Synthesis and selective anticancer activity of steroidal glycoconjugates

Article abstract of DOI:10.1016/j.ejmech.2012.06.027

The synthesis of glucosamine derivatives of the steroidal sapogenins diosgenin and hecogenin using the N-phthaloyl protected trichloroacetimidate of d-glucosamine as donor and TMSOTf as promoter is reported. The corresponding glycoconjugates were transformed into their acetamido derivatives and the hydrochloride salt (from diosgenin) and tested against HeLa, CaSki, and ViBo cervicouterine cancer cells. These compounds showed low cytotoxicity values on tumor cells and human lymphocytes, indicating that the main cell death process is presumably not necrosis. Significantly, the antiproliferative activity of these compounds on tumor cells did not affect the proliferative potential of peripheral blood lymphocytes.

Full text of DOI:10.1016/j.ejmech.2012.06.027

European Journal of Medicinal Chemistry 54 (2012) 721e727
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and selective anticancer activity of steroidal glycoconjugates
María A. Fernández-Herrera a, Hugo López-Muñoz b, José M.V. Hernández-Vázquez b,
,
c
a
, ,
,
*
Luis Sánchez-Sánchez b , María L. Escobar-Sánchez , B. Mario Pinto 1, Jesús Sandoval-Ramírez d
*
*
a Department of Chemistry, Simon Fraser University, 8888 University Drive, Burnaby, British Columbia, Canada V5A 1S6
b Facultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma de México, 09230 México, D.F., Mexico
c Departamento de Biología Celular, Facultad de Ciencias, Universidad Nacional Autónoma de México, Ciudad Universitaria, 04510 México, D.F., Mexico
d Facultad de Ciencias Químicas, Benemérita Universidad Autónoma de Puebla, Ciudad Universitaria, 72570 Puebla, Pue., Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
The synthesis of glucosamine derivatives of the steroidal sapogenins diosgenin and hecogenin using the
N-phthaloyl protected trichloroacetimidate of -glucosamine as donor and TMSOTf as promoter is
Received 26 February 2012
Received in revised form
8 June 2012
Accepted 13 June 2012
Available online 21 June 2012
D
reported. The corresponding glycoconjugates were transformed into their acetamido derivatives and the
hydrochloride salt (from diosgenin) and tested against HeLa, CaSki, and ViBo cervicouterine cancer cells.
These compounds showed low cytotoxicity values on tumor cells and human lymphocytes, indicating
that the main cell death process is presumably not necrosis. Significantly, the antiproliferative activity of
these compounds on tumor cells did not affect the proliferative potential of peripheral blood
lymphocytes.
Keywords:
b-D-Glucosamine steroidal derivatives
Diosgenin
Ó 2012 Elsevier Masson SAS. All rights reserved.
Hecogenin
Cervicouterine cancer cells
Antiproliferative activity
Cytotoxicity
Selective antitumor activity
1. Introduction
a constituent of many naturally occurring poly- and oligosaccha-
rides, glycoproteins, and glycolipids. Its presence in steroidal sapo-
Saponins constitute a group of structurally related compounds
widely distributed in the animal, plant, and fungal kingdoms. The
medicinal chemistry of steroidal saponins has generated a large
variety of structures and biological activities [1]. This group of
natural products has been extensively studied in the development of
new drugs for the treatment of infections, diabetes, and cancer,
among others. Steroidecarbohydrate conjugates are involved in
a wide variety of biological processes, and their synthesis and bio-
logical evaluation is of interest [2]. The carbohydrate moiety of
steroidal saponins plays a very important role in the biological
activity [3], and it is of interest, therefore, to search for new variants
of steroidal glycosides with selective anticancer activity. The most
common monosaccharide units present in natural and synthetic
nins opens up a broad spectrumof possibilities in biological activities
since the nature of the amino group confers specific properties on
different types of molecules [5]. We have described in previous work
the glycosylation of several steroidal derivatives along with their
corresponding anticancer activities [6]. In an extension of this work,
we now report the synthesis, characterization, and biological eval-
uation of diosgenin and hecogenin glycosides containing
b-D-
glucosamine and/or -N-acetylglucosamine at C-3 and their anti-
b-D
cancer activities versus HeLa, CaSki, and ViBo cell lines.
2. Results and discussion
2.1. Synthesis
steroidal saponins are
b-D-glucopyranose, a-L-rhamnopyranose
and -galactopyranose [1,4]. Glucosamine is an aminosugar
of great biological importance that is frequently encountered as
b-D
The synthetic strategy was based on the use of anomeric tri-
chloroacetimidates of peracetylated N-phthalimido glucosamine.
