30), 71.05 (C-300),
70.41 (C-500), 68.88 (C-60), 68.73 (C-200), 66.88 (C-400),
66.88 (C-26), 65.50 (C-50), 62.15 (C-17), 60.9 (C-600),
56.50 (C-14), 50.07 (C-9), 41.63 (C-20), 40.28 (C-13),
39.77 (C-12), 38.79 (C-4), 37.07 (C-1), 36.84 (C-10),
32.06 (C-7), 31.85 (C-2), 31.47 (C-8), 31.41 (C-15),
30.30 (C-25), 29.51 (C-23), 28.82 (C-24), 27.16 (CH3-
Piv), 20.86 (C-11), 20.73, 20.65 and 20.54 (CH3-Ac),
19.40 (C-19), 17.12 (C-27), 16.28 (C-18), 14.51 (C-21).
HRFABMS: 1079 (M+H)+, 1078.5470 (M+) (calcd. for
C59H83O18, 1078.5501) (5), 665 (14), 398 (17), 397 (57),
331 (74). Anal. Calcd for C59H82O18: C, 65.66; H,
7.66. Found: C, 65.91; H, 7.40.
tained on Bruker AMX-400, Avance 400, and Avance
300 spectrometers with standard pulse sequences operat-
ing at 400, 300 MHz in H NMR and 100, 75 MHz in
1
13C NMR. Chemical shifts are given in d values (ppm)
using tetramethylsilane as the internal standard. EIMS,
HREIMS, FABS, and HRFABS were taken on a
Micromass Autospec (70 eV) spectrometer. Column
chromatography was carried out on silica gel 60 (Merck
230–400 mesh), and preparative TLC on silica gel 60
PF254+366 plates (20 · 20 cm, 1 mm thickness), and
Sephadex LH-20 (Aldrich). IR data reported in cmꢁ1
were obtained using a Bruker IFS 55 spectrophotome-
ter. Elemental analyses (C, H, N) were performed on a
Fisons EA 1108 analyzer.
5.2. General conditions for coupling
To a mixture of diosgenyl 4,6-O-benzylidene-3-O-piva-
loyl-b-D-glucopyranoside 4a (360 mg, 0.48 mmol) and
˚
4 A MS (0.6 g) in dry CH2Cl2 (12 mL) at ꢁ78 ꢁC, under
N2, BF3ÆOEt2 (0.07 mL, 0.57 mmol) was added, fol-
lowed by a solution of imidate 5 (349 mg, 0.71 mmol)
in dry CH2Cl2 (4 mL). The mixture, warmed up to room
temperature for 7 h, was neutralized with Et3N
(ꢂ0.1 mL) to yield saponin 12 (Table 1).
5.2.1. Diosgenyl 2,3,4,6-tetra-O-acetyl-b-D-glucopyrano-
syl-(1 ! 2)-4,6-O-benzylidene-3-O-pivaloyl-b-D-glucopy-
20
D
ranoside (12). White amorphous solid, ½aꢃ ꢁ44ꢁ (c
1
0.100, CHCl3). H NMR (d, CDCl3, 400 MHz): 0.76
(6H, CH3-18, CH3-27), 0.95 (d, 3H, J = 6.7 Hz, CH3-
21), 1.00 (s, 3H, CH3-19), 1.22 (s, 9H, CH3-Piv), 1.97
(s, 3H, –OAc), 1.99 (s, 3H, –OAc), 2.03 (s, 3H, –OAc),
5.2.3. Diosgenyl 2,3,4,6-tetra-O-acetyl-a-D-mannopyran-
osyl-(1 ! 2)-4,6-O-benzylidene-3-O-pivaloyl-b-D-glucopy-
20
ranoside (14). White amorphous solid, ½aꢃ ꢁ43ꢁ (c
D

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J. C. Hernandez et al. / Bioorg. Med. Chem. 16 (2008) 2063–2076
2071
0.020, CHCl3). 1H NMR (d, CDCl3, 400 MHz): 0.76 (m,
6H, CH3-18, CH3-27), 0.89 (d, 3H, J = 5.5 Hz, CH3-21),
0.99 (s, 3H, CH3-19), 1.16 (s, 9H, CH3-Piv), 3.35 (t, 1H,
J = 10.9 Hz, H-26), 3.44 (d, 1H, J = 3.4 Hz, H-26), 3.50
(m, 1H, H-50), 3.56 (t, 1H, J = 9.6 Hz, H-40), 3.61 (m,
1H, H-3), 3.70 (m, 1H, H-20), 3.56 (m, 1H, H-40), 3.74
(t, 1H, J = 10.2 Hz, H-60b), 3.99 (m, 1H, H-50), 4.33
(dd, 1H, J = 10.4; 4.8 Hz, H ꢁ 60a), 4.38 (dd, 1H,
J = 14.4, 7.1 Hz, H-16), 4.71 (d, 1H, J = 7.5 Hz, H-10),
5.22 (dd, 1H, J = 10.1; 2.7 Hz, H-300), 5.29 (m, 1H, H-
200), 5.30 (m, 1H, H-400), 5.33 (t, 1H, J = 9.7 Hz, H-30),
5.36 (br d, 1H, H-6), 5.44 (s, 1H, H-100), 5.45 (s, 1H,
PhCH), 7.33–7.40 (m, 5H, Ph). 13C RMN (d, CDCl3,
75 MHz): 177.0 (CO-Piv), 170.7, 169.7, 169.6 and
169.5 (CO-Ac), 140.0 (C-5), 125.8 and 128.2 (Ph),
122.1 (C-6), 109.3 (C-22), 101.5 (C-10), 101.1 (PhCH),
97.2 (C-100), 80.81 (C-16), 79.25 (C-40), 79.25 (C-3),
76.48 (C-20), 72.43 (C-30), 69.33 (C-200), 68.89 (C-300),
68.63 (C-60), 68.63 (C-500), 66.82 (C-26), 65.95 (C-50),
65.79 (C-400), 62.13 (C-17), 62.13 (C-600), 56.47 (C-14),
50.07 (C-9), 41.62 (C-20), 40.26 (C-13), 39.79 (C-12),
38.07 (C-4), 37.17 (C-1), 36.85 (C-10), 32.10 (C-7),
31.84 (C-2), 31.84 (C-15), 31.40 (C-8), 30.29 (C-25),
29.63 (C-23), 28.81 (C-24), 27.16 (CH3-Piv), 20.80
(C-11), 20.71 and 20.63 (CH3-Ac), 19.18 (C-19), 17.12
(C-27), 16.24 (C-18), 14.51 (C-21). HRFABMS:
1079.5623 (M+H)+ (calcd for C59H83O18, 1079.5579),
661 (6), 398 (9), 397 (39), 331 (32). Anal. Calcd for
C59H82O18: C, 65.66; H, 7.66. Found: C, 65.35; H,
7.61.
