Synthesis and biological evaluation of the glycoside (25R)-3β, 16β-diacetoxy-22-oxocholest-5-en-26-yl β-d-glucopyranoside: A selective anticancer agent in cervicouterine cell lines

Article abstract of DOI:10.1016/j.ejmech.2011.05.058

The synthesis and biological evaluation of the new cholestane glycoside (25R)-3β,16β-diacetoxy-22-oxocholest-5-en-26-yl β-d- glucopyranoside starting from diosgenin is described. This compound showed selective antiproliferative activity against CaSki, ViBo, and HeLa cervicouterine cancer cells. Its effect on the cell-cycle was determined. The cytotoxic effects of the title compound on cervicouterine cancer cell lines and human lymphocytes indicate that the main cell death process is not necrosis; hence it is not cytotoxic. The title compound induced apoptosis in cervicouterine cancer cells. Importantly, the antiproliferative activity on tumor cells did not affect the proliferative potential of peripheral blood lymphocytes. The title compound showed selective antitumor activity and greater antiproliferative activity than its aglycon, and therefore serves as a promising lead candidate for further optimization.

Full text of DOI:10.1016/j.ejmech.2011.05.058

European Journal of Medicinal Chemistry 46 (2011) 3877e3886
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of the glycoside (25R)-3
b
,16
b-diacetoxy
-22-oxocholest-5-en-26-yl
b-D
-glucopyranoside: A selective anticancer agent
in cervicouterine cell lines
María A. Fernández-Herrera a b, Hugo López-Muñoz c, José M.V. Hernández-Vázquez c,
,
,
Moisés López-Dávila c, Sankar Mohan b, María L. Escobar-Sánchez d, Luis Sánchez-Sánchez c
,
**
,
a,
***
B. Mario Pinto b , Jesús Sandoval-Ramírez
*
a Facultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla. Ciudad Universitaria, 72570 Puebla, Pue., México
b Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada V5A 1S6
c Facultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma de México, 09230 México D.F., México
d Departamento de Biología Celular, Facultad de Ciencias, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 México, D. F., México
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis and biological evaluation of the new cholestane glycoside (25R)-3b,16b-diacetoxy-22-
Received 15 March 2011
Received in revised form
11 May 2011
Accepted 13 May 2011
Available online 30 May 2011
oxocholest-5-en-26-yl
b-D-glucopyranoside starting from diosgenin is described. This compound
showed selective antiproliferative activity against CaSki, ViBo, and HeLa cervicouterine cancer cells. Its
effect on the cell-cycle was determined. The cytotoxic effects of the title compound on cervicouterine
cancer cell lines and human lymphocytes indicate that the main cell death process is not necrosis; hence
it is not cytotoxic. The title compound induced apoptosis in cervicouterine cancer cells. Importantly, the
antiproliferative activity on tumor cells did not affect the proliferative potential of peripheral blood
lymphocytes. The title compound showed selective antitumor activity and greater antiproliferative
activity than its aglycon, and therefore serves as a promising lead candidate for further optimization.
Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords:
Cholestane glycoside
CaSki
ViBo
HeLa
Antiproliferative activity
Apoptosis
1. Introduction
with selective antiproliferative and anticancer activity. Steroidal
sapogeninshavebecomeidealsynthonsfortheelaborationofdiverse
Steroidal saponins are naturally occurring glycosides. Some
saponin-containing plants have been widely employed in folk
medicine because of the extended properties, and consequently,
great interest has been shown in the pharmacological and biological
characterization, as well as the chemical synthesis of the active
components [1]. Some of the steroidal saponins isolated recently
have shown antidiabetic [2], platelet aggregation inhibitory [3],
antifungal [4], antiinflammatory [5], and anticancer [6] properties.
We have systematically focused on the antitumor activity of these
agents, since several compounds currently used in cancer therapies
display problems due to the lack of selectivity, and trigger unwanted
side effects. Consequently, it is of interest to search for new scaffolds
saponins and derivatives because of their rigid framework and
potential for varying levels of functionalization, broad biological
activity profile, and ability to penetrate cell membranes and bind to
specific receptors [7]. Transformations of the spiroketal moiety of
sapogenins have yielded interesting steroidal structures for partial
synthesis [8]. We have described in previous work an efficient
process to open spirostanic side-chains, the glycosylation of the
resultant product, and the anticancer activities of the glyco-adducts
[9]. In an extension of this work, we now report the synthesis, char-
acterization, and biological evaluation of a new cholestanic saponin
versus CaSki, ViBo, and HeLa cell lines, starting from diosgenin.
2. Results and discussion
* Corresponding author. Tel.: þ1 778 782 4152; fax: þ1 778 782 4860.
** Corresponding author.
2.1. Chemical synthesis
*** Corresponding author.
E-mail addresses: luisss@servidor.unam.mx (L. Sánchez-Sánchez), bpinto@sfu.ca
(B.M. Pinto), jsandova@siu.buap.mx (J. Sandoval-Ramírez).