The trichloroacetimidates were obtained as described in Scheme 1
[7], and were used immediately in the coupling reactions.
The glycosylation reactions of the donor 4 with diosgenin (5)
and hecogenin (6) promoted by TMSOTf [6], afforded exclusively
* Corresponding authors.
E-mail addresses: luisss@unam.mx (L. Sánchez-Sánchez), bpinto@sfu.ca
(B.M. Pinto), jesus.sandoval@correo.buap.mx (J. Sandoval-Ramírez).
1
Tel.: þ1 778 782 4152; fax: þ1 778 782 4860.
the b-glycosides 7 and 8 (Scheme 2). The structure of the coupling
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.06.027

722
M.A. Fernández-Herrera et al. / European Journal of Medicinal Chemistry 54 (2012) 721e727
ViBo were treated in a range of concentrations. The antiproliferative
activity was determined after 24 h by crystal violet staining [8]. The
inhibitory effect of 9e11 on the proliferation of HeLa, CaSki, and
ViBo cells was observed to occur in a dose-dependent manner, as
shown in Fig. 1.
The IC50 values determined for the title compounds are shown in
Table 1. For compound 10, 50% of inhibition was not reached after
treatment with 90 mg/mL for ViBo cells, and precipitationwas observed
at higher concentrations. On the other hand, the hydrochloride of
compound 9 (compound 11) clearly showed greater activity.
2.2.2. Cytotoxic activity determination on cervicouterine cancer
cells and human lymphocytes
In order to determine if necrosis was induced, the cytotoxic
activity of compounds 9e11 was evaluated (Fig. 2). HeLa, CaSki, and
ViBo cultures were stimulated with the IC50 values determined for
9e11 (see Table 1). The amount of lactate dehydrogenase (LDH)
released in the culture supernatant was used as a measure of loss of
plasma-membrane integrity and Triton X-100 was used to induce
mortality of cells by lysis [9]. Surprisingly, null cytotoxicity values
were found in all cases, even with regard to the cytotoxicity on non-
tumoral cells (human lymphocytes). These results suggest that the
observed cell decrease in treated cultures is not a necrotic process,
and another event different from necrosis decreases the cell
number observed in cultures. It is well known, for example, that
inducing cells to cell-cycle arrest constitutes one of the most
prevalent strategies used to stop or limit cancer spreading [10]. The
decrease in the evaluated cultures suggests that compounds 9e11
presumably affect the cell-cycle in one of the phases (check-
points), causing cell death. This is a subject worthy of in depth
study in future work.
Scheme 1. Reagents and conditions: a) NaOMe/MeOH 0.5 M, phthalic anhydride, then
Ac2O, pyridine. b) Hydrazine acetate, DMF. c) CCl3CN, DBU, CH2Cl2.
products was confirmed by 2D NMR and HRMS analyses. The
deprotection of glycosides 7 and 8 was achieved using 85% hydra-
zine hydrate in refluxing ethanol which removed in one-pot the O-
acetyl protecting groups and yielded the free amine. It is note-
worthy that in the case of the hecogenin derivative 8, the carbonyl
group at C-12 did not react in the presence of the hydrazine
hydrate, so that, high chemoselectivity was found in this depro-
tection procedure. N-acetylation was carried out immediately using
acetic anhydride in methanol and triethylamine at room temper-
ature, which afforded the compounds 9 and 10 (Scheme 2). In the
case of 7, the amine was converted to its hydrochloride salt 11, after
removal of the protecting groups in the sugar moiety; the crude
amino compound was treated with HCl, obtained from an equiva-
lent amount of AcCl in dry MeOH. This procedure was attempted
with the hecogenin derivative 8 but decomposition of the free
amine was obtained instead. The structures of the target products
9e11 were confirmed by 2D NMR and HRMS analyses.
2.2.3. Evaluation of antiproliferative activity on non-tumoral cells
Major compounds used currently in chemotherapy present
problems for selective activity towards malignant cells and produce
undesirable secondary effects. It is crucial to determine the selec-
tivity of tested compounds along with the antiproliferative and
cytotoxicity assays in order to derive any conclusions on anticancer
activity. For this reason, the effect of 9e11 on the proliferation of
2.2. Biological activity
2.2.1. In vitro antiproliferative activity on cervical cancer cell lines
In order to determine the antiproliferative activity of
compounds 9e11, cervicouterine cancer cell lines HeLa, CaSki, and
Scheme 2. Reagents and conditions: a) TMSOTf, 4 Å MS, CH2Cl2. b) Hydrazine hydrate 85%, ethanol, reflux, then MeOH, Ac2O, Et3N. c) Hydrazine hydrate 85%, ethanol, reflux, then
AcCl/MeOH. *This reaction was performed with 7 to obtain 11.