5.2.5. Diosgenyl 2,3,4-tri-O-acetyl-b-L-arabinopyranosyl-
(1 ! 2)-4,6-O-benzylidene-3-O-pivaloyl-b-D-glucopyrano-
20
side (16). White amorphous solid, ½aꢃ ꢁ33ꢁ (c 0.105,
D
1
CHCl3). H NMR (d, CDCl3, 400 MHz): 0.75 (m, 6H,
CH3-18, CH3-27), 0.94 (d, 3H, J = 6.7 Hz, CH3-21),
0.98 (s, 3H, CH3-19), 1.17 (s, 9H, CH3-Piv), 2.00 (s,
3H, –OAc), 2.03 (s, 3H, –OAc), 2.05 (s, 3H, –OAc),
3.35 (t, 1H, J = 10.9 Hz, H-26), 3.43–3.60 (m, 5H, H-
26, H-3, H-50, H-500, H-40), 3.66–3.73 (m, 2H, H-20, H-
60), 4.12 (dd, 1H, J = 12.7; 4.8 Hz, H-500), 4.29 (dd, 1H,
J = 10.5; 4.9 Hz, H-60), 4.40 (dd, 1H, J = 14.7; 6.9 Hz,
H-16), 4.63 (d, 1H, J = 7.0 Hz, H-10), 4.65 (d, 1H,
J = 5.7 Hz, H-100), 4.98 (dd, 1H, J = 8.2; 3.4 Hz, H-300),
5.04 (dd, 1H, J = 8.1; 5.8 Hz, H-200), 5.16 (m, 1H, H-
400), 5.25 (t, 1H, J = 8.5 Hz, H-30), 5.36 (br d, 1H, H-
6), 5.42 (s, 1H, Ph-CH), 7.29–7.35 (m, 5H, Ph). 13C
RMN (d, CDCl3, 75 MHz): 177.0 (CO-Piv), 170.2 and
170.1 (CO-Ac), 140.5 (C-5), 128.2 (Ph) and 125.8 (Ph),
121.8 (C-6), 109.3 (C-22), 101.1 (PhCH), 100.2 (C-10),
99.9 (C-100), 80.78 (C-16), 79.70 (C-3), 78.87 (C-40),
77.40 (C-20), 73.63 (C-30), 69.46 (C-300), 69.10 (C-200),
68.87 (C-60), 66.99 (C-400), 66.81 (C-26), 65.54 (C-50),
62.10 (C-17), 61.73 (C-500), 56.47 (C-14), 50.07 (C-9),
41.60 (C-20), 40.25 (C-13), 39.74 (C-12), 38.79 (C-4),
37.09 (C-1), 36.83 (C-10), 32.05 (C-7), 31.82 (C-2),
31.43 (C-8), 31.38 (C-15), 30.27 (C-25), 29.51 (C-23),
28.79 (C-24), 27.16 (CH3-Piv), 20.86 (C-11), 20.74 and
20.63 (CH3-Ac), 19.34 (C-19), 17.13 (C-27), 16.29 (C-
18), 14.52 (C-21) FABMS: 1008 (M+H)+ (C56H79O16),
397 (64). Anal. Calcd for C56H79O16: C, 66.78; H,
7.81. Found: C, 66.85; H, 7.43.
5.2.4. Diosgenyl 2,3,4-tri-O-acetyl-b-D-xilopyranosyl-
(1 ! 2)-4,6-O-benzylidene-3-O-pivaloyl-b-D-glucopyrano-
20
D
5.2.6. Diosgenyl 2,3,4-tri-O-acetyl-b-L-fucopyranosyl-
side (15). White amorphous solid, ½aꢃ ꢁ57ꢁ (c 0.100,
1
(1 ! 2)-4,6-O-benzylidene-3-O-pivaloyl-b-D-glucopyrano-
CHCl3). H NMR (d, CDCl3, 400 MHz): 0.77 (m, 6H,
CH3-18, CH3-27), 0.96 (d, 3H, J = 6.7 Hz, CH3-21),
1.02 (s, 3H, CH3-19), 1.21 (s, 9H, CH3-Piv), 2.05 (s,
3H, –OAc), 2.08 (s, 3H, –OAc), 2.16 (s, 3H, –OAc),
3.35 (m, 1H, H-26), 3.38 (1H, H-500), 3.44–3.55 (m,
4H, H-26, H-3, H-50, H-40), 3.69–3.76 (m, 2H, H-20,
H-60), 4.25 (dd, 1H, J = 12.2; 4.7 Hz, H-500), 4.31 (dd,
1H, J = 10.5; 4.9 Hz, H-60), 4.40 (dd, 1H, J = 14.7;
6.9 Hz, H-16), 4.62 (d, 1H, J = 7.2 Hz, H-10), 4.75 (d,
1H, J = 6.1 Hz, H-100), 4.85–4.93 (m, 2H, H-200, H-400),
5.06 (t, 1H, J = 7.9 Hz, H-300), 5.29 (t, 1H, J = 8.9 Hz,
H-30), 5.37 (br d, 1H, H-6), 5.44 (s, 1H, Ph-CH), 7.29–
7.35 (m, 5H, Ph). 13C RMN (d, CDCl3, 75 MHz):
177.0 (CO-Piv), 169.9 and 169.6 (CO-Ac), 140.2 (C-5),
128.2 and 125.8 (Ph), 121.9 (C-6), 109.3 (C-22), 101.1
(PhCH), 101.0 (C-100), 100.1 (C-10), 80.80 (C-16), 79.73
(C-3), 78.91 (C-40), 77.25 (C-20), 73.74 (C-30), 70.89
(C-300), 70.19 (C-200), 69.05 (C-400), 68.79 (C-60), 66.81
(C-26), 65.59 (C-50), 62.06 (C-17), 61.78 (C-500), 56.47
(C-14), 50.04 (C-9), 41.58 (C-20), 40.24 (C-13), 39.74
(C-12), 38.63 (C-4), 37.12 (C-1), 36.82 (C-10), 32.04
(C-7), 31.81 (C-2), 31.37 (C-8), 31.37 (C-15), 30.27 (C-
25), 29.53 (C-23), 28.77 (C-24), 27.16 (CH3-Piv), 20.83
(C-11), 20.77, 20.70 and 20.63 (CH3-Ac), 19.40 (C-19),
17.10 (C-27), 16.27 (C-18), 14.49 (C-21). FABMS:
1008 (M+H)+(C56H79O16), 1005 (MꢁH)+, 604 (10),
593 (16), 523 (24), 398 (24), 397 (54), 396 (21). Anal.
Calcd for C56H79O16: C, 66.78; H, 7.81. Found: C,
66.91; H, 7.35.
20
side (17). White amorphous solid, ½aꢃ ꢁ34ꢁ (c 0.095,
D
1
CHCl3). H NMR (d, CDCl3, 400 MHz): 0.73 (m, 6H,
CH3-18, CH3-27), 0.91 (d, 3H, J = 6.7 Hz, CH3-21), 1.00
(s, 3H, CH3-19), 1.11 (d, 3H, J = 6.2 Hz, H-600), 1.19 (s,
9H, CH3-Piv), 1.91 (s, 3H, –OAc), 1.97 (s, 3H, –OAc),
2.05 (s, 3H, –OAc), 3.31 (t, 1H, J = 10.9 Hz, H-26), 3.41–
3.56 (m, 4H, H-26, H-3, H-50, H-40), 3.66–3.73 (m, 3H,
H-20, H-60, H-500), 4.26 (dd, 1H, J = 10.5; 4.8 Hz, H-60),
4.36 (dd, 1H, J = 14.7; 6.9 Hz, H-16), 4.59 (d, 1H,
J = 7.9 Hz, H-10), 4.86 (d, 1H, J = 7.6 Hz, H-100), 4.90–
4.98 (m, 2H, H-300, H-400), 5.12–5.19 (m, 2H, H-200, H-30),
5.32 (br d, 1H, H-6), 5.41 (s, 1H, Ph-CH), 7.29–7.35 (m,
5H, Ph). 13C RMN (d, CDCl3, 75 MHz) 177.0 (CO-Piv),
170.7, 170.2 169.8 and 169.4 (CO-Ac), 140.4 (C-5), 128.2
and 125.8 (Ph). 121.8 (C-6), 109.3 (C-22), 101.1 (PhCH),
100.97 (C-10), 99.08 (C-100), 80.80 (C-16), 79.90 (C-3),
78.92 (C-40), 75.49 (C-20), 71.79 (C-400), 70.94 (C-200),
70.28 (C-30), 69.56 (C-300), 68.80 (C-500), 68.66 (C-60),
66.81 (C-26), 66.11 (C-50), 62.10 (C-17), 56.51 (C-14),
50.07 (C-9), 41.62 (C-20), 40.27 (C-13), 39.78 (C-12),
38.80 (C-4), 37.10 (C-1), 36.81 (C-10), 32.05 (C-7), 31.79
(C-2), 31.42 (C-8), 31.42 (C-15), 30.30 (C-25), 29.56
(C-23), 28.81 (C-24), 27.16 (CH3-Piv), 20.97 (C-600),
20.86 (C-11), 20.76 and 20.55 (CH3-Ac), 19.39 (C-19),
17.12 (C-27), 16.28 (C-18), 14.51 (C-21). HRFABMS:
1021.5515 (M+H)+ (calcd. for C57H81O16, 1021.5525) (5),
607 (14), 398 (14), 397 (50), 396 (13). Anal. Calcd for
C57H80O16: C, 67.04; H, 7.90. Found: C, 67.05; H, 7.93.

