Solvent-free synthesis of 6β-phenylamino-cholestan-3β,5α-diol and (25R)-6β-phenylaminospirostan-3β,5α-diol as potential antiproliferative agents

Article abstract of DOI:10.1016/j.steroids.2017.08.008

In this paper is described a synthetic route to 6β-phenylamino-cholestan-3β,5α-diol and (25R)-6β-phenylaminospirostan-3β,5α-diol, starting from cholesterol and diosgenin, respectively. The products were obtained in two steps by epoxidation followed by aminolysis, through an environmentally friendly and solvent-free method mediated by SZ (sulfated zirconia) as catalyst. The use of SZ allows chemo- and regioselective ring opening of the 5,6α-epoxide during the aminolysis reaction eliminating the required separation of the epoxide mixture. The products obtained were spectroscopically characterized by ¹H, PENDANT ¹³C NMR and HETCOR experiments, and complemented with FTIR-ATR and HRMS. The antiproliferative effect of the β-aminoalcohols was evaluated on MCF-7 cells after 48 h of incubation, by MTT and CVS assays. These methodologies showed that both compounds have antiproliferative activity, being more active the cholesterol analogue. Additionally, the cell images obtained by Harris’ Hematoxylin and Eosin (H&E) staining protocol, evidenced formation of apoptotic bodies due to the presence of the obtained β-aminoalcohols in a dose-dependent manner.

Full text of DOI:10.1016/j.steroids.2017.08.008

Steroidsxxx(xxxx)xxx–xxx
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Solvent-free synthesis of 6β-phenylamino-cholestan-3β,5α-diol and (25R)-
6β-phenylaminospirostan-3β,5α-diol as potential antiproliferative agents
Delia Soto-Castroa, Roberto Carlos Lara Contrerasb, María Pina-Cansecoc, Rosa Santillánd,
María Teresa Hernández-Huertae, Guillermo E. Negrón Silvaf, Eduardo Pérez-Camposc,e
,
Susana Rincónb,⁎
a
CONACyT-Instituto Politécnico Nacional, CIIDIR Unidad Oaxaca, Hornos 1003, Santa Cruz Xoxocotlán, Oaxaca C.P. 771230, Mexico
Departamento de Ingeniería Química-Bioquímica, Instituto Tecnológico de Mérida, Av. Tecnológico S/N, 97118 Mérida, Yucatán, Mexico
b
c
Centro de Investigación Facultad de Medicina UNAM-UABJO, Facultad de Medicina y Cirugía, Universidad Autónoma “Benito Juárez” de Oaxaca, Ex Hacienda de
Aguilera S/N, Carretera a San Felipe del Agua, C.P. 68020 Oaxaca, Mexico
d
Departamento de Química, Centro de Investigación y de Estudios Avanzados del IPN, México, D.F, Apdo. Postal 14-740, 07000, Mexico
Unidad de Bioquímica e Inmunología, División de Estudios de Posgrado e Investigación, Instituto Tecnológico de Oaxaca, Av. Ing. Víctor Bravo Ahuja #125 esq, Clz.
e
Tecnológico, C.P. 68030 Oaxaca, Mexico
f
Departamento de Ciencias Básicas y Departamento de Química, UAM, Av. San Pablo No 180, C.P. 02200 México D.F., Mexico
A R T I C L E I N F O
A B S T R A C T
Keywords:
Diosgenin
Cholesterol
β-Aminoalcohols
Sulfated zirconia
Antiproliferative activity
In this paper is described a synthetic route to 6β-phenylamino-cholestan-3β,5α-diol and (25R)-6β-phenylami-
nospirostan-3β,5α-diol, starting from cholesterol and diosgenin, respectively. The products were obtained in two
steps by epoxidation followed by aminolysis, through an environmentally friendly and solvent-free method
mediated by SZ (sulfated zirconia) as catalyst. The use of SZ allows chemo- and regioselective ring opening of the
5,6α-epoxide during the aminolysis reaction eliminating the required separation of the epoxide mixture. The
products obtained were spectroscopically characterized by 1H, PENDANT 13C NMR and HETCOR experiments,
and complemented with FTIR-ATR and HRMS. The antiproliferative effect of the β-aminoalcohols was evaluated
on MCF-7 cells after 48 h of incubation, by MTT and CVS assays. These methodologies showed that both
compounds have antiproliferative activity, being more active the cholesterol analogue. Additionally, the cell
images obtained by Harris’ Hematoxylin and Eosin (H & E) staining protocol, evidenced formation of apoptotic
bodies due to the presence of the obtained β-aminoalcohols in a dose-dependent manner.