M.A. Fernández-Herrera et al. / European Journal of Medicinal Chemistry 54 (2012) 721e727
723
Dose-response curves for compound9
Dose-response curves for compound10
120
100
80
60
40
20
0
120
100
80
60
40
20
0
CaSki
HeLa
ViBo
CaSki
HeLa
ViBo
0
10 20 30 40 50 60 70 80 90 100
0
10
20
30
µg/mL
40
50
60
µg/mL
Dose-response curves for compound11
120
100
80
60
40
20
0
CaSki
HeLa
ViBo
0
10
20
30
g/mL
40
50
60
µ
Fig. 1. Dose-response curves of the antiproliferative effect of compounds 9e11 on cervicouterine cancer cell lines HeLa, CaSki, and ViBo.
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 compounds 9e11. ELPs from a normal blood donor
were labeled with 5(6)-carboxyfluorescein diacetate N-succini-
midyl ester (CFSE) and stimulated with PHA, and/or treated with
9e11 at the level of their determined IC50 values, and cultured for
72 h [11]. Cells were harvested and their proliferative potential was
analyzed by flow cytometry. The effect of 9e11 on the proliferative
potential of ELPs, shown in Fig. 3, indicates that under the influence
of 9e11 the proliferative potential was not negatively affected.
These results on lymphocytes indicate that the inhibition of cancer
cell proliferation was selective.
cultures is not a necrotic process and a cell-cycle arrest event might
be implicated. In addition, the proliferation of peripheral blood
lymphocytes was not affected. These results of selective anticancer
activity certainly augur well for testing in other cancer cell lines and
with different combinations of oligosaccharideesteroid conjugates.
4. Experimental section
4.1. Materials
Optical rotations were measured at 24 ꢀC on a Rudolph Research
Autopol II polarimeter using CHCl3 or MeOH as solvents. 1H and 13
C
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 two-
dimensional experiments (COSY, HSQC, and HMBC). Acquired
data were processed and analyzed using MestReNova 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 performed using
Merck Silica Gel 60 (230e400 mesh), and analytical thin-layer
chromatography (TLC) was performed on aluminum plates pre-
coated with Silica Gel 60F-254.
3. Conclusions
In summary, the glucosamine steroidal derivatives 9e11 were
synthesized from the steroidal sapogenins diosgenin and hecogenin,
and a protected glucosamine donor. The target compounds showed
antiproliferative and selective activity against HeLa, CaSki, and ViBo
cultures. The hydrochloride 11 showed better IC50 values and more
selectivity than its corresponding acetamido analog. The null cyto-
toxic consequence implies that the observed cell decrease in treated
Table 1
4.2. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-phthalimido-a,b-D-
glucopyranose (2)
Antiproliferative activities of compounds 9e11 (IC50 values in mM) in cervicouterine
cancer HeLa, CaSki, and ViBo cell lines.
Entry
Compound
IC50 (mM)
D
-Glucosamine hydrochloride (5.0 g, 23.2 mmol) was added to
HeLa
CaSki
ViBo
a 1 M solution of NaOMe/MeOH (30 mL). The mixture was stirred
for 15 min at room temperature and the resulting NaCl was filtered.
Then, MeOH (15 mL) was removed by rotatory evaporation and
phthalic anhydride (3.5 g, 23.6 mmol) was added. Stirring was
1
2
3
9
10
11
81
129
41
80
136
41
81
Not reached after 142
82

724
M.A. Fernández-Herrera et al. / European Journal of Medicinal Chemistry 54 (2012) 721e727
Cytotoxic activity on HeLa
Cytotoxic activity on CaSki
120
100
80
60
40
20
0
120
100
80
60
40
20
0
*
*
Triton X- Control DMSO
100
9
11
10
Triton X- Control DMSO
100
9
11
10
Cytotoxic activity on ViBo
Cytotoxic activity on lymphocytes
120
120
100
80
60
40
20
0
*
*
100
80
60
40
20
0
*
Triton X- Control DMSO
9
11
10
Triton X- Control DMSO
100
9
11
10
100
Fig. 2. Evaluation of cytotoxicity of compounds 9e11 on HeLa, CaSki, and ViBo cultures. 7500 cells/well were seeded in 96-well tissue culture plates, after 24 h the medium was
removed and cells were stimulated at the level of IC50 values for 9e11 or DMSO (10 L/mL) and evaluated after 24 h by the amount of LDH released in the culture supernatant.