For the synthesis of the aglycon, the side chain of diosgenin (1)
was opened, as previously described [9a] to give (25R)-26-hydroxy-
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.05.058

3878
M.A. Fernández-Herrera et al. / European Journal of Medicinal Chemistry 46 (2011) 3877e3886
Aglycon
O
O
OH
OAc
O
a
AcO
HO
diosgenin (1)
2
Sugar moiety
OH
OH
OBn
O
OBn
O
OBn
O
e
c
b
d
O
O
HO
HO
HO
HO
BnO
BnO
BnO
BnO
BnO
OH
OMe
OMe
OBn
OH
OBn
O
CCl
3
BnO
OH
OH
OBn
NH
6
7
5
4
3
Coupling
OH
HO
OBn
OBn
OH
O
O
O
BnO
O
O
O
OH
OBn
f
pure β
g
OAc
2 + 7
OAc
8
9
AcO
.
Reagents and conditions: (a) Ac2O, BF3 OEt2, 0 °C, 84% [9a]; (b) AcCl, MeOH, room temperature, quantitative [9b]; (c) BnBr,
NaH, DMF, room temperature, 87% [9b]; (d) AcOH, 1 M H2SO4, reflux, 55% [9b]; (e) CCl3CN, DBU, CH2Cl2, room temperature,
89% [10]; (f) TMSOTf (0.1 equiv), 4 Å MS, CH2Cl2, -20 °C, 89% [11]; (g) 10% Pd-C, H2, AcOEt-MeOH (v:v, 7:3), 89%.
Scheme 1. Synthesis of the glycoside 9.
22-oxocholest-5-en-3
b
,16
b
-diyl diacetate (2) in excellent yield (84%,
crystallographic data [9c], demonstrated that the substituent at C-16
is beta oriented. The assignment is also supported by the mechanism
of opening of the side chain [9a,c]. The synthesis of the sugar moiety
was performed starting from glucose (3) as described for the corre-
sponding hecogenin analogue [9b] (Scheme 1). The hemiacetal 6,
Scheme 1). With regard to the configuration at C-16, the substituent
at this position is attached to a five-membered ring, this can be only
alpha or beta. A recent study based on ROESY correlations (interac-
tion between H-16a and H-17a), and supported by X-ray
Table 1
1H (600 MHz) and 13C (150 MHz) NMR spectral data ( in ppm, d4-methanol) for the glycoside 9.
d
Position
13C
1H
Mult.
J (Hz)
HMBC
COSY
Aglycon
3
75.1
4.53
m
C-1, C-4,
H-2eq, H-2ax,
H-4eq, H-4ax
H-2eq, H-2ax,
H-3
C¼O-acetate-3
4
5
6
38.8
140.6
123.1
2.31
2.31
d
m
7.6
C-1, C-2,
C-3, C-5, C-6
H-4eq, H-4ax,
CH3ꢀ19
5.37
mbroad
H-4eq, H-4ax,
C-7, C-8, C-10
C-13, C-17
H-4eq, H-4ax,
H-7eq, H-7ax
H-16
15
16
35.5
76.7
2.36
4.98
dd
m
6.8, 6.8
C-13, C-14,
H-15, H-17
C¼O-acetate-16
18
13.7
0.90
s
C-12, C-13,
C-14, C-17
19
20
21
22
23
19.7
44.5
17.3
216.0
39.4
1.04
3.01
1.15
s
dq
d
C-5, C-9, C-10
C-17, C-21, C-22
C-17, C-20, C-22
H-20, CH3ꢀ21,Hꢀ23
C-22, C-24, C-25
10.5, 7.1
7.1
H-17, H-21
H-20
2.72
2.42
3.73
3.35
0.92
2.01
1.97
m
m
dd
m
d
H-24
H-25
H-25
26
75.7
9.6, 6.0
6.3
C-27, C-10
27
17.1
21.5
21.4
172.2
171.6
C-24, C-25, C-26
C¼O-acetate-3
C¼O-acetate-16
Me-acetate-3
Me-acetate-16
C[O-acetate-3
C[O-acetate-16
s
s
26-O-
b-D-glucopyranoside
10
104.2
74.7
77.7
71.3
77.4
62.5
4.23
3.20
3.35
3.31
3.26
3.86
3.66
d
7.7
8.4, 7.7
C-26, H-26
H-20
20
dd
m
m
m
d
H-10, H-30
H-20, H-40
H-30, H-50
H-40, H-60
H-50
30
40
50
60
12.0
12.0, 5.4
dd

M.A. Fernández-Herrera et al. / European Journal of Medicinal Chemistry 46 (2011) 3877e3886
3879
O
HMBC
H20
22
Me21
Me18
24
Me19
12
8
26
25
13
14
1
23
O
17
10
5
4
9
O
O
O
Me27
O
7
O
H16
H1'
H3
H6
OH
HO
OH
HO
COSY
O
H20
Me21
Me18
Me19
24
26
25
17
23
2
3
O
O
O
O
15
Me27
4
O
7
O
H16
2'
5'
3'
6'
H1'
H6
OH
4'
HO
OH
HO
Fig. 1. Selected HMBC and COSY correlations for the glycoside 9.
