Pregnane steroidal glycosides and their cytostatic activities

Article abstract of DOI:10.1093/glycob/cwq203

Four new steroidal glycosides such as 3-O-6-deoxy-3-O-methyl β- d-allopyranosyl-(1 → 4) β- d-oleandropyranosyl-(1 → 4) - d-cymaropyranosyl-(1 → 4) β- d-cymaropyranoside-12 - tigloyl-14 - hydroxy-17 - pregnane (1), 3-O-6-deoxy-3-O-methyl β- d-allopyranosyl-(1 → 4) β- d-oleandropyranosyl-(1 → 4) β- d-cymaropyranosyl-(1 → 4) β- d-cymaropyranoside-12 - (2'-amino)-benzoyl-14 - hydroxy-17 - pregnane (2), 3-O-6-deoxy-3-O-methyl β- d-allopyranosyl-(1 → 4) β- d-oleandropyranosyl-(1 → 4) β- d-cymaropyranosyl-(1 → 4) β- d-cymaropyranoside-12 - 14 - dihydroxy-17 - pregnane (3) and 3-O-6-deoxy-3-O-methyl β- d-allopyranosyl-(1 → 4) β- d-oleandropyranosyl-(1 → 4) β- d-cymaropyranosyl-(1 → 4) β- d-cymaropyranoside-12 - 14 - dihydroxy-17 - pregnane (4) were isolated from the aerial parts of Ceropegia fusca Bolle (Asclepiadaceae), a crassulacean acid metabolism plant, an endemic species to the Canary Islands that has been used in traditional medicine as a cicatrizant, vulnerary and disinfectant. The dichloromethane extract exhibited significant cytostatic activity against HL-60, A-431 and SK-MEL-1 cells, human leukemic, epidermoid carcinoma and melanoma cells, respectively. As shown in Table I, compounds 1 and 2 showed very similar IC50 values. The acetylation of 1 to give the diacetate 5 increases 5-fold the cytotoxicity against HL-60 cells. Compounds 3 and 4 did not show cytotoxicity at the assayed concentrations. With respect to the compounds containing only the steroid ring (6-8), the presence of a charged O-amino-benzoyl but not a tigloyl group improved the cytotoxicity.

Full text of DOI:10.1093/glycob/cwq203

Glycobiology vol. 21 no. 5 pp. 619–624, 2011
doi:10.1093/glycob/cwq203
Advance Access publication on December 8, 2010
Pregnane steroidal glycosides and their cytostatic activities
Víctor P García1,2, Jaime Bermejo2, Sara Rubio3,
José Quintana3, and Francisco Estévez3
2Departamento de Química de Productos Naturales y Biotecnología, Instituto
de Productos Naturales y Agrobiología de Canarias, Francisco Sánchez, 3,
38206 La Laguna, Tenerife, Canary Islands and 3Departamento de
Bioquímica, Biología Molecular y Fisiología, Universidad de Las Palmas de
Gran Canaria, Plaza Dr Pasteur s/n, 35016 Las Palmas de Gran Canaria,
Canary Islands
Introduction
In the course of a drug-screening project on medicinal plants
for biologically active compounds (Darias et al. 1996; Herrera
et al. 1996), we now report the isolation, structure determi-
nation and cytostatic activities of tetra-deoxypyranosides (1)
(4.84%), (2) (2.3%), (3) (0.9%), (4) (0.7%) and their deriva-
tives (5), 12-tigloyl-14-hydroxy-17 pregnane (6), 12-(2′-
amino)-benzoyl-14-hydroxy-17 pregnane (7) and ramanone
(8) in an attempt to establish a structure–activity relationship.