Experimental data is presented as the mean ꢂ SD of three independent experiments with three repetitions. *p < 0.05 versus DMSO (ANOVA followed by Tukey’s test).
m
continued for 60 min until the phthalic anhydride was completely
dissolved and Et3N (5 mL) was added. The mixture was stirred for
20 min at room temperature and 20 min more at 45 ꢀC and after-
wards the resulting mixture was cooled, and the precipitate
collected and dried. Subsequently, the solid was acetylated by
means of acetic anhydride (10 mL), pyridine (15 mL) and a catalytic
amount of DMAP. The residue was stirred for 2 h at room temper-
ature, diluted in CH2Cl2, washed with a saturated solution of
NaHCO3 (4 ꢁ 50 mL), 5% aq. HCl (4 ꢁ 50 mL), and brine (4 ꢁ 50 mL),
dried over Na2SO4, and evaporated under reduced pressure. The
crude product was purified by chromatography on silica gel, with
hexanes/EtOAc (6:4) as eluent, to afford compound 2 (mixture of
(C-3), 68.3 (C-4), 72.6 (C-5), 61.5 (C-6), 20.7e20.3 (Ac), 170.6e168.6
(C]O, Ac), 167.3 (C]O, Phth), 134.4, 131.2, 123.7 (Ar). HRMS Calcd.
for C22H23NO11: 477.1271. Found: 478.1279 [M þ H]þ.
4.3. 3,4,6-Tri-O-acetyl-2-deoxy-2-phthalimido-a,b-D-glucopyranosyl
trichloroacetimidate (4)
Compound 2 (500 mg, 1.05 mmol) was dissolved in DMF and
hydrazine acetate (135 mg, 1.47 mmol) was added portionwise, the
reaction mixture was stirred under nitrogen at room temperature
for 2 h. After the completion of the reaction, the crude product was
diluted in EtOAc (30 mL), washed with water (3 ꢁ 50 mL) and brine
(3 ꢁ 50 mL), dried over anhydrous Na2SO4, and concentrated under
reduced pressure. The resulting syrup was dried overnight under
high vacuum and then dissolved in CH2Cl2. A catalytic amount of
DBU was added and CCl3CN (0.5 mL, 5.0 mmol) was added drop-
wise. The mixture was stirred under nitrogen at room temperature
for 3 h, concentrated under vacuum, and the resulting residue was
purified by flash column chromatography with hexanes/EtOAc/
a
and
b
anomers) as a colorless solid (42%) [7]. 1H NMR (CDCl3,
7.85 (2H, m, Ar), 7.74 (2H, m, aromatics), 6.50 (1H, d,
b-
anomer):
d
J1,2 ¼ 8.9 Hz, H-1), 5.87 (1H, dd, J3,2 ¼ 10.5 Hz, J3,4 ¼ 9.2, H-3), 5.19
(1H, dd, J4,3 ¼ J4,5 ¼ 9.7 Hz, H-4), 4.45 (1H, dd, J2,3 ¼ 10.5 Hz,
J2,1 ¼ 8.9 Hz, H-2), 4.35 (1H, dd, J6a,5 ¼ Jgem ¼ 12.4 Hz, H-6a), 4.13
(1H, dd, J6b,5 ¼ 7.9 Hz, Jgem ¼ 12.4 Hz, H-6b), 4.01 (1H, m, H-5),
2.09e1.85 (12H, Ac). 13C NMR (CDCl3):
d 89.7 (C-1), 53.5 (C-2), 70.5
Fig. 3. Effect of compounds 9e11 on lymphocyte proliferation.

M.A. Fernández-Herrera et al. / European Journal of Medicinal Chemistry 54 (2012) 721e727
725
Et3N (44:55:1) as eluent to give the corresponding tri-
chloroacetimidate 4 as a syrup (575 mg, 71% over two steps). This
compound was used immediately in the glycosylation reactions.
added dropwise. The reaction mixture was stirred under nitrogen
at reflux for 20 h. After the completion of the reaction, the solvent
was evaporated, and the amorphous solid was dried under high
vacuum to remove traces of hydrazine. Due to the instability of the
free amine, this compound was immediately dissolved in MeOH,
and the amine was selectively acylated at room temperature stir-
ring for 5 min with Ac2O (0.5 mL) and Et3N (0.1 mL). After this time,
water was added and the organic phase was extracted with EtOAc,
washed with a saturated solution of NaHCO3 (3 ꢁ 30 mL), water
(2 ꢁ 30 mL), and dried over Na2SO4. Evaporation of the solvent and
chromatographic purification (CH2Cl2/MeOH 9:1) yielded
compound 9 (quantitative) as a white solid: mp 244e246 ꢀC (dec);
4.4. Diosgenin 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-D-
glucopyranoside (7)
A mixture of the donor 4 (100 mg, 0.17 mmol), diosgenin 5
(60 mg, 0.15 mmol) and 4 Å MS (100 mg) in dry CH2Cl2 (3 mL) was
stirred at room temperature for 15 min and then cooled to ꢃ20 ꢀC.