Fig. 2. Effect of compound 9 on lymphocyte proliferation. M1 is the proliferating cells region, and M2 is the non-proliferating cells region. (A) ELPs untreated. (B) ELPs in the
presence of PHA stimulation. (C) ELPs treated with 10 L of ethanol (1%). (D) ELPs treated with 33.9 M of 9.
m
m

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M.A. Fernández-Herrera et al. / European Journal of Medicinal Chemistry 46 (2011) 3877e3886
obtained via 4 and 5, was treated with CCl3CN [10] to give the
trichloroacetimidate donor 7 which was used directly in the
glycosylation reaction. Trimethylsilyl trifluoromethanesulfonate
(TMSOTf)-promoted glycosylation [11] of the trichloroacetimidate 7
with the cholestanic alcohol 2, proceeded smoothly to afford thepure
diacetate N-succinimidyl ester (CFSE), stimulated with phytohe-
magglutinin (PHA), and/or treated with 9, and cultured for 72 h
[14]. Cells were harvested and their proliferative potential was
analyzed by flow cytometry. The effect of 9 on the proliferative
potential of ELPs is shown in Fig. 2, indicating that under normal
conditions, proliferating cells were 58% of the total population
b-anomer (8) in 89% yield. Such glycosylation reaction conditions
generally afford a mixture of and anomers. Interestingly, the
a
b
a-
(Fig. 2B). When lymphocytes were treated with 33.9 mM of 9 (the
anomer was not formed despite the absence of a participating group
at O-2, presumably because of the steric requirements of the bulky
aglycon. Debenzylation of compound 8 with 10% PdeC and hydrogen
at atmospheric pressure afforded compound 9. It is significant that
cytotoxicity bioassays of some steroidal glycosides have shown that
highest concentration found and described in the section 3.1),
proliferating cells were 72% (Fig. 2D). Thus, in the presence of
compound 9, proliferative potential was surprisingly not negatively
affected. These results suggest a greater degree of antiproliferative
selectivity towards malignant cell lines than with lymphocytes.
This selective promotion in lymphocyte population will be bene-
ficial in in vivo assays.
the b-anomers exhibited higher activities than the corresponding a-
anomers [12].
2.2. NMR analysis
3.3. Effect on cell-cycle
Analysis of the 1H NMR spectrum of 9 showed the signal for the
anomeric hydrogen H-10 at
4.26 ppm presenting a coupling
Cell-cycle regulation ensures the fidelity of genomic replication
and cell division in order to avoid impaired transmission of genetic
information. Cells progress through the cell-cycle in several well-
d
constant of 7.7 Hz, indicative of a
b-linkage between the sugar
moiety and the aglycon. Table 1 shows selected 1H and 13C chemical
shifts observed for the glycoside 9.
In addition, selected HMBC and COSY correlations are displayed
in Fig. 1. Correlations in the HMBC spectrum were found between
H-3 and H-16 with their corresponding acetate carbonyl groups.
H-20, Me-21 and H-23 showed correlations with the carbonyl
carbon at C-22. Correlations between the anomeric carbon (C-10)
and both protons H-26, and between H-10 and C-26, established the
aglycon-sugar linkage. 1H-1H-COSY correlations were observed
from H-3 to H-2 and H-4, from the vinylic H-6 to H-4 and H-7, from
CaSki
A
100
Control
Ethanol
Compound 9
80
60
40
20
0
H-16 to peaks at
d 2.36, 1.06 and 1.87 ppm, corresponding to three
different proton signals from CH2-15 and CH-17. In addition, Me-21
and Me-27 showed typical correlations with H-20 and H-25,
respectively.
*
G1
S
G2/M
SubG1
3. Biological evaluation
ViBo
B
3.1. In vitro antiproliferative activity on CaSki, ViBo, and HeLa
cervical cancer cell lines
100
80
60
40
20
0
Control
Ethanol
Compound 9
In order to determine the antiproliferative activity of comp-
ound 9, cervicouterine cancer cell lines CaSki, ViBo, and HeLa were
treated in a range of concentrations using ethanol as a vehicle, as
measured by a decrease in cell population (IC50). The anti-
proliferative activity was determined after 24 h by crystal violet
staining [13]. The inhibitory effect of 9 on the proliferation of CaSki,
ViBo, and HeLa cells was observed to occur in a dose-dependent
*
manner with an IC50 value of 33.9
29.5 M (20 g/mL) for ViBo and HeLa cells. Compared to the IC50 of
the aglycon itself (compound 2) in CaSki cells [9a], the activity of
the glycoside 9, was enhanced (47.1 vs. 33.9 M). It is well known
mM (23 mg/mL) for CaSki, and
G1
S
G2/M
SubG1
m
m
HeLa
C
m
100
80
60
40
20
0
that unlike sapogenins, saponins more easily permeate the cell wall
[1def], and presumably, glucosylation of 2 is also beneficial by the
same mechanism.