The percentages were determined from pure samples (see the
“Extraction and isolation” section) drying the compounds in
vacuo, at 40°C until achieving a constant weight. This process
takes ꢀ72 h. To assess whether these compounds display
cytostatic activities, we also studied their effects on the viabi-
lity of the human promyelocytic cell line HL-60 and human
melanoma SK-MEL-1 cells. The semisynthetic derivative 5,
which we named fuscastatin, showed cytotoxicity against the
human myeloid leukemia cell line HL-60, which was caused
by the induction of apoptosis as determined by flow cytome-
try. On the basis of bioassay-directed fractionation using the
growth inhibition of tumor cells, we described the extraction
and purification of novel pregnane steroidal glycosides from
the aerial parts of the endemic plant Ceropegia fusca. Similar
IC50 values for compounds 1 and 5 were obtained on human
melanoma SK-MEL-1 cells (31.3 0.5 and 26.2 0.7 μM,
respectively).
Received on August 28, 2010; revised on December 2, 2010; accepted on
December 3, 2010
Four new steroidal glycosides such as 3-O-6-deoxy-3-O-
methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-
(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside-
12-β-tigloyl-14-β-hydroxy-17-β-pregnane (1), 3-O-6-deoxy-
3-O-methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-
(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-
cymaropyranoside-12-β-(2’-amino)-benzoyl-14-β-hydroxy-
17-β-pregnane
(2),
3-O-6-deoxy-3-O-methyl-β-D-
allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-(1 → 4)-β-D-
cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside-12-β-14-
β-dihydroxy-17-α-pregnane (3) and 3-O-6-deoxy-3-O-
methyl-β-D-allopyranosyl-(1 → 4)-β-D-oleandropyranosyl-
(1 → 4)-β-D-cymaropyranosyl-(1 → 4)-β-D-cymaropyranoside-
12-β-14-β-dihydroxy-17-β-pregnane (4) were isolated from
the aerial parts of Ceropegia fusca Bolle (Asclepiadaceae),
a crassulacean acid metabolism plant, an endemic species
to the Canary Islands that has been used in traditional
medicine as a cicatrizant, vulnerary and disinfectant. The
dichloromethane extract exhibited significant cytostatic
activity against HL-60, A-431 and SK-MEL-1 cells,
human leukemic, epidermoid carcinoma and melanoma
cells, respectively. As shown in Table I, compounds 1 and
2 showed very similar IC50 values. The acetylation of 1 to
give the diacetate 5 increases 5-fold the cytotoxicity
against HL-60 cells. Compounds 3 and 4 did not show
cytotoxicity at the assayed concentrations. With respect to
the compounds containing only the steroid ring (6–8), the
presence of a charged O-amino-benzoyl but not a tigloyl
group improved the cytotoxicity.
Results
The extract showed a positive Liebermann–Burchard reaction,
indicating the presence of a steroid skeleton. The structure of
the intact glycoside can be established unambiguously by
NMR spectroscopic methods (Supplementary data, Tables SI–
SIV; Agrawal 1992; Coxon 2009). The chemical shift values
for C-2 of the sugar moieties show that the two β-linked cym-
aroses have the D-configuration (δC 35.4) like oleandrose (δC
35.4). The anomeric carbon of 6-deoxy-3-O-methyl-
β-D-allopyranoside resonates at δC 104.0 when this sugar is
linked to the 4-OH of β-D-cymarose, in contrast resonates at
101.8 in C5D5N, 101.3 in Me2CO-d6 and 99.1 in CDCl3
(Supplementary data, Table SIV), respectively, when it is
linked to the 4-OH of D-oleandrose (Vlegaar et al. 1993;
Hayashi et al. 1988). High-resolution 1H-NMR spectra of
21-alkylpregnanes very often display well-resolved signals
that can be used as starting points in several selective NMR
experiments to study scalar (J coupling), and dipolar (NOE)
interactions. Selective excitation was carried out using a
Keywords: apoptosis / cytostatic compounds / glycosides /
steroids
1To whom correspondence should be addressed. Tel: +34-922-318584;
Fax: +34-922-318571; e-mail: vpergarw@gobiernodecanarias.org
© The Author 2010. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
619

VP García et al.
Fig. 2. Effect(s) of extracts on human cell lines viability. HL-60 and A-431
cells were cultured in the presence of the indicated concentrations of extracts
for 72 h. Control cells were cultured under the same conditions and received
vehicle (DMSO) in the same proportion used in the other groups. Cell
viability was determined following the MTT procedure. The results shown are
the mean SE of triplicate determinations from a single representative
experiment and are expressed as the percentage of viability with respect to the
control (100%).
elementary analysis, high-resolution ESI, FABMS and 13C
DEPT NMR.