A solution of TMSOTf in CH2Cl2 (3.1 mL, 0.017 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 which
was purified by flash column chromatography with hexanes/EtOAc
[a
]
D ꢃ115ꢀ (c 1.0, CHCl3). 1H NMR (CDCl3/MeOD):
d 5.30 (1H, m, H-
6), 4.58 (1H, d, J1,2 ¼ 7.8 Hz, H-10), 4.37 (1H, dd, J16,17 ¼ 7.4 Hz,
J16,15 ¼15.2, H-16), 3.80 (1H, m, H-60a), 3.74 (1H, m, H-60b), 3.59 (1H,
m, H-30), 3.43 (1H, m, H-40), 3.43 (1H, m, H-3), 3.43 (1H, m, H-26eq),
3.36 (1H, m, H-20), 3.33 (1H, m, H-26ax), 3.27 (1H, m, H-50), 2.25
(1H, m, H-4eq), 2.12 (1H, m, H-4ax), 1.99 (3H, NHAc), 0.96 (3H, s,
CH3-19), 0.93 (3H, d, J21,20 ¼ 7.0 Hz, CH3-21), 0.75 (3H, d,
J27,25 ¼ 6.8 Hz, CH3-27), 0.75 (3H, s, CH3-18). 13C NMR (CDCl3/
(75:25) as eluent to afford 7 as the pure
b
-anomer (116 mg, 96%).
7.86 (2H, m, Ar), 7.74
[
a
]
ꢃ10.4ꢀ (c 1.0, CHCl3). 1H NMR (CDCl3):
d
D
(2H, m, Ar), 5.77 (1H, dd, J3,2 ¼ 10.7 Hz, J3,4 ¼ 9.9, H-30), 5.47 (1H, d,
J1,2 ¼ 8.5 Hz, H-10), 5.22 (1H, m, H-6), 5.16 (1H, dd, J4,3 ¼ J4,5 ¼ 9.9 Hz,
H-40), 4.38 (1H, dd, J16,17 ¼ 7.5 Hz, J16,15 ¼15.0, H-16), 4.32 (1H, m, H-
60a), 4.30 (1H, m, H-20), 4.15 (1H, dd, J6 b,5 ¼ 2.5 Hz, Jgem ¼ 12.1 Hz,
MeOD): d 37.6 (C-1), 29.8 (C-2), 79.6 (C-3), 40.1 (C-4), 140.9 (C-5),
0
H-60b), 3.85 (1H, m, H-50), 3.47 (1H, m, H-3), 3.47 (1H, m, H-26eq),
3.36 (1H, dd, J26ax,25 ¼ Jgem ¼ 11.0 Hz, H-26ax), 2.10e1.85 (9H, Ac),
0.95 (3H, d, J21,20 ¼ 6.9 Hz, CH3-21), 0.88 (3H, s, CH3-19), 0.77 (3H, d,
122.1 (C-6), 31.7 (C-7), 31.9 (C-8), 50.6 (C-9), 37.2 (C-10), 21.2 (C-11),
39.2 (C-12), 40.7 (C-13), 56.9 (C-14), 32.5 (C-15), 81.5 (C-16), 62.4
(C-17), 17.3 (C-18), 19.6 (C-19), 42.1 (C-20), 14.6 (C-21), 110.1 (C-22),
32.1 (C-23), 29.0 (C-24), 30.6 (C-25), 67.3 (C-26), 16.6 (C-27), 100.1
(C-10), 71.3 (C-20), 74.9 (C-30), 57.0 (C-40), 76.4 (C-50), 62.1 (C-60), 23.0
(NHAc), 173.0 (C]O, NHAc). IR 3604 (OeH), 2941 (CeH), 1655 (C]
O), 1564 (C]C), 1564 (NeH), 1042, 904 (OeCeO). HRMS Calcd. for
C35H55NO8: 617.3928. Found: 618.4000 [M þ H]þ.