Control
Ethanol
Compound 9
3.2. In vitro antiproliferative activity on human lymphocytes
Major compounds used currently in chemotherapy present
problems for selective activity towards malignant cells and produce
undesirable secondary effects. For this reason, the effect of 9 on the
proliferation of peripheral blood lymphocytes was assessed. It is
well known that during chemotherapy the immune system is
usually affected; thus, the proliferation of enriched lymphocyte
population (ELP) was evaluated with compound 9. ELPs from
a healthy blood donor were labeled with 5(6)-carboxyfluorescein
*
G1
S
G2/M
SubG1
Fig. 3. Cell-cycle analysis of (A) CaSki, (B) ViBo, and (C) HeLa cells upon treatment with
compound 9 after 24 h. Values are expressed in distribution of % DNA, *p < 0.05 versus
ethanol.

M.A. Fernández-Herrera et al. / European Journal of Medicinal Chemistry 46 (2011) 3877e3886
3881
controlled phases [15]. Entry into each phase of the cell-cycle is
carefully regulated by cell-cycle checkpoints (G1/S and G2/M tran-
sitions) [16].
One theme emerging in drug discovery is the development of
agents that target the cell-cycle checkpoints that are responsible for
the control of cell-cycle phase progression. It is clear that cell-cycle
checkpoints can regulate the quality and rate of cell division and
several agents are now under development [17]. In cancer cell lines,
inducing cells to leave the cycle or cell-cycle arrest constitutes one
of the most prevalent strategies used to stop or limit cancer
spreading. In CaSki cells, compound 9 did not affect the cell-cycle,
implying that its antiproliferative activity is independent of the
cell-cycle phases (Fig. 3A), only showing an increase in the
percentage of cell nuclei from broken cells with a lower amount of
DNA in the so-called sub-G1 phase, thus indicating cell death.
Regarding ViBo (Fig. 3B) and HeLa (Fig. 3C) cells, arrest in the G2/M
and S phases respectively was identified, suggesting that the
activity is slightly dependent on the phases [18].
CaSki
A
100
75
50
25
0
3.4. Determination of cytotoxic activity of compound 9 on
cervicouterine cancer cells and human lymphocytes
It is crucial to determine the selectivity using the cytotoxicity
and apoptosis assays in order to derive any conclusions on the
potential for anticancer treatment. In order to evaluate if necrosis
was induced, the cytotoxic activity of compound 9 was evaluated.
CaSki, ViBo, and HeLa cultures were exposed to compound 9 at
the respective IC50 concentration, and the amount of lactate
dehydrogenase (LDH) released in the culture supernatant was
used as a measure of loss of plasma-membrane integrity. The
three cancer lines were treated with the nonionic surfactant
Triton X-100 in independent experiments, and the released LDH
was adjusted to 100% as a control [19]. Compound 9 induced
weak cytotoxicity, 1% of the cases for CaSki, 11% for ViBo, and 6%
for HeLa cells (Fig. 4AeC). These results suggest that compound 9
presents null cytotoxicity in all three lines, especially CaSki and
HeLa, indicating that the cell death induced by compound 9 is
a process different from necrosis.
Control
Triton X-100
Ethanol Compound 9
-25
ViBo
B
100
75
50
25
0
Cytotoxicity was evaluated next on human lymphocytes in order
to determine if necrosis is induced by 9 in non-tumor cells (Fig. 5).
Lymphocytes were activated with PHA and stimulated with
33.9 mM of 9. Results indicated that such concentrations are not
cytotoxic to human lymphocytes, and suggested a selective activity
through a different pathway than necrosis. Fig. 5.
*
3.5. Apoptosis
Control
Triton X-100
Ethanol
Compound 9
Apoptosis is an important and well controlled form of cell death
observed under a variety of physiological and pathological condi-
tions. Inappropriate apoptosis may be involved in many diseases
-25
HeLa
C
100
100
75
50
25
0
75
50
25
0
Control
Triton X-100
Ethanol
Compound 9
Control
Triton X-100
Ethanol
Compound 9
-25
-25
Fig. 4. Evaluation of cytotoxicity of compound 9 on (A) CaSki, (B) ViBo, and (C) HeLa
cultures. 7500 cells/well were seeded in 96-well tissue culture plates. After 24 h the
medium was removed and cells were exposed to compound 9 at the respective IC50
Fig. 5. Evaluation of cytotoxicity of compound 9 on human lymphocyte cultures.
