Synthesis and in vitro anticancer activity of 23(23′)E-benzylidenespirostanols derived from steroid sapogenins

Article abstract of DOI:10.1016/j.steroids.2017.08.017

Benzylidenespirostanols were prepared by two-step synthesis including BF₃·Et₂O-catalyzed aldol condensation of several acetylated steroid sapogenins with benzaldehyde followed by saponification. The obtained compounds showed moderate

Full text of DOI:10.1016/j.steroids.2017.08.017

Accepted Manuscript
Synthesis and in vitro anticancer activity of 23(23’)E-benzylidenespirostanols
derived from steroid sapogenins
Manuel A. Ramos-Enríquez, Katherine Vargas-Romero, Lucie Rárová,
Miroslav Strnad, Martin A. Iglesias Arteaga
PII:
S0039-128X(17)30159-9
http://dx.doi.org/10.1016/j.steroids.2017.08.017
STE 8151
DOI:
Reference:
Steroids
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19 June 2017
22 August 2017
30 August 2017
Please cite this article as: Ramos-Enríquez, M.A., Vargas-Romero, K., Rárová, L., Strnad, M., Iglesias Arteaga,
M.A., Synthesis and in vitro anticancer activity of 23(23’)E-benzylidenespirostanols derived from steroid
sapogenins, Steroids (2017), doi: http://dx.doi.org/10.1016/j.steroids.2017.08.017
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Graphical Abstract
Synthesis and in vitro anticancer activity of 23(23´)E-benzylidenespirostanols derived
from steroid sapogenins
Manuel A. Ramos-Enríquez, Katherine Vargas-Romero, Lucie Rárová,
Miroslav Strnad, Martin A. Iglesias-Arteaga
different substition pattern
O
O
O
O
i, ii
H3CCOO
HO
5 examples
cytotoxicity against
CEM, MCF7 , Hela and BJ
H
H
i) Benzaldehyde, BF3•Et2O, CH2Cl2; ii) KOH, CH3OH

Synthesis and in vitro anticancer activity of 23(23´)E-benzylidenespirostanols derived
from steroid sapogenins
Manuel A. Ramos-Enríquez,a Katherine Vargas-Romeroa Lucie Rárová,b Miroslav Strnad,c
Martin A. Iglesias Arteagaa*
a) Facultad de Química, Universidad Nacional Autónoma de México,
Ciudad Universitaria, 04510 México, D.F.,
b) Department of Chemical Biology and Genetics, Centre of the Region Haná for
Biotechnological and Agricultural Research, Faculty of Science, Palacký University,
Šlechtitelů 27, CZ-783 71, Olomouc, Czech Republic.
c) Laboratory of Growth Regulators, Centre of the Region Haná for Biotechnological and
Agricultural Research, Palacký University & Institute of Experimental Botany ASCR,
Šlechtitelů 27, CZ-783 71, Olomouc, Czech Republic.
Corresponding author: Martin A. Iglesias-Arteaga
Facultad de Química, Universidad Nacional Autónoma de México, Ciudad Universitaria,
04510 México, D.F., México
Telephone: 52(55) 56223899 ext 44417
Fax: 52(55) 56223899 ext 44417
e-mail martin.iglesias@unam.mx

Abstract: Benzylidenespirostanols were prepared by two-step synthesis including
BF3•Et2O-catalyzed aldol condensation of several acetylated steroid sapogenins with
benzaldehyde followed by saponification. The obtained compounds showed moderate
cytotoxicity against three cancer cell lines (T-lymphoblastic leukemia cell line CEM, breast
carcinoma cell line MCF7 and cervical carcinoma cell line HeLa) and normal human
fibroblasts (BJ). The most active of the five tested substances was 3c (lowest IC50 for
MCF7 cells 19.9 ± 0.1 µM) without any selectivity towards human cancer and normal cells,
respectively.
Keywords: Aldol condensation; Benzylidenespirostanols; Cytotoxicity; Cancer cell lines,
Fibroblasts
Introduction
Cytotoxic steroids have been the focus of intensive research for more than 30 years. In the
main, a large number of naturally occurring steroids is biologically tested to assess their
antiproliferative and/or cytotoxic activity against different cancer cell lines in vitro. The
already large number of naturally occurring cytotoxic steroids is thus continuously
increasing. The diversity of these compounds is vast and includes polyhydroxy sterols [1],
saponins [2], monomeric spiroketals like steroid sapogenins (SS) [3] and hippuristanols [4]
as well as the dimmeric spiroketals, cephalostatins and ritterazines [5] amongst many
others.
The comprehensive research directed to identifying potential antitumor agents includes the
synthesis of non-natural cytotoxic steroids by introduction of structural modifications into