1. Introduction
aminoalcohol groups at several positions of the steroidal core with
hydroxy-phenylmethylamine,
5-chloro-2-hydroxyl-phenylmethylamine,
Cancer is a major public health problem in many parts of the world
and according to the last statistics published; it is a leading cause of
death worldwide, accounting for 8.2 million deaths in 2012. The
number of new cancer cases is expected to rise by about 70% over the
next two decades [1], being breast cancer the major cause of hospital
morbidity by malignant tumors in the population aged 20 and over
(18.7%) for 2013.
Steroids are important natural products showing a wide range of bio-
logical activities and physiological effects, some important members of this
class of compounds are sexual hormones, bile acids, adrenocortical and
vitamin D, among others [2]. Throughout history, these molecules have
been raw materials for commercially important steroidal drugs [3,4]. Spe-
cially, steroidal β-aminoalcohol derivatives have been refocused as study
element due to their biological properties. For example, insertion of β-
among other amines, has led to antiparasitic, antibacterial, antimycotic
and/or antiviral drugs. Although the patented methodologies to obtain
them implied a long reaction sequence [5], the steroidal β-aminoalcohols
showed lower hormonal activity and better permeability due to the pre-
sence of the basic nitrogen group. Other works have reported the anti-
proliferative activity against leukemia by 2β-(N-substituted piperazino)-5α-
androstane-3α,17β-diols [6–8]. Yousuf et al. [9] described the chemical
modification of Withaferin A, a natural steroid, to obtain a 3β-amino-4β-
hydroxy derivative. The compound showed good in vitro cytotoxicity at
100 µM against Lung (A-549), Ovary (IGR-OV-1), Colon (CoLo-205) and
Prostate (PC-3) cancer cell lines.
Hewet et al. [10] reported the synthesis of several 5α-hydroxy-6β-
amino steroids, where the amino groups were derived from morpholine,
piperidine, ammonia or butylamine. The β-aminoalcohols showed
⁎
Corresponding author.
E-mail address: susana74a@yahoo.com (S. Rincón).
http://dx.doi.org/10.1016/j.steroids.2017.08.008
Received 7 May 2017; Received in revised form 30 July 2017; Accepted 15 August 2017
0039-128X/©2017PublishedbyElsevierInc.
Pleasecitethisarticleas:Soto-Castro,D.,Steroids(2017),http://dx.doi.org/10.1016/j.steroids.2017.08.008

D. Soto-Castro et al.
Steroidsxxx(xxxx)xxx–xxx
anesthetic, sedative or hypotensive properties. Nonetheless, the synth-
esis of them implied several hours or days under reflux in the corre-
sponding amine. These types of compounds were reinvestigated by
Anderson et al., where the proposed methodology greatly reduces the
time reaction to 4.5 h at 120–130 °C in ethylene glycol [11].
2.2. General procedure for the synthesis of sulfated zirconia
The SZ was synthesized as previously reported by Negrón et al. [16].
Zirconium n-propoxide (20 mL, 70% n-propanol) was mixed with n-
propanol (30 mL) and stirred with a magnetic bar. Then, an acid so-
lution (1 mL 98% sulfuric acid in 3.2 mL of distilled water) was added
dropwise in order to cause the hydrolysis and gelation of the zirconium
n-propoxide. The solid was filtered and dried at 80 °C until complete
alcohol evaporation, then calcinated in air at 600 °C for 6 h.