Acid hydrolysis of 1 and 2 gave the semisynthetic agly-
cones 6 and 7, respectively. The hydrolyzation shifts of the
aglycone carbon of 1 and 2 were observed at C-2 (+2.0 ppm),
C-3 (−6.0) and C-4 (+3.4, +3.3), respectively. The stereo-
chemistry of the 14β-hydroxyl group was further corroborated
by synthesis (Mitsuhashi and Nomura 1965). The alkaline
hydrolysis and subsequent acid hydrolysis of these two sapo-
nins gave a known aglycone, ramanone (8) (see Experimental
Section in Supplementary data). As shown in Supplementary
data, Table SIII, this aglycone had four oxygenated carbons
signal at δ 71.5 (C-3), 68.2 (C-12), 86.9 (C-14) and 214.5
(C-20) in a C/D cis-pregnane system. We can find a remark-
able difference in the carbon chemical shifts, especially in
those assignable to C-13 and C-14, at δ 53.6–55.2 and 85.6–
87.5 in a C/D cis junction, while a chemical shift for the
same carbons has been reported at δ 41.6–42.7 and 58.7–59.2
for pregnanes with C/D trans junction, respectively (Kimura
et al. 1982). The 6, 7 and 8 aglycones, 17α-epimers, were
more stable and were obtained as the main products
(Mitsuhashi et al. 1964), whereas the ROESY experiment for
17β compounds showed an H-12α and an H-17α correlation.
The 17α configuration was also confirmed by the observation
that the carbonyl carbon of α-linked methyl ketone at C-17
Fig. 1. Natural 21-alkylpregnanes and their derivatives.
double pulsed-field gradient spin-echo sequence in which
180° Gaussian pulses are sandwiched between sine shaped z-
gradients (Figure 1). The CH2Cl2 extract showed cytostatic
activity against human leukemia HL-60 cells and human
epidermoid tumor A-431 cells, while the butanol extract did
not exhibit cytotoxicity (Figure 2).
The tigloyl pregnane glycoside (1) was found to have the
molecular formula C54H86O18 on the basis of its elementary
analysis, high-resolution ESIMS and 13C DEPT NMR spec-
troscopy. The acetylation of 1 gave the diacetate 5. After the
acetylation the H-2 of 3-O-methyl-6-deoxy-β-D-allopyranosyl
1
moved downfield to 4.67 in the H-NMR spectrum (dd, J =
8.4, 2.8 Hz, 1H) and the H-4 moved to 4.05 ppm (dd, J = 9.9,
2.7 Hz, 1H). The 13C-NMR spectrum shows signals at δ 20.9
(X2), 169.6 and 169.8. On the basis of the above results, the
hydroxyl groups at C-2 and C-4 of 1 were acetylated.
The (2′-amino)-benzoyl pregnane glycoside (2) was shown
to have the molecular formula C56H85NO18 on the basis of its
620

Structure activity relationship studies of steroidal glycosides
appears at δ 209.2, 209.3 and 214.5 ppm for the aglycones 6,
7 and 8, respectively, when compared with δ 217 ppm for the
β configuration (Supplementary data, Table SIII; Warashina
and Noro 2000; Pawar et al. 2007).