Lymphocytes were seeded in 96-well tissue culture plates, exposed to compound 9 at
concentrations or with ethanol (10
m
L/mL) and evaluated after 24 h by the amount of
the respective IC50 concentrations or with ethanol (10
by the amount of LDH released in the culture supernatant. Activated lymphocytes with
PHA (15 L/mL) were used as a control. Experimental data are presented as the
mean ꢁ S.D. of three independent experiments with three repetitions.
mL/mL) and evaluated after 72 h
LDH released in the culture supernatant. Experimental data are presented as the
mean ꢁ S.D. of three independent experiments with three repetitions, *p < 0.05
ethanol versus compound 9 (Student’s t-test).
m

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M.A. Fernández-Herrera et al. / European Journal of Medicinal Chemistry 46 (2011) 3877e3886
such as Alzheimer’s disease, immune deficiency and autoimmune
disorders, leukemias, lymphomas, and other malignancies. There-
fore, the control of apoptosis is an important potential target for
therapeutic intervention [20].
also required for some typical hallmarks of apoptosis [21]. Active
caspase-3 expression was determined by immunocytochemistry.
Fig. 6 shows that 9 induced the expression of active caspase-3 in all
cell lines, implying that apoptosis could be triggered.
3.5.1. Expression of active Caspase-3
3.5.2. DNA fragmentation assessments
Caspases, or cysteine-aspartic proteases, are a family of cysteine
proteases, which are crucial mediators of apoptosis. Among them,
caspase-3 is probably the best understood of the mammalian cas-
pases in terms of its specificity and roles in apoptosis. Caspase-3 is
One of the characteristics of apoptosis is the degradation of
DNA. The TUNEL method identifies apoptotic cells in situ by
using terminal deoxynucleotidyl transferase (TdT) to transfer
biotin-dUTP to these strand breaks of cleaved DNA [22]. Fig. 7
Fig. 6. Immunodetection of active caspase-3 on CaSki (I), ViBo (II), and HeLa (III) cultures. (A), (D) and (G) Cells in phase contrast. (B), (E) and (H) Blue fluorescence indicates cells
counterstained with DAPI. (C), (F) and (I) Green fluorescence indicates the presence of active caspase-3 distributed in the cytoplasm of apoptotic cells, induced by the glycoside 9.
The images were obtained using an epifluorescence microscope, and correspond to an experiment representative of three independent assays. (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)

M.A. Fernández-Herrera et al. / European Journal of Medicinal Chemistry 46 (2011) 3877e3886
3883
Fig. 7. Compound 9 induces apoptotic death. CaSki (I), ViBo (II) and HeLa (III) cells were cultured with and without compound 9 (IC50) for 24 h. Ethanol (10
m
L/mL) was used as
a control. DNA fragmentation was detected by TUNEL assay as described in Experimental procedures. (A), (D) and (G) Cells in phase contrast. (B), (E) and (H) Blue fluorescence
indicates cells counterstained with DAPI. (C), (F) and (I) Red fluorescence indicates cells positive to the technique. The images were obtained using an epifluorescence microscope,
and correspond to an experiment representative of three independent assays. (For interpretation of the references to colour in this figure legend, the reader is referred to the web
version of this article.)
shows that fragmentation of DNA in CaSki, ViBo, and HeLa cells
is induced by compound 9. These results indicate that the
apoptotic event induced by 9 is completed in a 24 h period,
suggesting a short period for the completion of the apoptosis
induced by 9.
showed anticancer and selective activity against CaSki, ViBo, and
HeLa cervicouterine cancer cell lines, with greater antiproliferative
activity against CaSki cells than the de-O-glucosylated compound 2.
The biological evaluations suggest that 9 is a potent apoptosis
inducer with a null cytotoxic consequence. In addition, the prolif-
eration of peripheral blood lymphocytes was not affected, indica-
tive of an immunostimulatory effect. These in vitro results certainly
augur well for testing in other cancer cell lines and in vivo assays in
the next stage of the research. We believe, therefore, that
this glycoside serves as a promising lead candidate for further
evaluation.
4. Conclusions
In summary, the cholestanic 22-keto-26-glycoside
9 was
synthesized from the steroidal sapogenin diosgenin, through an
excellent method to open spirostanic skeletons. Compound 9

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M.A. Fernández-Herrera et al. / European Journal of Medicinal Chemistry 46 (2011) 3877e3886
5. Experimental section
27.1 (C-24), 33.2 (C-25), 74.8 (C-26), 17.0 (C-27), 103.6 (C-10), 82.2 (C-
20), 84.7 (C-30), 77.9 (C-40), 74.8 (C-50), 68.9 (C-60), 170.5 (CH3CO2-3),
169.7 (CH3CO2-16), 21.4 (CH3CO2-3), 21.1 (CH3CO2-16), 128.3e127.5
(CH-aromatics), 138.6 (Cipso-PhCH2-O-30), 138.4 (Cipso-PhCH2-O-20),
138.1 (Cipso-PhCH2-O-60), 138.0 (Cipso-PhCH2-O-40), 74.8 (PhCH2-O-
20), 75.6 (PhCH2-O-30), 75.0 (PhCH2-O-40), 73.4 (PhCH2-O-60). HRMS
Calcd for C65H83O11: 1039.5935, [M þ H]þ. Found: 1039.5927.