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5.2.7. Diosgenyl 2,3,4-tri-O-acetyl-a-L-rhamnopyranosyl-
62.84 (C-17), 62.84 (C-600), 62.62 (C-60), 56.65 (C-14),
50.24 (C-9), 41.97 (C-20), 40.45 (C-13), 39.89 (C-12),
39.22 (C-4), 37.32 (C-1), 37.02 (C-10), 32.18 (C-2),
32.18 (C-7), 31.79 (C-8), 31.61 (C-15), 30.58 (C-25),
30.21 (C-23), 29.24 (C-24), 21.11 (C-11), 19.49 (C-19),
17.34 (C-27), 16.37 (C-18), 15.05 (C-21). FABMS m/z
761 (M+Na)+, 413, 397. HRFABMS m/z 761.4117,
calcd for C39H62O13Na, 761.4088; Anal. Calcd for
C39H62O13: C, 63.39; H, 8.46. Found: C, 63.32; H, 8.74.
(1 ! 2)-4,6-O-benzylidene-3-O-pivaloyl-b-D-glucopyrano-
20
side (18). White amorphous solid, ½aꢃ ꢁ45ꢁ (c 0.090,
D
1
CHCl3). H NMR (d, CDCl3, 400 MHz): 0.77 (m, 6H,
CH3-18, CH3-27), 0.96 (d, 3H, J = 6.7 Hz, CH3-21),
1.01 (s, 3H, CH3-19), 1.19 (d, 3H, J = 6.1 Hz, CH3-60),
1.12 (s, 9H, CH3-Piv), 1.95 (s, 3H, –OAc), 2.00 (s, 3H,
–OAc), 2.09 (s, 3H, –OAc), 3.36 (t, 1H, J = 10.8 Hz,
H-26), 3.45–3.54 (m, 3H, H-26, H-50, H-40), 3.63 (m,
1H, H-3), 3.71–3.80 (m, 2H, H-20, H-60), 4.32 (dd, 1H,
J = 10.5; 4.8 Hz, H-60), 4.37–4.48 (m, 2H, H-16, H-500),
4.70 (d, 1H, J = 7.6 Hz, H-10), 4.93 (s, 1H, H-100), 5.04
(t, 1H, J = 9.9 Hz, H-400), 5.17 (d, 1H, J = 1.4 Hz, H-
200), 5.23 (dd, 1H, J = 9.9; 3.3 Hz, H-300), 5.37–5.41 (m,
2H, H-6, H-30), 5.43 (s, 1H, Ph-CH), 7.29–7.35 (m,
5H, Ph). 13C RMN (d, CDCl3, 75 MHz): 177.0 (CO-
Piv), 170.55, 170.27 and 169.27 (CO-Ac), 140.09 (C-5),
128.2 and 125.8 (Ph), 122.03 (C-6), 109.22 (C-22),
101.1 (PhCH), 99.6 (C-10), 97.6 (C-1000), 80.74 (C-16),
79.04 (C-40), 78.61 (C-3), 75.39 (C-2 ), 73.84 (C-30),
71.22 (C-400), 69.60 (C-200), 68.84 (C-300), 68.65 (C-60),
66.77 (C-26), 66.40 (C-500), 66.02 (C-50), 62.09 (C-17),
56.44 (C-14), 50.06 (C-9), 41.57 (C-20), 40.26 (C-13),
39.72 (C-12), 38.15 (C-4), 37.16 (C-1), 36.82 (C-10),
32.03 (C-7), 31.83 (C-2), 31.40 (C-15), 31.35 (C-8),
30.24 (C-25), 29.74 (C-23), 28.78 (C-24), 27.16 (CH3-
Piv), 20.89 (C-11), 20.97, 20.89 and 20.52 (CH3-Ac),
19.41 (C-19), 17.21 (C-600), 17.13 (C-27), 16.00 (C-18),
14.52 (C-21). HRFABMS: 1021.5549 (M+H)+ (calcd
for C57H81O16, 1021.5525) (4), 607 (17), 398 (19), 397
(65), 396 (17). Anal. Calcd for C57H81O16: C, 67.04; H,
7.90. Found: C, 67.09; H, 7.89.
5.3.2. Diosgenyl b-D-galactopyranosyl-(1 ! 2)-b-D-gluco-
20
pyranoside (20). White amorphous solid, ½aꢃ ꢁ27ꢁ (c
D
0.041, EtOH); IR mmax (film, NaCl) 3363, 2925, 1462,
1
1080, 897 cmꢁ1; H NMR (d, pyridine, 400 MHz): 0.65
(d, 3H, J = 4.7 Hz, CH3-27), 0.78 (s, 3H, CH3-18),
0.94 (s, 3H, CH3-19), 1.09 (d, 3H, J = 6.8 Hz, CH3-
21), 3.45 (t, 1H, J = 9.9 Hz, H-26), 3.53 (m, 1H, H-26),
3.79–3.84 (m, 2H, H-3, H-50), 3.95 (m, 1H, H-500), 4.08
(m, 2H, H-20, H-200), 4.15 (m, 1H, H-300), 4.19 (m, 1H,
H-40), 4.27–4.33 (m, 2H, H-30, H-600), 4.44–4.54 (m,
3H, H-60, H-600) 4.55 (m, 1H, H-500), 4.58 (m, 1H, H-
200), 5.02 (d, 1H, J = 7.5 Hz, H-10), 5.11 (d, 1H,
J = 7.7 Hz, H-100), 5.33 (br d, 1H, H-6). 13C NMR (d,
pyridine, 75 MHz): 140.8 (C-5), 121.3 (C-6), 109.0
(C-22), 107.3 (C-100), 101.2 (C-10), 85.00 (C-20), 81.87
(C-1060 ), 79.12 (C-3), 77.93 (C-50), 77.67 (C-30), 76.97
(C-4 ), 74.80 (C-300), 74.35 (C-200), 71.12 (C-40), 69.64
(C-500), 62.65 (C-17), 66.64 (C-26), 62.64 (C-600), 62.33
(C-60), 56.42 (C-14), 50.01 (C-9), 41.73 (C-20), 40.21
(C-13), 39.65 (C-12), 39.05 (C-4), 37.18 (C-1), 36.81
(C-10), 32.04 (C-2), 32.04 (C-7), 31.58 (C-8), 31.40 (C-
15), 30.36 (C-25), 30.00 (C-23), 29.03 (C-24), 20.88 (C-
11), 19.27 (C-19), 17.10 (C-27), 16.12 (C-18), 14.80 (C-
21). FABMS m/z 761 (M+Na)+, 397; HRFABMS m/z
761.4053, calcd for C39H62O13Na, 761.4088; Anal. Calcd
for C39H62O13: C, 63.39; H, 8.46. Found: C, 63.34; H,
8.71.