bioactive natural compounds and de novo design and synthesis of new steroid candidate
molecules. Both approaches have produced a plethora of synthetic cytotoxic steroids [6].
Previous studies reported that side chain modified steroid sapogenins (SS) showed
cytotoxic activity against a wide spectrum of cancer cell lines [7]. Sandoval et. al. found
that while diosgenin exhibited cytotoxic activity against cervical carcinoma cell line HeLa
(IC50 36.17 M) and CaSki (IC50 31.35 M), the oxime of 23-acetyldiosgenin showed an
increased activity against HeLa (IC50 10.9 M) and CaSki (IC50 10.51 M) [7a]. On the
other hand Santillan et. al. described a set of transformations on the SS side chain that
resulted in the synthesis of a family of 23-ethylidene-26-hydroxy-22-oxo-cholestanes that
exhibited cytotoxic activity against CEM cells (IC50 5.9 – 40.9 M) [7b].
Due to the vast structural diversity of cytotoxic steroids, finding accurate SAR is a
complicated, if not impossible, task. Nevertheless, the gathered evidence of anticancer
activities allows the association of the cytotoxicity with the presence of certain
substructures. Fuchs et. al. suggested that oxocarbenium ions, formed as a result of the
cleavage of spiroketal moieties, may act as alkylating agents of nucleophilic biomolecules
and that this underlies the cytotoxicity observed in cephalostatins and ritterazines[8].
Although this hypothesis is yet not confirmed, the large number of cytotoxic steroid
spiroketals, as well as the inverse relationship between the cytotoxicity and the enthalpy of
oxocarbenium ion formation [8b] suggest that this putative explanation may be, at least,
well oriented.
23E-Benzylidene spirostanes, a new family of steroid spiroketals derived from the
BF3•Et2O catalyzed aldol condensation of SS and benzaldehyde, that we recently described

[9a] exhibit a reactivity profile that differ from that of the parent SS and leads to different
mode of cleavage of the spiroketal moiety [9b,c]. Bearing in mind that the native steroid
sapogenins demonstrated cytotoxicity and that the presence of the 23E-benzylidene moiety
in the spiroketal side chain produces drastic changes in the reactivity of this fragment, we
decided to explore the cytotoxicity of some members of this new derivatives of SS. In this
paper, we describe the synthesis and cytotoxic activity against human cancer cells and
normal fibroblasts of 23E-benzylidenespirostanols derived from various steroid sapogenins.
Experimental
Reactions were monitored by TLC on ALUGRAM SIL G/UV254 plates from
MACHEREY-NAGEL. Chromatographic plates were sprayed with a 1% solution of
vanillin in 50% HClO4 and heated until color developed. Melting points were measured on
a Melt-Temp II instrument . Mass spectra were obtained from a Thermo-Electron
spectrometer model DFS (Double Focus Sector). NMR spectra were recorded in CDCl3 or
DMSO-d6 solutions on a Varian INOVA 400 spectrometer using the solvent signals as
reference. NMR signal assignments were carried out with the aid of a combination of 1D
1
and 2D NMR techniques that included H, 13C, COSY, Nuclear Overhauser Effect
Spectroscopy (NOESY), Heteronuclear Single Quantum Correlation (HSQC) and
Heteronuclear Multiple Bond Correlation (HMBC). Compounds 2a to 2d were obtained as
previously described [9a].
[23(23´)E,25R]-23(23´)-benzylidenspirost-7-en-3β-ol acetate (2e). Freshly distilled
BF3•Et2O (40.56 ml) was added to solution of the steroid sapogenin 2e (3.10 g, 6.8 mmol)