Recently, de Medina et al. [12] reported the potent biological
properties of a set of products obtained by catalytic aminolysis of 5,6α-
epoxy-cholesterol with different polyamines. In the anticancer line, 5α-
hydroxy-6β-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3β-ol known
as dendrogenin A is a β-aminoalcohol that, at a nanomolar concentra-
tion, induces the dead of tumor cell lines of metastatic melanoma and
breast cancer. Additionally, Poirot’s group showed [13] that den-
drogenin A is a natural metabolite occurring in mammals as an enzy-
matic product of the conjugation of 5,6α-epoxycholesterol and hista-
mine. Its activity, as tumor suppressor metabolite, was found to be
related to the selective inhibition of cholesterol epoxide hydrolase and
it triggered tumor re-differentiation and growth control in mice, and
hence it improved animal survival. Although these compounds could be
natural, they were synthesized by regioselective aminolysis of 5,6α-
epoxycholesterol with lithium perchlorate in anhydrous ethanol under
reflux for 4 days [12].
In this context, the classical synthesis to obtain β-aminoalcohols [14]
involves epoxide aminolysis at high temperatures, the use of expensive
chemicals, long reaction times, and usually high selectivity is difficult to
attain, as described before. Because of this, the research has been directed to
remedying these problems using solid catalysts. In this context, sulfated
zirconia has received much attention as solid catalyst, due to its acid and
basic catalytic properties and because the reactions can be carried out under
solvent free conditions avoiding environmental problems. For this purpose,
sulfated zirconia (SZ) has been used in selective epoxide aminolysis, a
methodology showing experimental simplicity, solvent free and mild reac-
tion conditions. Also, SZ is an inexpensive and reusable catalyst that pro-
vides products in high yields [15,16].
Several cell lines that originated from carcinomas of human patients
are available for use in basic studies of human cancer. Among them, the
MCF-7 cell line is used in apoptosis studies because it has a genetically
defective form of caspase 3, an important apoptosis- inducing effector
molecule [17]. MCF-7 is a stable epithelial cell line originally obtained
from the pleural effusion of a female patient with metastatic breast
cancer whose disease responded to hormone therapy. These cells have
substantial amounts of estrogen receptor [18].
Due to the wide range of biological activities that steroidal ami-
noalcohols have and taking in consideration the harsh condition and
long reaction times to obtain them, there is a need to implement novel
synthetic methodologies to obtain this kind of compounds. So, this
work was focused on the development of an alternative route for ami-
nolysis by using SZ, which provided selective α-epoxide cleavage
under solvent-free conditions making it environmentally friendly.
Additionally, in vitro antiproliferative activity was evaluated for β-
aminoalcohol derivatives from cholesterol 5 and diosgenin 6 against
MCF-7 cancer line by Crystal violet staining (CVS) and 3-(4,5-di-
methylthiazo-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays.
2.3. Synthesis of compounds
2.3.1. General procedure for the synthesis of steroidal epoxides
The compounds were prepared according to the protocols reported
by Yadav et al. [19] and de Medina et al. [12] with slight modifications.
To a 150 mL round-bottomed flask equipped with a magnetic stirrer,
were added 1.00 g of steroid and 20 mL of CH2Cl2. The solution was
poured onto an ice bath at 0
2 °C. Afterwards, 1.5 equivalents of m-
chloroperbenzoic acid (m-CPBA) were added and the reaction was
stirred 30 min. Crushed ice was added and the aqueous layer was ex-
tracted 3 times with CH2Cl2. The organic layer was washed with
NaHCO3 solution until pH 7 was reached, dried with Na2SO4 and
concentrated in vacuum.
2.3.1.1. 5,6α-Epoxy-5α-cholestan-3β-ol (3a):5,6β-epoxy-5β-cholestan-3β-
ol (3b). Cholesterol (1) (1.50 g, 3.88 mmol) was reacted with 1.00 g
(5.79 mmol) of m-CPBA according to the general procedure and 1.53 g
(3.79 mmol, 98%) of white solid product were obtained as a mixture of
isomers in a 5:1 ratio of α:β epoxides. The structure of the compounds
was confirmed by comparison with NMR data previously reported [12]
(S1.1).