5.1. Materials
Optical rotations were measured at 24 ꢂC on a PerkineElmer 241
polarimeter. 1H and 13C NMR spectra were recorded at 600 and
150 MHz, respectively on a Bruker AVANCE NMR instrument. The
spectra were referenced to residual protonated solvent. Coupling
constants are expressed in Hertz (Hz). All assignments were
confirmed with the aid of 1D and 2D experiments (APT, COSY,
HSQC, and HMBC). Processing of the spectra was performed using
MestRec software. High resolution mass spectra were obtained by
the Electrospray Ionization (ESI) technique, using an Agilent 6210
TOF LC/MS mass spectrometer. Column chromatography was per-
formed using Merck silica gel 60 (230e400 mesh), and analytical
thin layer chromatography (TLC) was performed on aluminum
plates precoated with silica gel 60F-254.
5.2.2. (25R)-3b,16b-Diacetoxy-22-oxocholest-5-en-26-yl b-D-
glucopyranoside (9)
To a solution of 8 (300 mg, 0.29 mmol) in MeOH:AcOEt (7:3)
was added 10% Pd on carbon (200 mg) and the mixture was
stirred under hydrogen for 2 h at room temperature. The catalyst
was removed by filtration and the filtrate was evaporated to
dryness. The residue was purified by flash column chromatog-
raphy using CHCl3/MeOH (93:7) as eluent to give 9 as a white
powder (196 mg, 89%). [
a]
þ 10.0ꢂ (c 0.3, CHCl3). IR: 3390 (OH),
D
2939 (CH, aliphatic), 1724 (C]O, acetate), 1711 (C]O, ketone). 1H
5.2. Chemical synthesis
NMR (CD3OD): d 5.37 (1H, mbroad, H-6), 4.98 (1H, m, H-16), 4.53
(1H, m, H-3), 4.23 (1H, d, J1 ,2 ¼ 7.7 Hz, H-10), 3.86 (1H, d,
0
0
5.2.1. (25R)-3
tetra-O-benzyl-
A mixture of 2,3,4,6-tetra-O-benzyl-
0.95 mmol) [9b,10], CCl3CN (0.48 mL, 4.74 mmol), and DBU (15
b,16b
-Diacetoxy-22-oxocholest-5-en-26-yl 2,3,4,6-
Jgem ¼ 12.0 Hz, H-60a), 3.73 (1H, dd, J26a,25 ¼ 6.0 Hz, Jgem ¼ 9.6 Hz,
H-26a), 3.66 (1H, dd, Jgem ¼ 12.0 Hz, J6’a,5 ¼ 5.4 Hz, H-60b), 3.35
0
b
-D-glucopyranoside (8)
D
-glucopyranose 6 (513 mg,
(1H, m, H-30), 3.35 (1H, m, H-26b), 3.31 (1H, m, H-40), 3.26 (1H, m,
0
0
0
0
mL,
H-50), 3.20 (1H, dd, J2 ,3 ¼ 8.4 Hz, J2 ,1 ¼ 7.7 Hz, H-20), 3.01 (1H,
dq, J20,21 ¼ 7.1 Hz, J20,17 ¼ 10.5 Hz, H-20), 2.72 (1H, m, 23a), 2.42
(1H, m, H-23b), 2.36 (1H, dd, J15a,16 ¼ J15a,14 ¼ 6.8 Hz, H-15a), 2.31
(1H, d, J4ax,3 ¼ 7.6 Hz, H-4ax), 2.31 (1H, m, H-4eq), 2.01 (3H, s,
CH3CO2-3), 1.97 (3H, s, CH3CO2-16), 1.15 (3H, d, J21,20 ¼ 7.1 Hz,
CH3-21), 1.04 (3H, s, CH3-19), 0.92 (3H, d, J27,25 ¼ 6.3 Hz, CH3-27),
0.1 mmol) in CH2Cl2 (5 mL) was stirred at room temperature for 3 h.
The mixture was concentrated under vacuum and the resulting
residue was purified by flash column chromatography with hexane/
ethyl acetate/triethylamine (90:9:1) as eluent to give the corre-
sponding trichloroacetimidate 7 as a syrup (575 mg, 89%). This
compound was used immediately in the glycosylation reaction
without any further purification. A mixture of the donor 7 (409 mg,
0.60 mmol), aglycon 2 (258 mg, 0.50 mmol) and 4 Å MS (200 mg) in
dry CH2Cl2 (10 mL) was stirred at room temperature for 15 min and
0.90 (3H, s, CH3-18). 13C NMR (CD3OD):
d 37.8 (C-1), 28.5 (C-2),
75.1 (C-3), 38.8 (C-4), 140.6 (C-5), 123.1 (C-6), 32.5 (C-7), 32.2 (C-
8), 50.9 (C-9), 37.4 (C-10), 21.6 (C-11), 40.6 (C-12), 42.8 (C-13),
54.9 (C-14), 35.5 (C-15), 76.7 (C-16), 56.3 (C-17), 13.7 (C-18), 19.7
(C-19), 44.5 (C-20), 17.3 (C-21), 216.0 (C-22), 39.4 (C-23), 28.0 (C-
24), 33.9 (C-25), 75.7 (C-26), 17.1 (C-27), 104.2 (C-10), 74.7 (C-20),
77.7 (C-30), 71.3 (C-40), 77.4 (C-50), 62.5 (C-60), 172.2 (CH3CO2-3),
171.6 (CH3CO2-16), 21.5 (CH3CO2-3), 21.4 (CH3CO2-16). HRMS
Calcd for C37H59O11: 679.4057, [M þ H]þ. Found: 679.4051.