and benzaldehyde (1.43 g, 13.5 mmol) in dry CH2Cl2 (200 ml) and the mixture was stirred
under argon at room temperature for 4 h. The reaction mixture was washed with water (10
x 75 ml), dried (anh. Na2SO4) and evaporated to produce a syrupy residue. The obtained oil
was leached with hot methanol and the solid precipitate was washed with methanol to
1
obtain the desired product. Yield: 1.73 g, 47 %. Mp 177.9 – 179.7 °C(from acetone) H
NMR (CDCl3, 400 MHz) δ ppm 7.33 (dd, J=8.6, 6.7 Hz, 2H, H-meta), 7.25 – 7.19 (m, 3H,
H-ortho H-para), 6.57 (d, J=1.9 Hz, 1H, H-23´), 5.15 (dd, J=4.7, 2.3 Hz, 1H, H-7), 4.70 (tt,
J=11.3, 4.6 Hz, 1H, H-3), 4.52 (m, 1H, H-16), 3.65 – 3.51 (m, 2H, H-26 ax. and H-26
eq.), 2.79 (ddd, J=13.4, 4.2, 1.6 Hz, 1H, H-24 eq.), 2.58 (p, J=7.0 Hz, 1H, H-20), 2.10 (m,
1H, H-24 ax.), 1.12 (d, J=7.0 Hz, 3H, H-21), 0.84 (s, 3H, H-19), 0.80 (d, J=6.6 Hz, 3H, H-
27), 0.72 (s, 3H, H-18). 13C NMR(CDCl3, 100.53 MHz) δ ppm: δ 36.8 C-1; 27.4 C-2; 73.3
C-3, 33.8 C-4, 40.1 C-5, 29.4 C-6, 117.9 C-7, 138.4 C-8, 48.9 C-9, 34.3 C-10, 21.4 C-11,
39.6 C-12, 41.6 C-13, 54.8 C-14, 31.0 C-15, 80.2 C-16, 61.5 C-17, 16.4 C-18, 13.0 C-19,
37.7 C-20, 14.8 C-21, 110.6 C-22; 137.1 C-23, 32.9 C-24, 33.1 C-25, 66.1 C-26, 17.2 C-27,
170.7 C=O acetyl, 21.4 CH3 acetyl, 137.1 ipso, 129.1 ortho, 128.1 meta, 126.5 para, 122.9
C-23´. ESIMS m/z: 545 M++1 (2.5), 454 (26.8), 440 (13.6), 99(13.9), 85 (100.0), 69 (15.0),
55 (10.8).
General procedure for the saponification of benzylidene spirostanes: KOH (600 mg) was
added to a solution of the benzylidene spirostane 2a-e (200 mg) in methanol (10 mL) and
dioxane (5 mL) and the mixture was stirred until the starting material was consumed
(TLC). The volatile solvent was evaporated under reduced pressure and ethyl acetate (50
mL) was added. The organic phase was washed with H2O (6x25 mL) and brine (2x25 mL),
dried (anhydrous Na2SO4) and evaporated. The produced solid was purified in a