2.3.1.2. (5,6α,25R)Epoxy-spirostan-3β-ol (4a):(5,6β,25R)epoxy-spirostan-3β-
ol (4b). Diosgenin (2) (1.50 g, 3.61 mmol) was reacted with 0.93 g
(5.41 mmol) of m-CPBA according to the general procedure and 1.49 g
(3.46 mmol, 96%) of white solid product were obtained as a mixture of
isomers in a 5:1 ratio of α to β epoxides. The structure of the compounds
was confirmed by comparison with NMR data previously reported [20,21]
(Supporting information S1.2).
2.3.2. General procedure for the synthesis of steroidal β-aminoalcohols
The steroidal mixture of α and β epoxides (5:1 ratio) and sulfated
zirconia (SZ, previously heated to 110 °C for 12 h), in a 2:1 wt ratio,
were placed in a 25 mL round-bottom flask, which was purged with N2.
Over these, 4 equivalents of aniline were added and the reaction was
stirred 6 h at 120 °C. Once the reaction was completed and room tem-
perature was reached, the mixture was diluted with CH2Cl2 and filtered
to recover the SZ. The crude product was purified by column chroma-
tography on silica gel using increasing polarity from hexane to hexane/
EtOAc.
2.3.2.1. 6β-Phenylamino-cholestan-3β,5α-diol (5). 300 mg (0.75 mmol)
of epoxide mixture (3a, 3b) were reacted with 0.27 mL (3.00 mmol) of
aniline catalyzed by 150 mg of SZ. After purification by column
chromatography (product 5 eluted with hexane/EtOAc 8:2), 298 mg
(0.60 mmol, 97%) of a white solid was obtained. m.p. 191.3–192.5 °C,
lit. m.p. 202 °C [22] FT-IR (ATR, cm−1): 3524 (NH); 3407 (OH); 2945
(CH). 1H NMR (500 MHz, CDCl3) δ (ppm)): 7.15 (2H, t, J = 7.8, H-29);
6.66 (1H, t, J = 7.3, H-31); 6.58 (2H, d, J = 7.9, H-30); 4.07 (1H, m, H-
3); 3.69 (1H, d, J = 8.5, H-NH); 3.35 (1H, s, H-6); 2.02 (2H, m, H-4);
1.22 (3H, s, H-19); 0.9 (3H, d, J = 6.4, H-21); 0.86 (3H, d, J = 6.4, H-
26); 0.85 (3H, d, J = 6.4, H-27); 0.68 (3H, s, H-18). 13C NMR
(126 MHz, CDCl3) δ (ppm): 147.8 (C-28); 129.4 (C-30 and C-32);
117.2 (C-31); 113.0 (C-29); 113.0 (C-33); 77.1 (C-5); 67.8 (C-3); 59.5
(C-6); 56.3 (C-17); 55.9 (C-14); 46.0 (C-9); 43.8 (C-13); 41.8 (C-4); 39.6
(C-24); 40.0 (C-12); 38.8 (C-10); 36.2 (C-22); 35.9 (C-20); 31.8 (C-1);
2. Experimental
2.1. General
All chemicals were obtained from commercial sources. Unless stated
otherwise, these were used without further purification. CH2Cl2 was
dried by distillation over CaH2. NMR spectra were recorded on a JEOL
ECA+500 instrument in CDCl3 as solvent. Chemical shifts are reported
in parts per million (ppm) and coupling constants are expressed in Hz.
Mass spectra were recorded with an Agilent Technologies MS TOF using
the ESI (+) technique. The absorbance of 96-well plates for crystal
violet and MTT was determined in a plate reader (Thermo Scientific,
USDA) at 595 and 570 nm, respectively.