J27,25 ¼ 6.3 Hz, CH3-27), 0.74 (3H, s, CH3-18). 13C NMR (CDCl3):
d 37.1
(C-1), 29.3 (C-2), 79.4 (C-3), 38.5 (C-4), 140.2 (C-5), 121.8 (C-6), 32.0
(C-7), 31.4 (C-8), 50.0 (C-9), 36.7 (C-10), 20.8 (C-11), 39.7 (C-12),
40.2 (C-13), 56.4 (C-14), 31.8 (C-15), 80.8 (C-16), 62.0 (C-17), 16.2 (C-
18), 19.3 (C-19), 41.6 (C-20), 14.5 (C-21), 109.3 (C-22), 31.3 (C-23),
28.8 (C-24), 30.3 (C-25), 66.8 (C-26), 17.1 (C-27), 96.8 (C-10), 54.8 (C-
20), 70.9 (C-30), 69.1 (C-40), 71.7 (C-50), 62.2 (C-60), 20.8e20.5 (Ac),
170.7e169.5 (C]O, eAc),167.8 (C]O, Phth), 134.3, 131.4, 123.6 (Ar).
HRMS Calcd. for C47H61NO12: 831.4194. Found: 832.4199 [M þ H]þ.
4.7. Hecogenin 2-acetamido-2-deoxy-b-D-glucopyranoside (10)
The same procedure described above for the synthesis of 9 was
followed starting with 8 (132 mg, 0.16 mmol) to give compound 10,
after chromatography with CH2Cl2/MeOH (9:1), as a white solid
4.5. Hecogenin 3,4,6-tri-O-acetyl-2-deoxy-2-phthalimido-b-D-
glucopyranoside (8)
(71 mg, 72%): mp 249e251 ꢀC (dec); [
a]
ꢃ86ꢀ (c 1.0, CHCl3). 1H
D
0
0
0
NMR (CDCl3/MeOD): 4.56 (1H, d, J1 ,2 ¼ 7.8 Hz, H-1 ), 4.29 (1H, ddd,
The same procedure described above for the synthesis of 7 was
followed, starting from hecogenin (6, 61 mg, 0.14 mmol) to obtain
J16,17 ¼ J16,15a ¼ 14.6 Hz, J16,15b ¼ 7.3 Hz, H-16), 3.78 (1H, dd,
Jgem ¼ 11.9 Hz, J6 a,5 ¼ 2.7 Hz, H-60a), 3.72 (1H, dd, Jgem ¼ 11.9 Hz,
0
0
4.2 Hz, H-60b), 3.59 (1H, m, H-3), 3.43 (1H, ddd,
0
0
compound 8 starting from hecogenin (6), as the pure
b
-anomer
J6 b,5
¼
(98 mg, 82%) after chromatography with hexanes/EtOAc (1:1).
J26eq,25 ¼ 2.2 Hz, Jgem ¼ 11.0 Hz, J26eq,24eq ¼ 1.8 Hz, H-26eq), 3.41 (1H,
D þ26.1ꢀ (c 0.5, CHCl3). 1H NMR (CDCl3):
d
7.86 (2H, m, Ar), 7.74
dd, J4 ,5 ¼ J4 ,3 ¼ 8.4 Hz, H-4 ), 3.40 (1H, dd, J3 ,4 ¼ J3 ,2 ¼ 8.4 Hz, H-
0
0
0
0
0
0
0
0
0
[
a
]
(2H, m, Ar), 5.75 (1H, dd, J3,2 ¼ 10.5 Hz, J3,4 ¼ 9.6, H-30), 5.45 (1H, d,
J1,2 ¼ 8.4 Hz, H-10), 5.14 (1H, dd, J4,3 ¼ J4,5 ¼ 9.6 Hz, H-40), 4.32 (1H, m,
H-16), 4.30 (1H, m, H-60a), 4.28 (1H, m, H-20), 4.14 (1H, dd,
30), 3.37 (1H, dd, J2 ,3 ¼ 8.4 Hz, J2 ,1 ¼ 7.8 Hz, H-20), 3.13 (1H, dd,
J26ax,25 ¼ Jgem ¼ 11.0 Hz, H-26ax), 3.22 (1H, m, H-50), 2.45 (1H, dd,
J17,16 ¼ 6.6 Hz, J17,20 ¼ 8.4 Hz, H-17), 2.35 (1H, dd, J11ax,9 ¼ 13.2 Hz,
Jgem ¼ 14.4 Hz, H-11ax), 2.15 (1H, dd, J11eq,9 ¼ 4.8 Hz, Jgem ¼ 14.4 Hz,
H-11eq), 2.06 (1H, m, H-15a),1.99 (3H, NHAc),1.86 (1H, m, H-8),1.83
(1H, m, 2eq),1.70 (1H, m, H-20),1.05 (1H, m, H-9).1.05 (1H, m, H-14),
1.00 (3H, d, J21,20 ¼ 7.2 Hz, CH3-21), 0.99 (3H, s, CH3-18), 0.85 (3H, s,
CH3-19), 0.74 (3H, d, J27,25 ¼ 6.3 Hz, CH3-27). 13C NMR (CDCl3/
0
0
0
0
J6 b,5 ¼ 2.2 Hz, Jgem ¼ 12.1 Hz, H-60b), 3.85 (1H, m, H-50), 3.51 (1H, m,
0
H-3), 3.47 (1H, dd, J26eq,25 ¼ 6.4 Hz, Jgem ¼ 11.0, H-26eq), 3.32 (1H, dd,
J26ax,25 ¼ Jgem ¼ 11.0 Hz, H-26ax), 2.48 (1H, dd, J17,16 ¼ 6.9 Hz,
J17,20 ¼ 8.5 Hz, H-17), 2.33 (1H, dd, J11ax,9 ¼ Jgem ¼ 13.8 Hz, H-11ax),