chromatographic column packed with silica gel (10 g) employing hexane/ethyl acetate (6/1
for 3a, 3b and 3d; 4/1 for 3d; 3/2 for 3c) as eluent, to afford the desired compound.
[23(23´)E,25R]-23(23´)-Benzylidene-5-spirostan-3-ol (3a) Yield 183.9 mg (99%). Mp
1
200.8 – 201.8 °C (from ethyl acetate). H NMR(CDCl3, 400 MHz) δ ppm: 7.33 (dd, J=8.2,
7.0 Hz, 2H, H-meta), 7.26 – 7.20 (m, 3H, H-ortho and H-para), 6.57 (d, J=2.2 Hz, 1H, H-
23´), 4.43 (dt, J=9.0, 7.1 Hz, 1H, H-16), 3.66 – 3.47 (m, 3H, H-3, H-26 ax. and H-26
eq.), 2.78 (ddd, J=13.3, 4.4, 1.5 Hz, 1H, H-24 eq.), 2.60 (q, J=6.9 Hz, 1H, H-20), 2.10
(ddd, J=13.5, 11.7, 1.9 Hz, 1H, H-24 ax.), 2.00 (ddd, J=12.2, 7.5, 5.2 Hz, 1H, H-15), 1.89
(dd, J=9.0, 7.1 Hz, 1H, H-17), 1.10 (d, J=6.9 Hz, 3H, H-21), 0.84 (s, 3H, H-18), 0.83 (s,
13
3H, H-19), 0.80 (d, J=6.6 Hz, 3H, H-27), 0.67 (ddd, J=12.2, 10.3, 4.1 Hz, 1H, H-9). C
NMR(CDCl3, 100.52 MHz) δppm: δ36.9 C-1; 31.5 C-2; 71.3 C-3, 38.2 C-4, 44.8 C-5, 28.6
C-6, 32.3 C-7, 35.0 C-8, 54.4 C-9, 35.6 C-10, 21.1 C-11, 40.3 C-12, 40.8 C-13, 56.3 C-14,
31.6 C-15, 80.5 C-16, 61.6 C-17, 16.6 C-18, 12.4 C-19, 37.3 C-20, 14.8 C-21, 110.6 C-22;
137.1 C-23, 32.8 C-24, 33.1 C-25, 66.1 C-26, 17.2 C-27, 137.1 ipso, 129.1 ortho, 128.1
meta, 126.6 para, 122.9 C-23´. ESIMS m/z: 505 M++1 (9.3), 454 (42.8), 440 (22.3), 99
(14.3), 98 (11.5), 85 (100), 69 (15.9), 55 (12.1).
[23(23´)E,25S]-23(23´)-Benzylidene-5-spirostan-3-ol (3b) Yield 153.8 mg (83%). Mp
1
129.6 – 130.8 °C (from CH2Cl2/acetone) H NMR(CDCl3, 400 MHz) δ ppm: 7.36 – 7.29
(m, 2H, H-meta), 7.23 (d, J=7.5 Hz, 3H, H-ortho and H-para), 6.72 (s, 1H, H-23´), 4.47 (dt,
J=8.9, 7.1 Hz, 1H, H-16), 4.15 – 4.09 (m, 1H, H-3), 4.05 (dd, J=11.0, 4.2 Hz, 1H, H-26
ax.), 3.32 (dt, J=11.2, 1.7 Hz, 1H, H-26 eq.), 2.72 (ddd, J=13.7, 5.8, 1.7 Hz, 1H, H-24 eq.),

2.49 (p, J=6.9 Hz, 1H, H-20), 2.29 (dd, J=13.7, 5.2 Hz, 1H, H-24 ax.), 1.11 (d, J=6.9 Hz,
13
3H, H-21), 0.99 (s, 3H, H-19), 0.99 (d, J=6.9 Hz, 3H, H-27), 0.85 (s, 3H, H-18). C
NMR(CDCl3, 100.52 MHz) δ ppm: δ29.9 C-1; 27.8 C-2; 67.1 C-3, 33.5 C-4, 36.5 C-5, 26.5
C-6, 26.5 C-7, 35.2 C-8, 39.8 C-9, 35.3 C-10, 20.9 C-11, 40.4 C-12, 40.9 C-13, 56.4 C-14,
31.6 C-15, 81.0 C-16, 61.8 C-17, 16.7 C-18, 23.9 C-19, 39.7 C-20, 14.7 C-21, 111.5 C-22;
136.7 C-23*, 30.8 C-24, 30.3 C-25, 65.1 C-26, 18.3 C-27, 137.2 ipso*, 129.0 ortho, 128.0
meta, 126.5 para, 125.0 C-23´. ESIMS m/z: 505 M++1 (7.9), 454 (36.7), 440 (18.8), 99
(14.5), 98 (10.1), 85 (100), 69 (15.7), 55 (12.4). * interchangeable.
[23(23´)E,25R]-23(23´)-Benzylidene-3-hydroxy-5-spirostan-12-one (3c) Yield 135.4 mg
(73%). Mp 126.2 - 127.1 °C (from CH2Cl2/MeOH).1H NMR(CDCl3, 400 MHz) δ ppm:
7.33 (t, J=7.5 Hz, 2H, H-meta), 7.26 – 7.15 (m, 3H, H-ortho and H-para), 6.55 (d, J=1.9
Hz, 1H, H-23´), 4.38 (dt, J=8.9, 6.8 Hz, 1H, H-16), 3.69 – 3.51 (m, 1H, H-3), 2.79 (dd,
J=13.4, 3.7 Hz, 1H, H-24 eq.), 2.67 (dd, J=9.1, 7.0 Hz, 1H, H-17), 2.51 – 2.37 (m, 1H, H-
20), 2.25 (dd, J=14.4, 5.0 Hz, 1H, H-11eq.), 2.12 – 2.05 (m, 1H, H-24 ax.), 1.20 (d, J=6.9
Hz, 3H, H-21), 1.11 (s, 3H, H-18), 0.91 (s, 3H, H-19), 0.80 (d, J=6.6 Hz, 3H, H-27). ). 13C
NMR(CDCl3, 100.52 MHz) δ ppm: δ 36.5 C-1; 31.2 C-2; 70.9 C-3, 37.8 C-4, 44.6 C-5,
28.3 C-6, 31.0 C-7, 34.2 C-8, 55.5 C-9, 36.1 C-10, 37.8 C-11, 213.4 C-12, 55.3 C-13, 55.7
C-14, 31.6 C-15, 78.8 C-16, 52.9 C-17, 16.2 C-18, 12.0 C-19, 37.8 C-20, 13.6 C-21, 110.6
C-22; 137.0 C-23, 32.9 C-24, 33.0 C-25, 66.1 C-26, 17.2 C-27, 137.0 ipso, 129.0 ortho,
128.0 meta, 126.6 para, 123.0 C-23´. ESIMS m/z: 541 M+Na (<1), 454 (36.1), 440 (18.3),
99 (14.0), 85 (100.0), 69 (14.8), 55 (11.2).