5.3. General procedure to obtain compounds 19–25
A solution of 12 (269.5 mg, 0.25 mmol) in AcOH
(20 mL, 80%) was stirred at 70 ꢁC for 2 h, then concen-
trated and dissolved in CH2Cl2/MeOH (1:1) (8 mL) and
NaOH (0.1 equiv for each ester) was added and stirred
overnight at 40 ꢁC. The mixture was neutralized with
acid resin Dowex-50 (H+), the residue was filtered, and
the solvent was evaporated to yield compound 19
(Table 1).
5.3.3. Diosgenyl a-D-mannopyranosyl-(1 ! 2)-b-D- gluco-
20
pyranoside (21). White amorphous solid, ½aꢃ ꢁ34 ꢁ (c
D
0.013, EtOH); IR mmax (film, NaCl) 3427, 2930, 1645,
1
979 cmꢁ1; H NMR (d, pyridine, 400 MHz): 0.65 (d,
3H, J = 4.3 Hz, CH3-27), 0.79 (s, 3H, CH3-18), 0.81 (s,
3H, CH3-19), 1.09 (d, 3H, J = 6.9 Hz, CH3-21), 3.45 (t,
1H, J = 10.4 Hz, H-26), 3.54 (m, 1H, H-26), 3.76 (m,
1H, H-50), 3.85 (m, 1H, H-3), 3.95 (m, 1H, H-500),
4.10–4.17 (m, 3H, H-20, H-30, H-40), 4.27–4.55 (m, 3H,
H-600, H-60, H-16), 4.68 (dd, 1H, J = 3.2; 9.1 Hz, H-
300), 4.75–4.79 (m, 2H, H-200, H-400), 4.94 (d, 1H,
J = 7.3 Hz, H-10), 5.13 (m, 1H, H-500), 5.19 (br d, 1H,
J = 4.8 Hz, H-6), 6.07 (s, 1H, H-100). 13C NMR (d, pyri-
dine, 75 MHz): 141.0 (C-5), 121.6 (C-6), 109.3 (C-22),
106.5 (C-100), 101.4 (C-10), 84.42 (C-20), 81.83 (C-16),
79.35 (C-3), 78.73 (C-50), 78.16 (C-500), 77.96 (C-300),
77.84 (C-30), 76.95 (C-200), 71.42 (C-40), 71.36 (C-400),
66.84 (C-26), 62.84 (C-17), 62.84 (C-600), 62.80 (C-60),
56.65 (C-14), 50.24 (C-9), 41.97 (C-20), 40.45 (C-13),
39.89 (C-12), 39.22 (C-4), 38.32 (C-1), 37.02 (C-10),
32.18 (C-2), 32.18 (C-7), 31.79 (C-8), 31.61 (C-15),
30.58 (C-25), 30.21 (C-23), 29.24 (C-24), 21.11 (C-11),
19.49 (C-19), 17.34 (C-27), 16.37 (C-18), 15.05 (C-21).
FABMS m/z 761 (M+Na)+, 513, 397; HRFABMS m/z
5.3.1. Diosgenyl b-D-glucopyranosyl-(1 ! 2)-b-D-gluco-
20
pyranoside (19). White amorphous solid, ½aꢃ ꢁ38ꢁ (c
D
0.011, EtOH); IR mma1x (film, NaCl) 3363, 2926, 1376,
1075, 982, 898 cmꢁ1; H NMR (d, pyridine, 400 MHz):
0.66 (d, 3H, J = 4.3 Hz, CH3-27), 0.78 (s, 3H, CH3-
18), 0.95 (s, 3H, CH3-19), 1.10 (d, 3H, J = 6.8 Hz,
CH3-21), 1.77 (m, 1H, H-17), 3.45 (t, 1H, J = 9.9 Hz,
H-26), 3.54 (m, 1H, H-26), 3.80 (m, 1H, H-3), 3.87 (m,
1H, H-50), 3.95 (m, 1H, H-500), 4.11 (m, 2H, H-20, H-
200), 4.21 (m, 2H, H-300, H-400), 4.31 (m, 2H, H-30, H-
40), 4.31–4.50 (dd, 1H, J = 14.4, 7.1 Hz, H-16), 4.51
(m, 2H, H-60, H-600), 5.03 (d, 1H, J = 7.5 Hz, H-10),
5.24 (d, 1H, J = 7.6 Hz, H-100), 5.30 (br d, 1H,
J = 4.6 Hz, H-6). 13C NMR (d, pyridine, 75 MHz):
141.0 (C-5), 121.6 (C-6), 109.3 (C-22), 106.5 (C-100),
101.4 (C-10), 84.42 (C-20), 81.83 (C-16), 79.35 (C-3),
78.73 (C-50), 78.16 (C-500), 77.96 (C-300), 77.84 (C-30),
76.95 (C-200), 71.42 (C-40), 71.36 (C-400), 66.84 (C-26),

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J. C. Hernandez et al. / Bioorg. Med. Chem. 16 (2008) 2063–2076
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761.4088, calcd for C39H62O13Na, 761.4088; Anal. Calcd
for C39H62O13: C, 63.39; H, 8.46. Found: C, 63.41; H,
8.42.
CH3-21), 1.45 (d, 3H, J = 6.2 Hz, CH3-600), 3.45 (t, 1H,
J = 9.5 Hz, H-26), 3.55 (m, 1H, H-26), 3.78–3.86 (m,
3H, H-500, H-50, H-3), 3.98–4.02 (m, 2H, H-20, H-200),
4.05 (m, 1H, H-300), 4.13 (t, 1H, J = 8.8 Hz, H-30), 4.21
(t, 1H, J = 8.9 Hz, H-40), 4.31–4.38 (m, 1H, H-16),
4.47–4.58 (m, 2H, H-16, H-60), 4.96 (d, 1H, J = 7.7 Hz,
H-10), 5.14 (d, 1H, J = 7.7 Hz, H-100), 5.31 (br d, 1H,
H-6). 13C NMR d (d, pyridine, 75 MHz): 140.9 (C-5),
121.4 (C-6), 109.0 (C-22), 104.6 (C-100), 100.4 (C-10),
82.30 (C-20), 81.86 (C-16), 78.94 (C-3), 77.95 (C-50),
76.63 (C-30), 74.95 (C-300), 72.33 (C-400), 72.23 (C-200),
71.49 (C-500), 71.16 (C-40), 62.65 (C-17), 66.62 (C-26),
62.36 (C-60), 56.46 (C-14), 50.06 (C-9), 41.73 (C-20),
40.23 (C-13), 39.66 (C-12), 39.01 (C-4), 37.21 (C-1),
36.82 (C-10), 32.07 (C-2), 31.96 (C-7), 31.58 (C-8),
31.45 (C-15), 30.36 (C-25), 30.08 (C-23), 29.02 (C-24),
20.89 (C-11), 19.25 (C-19), 17.09 (C-27), 16.90 (C-600),
16.14 (C-18), 14.81 (C-21); FABMS m/z 723 (M+H)+,
416, 397. HRFABMS m/z 723.4362, calcd for C39H63
O12, 723.4320; Anal. Calcd for C39H62O12: C, 64.80;
H, 8.64. Found: C, 64.39; H, 8.98.