Studies of aqueous solutions of carbohydrates are compli-
cated because they are present in solution in several forms
derived from free sugar mutarotation: a specific optical
rotation of aqueous sugar solution changes with time
(Junquera et al. 2002). Acid hydrolysis was conducted in 1 M
HCl solution (1:1 H2O–MeOH). Acid hydrolysis of the com-
pounds 1 and 2 afforded as main products after the acetylation
cymaropyranose diacetate (9) and the disaccharide pachibiose
triacetate (10). The secondary methyl signal in the 500-MHz
1H-NMR spectrum of 9 at δ 1.19 (3H, d, J = 6.3) suggest the
presence of one 6-deoxy sugar in the molecule. The
2,6-deoxypyranose moiety is a 3-methyl ether, indicated by
one singlet signal at δ 3.49 (3H, s). The disaccharide 10 was
suggested to have the molecular formula C20H32O11 based on
Fig. 3. Effect(s) of compound 5 on HL-60 cell viability and morphology. (A)
Cells were cultured in the presence of the indicated doses of compound 5 for
72 h, and thereafter cell viability was determined by the MTT assay as
described in Experimental Section in Supplementary data. The results of a
representative experiment are shown. Each point represents the mean SE of
triplicate determinations. (B) Cells were incubated in the absence (control) or
the presence of 30 μM of compound 5 for 24 h, and morphology was
visualized by phase contrast microscopy. Control cells were cultured under
the same conditions and received vehicle (DMSO) in the same proportion
used in the other groups.
1
high-resolution FABMS. The H-NMR spectrum of 10 shows
two secondary methyl signals at δ 1.21 (3H, d, J = 6.2) and
1.28 (3H, d, J = 6.5), and two 3-methyl ethers at δ 3.48 (3H,
s) and 3.56 (3H, s), respectively (see Experimental Section in
Supplementary data). Cymarose and pachibiose were also
obtained by Yoshimura et al. (1985) from the acid hydrolysis
of dregeoside Dp1.
Table I. Effects of the isolated compounds on the growth of HL-60 cell linea
Compound
IC50 (μM)
The pregnane glycosides 3 and 4 were assigned the mol-
ecular formula C49H80O17 on the basis of their elementary
analysis, high-resolution ESIMS and 13C DEPT NMR.
We report here on the in vitro activity of natural and syn-
thetic derivatives obtained from C. fusca against human
HL-60 leukemia cells. Bioassay-directed fractionation using
cytotoxicity with human tumor cell lines yielded an active
mixture from the aerial parts of the endemic plant C. fusca.
Fractionation of the CH2Cl2 extract yielded compounds 1–4
and their cytostatic activities against HL-60 cells were
assayed. Moreover, semisynthetic derivatives from the above
compounds 5–8 were also tested. Also, the effect of com-
pound 5 on HL-60 cells viability was studied. Cells were cul-
tured in the presence of the indicated doses of compound
5 for 72 h, and thereafter cell viability was determined by
the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium
bromide assay as described in Experimental Section in
Supplementary data. Antiproliferative studies on 5 indicate
that this compound displays cytotoxic properties in human
myeloid HL-60 leukemia cells with an IC50 value of 10
1.5 μM (Figure 3A). Analysis by phase contrast microscopy at
24 h revealed the evidence of morphological changes and
cells appeared to be unhealthy (Figure 3B).
1
2
3
4
5
6
7
8
49 0.6
63 0.2
ND
ND
10 1.5
ND
44 0.6
ND
aThe data shown represent the mean SEM of two independent experiments
with three determinations in each. The IC50 values were calculated from
experiments such as those shown in Figure 2A using the methodology
described in the Experimental Section in Supplementary data. ND, growth
inhibition not detected at the highest dose tested.
which revealed that compound 5 caused G1 arrest in cell
cycle progression in a dose- and a time-dependent manner.
Concomitant with this increase, a decrease was observed in
the percentage of cells in the synthesis (S) and G2/M phases
of the cell cycle (Table II).
Discussion
The results indicate that the natural compounds 1 and 2
showed very similar IC50 values (49 and 63 μM, respectively),
indicating that the tigloyl group or the O-amino-benzoyl
group at C-12 of the steroid ring does not play a significant
role in cytostatic activity. The aglycon itself is not active.
Usually, the glycan moiety has been viewed as a molecular
structure that controls the pharmacokinetics of a drug. The
introduction of two acetyl groups in the oligosaccharide
moiety of compound 1 to give compound 5 increases 5-fold
the cytotoxicity against HL-60 cells (IC50 49 μM vs. IC50
10 μM; Table I, Figure 3A). One possibility is that the intro-
duction of the acetyl groups facilitates entry into the cell and
In order to determine whether the decrease in viability
observed after treatment with compound 5 occurred by apop-
tosis (Table I, Figure 3), cells were stained with propidium
iodide and subjected to flow cytometry. An increasing number
of hypodiploid cells (i.e. apoptosis) were identified in
response to compound 5 (Figure 4A). These data suggest that
the cytostatic activity of compound 5 in HL-60 cells occurred
via apoptosis. The percentage of apoptotic cells increased in a
dose- and a time-dependent manner (Figure 4B).