[23(23´)E,25R]-23(23´)-Benzylidene-5-spirost-5-en-3-ol (3d) Yield 171.8 mg (91%).
1
Mp 148.3 – 150.6 °C (from CH2Cl2/MeOH). H NMR(CDCl3, 400 MHz) δ ppm: 7.36 –
7.30 (m, 2H, H-meta), 7.25 – 7.20 (m, 3H, H-otro and H-para), 6.58 (s, 1H, H-23´), 5.35
(m, 1H, H-6), 4.46 (dt, J=9.0, 7.0 Hz, 1H, H-16), 3.68 – 3.44 (m, 3H, H-3, H-26 ax and H-
26 eq.), 2.78 (dd, J=13.3, 3.5 Hz, 1H, H-24 eq.), 2.60 (p, J=7.0 Hz, 1H, H-20), 2.10 (ddd,
J=13.5, 11.8, 2.0 Hz, 1H, H-24 ax.), 1.11 (d, J=6.9 Hz, 3H, H-21), 1.03 (s, 3H, H-19), 0.86
(s, 3H, H-18), 0.80 (d, J=6.5 Hz, 3H, H-27). 13C NMR(CDCl3, 100.52 MHz) δ ppm: δ 37.2
C-1; 31.6 C-2; 71.7 C-3, 42.3 C-4, 140.8 C-5, 121.4 C-6, 32.1 C-7, 31.4 C-8, 50.1 C-9, 36.7
C-10, 20.9 C-11, 40.0 C-12, 40.5 C-13, 56.5 C-14, 31.7 C-15, 80.4 C-16, 61.5 C-17, 16.5
C-18, 19.4 C-19, 37.3 C-20, 14.9 C-21, 110.6 C-22; 137.1 C-23, 32.9 C-24, 33.1 C-25, 66.1
C-26, 17.2 C-27, 137.1 ipso, 129.1 ortho, 128.1 meta, 126.5 para, 122.9 C-23´. ESIMS m/z:
541 M+K (<1), 454 (39.8), 440 (25.9), 99 (15.1), 98 (17.0), 97 (13.7), 95 (10.6), 85 (100.0),
69 (15.7), 55 (12.1).
[23(23´)E,25R]-23(23´)-Benzylidene-5-spirost-7-en-3-ol (3e) Yield 124.4 mg (67%).
Mp 156.1 - 158.3 (from CH2Cl2/hexane).1H NMR(CDCl3, 400 MHz) δ ppm: 7.33 (t, J=7.6
Hz, 2H, H-meta), 7.25 – 7.20 (m, 3H, H-ortho and H-para), 6.57 (s, 1H, H-23´), 5.16 (m,
1H, H-7), 4.52 (m, 1H, H-16), 3.68 – 3.43 (m, 2H, H-26 ax. and H-26 eq.), 2.79 (m, 1H,
H-24 eq.), 2.58 (q, J=7.0 Hz, 1H, H-20), 1.12 (d, J=6.8 Hz, 3H. H-21), 0.82 (s, 3H, H-19),
0.80 (d, J=6.9 Hz, 3H, H-27), 0.72 (s, 3H, H-18). 13C NMR(CDCl3, 100.52 MHz) δ ppm: δ
37.1 C-1; 31.4 C-2; 71.0 C-3, 37.9 C-4, 40.3 C-5, 29.6 C-6, 118.0 C-7, 138.5 C-8, 49.1 C-9,
34.3 C-10, 21.5 C-11, 39.6 C-12, 41.6 C-13, 54.9 C-14, 31.0 C-15, 80.2 C-16, 61.5 C-17,
16.4 C-18, 13.1 C-19, 37.8 C-20, 14.8 C-21, 110.6 C-22; 137.1 C-23*, 32.9 C-24, 33.1 C-