then cooled to ꢀ20 ꢂC. A solution of TMSOTf in CH2Cl2 (10
mL,
0.05 mmol) was added slowly to the reaction mixture. After stirring
for 1 h, the reaction was quenched by addition of triethylamine
(0.1 mL), and filtered. The filtrate was concentrated under vacuum to
give a residue of the crude b-anomer 8 which was purified by flash
column chromatography with hexane/ethyl acetate (7:3) as eluent
to afford a colorless foam (462 mg, 89%). [
a]
D þ 7.0ꢂ (c 1.1, CHCl3). IR:
5.3. Biological activity
2938 (CH, aliphatic), 1727 (C]O, acetate), 1710 (C]O, ketone), 1505,
1086 and 3067 (CH aromatics). 1H NMR (CDCl3):
d
7.34-7.12 (20H, m,
5.3.1. Cell culture
aromatics), 5.34 (1H, d, J6,7eq ¼ 4.8 Hz, H-6), 4.95 (1H, m, H-16), 4.93
(1H, d, Jgem ¼ 11.0 Hz, PhCH2-O-20a), 4.90 (1H, d, Jgem ¼ 10.9 Hz,
PhCH2-O-30a), 4.79 (1H, d, Jgem ¼ 10.8 Hz, PhCH2-O-40a), 4.76 (1H, d,
Jgem ¼ 10.9 Hz, PhCH2-O-30b), 4.69 (1H, d, Jgem ¼ 11.0 Hz, PhCH2-O-
20b), 4.59 (1H, d, Jgem ¼ 12.6 Hz, PhCH2-O-60a), 4.58 (1H, m, H-3),
4.53 (1H, d, Jgem ¼ 12.6 Hz, PhCH2-O-60b), 4.51 (1H, d, Jgem ¼ 10.8 Hz,
CaSki, ViBo, and HeLa cell lines were purchased from the
American Type Culture Collection (ATCC Rockville, MD) and were
cultured in RPMI-1640 medium (GIBCO, USA) containing 5%
Newborn Calf Serum (NCS, GIBCO, USA) with phenol red supple-
mented by benzylpenicillin. Cultures were maintained in a humid-
ified atmosphere with 5% CO2 at 37 ꢂC. All cell-based assays were
performed using cells in the exponential growth phase.
PhCH2-O-40b), 4.35 (1H, d, J1 ,2 ¼ 7.8 Hz, H-10), 3.75 (1H, dd,
J26a,25 ¼ 6.2 Hz, Jgem ¼ 9.5 Hz, H-26a), 3.72 (1H, dd, Jgem ¼ 10.8 Hz,
0
0
J6’a,5 ¼ 1.9 Hz, H-60a), 3.66 (1H, dd, Jgem ¼ 10.8 Hz, J6’b,5 ¼ 4.9 Hz, H-
5.3.2. Cell proliferation assay
0
0
60b), 3.62 (1H, dd, J3 ,2 ¼ 9.0 Hz, J3 ,4 ¼ 11.2 Hz, H-30), 3.56 (1H, dd,
Assays were performed by seeding 7500 cells/well in 96-well
0
0
0
0
J4 ,3 ¼ 11.2 Hz, J4 ,5 ¼ 9.6 Hz, H-40), 3.43 (1H, m, H-50), 3.42 (1H, dd,
tissue culture plates in a volume of 100
supplemented with 5% NCS per well. Cells were allowed to grow for
24 h in culture medium prior to exposure to 33.9 M or 29.5 M of
m
L of RPMI-1640 medium
0
0
0
0
J2 ,3 ¼ 9.0 Hz, J2 ,1 ¼ 7.8 Hz, H-20), 3.38 (1H, dd, J26b,25 ¼ 5.5 Hz,
Jgem ¼ 9.5 Hz, H-26b), 2.91 (1H, dq, J20,21 ¼7.1 Hz, J20,17 ¼ 10.9 Hz, H-
20), 2.61 (1H, ddd, Jgem ¼ 17.7 Hz, J23a,24b ¼ 10.0 Hz, J23a,24a ¼ 5.5 Hz,
23a), 2.39 (1H, m, H-15b), 2.33 (1H, m, H-23b), 2.29 (2H, m, 4ax and
4eq), 2.01 (3H, s, CH3CO2-3), 1.92 (3H, s, CH3CO2-16), 1.35 (1H, m, H-
24b), 1.24 (1H, ddd, J12ax,11ax ¼ Jgem ¼ 12.7 Hz, J12ax,11eq ¼ 4.0 Hz, H-
12ax), 1.08 (3H, d, J21,20 ¼ 7.2 Hz, CH3-21), 1.00 (3H, s, CH3-19), 0.95
(3H, d, J27,25 ¼ 6.6 Hz, CH3-27), 0.83 (3H, s, CH3-18). 13C NMR (CDCl3):
0
0
0
0
m
m
9. 1% of vehicle (ethanol) was added to control cells. Anti-
proliferative activity (IC50) was determined after 24 h by crystal
violet staining [13]. Growth inhibition was determined by
measuring the absorbance at 590 nm in an Enzyme-Linked
ImmunoSorbent Assay (ELISA) plate reader (Tecan, USA).