2

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30.8 (C-7); 30.7 (C-8); 33.2 (C-2); 28.3 (C-16); 28.1 (C-25); 24.3 (C-15);
24.0 (C-23); 22.9 (C-27); 22.7 (C-26); 21.9 (C-11); 18.7 (C-21); 18.3 (C-
48 h after incubation with six concentrations of compound 5 or 6.
Briefly, the medium was removed and replaced with 50 µL of glutar-
aldehyde. After 10 min incubation at room temperature, cells were
washed with water, dried and incubated with 50 µL of 0.5% crystal
violet for 20 min at room temperature. Cells were washed with water,
dried and then 50 µL of 10% acetic acid was added. Stained cells were
subjected to spectrophotometric quantification (OD 620 nm) using a
plate reader (Thermo Scientific™ Multiskan™ FC microplate photometer
with incubator). All data were obtained from 9 independent experi-
19); 12.4 (C-18). HR-ESI- TOF (m/z): calcd for C33H53NO2+H+
,
496.4154; found 496.4150.
2.3.2.2. (25R)-6β-Phenylaminospirostan-3β,5α-diol (6). 300 mg (0.70 mmol)
of epoxide mixture (4a, 4b) were reacted with 0.25 mL (2.80 mmol) of
aniline catalyzed by 150 mg of SZ. After purification by column
chromatography (product
6 eluted with hexane/EtOAc 8:2), 205 mg
(0.39 mmol, 68%) of white solid was obtained. m.p. 222.8–223.8 °C, lit.
m.p. 243 °C [22]. FT-IR (ATR, cm−1): 3323 (NH); 2929 (CH). 1H NMR
(500 MHz, CDCl3) δ (ppm)): 7.14 (2H, t, J = 7.8, H-29); 6.67 (1H, t, J = 7.3,
H-31); 6.56 (2H, d, J = 8.0, H-30); 4.37 (1H, dd, J = 14.7, 7.6, H-16); 4.02
(1H, m, H-3); 3.66 (1H, s, H-NH); 3.46 (1H, dd, J = 19.6, 8.4, H-26eq); 3.37
(1H, t, J = 11.2, H-26ax); 3.31 (1H, s, H-6); 1.20 (3H, s, H-19); 0.95 (3H,
J = 6.9, H-21); 0.79 (3H, s, H-18); 0.77 (3H, d, J = 6.3, H-27). 13C NMR
(126 MHz, CDCl3) δ (ppm): 147.9 (C-28); 129.4 (C-30); 117.4 (C-31); 113.2
(C-29); 109.3 (C-22); 80.8 (C-16); 67.7 (C-3); 66.9 (C-26); 77.0 (C-5); 62.2 (C-
17); 59.6 (C-6); 55.7 (C-14); 45.8 (C-9); 40.0 (C-4); 41.7 (C-20); 40.8 (C-13);
41.7 (C-12); 38.8 (C-10); 32.2 (C-1); 30.8 (C-7); 31.8 (C-15); 31.8 (C-2); 31.5
(C-23); 30.4 (C-8); 30.4 (C-25); 28.8 (C-24); 21.1 (C-11); 18.3 (C-19); 17.2 (C-
27); 16.8 (C-18); 14.6 (C-21). HR-ESI-TOF (m/z): calcd for C33H49NO4+H+,
524.3739; found 524.3734.
ments and expressed as the mean
standard deviation.
2.4.4. MTT assay
In vitro cytotoxicity of compounds 5 and 6 was investigated using
the MTT assay. MTT is reduced by metabolically active cells and is
therefore a measure for cell viability. In this investigation, MTT assay
was performed using a slight modification of Mossman method [25]. In
brief, 48 h after treating MCF-7 cells with the six concentrations of
compound 5 or 6 in 96 well plates, 20 µL of MTT solution at 1 mg/ml in
PBS were added into each well containing treated and control cells
(only medium added). Cells were then incubated for 4 h at 37 °C with
6% CO2, 95% air and complete humidity. After 4 h, the MTT solution
was remove and replaced with 50 µL of acidified isopropanol into each
well. The optical density (OD) of the well was determined using a plate
reader at a test wavelength of 595 nm. The relative cell viability (%)
related to control cells was calculated by [A] samples/[A] con-
trol × 100, where [A] samples was the absorbance of the test sample
and [A] control was the absorbance of wells with only medium added.