2.16 (1H, dd, J11eq,9 ¼ 4.9 Hz, Jgem ¼ 13.8 Hz, H-11eq), 2.09e1.85 (9H,
Ac),1.04 (3H, d, J21,20 ¼ 7.0 Hz, CH3-21),1.00 (3H, s, CH3-18), 0.77 (3H,
d, J27,25 ¼ 6.4 Hz, CH3-27), 0.76 (3H, s, CH3-19). 13C NMR (CDCl3):
MeOD):
d 36.4 (C-1), 29.0 (C-2), 78.1 (C-3), 34.0 (C-4), 55.6 (C-5),
28.6 (C-6), 31.4 (C-7), 34.2 (C-8), 44.3 (C-9), 36.1 (C-10), 37.7 (C-11),
214.1 (C-12), 55.0 (C-13), 55.4 (C-14), 30.9 (C-15), 79.1 (C-16), 53.3
(C-17), 15.9 (C-18), 11.7 (C-19), 44.3 (C-20), 13.0 (C-21), 109.3 (C-22),
31.2 (C-23), 28.2 (C-24), 30.0 (C-25), 66.8 (C-26),16.9 (C-27), 99.2 (C-
10), 71.3 (C-20), 74.9 (C-30), 57.0 (C-40), 76.4 (C-50), 62.1 (C-60), 23.3
(NHAc),172.0 (C]O, NHAc). IR 3385 (OeH), 2953 (CeH),1708 (C]O
ketone), 1649 (C]O amide), 1560 (NeH), 1039, 987, 901 (OeCeO).
HRMS Calcd. for C35H55NO9: 633.3877. Found: 634.3939 [M þ H]þ.
d
36.4 (C-1), 29.0 (C-2), 79.1 (C-3), 34.2 (C-4), 55.4 (C-5), 28.7 (C-6),
31.4 (C-7), 34.2 (C-8), 44.4 (C-9), 36.0 (C-10), 37.7 (C-11), 213.4 (C-
12), 55.1 (C-13), 55.7 (C-14), 31.1 (C-15), 79.1 (C-16), 53.5 (C-17),15.9
(C-18), 11.8 (C-19), 42.2 (C-20), 13.2 (C-21), 109.2 (C-22), 31.4 (C-23),
28.1 (C-24), 30.2 (C-25), 66.9 (C-26), 17.1 (C-27), 97.0 (C-10), 54.9 (C-
20), 70.9 (C-30), 69.1 (C-40), 71.7 (C-50), 62.2 (C-60), 20.8e20.4 (Ac),
170.7e169.5 (C]O, Ac), 167.8 (C]O, Phth), 134.3, 131.4, 123.6 (Ar).
HRMS Calcd. for C47H61NO13: 847.4143. Found: 848.4216 [M þ H]þ.
4.8. Diosgenyl 2-amino-2-deoxy-b-D-glucopyranoside
4.6. Diosgenin 2-acetamido-2-deoxy-
b
-D-glucopyranoside (9)
hydrochloride (11)
Compound 7 (50 mg, 0.06 mmol) was suspended in ethanol
(4 mL) and hydrazine hydrate (0.05 mL of an 85% solution) was
Compound 7 (50 mg, 0.06 mmol) was suspended in ethanol
(4 mL) and 50 L of hydrazine hydrate (85% solution) were added
m

726
M.A. Fernández-Herrera et al. / European Journal of Medicinal Chemistry 54 (2012) 721e727
dropwise. The reaction mixture was stirred under nitrogen at reflux
for 20 h. After the completion of the reaction the solvent was
evaporated, and the amorphous solid was dried under high vacuum
to remove traces of hydrazine. The resulting pale yellow solid was
immediately dissolved in dry MeOH (2 mL) and treated with an
equivalent amount of dry HCl (from AcCl/MeOH). The solvent was
evaporated and the resulting hydrochloride was purified by chro-
matography (CH2Cl2/MeOH 9:1) to yield compound 11 as a white
(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 the adherent cells (cells
were incubated at 37 ꢀC, 5% CO2 for 1 h, and non-adherent cells
were harvested). ELPs were re-suspended into RPMI-1640 medium
at a concentration of 1 ꢁ 106 cells/mL. CFSE (from SigmaeAldrich,
USA) was added to the cell suspension at a final concentration of
12 mM and incubated for 15 min at room temperature in the dark.
solid (28 mg, 76%): mp 229e231 ꢀC (dec); [
1H NMR (CDCl3/MeOD):
a
]
D ꢃ63ꢀ (c 1.0, CHCl3).