25, 66.1 C-26, 17.2 C-27, 137.0 ipso*, 129.1 ortho, 128.1 meta, 126.5 para, 122.9 C-23´.
ESIMS m/z: 541 M+K (<1), 454 (46.7), 440 (25.8), 99 (14.0), 85 (100), 69 (15.5), 55
(11.3). * interchangeable.
Biological tests
Cell culture
Stock solutions (10 mmol/L) of the tested compounds were prepared by dissolving an
appropriate quantity of each substance in dimethylsulfoxide (DMSO). Dulbecco’s modified
Eagle’s medium (DMEM), fetal bovine serum (FBS), L-glutamine, penicillin and
streptomycin were purchased from Sigma Aldrich (MO, USA). Calcein AM was obtained
from Molecular Probes (ThermoFisher, MA, USA). The screening cell lines (T-
lymphoblastic leukemia cell line CEM, breast carcinoma cell line MCF7, cervical
carcinoma cell line HeLa and human fibroblasts BJ) were obtained from the American
Type Culture Collection (Manassas, VA, USA). CEM cell line was cultured in RPMI 1640
medium with 20 % fetal bovine serum and all other cells were cultured in DMEM medium
(Sigma Aldrich, MO, USA), supplemented with 10% fetal bovine serum, L-glutamine (2
mmol/L), penicillin and streptomycin (1 %). The cell lines were maintained under standard
cell culture conditions at 37 ºC and 5% CO2 in a humid environment. Cells were
subcultured twice or three times a week using the standard trypsinization procedure.
Calcein AM assay
Suspensions of tested cell lines (ca. 1.0 × 105 cells/mL) were placed in 96-well microtiter
plates and after 24 h of stabilization (time zero) the tested compounds were added (in three
20 µL aliquots) in serially diluted concentrations in dimethylsulfoxide (DMSO). Control

cultures were treated with DMSO alone, and the final concentration of DMSO in the
incubation mixtures never exceeded 0.6%. The test compounds were typically evaluated at
six 3-fold dilutions and the highest final concentration was generally 50 µM. After 72 h
incubation, Calcein AM solution (100 µL, Molecular Probes, ThermoFisher, MA, USA)
was added, and incubation was continued for another hour. The fluorescence of viable cells
was then quantified using a Fluoroskan Ascent instrument (Labsystems, Finland). The
percentage of surviving cells in each well was calculated by dividing the intensity of the
fluorescence signals from the exposed wells by the intensity of signals from control wells
and multiplying by 100. These ratios were then used to construct dose-response curves
from which IC50 values, the concentrations of the respective compounds that were lethal to
50% of the tumor cells, were calculated. All the experiments were repeated three times in
triplicates.
Results and discussion
In a previous communication we described that treatment of the native steroid sapogenins
with BF3•Et2O and benzaldehyde in CH2Cl2 afforded good yields of the acetylated
benzylidenespirostanols 2a-d [9a]. When this methodology was applied to 7-tigogenin
acetate (1e), a new acetylated 23E-benzylidenespirostane 2e was obtained in a moderate
yield. As previously reported for 2a-d [9a], the observation of the H-20↔H-23' NOE effect
allows the easy verification of the E configuration of the benzylidene in compound 2e.
Saponification of 2a-e with KOH in methanol led to the desired 23E-
benzylidenespirostanols 3a-e in good to excellent yields. (Scheme 1).