d
36.8 (C-1), 27.7 (C-2), 73.8 (C-3), 38.0 (C-4), 139.6 (C-5),122.3 (C-6),
5.3.3. CSFE labeling assay
31.5 (C-7), 31.2 (C-8), 49.7 (C-9), 36.5 (C-10), 20.7 (C-11), 39.6 (C-12),
41.8 (C-13), 53.8 (C-14), 34.8 (C-15), 75.7 (C-16), 55.0 (C-17), 13.2 (C-
18), 19.2 (C-19), 43.5 (C-20), 16.7 (C-21), 212.8 (C-22), 38.7 (C-23),
Heparinized blood samples were obtained from healthy human
volunteers. Peripheral blood mononuclear cells (PBMCs) were iso-
lated using standard Hypaque (Sigma-aldrich USA) density gradient

M.A. Fernández-Herrera et al. / European Journal of Medicinal Chemistry 46 (2011) 3877e3886
3885
centrifugation. PBMCs were washed twice with RPMI 1640 (GIBCO
USA) medium containing 10% NCS, penicillin (100 U/mL), and
streptomycin (100 U/mL). The lymphocyte population was further
enriched (ELP) by the elimination of adherent cells (cells were
incubated at 37 ꢂC, 5% CO2 for 1 h, and non-adherent cells were
harvested). ELPs were re-suspended in RPMI-1640 medium at
a concentration of 1 ꢃ 106 cells/mL. CFSE (from SigmaeAldrich,
USA) was added to the cell suspension at a final concentration of
washed three times, and labeled with biotin-dUTP by incubation
with reaction buffer containing terminal deoxynucleotidyl trans-
ferase enzyme for 1 h at 37 ꢂC. Biotinylated nucleotides were
detected using rhodamine-conjugated streptavidin. Cells were
counterstained using DAPI to determine DNA distribution. Cell
fluorescence was determined using an E600 Nikon Eclipse micro-
scope with red and blue filters.
12
mM and incubated for 15 min at room temperature in the dark.
5.3.8. Statistical analysis
Labeling was completed by adding, during 5 min at room temper-
ature, the same volume of NCS to quench the free CFSE. Labeled
cells were washed 5 times with sterile PBS containing 10% NCS,
counted, and re-suspended in RPMI-1640 medium at 1 ꢃ 106 cells/
mL [14]. Unstimulated, PHA-stimulated, or treated cells were plated
at 2 ꢃ 105 cells/well in 96-well flat-bottomed cell culture plates,
and five replicate samples for each treated amount were prepared.
The cells were incubated in a 5% CO2 incubator at 37 ꢂC for 72 h.
Cultured cells were harvested, washed twice with PBS, fixed with
1% formaldehyde, then analyzed using flow cytometry, acquiring
a minimal of 20,000 events from each sample; data analysis was
performed using CellQuest (BectoneDickinson) software.
The median and standard deviation (SD) were calculated using
Excel (Microsoft Office, Version 2007). Statistical analysis of
differences was carried out by analysis of variance (ANOVA) using
SPSS 10.0 for Windows. A p-value of less than 0.05 (Student’s t-test)
was considered to be significant.
Acknowledgments
The authors gratefully thank CONACYT for the PhD scholarships
to MAFH and HLM, for grant 84380 to JSR, and for the postdoctoral
fellowship 144219 to MAFH. We thank PAPIME for grant PE204609
and the Natural Sciences and Engineering Research Council of
Canada for a grant to BMP. We thank Dr. Dionisio Parra (Gyneco-
Perinatology Department) and Dr. René García (Teaching Depart-
ment) from Hospital General “Ignacio Zaragoza,” ISSSTE for
technical assistance, and PROQUINA for the donation of diosgenin.
5.3.4. Cell-cycle analysis
CaSki, ViBo, and HeLa cells were seeded at 105 cells/mL in 50 mm
tissue culture plates and allowed to grow for 24 h in culture medium
prior to exposure to 33.9 mM or 29.5 mM of 9. Cells were harvested
with versene solution. For DNA content analysis, cells were fixed and
permeabilized in 50% methanol in phosphate-buffered saline (PBS),
washed in PBS, treated with RNase (2.5 U/mL) and stained with
propidium iodide (0.2 mg/mL). Finally, samples were analyzed by
flow cytometry analysis (FACS Aria II, B.D., USA) [18].
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