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 phosphate-buffered saline
(PBS) containing 10% NCS, counted, and re-suspended into RPMI-
1640 medium at 1 ꢁ 106 cells/mL [11]. Unstimulated, PHA-
stimulated or treated cells were plated at 2 ꢁ 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 ꢀC 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 WinMDI software.
d
5.36 (1H, m, H-6), 4.40 (1H, m, H-10), 4.40
(1H, m, H-16), 3.85 (1H, m, H-60a), 3.70 (1H, m, H-60b), 3.59 (1H, m,
H-3), 3.45 (1H, m, H-26eq), 3.31 (1H, m, H-40), 3.31 (1H, m, H-3),
3.31 (1H, m, H-50), 3.31 (1H, m, H-26ax), 2.62 (1H, m, H-20), 2.41
(1H, m, H-4eq), 2.23 (1H, m, H-4ax), 1.03 (3H, s, CH3-19), 0.95 (3H, d,
J21,20 ¼ 7.0 Hz, CH3-21), 0.79 (3H, s, CH3-18), 0.78 (3H, d,
J27,25 ¼ 6.5 Hz, CH3-27). 13C NMR (CDCl3/MeOD):
d 38.0 (C-1), 30.4
(C-2), 79.4 (C-3), 40.5 (C-4), 141.2 (C-5), 122.5 (C-6), 32.0 (C-7), 32.3
(C-8), 51.1 (C-9), 37.6 (C-10), 21.6 (C-11), 39.4 (C-12), 41.1 (C-13), 57.3
(C-14), 32.8 (C-15), 81.8 (C-16), 63.0 (C-17), 16.7 (C-18), 19.8 (C-19),
42.5 (C-20), 14.8 (C-21), 110.4 (C-22), 32.4 (C-23), 29.4 (C-24), 31.0
(C-25), 67.6 (C-26), 17.4 (C-27), 100.2 (C-10), 71.5 (C-20), 76.9 (C-30),
62.5 (C-40), 77.4 (C-50), 54.3 (C-60). IR 3383 (OeH), 3042 (NeH),
2938 (CeH), 1550 (NeH), 1062, 1011, 897 (OeCeO). HRMS Calcd.
for C33H53NO7: 575.3822. Found: 576.3895 [M þ H]þ.
4.9.5. Statistical analysis
The median and standard deviation (SD) were calculated using
Excel (Microsoft Office, Version 2007). Statistical analysis of
differences was performed by analysis of variance (ANOVA) using
SPSS 10.0 for Windows. A p-value of less than 0.05 (Tukey’s t-test)
was considered to be significant.
4.9. Biological activity
4.9.1. Cell culture
HeLa, CaSki and ViBo 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 red phenol supple-
mented by benzylpenicillin. Cultures were maintained in a humid-
ified atmosphere with 5% CO2 at 37 ꢀC. All cell-based assays were
performed using cells in the exponential growth phase.
Acknowledgments
The authors thank CONACYT for the postdoctoral fellowship to
MAFH, for the doctoral fellowship to HLM and for grants 84380 and
166040 to JSR and MAFH, respectively. We thank VIEP-BUAP for
academic and financial support, PAPIME-UNAM for grant PE206812
to LSS and NSERC for a grant to BMP. We also thank Dr. Dionisio
Parra (Gyneco-perinatology Department) and Dr. René García
(Teaching Department) from Hospital General “Ignacio Zaragoza,”
ISSSTE for technical assistance.
4.9.2. Cell proliferation assay
Assays were performed by seeding 7500 cells/well in 96-well
tissue culture plates in a volume of 100 mL of RPMI-1640 medium
supplemented with 5% NCS per well. Cells were allowed to grow for
24 h in culture medium prior to exposure to the IC50 values
determined for 9e11. 1% of vehicle (DMSO) was added to control
cells. Antiproliferative activity (IC50) was determined after 24 h by
crystal violet staining [8]. Growth inhibition was determined by
measuring the absorbance at 590 nm in an Enzyme-linked immu-
nosorbent assay (ELISA) plate reader (Tecan, USA).
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2012.06.027.
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