R4
R4
R4
NOE
O
O
O
H
R3
R3
R3
R2
R2
R2
O
O
O
H
i
ii
H3CCOO
H3CCOO
HO
R1
R1
R1
1a R1= H, R2= H,H, R3=CH3, R4=H
1b R1= H, R2= H,H, R3=H, R4=CH3
1c R1= H, R2= O, R3=CH3, R4=H
2a R1= H, R2= H,H, R3=CH3, R4=H
2b R1= H, R2= H,H, R3=H, R4=CH3
2c R1= H, R2= O, R3=CH3, R4=H
3a R1= H, R2= H,H, R3=CH3, R4=H
3b R1= H, R2= H,H, R3=H, R4=CH3
3c R1= H, R2= O, R3=CH3, R4=H
NOE
O
O
O
H
O
O
O
H
i
ii
H3CCOO
H3CCOO
HO
1d
2d
3d
NOE
O
O
O
H
O
O
O
H
i
ii
H3CCOO
H3CCOO
HO
H
H
H
1e
2e
3e
i) Benzaldehyde, BF3•Et2O, CH2Cl2; ii) KOH, CH3OH
Scheme 1. Synthesis of benzylidene spirostanols 3a-e.
Antiproliferative activities in vitro
The in vitro antiproliferative activities of the studied compounds were tested in cancer cell
lines of various histopathological origins, including T-lymphoblastic leukaemia CEM,
breast adenocarcinoma MCF7, and cervical carcinoma HeLa lines. The results were
compared with the cytotoxicity towards normal human BJ fibroblasts. The cells of all of
these lines were exposed to six serial 3-fold dilutions of each drug for 72 h, proportions of
surviving cells were then estimated and IC50 values (50% inhibitory concentrations)
calculated. The results from Calcein AM assays are presented in Table 1. All tested
compounds showed moderate cytotoxic activity towards all cell lines including normal
human fibroblasts. The most active of the five substances was 3c (lowest IC50 for MCF7
cells 19.9 ± 0.1 µM). None of the benzylidenespirostanols was selective for any cell line.

Table 1 IC50 (µM) values obtained from Calcein AM assays using three cancer cell lines
(CEM, MCF7 and HeLa) and normal human fibroblasts (BJ) treated with tested substances
for 72 h.
IC50 (µM)
Compound
CEM
MCF7
HeLa
BJ
26.8 ± 2.4
31.5 ± 5.1
23.2 ± 0.4
28.0 ± 1.3
28.9 ± 2.8
33.0 ± 4.2
44.7 ± 1.6
19.9 ± 0.1
40.3 ± 3.3
36.6 ± 4.7
36.9 ± 3.1
42.6 ± 2.5
35.7 ± 3.5
41.0 ± 3.5
39.2 ± 7.1
41.9 ± 1.4
42.9 ± 0.1
45.3 ± 2.3
44.1 ± 1.2
41.6 ± 1.5
3a
3b
3c
3d
3e
Although these results are preliminary and the differences are small, a tendency can be
seen. Naturally occurring steroids bearing an oxygenated substituent in ring C like
cephalostatine-1, ritterazine-F [5b], porrigenin C [3e] and hippuristanols [4e] are among the
most cytotoxic steroids and compared with the partner bearing a non-oxygenated ring C,
they showed increased cytotoxicity. This suggests that in the natural products the presence
of an oxygenated substituent in ring C can lead to enhanced cytotoxicity. Interestingly the
most active of the studied compounds was 3c that bears a carbonyl group at position C-12
on ring C. Additionally, the obtained results suggest that compounds bearing a trans-fused
AB ring system with a saturated B ring (3a and 3c) exhibit higher activity than those with a
cis-fused AB core (3b) or an unsaturated B ring (3d and 3e).
Conclusions
We found that benzylidenespirostanols readily obtained from steroid sapogenins by
condensation of spirostanes and benzaldehyde followed by saponification, exhibit moderate
cytotoxicity towards three different human cancer cell lines and normal human fibroblasts.
Further experiments to explore the effects of the modification of the of benzylidene moiety
on cytotoxicity are in progress.

Acknowledgments
The authors acknowledge the financial support provided by Dirección General de Asuntos
del Personal Académico (Project DGAPA- IN212417), the Faculty of Chemistry-UNAM
(PAIP-5000-9063) and grants 14-27669P and 17-14007S (LR and MS). Thanks are due to
CONACyT-México for the scholarship granted to M.A.R-E. We thank O. Hustáková for
the excellent technical assistance.
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Highlights




Condensation of spirostanes and benzaldehyde led to benzylidenspirostanes
Saponification of benzylidenspirostanes led to benzylidenspirostanols
New benzylidenspirostanols showed cytotoxicity against three cancer cell lines
The most active compound bears a trans AB core and a carbonyl group at C-12