To further explore the molecular mechanisms underlying
cell growth inhibition, we conducted a cell cycle analysis
621

VP García et al.
region of DNA than the charged monosaccharide daunosa-
mine in daunorubicin and doxorubicin (Yang and Wang
1994). However, the length of the sugar chain did not corre-
late with in vivo antitumor activity (Matsuzawa et al. 1981).
As shown in Table I, the presence of an ester group on the
hydroxyl group of C-12 in the steroid ring of compounds 1
and 2 seems to be important in cytostatic induction since
compound 4 did not show cytotoxicity at the assayed concen-
trations (up to 100 μM). The oligosaccharide moiety is not
involved in the cytotoxicity-inducing ability since compounds
2 and 7 display a similar cytotoxic potency. With respect to
the compounds containing only the steroid ring (compounds
6–8), 17α-epimers, the presence of a charged O-amino-
benzoyl (7) but not a tigloyl (6) group improved the
cytotoxicity.
Although the isomerization of C/D cis 17β-20-ketopreg-
nane to 17α- by alkaline hydrolysis is well known (Danieli
et al. 1962; Hasegawa et al. 1963) to our knowledge, this is
the first time that the epimerization of C/D cis
17β-20-ketopregnane to the more stable 17α configuration by
acid treatment has been reported.
The precise function of the carbohydrate (Křen 2008) and
ester moieties in most glycosylated metabolites has not been
elucidated.
Fig. 4. Apoptosis induction by compound 5 on HL-60 cells. (A) Cells were
cultured in the absence (control) or the presence of 30 μM of compound 5 for
24 h and the measurement of a cellular DNA content was determined by flow
cytometry. Percentage of apoptotic cells displaying a sub-G1 DNA content is
given. Representative histograms are shown. (B) Cells were cultured in the
presence of the indicated concentrations of compound 5, harvested at 12 and
24 h, and the percentage of apoptotic cells were determined by flow
cytometry. The results of a representative experiment are shown, and each
point represents the mean SE of duplicate determinations.
Material and methods
General experimental procedures
Melting points were determined on a Büchi B-540 apparatus
and are uncorrected. Optical rotations were recorded in a
Perkin-Elmer model 343 polarimeter. IR spectra were
recorded using a Bruker model IFS-55 spectrophotometer and
Table II. Effects of compound 5 on cell cycle distribution of HL-60 cellsa
a
Bruker model IFS-66/S spectrophotometer for ATR.
1H-NMR and 13C-NMR spectra were obtained on a Bruker
model AMX-500 spectrometer with the standard pulse
%G1
%S
%G2-M
1
sequences operating at 500 MHz in H-NMR and 125 MHz in
Control
10 μM comp-5
30 μM comp-5
48.7 0.7
56.5 1.1
58.4 1.8
28.3 0.4
22.5 1.4
21.7 0.3
23.1 0.3
21.0 0.6
19.8 0.7
13C-NMR. CDCl3 and CO (CD3)2 were used as solvents.
Chemical shifts were expressed in δ (ppm) with TMS as an
internal standard. FAB and EIMS were taken on a Micromass
model Autospec (70 eV) spectrometer. Column chromato-
graphy (CC) was carried out on silica gel (70–230 mesh,
Merck, Germany) and 0.05 silica gel (Aldrich Chemical
Company). Fractions obtained from CC were monitored by
TLC (silica gel 60 F254).
aThe cells were cultured with the indicated concentrations of compound 5 for
24 h and the cell cycle distribution was determined by flow cytometry. The
results of a representative experiment are expressed as the mean SE of
duplicate determinations.
improves binding to specific targets. When steroidal glyco-
sides are used topically as antineoplasic agents, they enter
cells via endogenous lectins that have specific receptors for
the sugar moiety of the glycoside (Cham 2007).