5.3.4. Diosgenyl b-D-xylopyranosyl-(1 ! 2)-b-D-glucopy-
20
ranoside (22). White amorphous solid, ½aꢃ ꢁ39 ꢁ (c
D
0.022, EtOH); IR mmax (film, NaCl) 3271, 2928, 1046,
1
982, 833 cmꢁ1; H NMR (d, pyridine, 400 MHz): 0.66
(d, 3H, J = 4.3 Hz, CH3-27), 0.80 (s, 3H, CH3-18),
0.92 (s, 3H, CH3-19), 1.10 (d, 3H, J = 6.8 Hz, CH3-
21), 3.45 (t, 1H, J = 9.5 Hz, H-26), 3.55 (m, 1H, H-26),
3.72 (t, 1H, J = 10.2 Hz, H-500), 3.86 (m, 2H, H-3, H-
50), 4.04–4.10 (m, 2H, H-20, H-200), 4.14–4.19 (m, 3H,
H-40, H-300, H-400), 4.29 (m, 2H, H-30, H-60), 4.40–4.51
(m, 3H, H-60, H-500, H-16), 5.00 (d, 1H, J = 7.4 Hz, H-
10), 5.18 (m, 1H, H-100), 5.29 (br d, 1H, H-6). 13C
NMR (d, pyridine, 75 MHz): 140.8 (C-5), 121.46 (C-6),
109.0 (C-22), 106.5 (C-100), 100.7 (C-10), 83.32 (C-20),
81.86 (C-16), 78.80 (C-3), 78.04 (C-50), 77.95 (C-30),
77.43 (C-300), 75.79 (C-200), 71.09 (C-40), 70.79 (C-400),
67.12 (C-500), 62.65 (C-17), 66.62 (C-26), 62.33 (C-60),
56.44 (C-14), 50.02 (C-9), 41.73 (C-20), 40.23 (C-13),
39.66 (C-12), 39.00 (C-4), 37.16 (C-1), 36.84 (C-10),
32.01 (C-2), 31.96 (C-7), 31.58 (C-8), 31.43 (C-15), 30.36
(C-25), 29.90 (C-23), 29.03 (C-24), 20.89 (C-11), 19.20
(C-19), 17.10 (C-27), 16.14 (C-18), 14.81 (C-21); FABMS
m/z 731 (M+Na)+, 415, 397. HRFABMS m/z 731.3962,
calcd for C38H60O12Na, 731.3982; Anal. Calcd for
C38H60O12: C, 64.38; H, 8.53. Found: C, 64.45; H, 8.46.
5.3.7. Diosgenyl
glucopyranoside (25). White amorphous solid, ½aꢃ
a-L-rhamnopyranosyl-(1 ! 2)-b-D-
20
D
ꢁ56ꢁ (c 0.034, EtOH); IR mmax (film, NaCl) 3365,
1
2936, 1455, 1050, 900, 873 cmꢁ1; H NMR (d, pyridine,
400 MHz) 0.66 (d, 3H, J = 3.8 Hz, CH3-27), 0.78 (s, 3H,
CH3-18), 1.01 (s, 3H, CH3-19), 1.10 (d, 3H, J = 6.7 Hz,
CH3-21), 1.73 (d, 3H, J = 6.7 Hz, CH3-600), 3.46 (t, 1H,
J = 9.5 Hz, H-26), 3.55 (m, 1H, H-26), 3.81–3.91 (m,
2H, H-3, H-50), 4.13 (m, 1H, H-30), 4.24–4.28 (m, 3H,
H-20, H-40, H-400), 4.45–4.53 (m, 2H, H-16, H-500), 4.59
(dd, 1H, J = 9.1; 3.1 Hz, H-300), 4.76 (br s, 1H, H-200),
5.00 (m, 1H, H-10), 5.27 (br d, 1H, J = 3.8 Hz, H-6),
6.34 (s, 1H, H-100). 13C NMR (d, pyridine, 75 MHz):
140.9 (C-5), 121.8 (C-6), 109.0 (C-22), 101.8 (C-100),
100.1 (C-10), 79.50 (C-16), 79.32 (C-20), 77.96 (C-50),
77.65 (C-30), 77.65 (C-3), 73.87 (C-400), 72.54 (C-300),
72.27 (C-200), 71.53 (C-40), 69.23 (C-500), 66.58 (C-26),
62.59 (C-17), 62.36 (C-60), 56.35 (C-14), 49.99 (C-9),
41.69 (C-20), 40.18 (C-13), 39.58 (C-12), 38.70 (C-4),
37.23 (C-1), 36.86 (C-10), 32.03 (C-2), 31.53 (C-7),
31.40 (C-8), 30.31 (C-15), 29.90 (C-25), 29.69 (C-23),
28.97 (C-24), 20.82 (C-11), 19.14 (C-19), 18.39 (C-600),
17.05 (C-27), 16.06 (C-18), 14.76 (C-21). FABMS m/z
723 (M+H)+, 413, 397; Anal. Calcd for C39H62O12: C,
64.80; H, 8.64. Found: C, 64.74; H, 8.67.
5.3.5. Diosgenyl b-L-arabinopyranosyl-(1 ! 2)-b-D-gluco-
20
pyranoside (23). White amorphous solid, ½aꢃ ꢁ21ꢁ (c
D
0.038, EtOH); IR mmax (film, NaCl) 3442, 2360, 1054,
1
835 cmꢁ1; H NMR (d, pyridine, 400 MHz): 0.72 (d,
3H, J = 4.3 Hz, CH3-27), 0.86 (s, 3H, CH3-18), 0.98 (s,
3H, CH3-19), 1.16 (d, 3H, J = 6.8 Hz, CH3-21), 3.52 (t,
1H, J = 9.5 Hz, H-26), 3.62 (m, 1H, H-26), 3.84–3.91
(m, 3H, H-50, H-600, H-3), 4.22 (t, 1H, J = 10.2 Hz, H-
40), 4.27–4.37 (m, 3H, H-30, H-300, H-60), 4.44–4.52 (m,
2H, H-60, H-600), 4.56–4.62 (m, 2H, H-200, H-16), 5.05
(d, 1H, J = 7.2 Hz, H-10), 5.30 (d, 1H, J = 5.8 Hz, H-
100), 5.34 (br d, 1H, H-6). 13C NMR (d, pyridine,
75 MHz): 140.9 (C-5), 121.76 (C-6), 109.3 (C-22),
105.7 (C-100), 100.9 (C-10), 82.85 (C-20), 81.12 (C-16),
78.92 (C-3), 78.13 (C-30), 78.13 (C-50), 74.06 (C-300),
73.09 (C-200), 71.30 (C-40), 68.58 (C-400), 66.85 (C-26),
66.14 (C-500), 62.79 (C-17), 62.48 (C-60), 56.64 (C-14),
50.22 (C-9), 41.94 (C-20), 40.44 (C-13), 39.86 (C-12),
39.12 (C-4), 37.40 (C-1), 37.02 (C-10), 32.15 (C-2),
32.15 (C-7), 31.76 (C-8), 31.62 (C-15), 30.55 (C-25),
29.94 (C-23), 29.21 (C-24), 21.09 (C-11), 19.42 (C-19),
17.31 (C-27), 16.36 (C-18), 15.02 (C-21). FABMS m/z
731 (M+Na)+ 415, 397. HRFABMS m/z 731.3977, calcd
for C38H60O12Na, 731.3982; Anal. Calcd for C38H60O12:
C, 64.38; H, 8.53. Found: C, 64.25; H, 8.59.
5.3.8.