Plant material
Ceropegia fusca Bolle aerial parts were collected in August
2007 at Arico, Tenerife (Canary Islands), and were identified
by Dr MC Alfayate (University of La Laguna). Voucher speci-
mens are deposited at the Herbarium of that institution.
Anthracyclines are among the most potent antineoplasic
agents, but their use in cancer treatment is limited by their
severe chronic cardiotoxicity. It has been reported that anthra-
cyclines which contain several sugars have, in general, fewer
side effects than other antitumor antibiotics (Jung et al. 1982;
Kremer and Caron 2004; Minotti et al. 2004; Menna et al.
2007). Aclacinomycins A and B are anthracyclines with
potent antitumor activity. It has been suggested that aclacino-
mycins may have different modes of biological activity com-
pared with daunomycin and doxorubicin. The more extensive
sugar of aclacinomycins projects further into the solvent
Extraction and isolation
The plant aerial parts were macerated in ethanol (8 L) for 30
days at room temperature and then subjected to reverse extrac-
tion with dichloromethane (3 × 4 L) and n-butanol in a Griffin
Flask Shaker (3 × 500 mL). Removal of the solvent from the
dichloromethane extraction gave a residue (26.97 g) which
was subjected to CC on silica gel using n-hexane-ETOAc of
622

Structure activity relationship studies of steroidal glycosides
increasing polarity and then with increasing percentages of
MeOH. The saponins fractions were re-chromatographed on
silica gel with 7:3 n-hexane/acetone.
Funding
This work was supported by grants from CajaCanarias,
Ministry of Science and Technology, Luciano Reverón e
Hijos S. L., Canary Islands Government, FSE, Ministry of
Education and Science (SAF2007-62536), Canarian Institute
of Cancer Research, and FEDER.
Cell culture
Human HL-60 myeloid leukemia cells were grown in RPMI
1640 (Sigma) supplemented with 10% (v/v) heat-inactivated
fetal bovine serum (Sigma) and 100 U/mL of penicillin and
100 μg/mL of streptomycin at 37°C in a humidified atmos-
phere containing 5% CO2. The cultures were passed twice
weekly exhibiting characteristic doubling times of ꢀ24 h. The
cell numbers were counted by a hemocytometer, and the via-
bility was always greater than 95% in all experiments as
assayed by the 0.025% trypan blue exclusion method. Stock
solutions of 100 mM compounds were made in dimethyl sulf-
oxide (DMSO), and aliquots were frozen at −20°C. Further
dilutions were made in culture media immediately prior to
use. In all experiments, the final concentration of DMSO did
not exceed 0.5% (v/v), a concentration which is nontoxic to
the cells. To ensure exponential growth, cells were resus-
pended in fresh medium 24 h before each treatment. The
A-431 cell line was grown as monolayers in plastic tissue
flasks containing Dulbecco’s modified Eagle’s medium sup-
plemented with 10% fetal bovine serum, 100 U/mL of peni-
cillin and 100 μg/mL of streptomycin. Human SK-MEL-1
melanoma cells (DSMZ N° ACC 303, DSMZ, German
Collection of Microorganisms and Cell Cultures,
Braunschweig, Germany) were grown in RPMI 1640 (Sigma)
supplemented with 10% (v/v) heat-inactivated fetal bovine
serum (Sigma) and 100 U/mL of penicillin and 100 μg/mL of
streptomycin at 37°C in a humidified atmosphere containing
5% CO2.
Acknowledgements
We would like to thank M. Carmen Mederos, Juan A. Suárez,
Manuel de León, Domingo Acosta and Sergio Suárez for
technical assistance, Dr John Lowry, Humboldt State
University, for helpful comments on the manuscript and
Pauline Agnew for assistance with language revision.
Abbreviation
CC, column chromatography; DMSO, dimethyl sulfoxide;
PBS, phosphate buffered saline.
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Supplementary data
Supplementary data for this article is available online at http://
glycob.oxfordjournals.org/.
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