Diosgenyl
4-O-acetyl-a-L-rhamnopyranosyl-
(1 ! 2)-6-O-acetyl-b-D-glucopyranoside (26)24. White
20
amorphous solid, ½aꢃ ꢁ29ꢁ (c 0.015, EtOH); IR mmax
D
(film, NaCl) 3421, 2934, 1734, 1041, 900, 872 cmꢁ1; H
NMR (d, pyridine, 400 MHz) 0.66 (d, 3H, J = 5.2 Hz,
CH3-27), 0.83 (s, 3H, CH3-18), 1.06 (s, 3H, CH3-19),
1.23 (d, 3H, J = 6.9 Hz, CH3-21), 1.46 (d, 3H,
J = 6.3 Hz, CH3-600), 1.88 (s, 3H, Ac), 2.06 (s, 3H, Ac),
3.46 (t, 1H, J = 9.5 Hz, H-26), 3.55 (m, 1H, H-26),
3.92–4.01 (m, 3H, H-3, H-40, H-50), 4.12–4.24 (m, 2H,
H-20, H-30), 4.53 (dd, 1H, J = 14.5; 7.4 Hz, H-16), 4.63
(dd, 1H, J = 9.5; 3.1 Hz, H-300), 4.69–4.84 (m, 3H, H-
60, H-200), 4.99–5.01 (m, 1H, H-10), 5.85 (t, 1H,
J = 9.8 Hz, H-400), 6.37 (s, 1H, H-100). 13C NMR (d, pyr-
idine, 75 MHz): 171.581 and 171.51 (CO-Ac), 140.50
1
5.3.6. Diosgenyl b-L-fucopyranosyl-(1 ! 2)-b-D-glucopy-
20
ranoside (24). White amorphous solid, ½aꢃ ꢁ54ꢁ (c
D
0.024, EtOH); IR mmax (film, NaCl) 3388, 2940, 1052,
1
900, 865 cmꢁ1; H NMR (d, pyridine, 400 MHz): 0.65
(d, 3H, J = 4.3 Hz, CH3-27), 0.79 (s, 3H, CH3-18),
0.94 (s, 3H, CH3-19), 1.10 (d, 3H, J = 6.7 Hz,

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J. C. Hernandez et al. / Bioorg. Med. Chem. 16 (2008) 2063–2076
2074
(C-5), 121.81 (C-6), 109.0 (C-22), 101.30 (C-10), 99.83
(C-100), 81.87 (C-16), 79.05 (C-20), 77.77 (C-3), 76.52
(C-30), 75.94 (C-400), 74.68 (C-40), 72.24 (C-200), 71.16
(C-50), 69.96 (C-300), 66.63 (C-26), 65.23 (C-500), 64.23
(C-60), 62.66 (C-17), 56.41 (C-14), 50.08 (C-9), 41.74
(C-20), 40.25 (C-13), 39.63 (C-12), 38.78 (C-4), 37.20
(C-1), 36.92 (C-10), 32.08 (C-2), 31.99 (C-7), 31.58 (C-
8), 31.50 (C-15), 30.36 (C-25), 29.03 (C-23), 29.03 (C-
24), 20.93 (C-11), 20.93 and 20.48 (–OAc), 19.18 (C-
19), 17.77 (C-600), 17.09 (C-27), 16.17 (C-18), 14.82 (C-
21). FABMS m/z 829 (M+Na)+ 413, 397. Anal. Calcd
for C43H66O14: C, 64.00; H, 8.24. Found: C, 64.10; H, 8.33.
(s, 3H, (CH3)2–C), 1.96 (s, 3H, –OAc), 2.03 (s, 3H, –
OAc), 2.11 (s, 3H, –OAc), 3.38 (t, 1H, J = 10.9 Hz, H-
26), 3.47–3.53 (m, 3H, H-26, H-50, H-200), 3.60–3.65
(m, 2H, H-3, H-40), 3.78–3.87 (m, 3H, H-20, H-60, H-
5000), 4.03 (d, 1H, J = 5.5 Hz, H-200), 4.14–4.17 (m, 2H,
H-300, H-500), 4.35 (dd, 1H, J = 10.9, 5.5 Hz, H-60), 4.41
(dd, 1H, J = 14.4, 7.1 Hz, H-16), 4.66 (d, 1H,
J = 7.6 Hz, H-10), 5.09 (t, 1H, J = 9.8 Hz, H-400), 5.010 5
(s, 1H, H-1000), 5.23 (dd, 1H, J = 10.0; 3.4 Hz, H-3 ),
5.28 (m, 1H, H-200), 5.33 (d, 1H, J = 1.5 Hz, H-100),
5.39 (t, 1H, J = 9.4 Hz, H-30), 5.44 (br d, 1H, H-6),
5.49 (s, 1H, Ph-CH), 7.33–7.42 (m, 5H, Ph). 13C NMR
(d, CDCl3, 75 MHz): 177.1 (CO-Piv), 169.7 and 169.3
(CO-Ac), 139.6 (C-5), 136.7, 128.6 and 125.6 (Ph),
122.1 (C-6), 109.2 ((CH3)2-C), 109.0 (C-22), 100.7
(PhCH), 99.7 (C-10), 97.4 (C-10000), 95.8 (C-100), 80.58
(C-16), 78.58 (C-3), 78.58 (C-400), 77.59 (C-300), 76.79
(C-50), 75.96 (C-20), 75.64 (C-4000), 73.81 (C-30), 70.90
(C-4000), 69.63 (C-2000), 69.03 (C-3 ), 66.53 (C-5000), 68.67
(C-60), 65.91 (C-200), 64.02 (C-500), 61.87 (C-17), 60.60
(C-26), 56.62 (C-14), 49.83 (C-9), 41.37 (C-20), 40.03
(C-13), 39.55 (C-12), 38.70 (C-Piv), 38.05 (C-4), 36.88
(C-1), 36.64 (C-10), 31.85 (C-2), 31.62 (C-7), 31.14 (C-
8), 31.14 (C-15), 30.06 (C-25), 29.30 (C-23), 28.56 (C-
24), 27.61 and 26.10 ((CH3)2–C), 26.86 (CH3-Piv), 20.8
(C-11), 20.72, 20.57 and 20.40 (CH3-Ac), 18.93 (C-19),
17.82 (C-6000), 17.13 (C-600), 16.89 (C-27), 15.99 (C-18),
14.28 (C-21). HRFABMS: 1207.6401 (M+H)+ (calcd
for C66H95O20, 1207.6417), 793 (6), 397 (17), 395 (9).
Anal. (C66H94O20) C, H. Anal. Calcd for C66H95O20:
C, 65.65; H, 7.85. Found: C, 65.70; H, 7.36.
5.3.9. Diosgenyl a-L-rhamnopyranosyl-(1 ! 2)-6-O-acet-
20
yl-b-D-glucopyranoside (27)24. White amorphous solid,
½aꢃ ꢁ22 ꢁ (c 0.009, EtOH); IR mmax (film, NaCl) 3277,
D
2934, 1648, 1419, 1052, 872 cmꢁ1; 1H NMR (d, pyridine,
400 MHz): 0.66 (d, 3H, J = 4.9 Hz, CH3-27), 0.85 (s, 3H,
CH3-18), 1.02 (s, 3H, CH3-19), 1.12 (d, 3H, J = 6.9 Hz,
CH3-21), 1.75 (d, 3H, J = 6.3 Hz, CH3-600), 1.91 (s, 3H,
Ac), 3.47 (t, 1H, J = 9.5 Hz, H-26), 3.56 (m, 1H, H-
26), 3.93–3.99 (m, 3H, H-3, H-40, H-50), 4.21–4.26 (m,
2H, H-20, H-30), 4.34 (t, 1H,J = 9.5 Hz, H-400), 4.53
(dd, 1H, J = 14.5; 7.4 Hz, H-16), 4.62 (dd, 1H, J = 9.3;
3.0 Hz, H-300), 4.75 (dd, 1H, J = 11.7; 5.1 Hz, H-60),
4.79 (br s, 1H, H-200), 4.84 (m, 1H, H-60), 4.95 (m, 1H,
H-10), 4.99 (m, 1H, H-500), 5.28 (d, 1H, J = 4.5 Hz, H-
6), 6.38 (s, 1H, H-100). 13C NMR (d, pyridine,
75 MHz): 171.0 (CO-Ac), 141.0 (C-5), 121.9 (C-6),
109.4 (C-22), 102.7 (C-10), 102.3 (C-100), 81.3 (C-16),
79.5 (C-20), 77.6 (C-3), 76.8 (C-30), 75.9 (C-40), 73.87
(C-400), 72.7 (C-200), 70.5 (C-50), 70.5 (C-300), 67.0 (C-
26), 67.0 (C-500), 64.7 (C-60), 61.5 (C-17), 56.8 (C-14),
50.4 (C-9), 40.6 (C-20), 40.0 (C-13), 39.5 (C-4), 39.2
(C-12), 37.2 (C-1), 37.2 (C-10), 32.0 (C-2), 32.0 (C-7),
31.9 (C-15), 31.8 (C-8), 30.0 (C-25), 29.4 (C-24), 29.3
(C-23), 21.3 (C-11), 20.9 (CH3-Ac), 19.6 (C-19), 17.5
(C-27), 117.5 (C-600), 6.5 (C-18), 15.2 (C-21). FABMS
m/z 787 (M+Na)+. HRFABMS m/z 787.4210, calcd
for C41H64O13Na, 787.4245; Anal. Calcd for
C41H64O13: C, 64.38; H, 8.43. Found: C, 64.05; H, 8.72.
5.3.11. Diosgenyl a-L-rhamnopyranosyl-(1 ! 4)-a-L-
rhamnopyranosyl-(1 ! 2)-b-D-glucopyranoside
(30).
120.0 mg (0.09 mmol) of 29 in 10 mL of AcOH
(80%) was heated for 3 h at 70 ꢁC. The residue was
concentrated, dissolved in a mixture of MeOH/THF/
H2O (1:1:1) and NaOH (38 mg, 0.95 mmol) was
added. The solution was heated at 40 ꢁC for 7 h, then
neutralized with an acid resin Dowex-50 (H+), filtered,
concentrated, and purified in CC using CH2Cl2/MeOH
(8:2) to yield 30 (15.1 mg, 0.017 mmol, 19%). White
20
amorphous solid, ½aꢃ ꢁ48ꢁ (c 0.015, EtOH); IR mmax
D
1
5.3.10. Diosgenyl 2,3,4-tri-O-acetyl-a-L-rhamnopyrano-
syl-(1 ! 4)-2,3-O-isopropylidene-a-L-rhamnopyranosyl-
(1 ! 2)-4,6-O-benzylidene-3-O-pivaloyl-b-D-glucopyrano-
side (29). To a stirred mixture of imidate (350 mg,
(film, NaCl) 3418, 2360, 1050, 891 cmꢁ1; H NMR (d,
pyridine, 400 MHz): 0.65 (d, 3H, J = 4.9 Hz, CH3-27),
0.76 (s, 3H, CH3-18), 1.10 (d, 3H, J = 6.9 Hz, CH3-
21), 1.15 (s, 3H, CH3-19), 1.59 (d, 3H, J = 6.3 Hz,
CH3-600), 1.69 (d, 3H, J = 6.3 Hz, CH3-6000), 3.46 (t,
1H, J = 9.5 Hz, H-26), 3.55 (m, 1H, H-26), 3.83–3.93
(m, 2H, H-3, H-50), 4.13 (t, 1H,J = 9.0 Hz, H-40),
4.21–4.25 (m, 2H, H-20, H-30), 4.29 (m, 1H, H-60),
4.39 (m, 1H, H-5000), 4.45–4.52 (m, 5H, H-60, H-300,
H-4000, H-3000, H-16), 4.65 (br s, 1H, H-200), 4.80 (br s,
1H, H-2000), 4.99 (m, 1H, H-10), 5.05 (m, 1H, H-50),
5.33 (d, 1H, J = 3.7 Hz, H-6), 6.34 (s, 1H, H-100),
6.36 (s, 1H, H-1000). 13C NMR (d, pyridine, 75 MHz):
140.48 (C-5), 121.66 (C-6), 109.0 (C-22), 102.71 (C-
1000), 101.24 (C-100), 99.71 (C-10), 80.88 (C-16), 79.36
(C-20), 79.05 (C-400), 78.05 (C-50), 77.72 (C-30), 77.48
(C-3), 73.80 (C-4000), 73.39 (C-2000), 72.93 (C-3000), 72.67
(C-300), 72.28 (C-200), 71.53 (C-40), 70.07 (C-500), 67.40
(C-5000), 66.63 (C-26), 62.65 (C-17), 62.37 (C-60),
56.50 (C-14), 50.20 (C-9), 41.74 (C-20), 40.22 (C-13),
0.80 mmol), 2820 (158 mg, 0.53 mmol), and 4 A MS in
˚
dry CH2Cl2 (12 mL) at ꢁ70 ꢁC under N2, TMSOTf
(0.05 M in CH2Cl2, 1.0 mL) was added dropwise. After
stirring for 1 h, compound 4a (258 mg, 0.34 mmol) was
added. The resulting mixture was stirred at ꢁ20 ꢁC for
1.5 h, before the addition of Et3N to quench the reac-
tion. The mixture was filtered through a pad of Celite.
The filtrate was concentrated and applied to a silica
gel column chromatography (CH2Cl2/acetone; 40:1) to
provide the desired saponin 29 as a white solid
(0.19 mmol, 55%). White amorphous solid, [a]D ꢁ88ꢁ
1
(c 0.095, CHCl3). H NMR (d, CDCl3, 400 MHz): 0.80
(m, 6H, CH3-18, CH3-27), 0.98 (d, 3H, J = 6.9 Hz,
CH3-21), 1.04 (s, 3H, CH3-19), 1.21 (d, 3H,
J = 6.3 Hz, CH3-6000), 1.22 (s, 9H, CH3-Piv), 1.33 (d,
3H, J = 6.2 Hz, CH3-600), 1.51 (s, 3H, (CH3)2–C), 1.67

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J. C. Hernandez et al. / Bioorg. Med. Chem. 16 (2008) 2063–2076
2075
39.67 (C-12), 38.73 (C-4), 37.26 (C-1), 36.92 (C-10),
32.07 (C-2), 31.96 (C-7), 31.60 (C-8), 30.39 (C-15),
30.36 (C-25), 29.90 (C-23), 29.03 (C-24), 20.94 (C-
11), 19.39 (C-19), 18.91 (C-6000), 18.25 (C-600), 17.09
(C-27), 16.22 (C-18), 14.76 (C-21). FABMS m/z 891
(M+Na)+, 413 (3), 397 (4). Anal. (C45H72O16) C, H.
Anal. Calcd for C45H72O16: C, 62.19; H, 8.35. Found:
C, 62.15; H, 8.54.
(Beckman–Coulter). Histograms were analyzed with
the Expo 32 ADC SoftwareTM (Beckman–Coulter). The
quantitative data presented are means SE of percent-
age of cells in different phases of cell cycle from triplicate
samples in each treatment, and were reproducible in 2–
3-independent experiments.
5.4.4. Immunoblot analysis of procaspase-3, procaspase-
9, and Bcl-2. HL-60 or U937 cells (1 · 106) were treated
with diosgenyl glycosides at the indicated concentra-
tions in RPMI 1640 medium. Cells were pelleted by
centrifugation, washed with phosphate-buffered saline
and lysed in lysis buffer containing 125 mM Tris–HCl,
pH 6.8, 2% sodium dodecyl sulfate, 5% glycerol, and
1% b-mercaptoethanol, and boiled for 5 min. The sam-
ples were separated on 12% sodium dodecyl sulfate–
polyacrylamide gel, electrotransferred to a polyvinyli-
dene difluoride membrane. The membrane was probed
first with a polyclonal anti-procaspase-3 (Stressgen,
1:2000 dilution), anti-procaspase-9 (Stressgen, 1:1000)
or anti-Bcl-2 (Santa Cruz Biotechnology, 1:1000) and
then with anti-rabbit (pro-caspase-3) or anti-mouse
(pro-caspase-9 and Bcl-2) antibody conjugated to
horseradish peroxidase (HRP). Protein bands were de-
tected by chemiluminescence (SuperSignal West Pico
Chemiluminescent Substrate, Pierce) using the manu-
facturer’s protocol.
5.4. Biological activity
5.4.1. Cell culture. Human HL-60 and U937 myeloid
leukemia cells and human SK-MEL-1 melanoma cells
were grown in RPMI 1640 (Sigma) supplemented with
10% (v/v) heat-inactivated fetal bovine serum (Sigma)
and 100 U/mL penicillin and 100 lg/mL streptomycin
at 37 ꢁC in a humidified atmosphere containing 5%
CO2. The cell numbers were counted by a hematocytom-
eter, and the viability was always greater than 95% in all
experiments as assayed by the 0.025% Trypan blue
exclusion method. Stock solutions of 100 mM diosgenyl
glycosides were made in dimethylsulfoxide (DMSO),
and aliquots were frozen at ꢁ20 ꢁC.
5.4.2. Assay for growth inhibition and cell viability. The
cytotoxicity of diosgenyl glycosides was assessed using
a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazo-
lium bromide (MTT) assay.28 Briefly, 1 · 104 exponen-
tially growing cells were seeded in 96-well microculture
plates with various diosgenyl glycoside concentrations
(0.3–100 lM) in a volume of 200 lL. DMSO concentra-
tion was the same in all the treatments and did not ex-
ceed 0.1% (v/v). After 72 h, surviving cells were
detected based on their ability to metabolize 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bro-
mide (Sigma) into formazan crystals. Optical density
was read with an ELISA reader at a wavelength of
570 nm and was used as a measure of cell viability.
The MTT dye-reduction assay measures mitochondrial
respiratory function and can detect the onset of cell
death earlier than dye-exclusion methods. Cell survival
was calculated as the fraction of cells alive relative to
control for each point: cell survival (%) = mean absor-
bance in treated cells/mean absorbance in control
wells · 100. Concentrations inducing a 50% inhibition
of cell growth (IC50) were determined graphically for
each experiment. Parameters describing the concentra-
tion–response curves (IC50) were determined using the
curve fitting routine of the computer software PrismTM
(GraphPad) and the equation derived by DeLean et al.29
5.4.5. Immunoblotting of poly(ADP-ribose) polymerase.
Induction of apoptosis was also examined by proteolytic
cleavage of poly(ADP-ribose) polymerase. Briefly,
1 · 106 exponentially growing HL-60 or U937 cells were
treated with diosgenyl glycosides at the indicated con-
centrations for 6 h at 37 ꢁC. Cells were pelleted by cen-
trifugation, washed twice with phosphate-buffered
saline resuspended in lysis buffer, and subjected to Wes-
tern blot analysis as described previously.28 Proteins
were separated on 7.5% sodium dodecyl sulfate–poly-
acrylamide mini gels and electrotransferred to polyvinyl-
idene difluoride membrane. The membrane was probed
with polyclonal anti-poly(ADP-ribose) polymerase
(Stressgen, 1:1000 dilution) and then with anti-rabbit
antibody conjugated to horseradish peroxidase (HRP).
Protein bands were detected by chemiluminescence
(SuperSignal West Pico Chemiluminescent Substrate,
Pierce) as described above.
5.4.6. Release of cytochrome c from mitochondria in
diosgenyl glycoside-treated cells. Untreated and diosge-
nyl glycosides-treated cells were harvested by centrifuga-
tion at 1000g for 5 min at 4 ꢁC. Cell pellets were washed
twice with ice-cold phosphate-buffered saline and resus-
pended with 20 mM Hepes–KOH (pH 7.5), 10 mM
KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA,
1 mM dithiothreitol, 0.1 mM phenylmethylsulfonylfluo-
ride (PMSF), and 250 mM sucrose. After 15-min incuba-
tion on ice, cells were lysed by pushing them several times
through a 22-gauge needle and the lysate spun down at
1000g for 5 min at 4 ꢁC. The supernatant fraction was
centrifuged at 105,000g for 45 min at 4 ꢁC and the result-
ing supernatant was used as the soluble cytosolic frac-
tion. Cytosolic proteins (50 lg) were resolved on a
sodium dodecyl sulfate/15% polyacrylamide gel and elec-
5.4.3. Flow cytometry analysis for cell cycle distribution.
After 16 h of treatment, cells were harvested and quickly
washed twice with ice-cold phosphate-buffered saline
(PBS), and cell pellets were collected and resuspended
in 50 lL of PBS. Following dropwise addition of 1 mL
of ice-cold 75% ethanol, fixed cells were stored at
ꢁ20 ꢁC for 1 h. Samples were then centrifuged at 500g
for 10 min at 4 ꢁC and washed with PBS before resus-
pension in 1 mL containing 50 lg/mL propidium iodide
and 100 lg/mL RNase A and incubation for 1 h at 37 ꢁC
in the dark. Cell cycle distribution was then analyzed by
flow cytometry using a Coulter EPICSTM cytometer

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J. C. Hernandez et al. / Bioorg. Med. Chem. 16 (2008) 2063–2076
2076
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trotransferred onto a polyvinylidene difluoride mem-
brane (Millipore). Membrane was blocked with 5% non-
fat milk in Tris-buffered saline containing 0.1% Tween 20
(TBST) for 1 h, followed by incubation with anti-cyto-
chrome c monoclonal antibody (BD Pharmingen;
1:1000) overnight. After washing and incubation with
horseradish peroxidase-conjugated anti-mouse antibody
(Amersham Biosciences), the antigen–antibody com-
plexes were visualized by SuperSignal West Pico Chemi-
luminescent Substrate (Pierce) as described above. The
same membrane was probed with monoclonal anti-b-ac-
tin (Sigma, 1:3000 dilution) as loading control.
´
´
´
5. Hernandez, J. C.; Leon, F.; Estevez, F.; Quintana, J.;
Bermejo, J. Bioorg. Med. Chem. 2004, 12, 4423–4429.
6. Ma, X.; Yu, B.; Hui, Y.; Miao, Y.; Ding, J. Bioorg. Med.
Chem. Lett. 2001, 11, 2153–2156.
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Acknowledgments
This work was supported in part by grants from the
Programa de Iniciativa Comunitaria INTERREG IIIB
Azores-Madeira-Canarias (04/MAC/3.5/C5), from the
13. Cheng, M. S.; Wang, Q. L.; Tian, Q.; Song, H. Y.; Liu, Y.
X.; Li, Q.; Xu, X.; Miao, H. D.; Yao, X. S.; Yang, Z.
J. Org. Chem. 2003, 68, 3658–3662.
´
Instituto Canario de Investigacion del Cancer (G-05-09
to J.B. and F.L.), from FEDER (Grant No. 1FD1997-
´
14. Cheng, M. S.; Wang, Q. L.; Tian, Q.; Song, H. Y.; Liu, Y.
X.; Li, Q.; Xu, X.; Miao, H. D.; Yao, X. S.; Yang, Z.
Chem. Lett. 2005, 34, 1220–1221.
15. Hou, S.; Xu, P.; Zhou, L.; Yu, D.; Lei, P. Bioorg. Med.
Chem. Lett. 2006, 16, 2454–2458.
´
1831 to J.B.), and from the Direccion General de
Universidades e Investigacion of the Canary Islands
´
Government (Grants TR2003/002 and GRUP-2004-44
to F.E.). J.C.H. thanks the Cabildo Insular de Tenerife
(Spain) and the Town Council of Icod de los Vinos
(Tenerife, Spain). F.T. was supported by a research stu-
´
´
´
16. Hernandez, J. C.; Leon, F.; Estevez, F.; Quintana, J.;
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Investigacion of the Canary Islands Government. We
´
´
´
thank Mr. Jose Estevez (Hospital Universitario Insular
de Gran Canaria) for his collaboration in the Western
blot assays and Mr. Lennart Loven for his encourage-
ment and support.
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Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmc.2007.10.089.
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