All data were obtained from 9 independent experiments and expressed
2.4. Cell culture
The breast cancer cell line MCF-7 was obtained from American Type
Collection (ATCC, Manassas, VA, USA). The cell line was cultivated
routinely in 25 T-flask in RPMI medium supplemented with 10% FBS,
100 U/mL penicillin and 100 µg/ml streptomycin. The T-flasks were
incubated at 37 °C in 6% CO2, 95% air and complete humidity. Once
the cells reached ∼90% confluency they were washed with PBS and
detached using 5 mL of trypsin-EDTA 0.05% for 5 min. Trypsin was
inactivated by the addition of 5 mL of medium. The suspension was
centrifuged at 1200 rpm for 5 min. The pellet was suspended in fresh
medium and sub-cultured at 1:4 split ratio or used in the experimental
investigation. Cell concentration and viability were routinely measured
by trypan blue exclusion using hemocytometer.
as the mean
standard deviation. The viability versus concentration
curves were graphed by the Microsoft Excel program.
2.4.5. Detection of apoptotic cells
Harris’ Hematoxylin and Eosin (H & E) staining protocol was carried
out for detection of apoptotic cells. Hematoxylin is used to dye cell
nuclei blue and eosin is used to stain cytoplasm and other non-nuclear
components in cells pink and orange [26,27]. The method was per-
formed using the method described by Sigma-Aldrich [28]. Approxi-
mately 30,000 cell/well were seeded in a 6-wells plate, a day before cell
treatment. In the day treatment, the six concentrations of compounds 5
and 6 were added and the plates incubated for 48 h at normal culture
conditions. The medium was then removed and replaced with absolute
alcohol for 3 min, 95% alcohol for 2 min and 70% alcohol for 3 min.
The wells were washed gently in distilled water, stained in Harris he-
matoxylin solution for 5 min and washed in running tap water for
5 min. Acid alcohol (3% hydrochloric acid in 95% ethanol) was then
added for 30 s and washed with running tap water for 1 min. Saturated
lithium carbonate solution was added for 1 min and washed in running
tap water for 5 min. The wells were rinsed with 95% alcohol for 2 min
and counterstain in eosin solution for 30–60 s. Following eosin staining,
the samples were dehydrated through 95% alcohol and two changes of
absolute alcohol, 5 min each. The cells were observed on a Leica mi-
croscope.
2.4.1. Tests compounds preparation
Six concentrations of both compound 5 and 6 were assessed. The
stocks were prepared by first dissolving the compounds in 100% di-
methyl sulfoxide (DMSO). Serial dilutions were then prepared in cul-
ture medium immediately prior to each assay. The final concentrations
in medium were 1.81, 0.90, 0.45, 0.22, 0.11 and 0.05 µM for compound
5 and 1.025, 0.51, 0.25, 0.12, 0.06 and 0.03 µM for compound 6. DMSO
concentration in all the solutions was adjusted to < 0.1%.
2.4.2. Treatment with test chemicals
Crystal violet staining and MTT assays were carried out to assess the
effects of β-aminoalcohol derivatives on proliferation of MCF-7 cells.
For both methods, the cells were trypsinized and seeded into 96-well
microplates at a density of 5000 cells/well (100 µl/well). Cells were
then incubated for 24 h under normal culture conditions to allow cell
adhesion. After the incubation, medium containing the six increasing
concentrations of the investigated compounds were added to the wells.
Control cells were exposed only with DMSO. The plates were then in-
cubated for 48 h under normal culture conditions until CVS or MTT
assays.
2.5. Statistical analysis
Data are presented as the mean value standard deviation (S.D.).
The study was conducted in at least nine independent runs. The sta-
tistical analysis for CVS and MTT assays was performed with Minitab 15
software using a Two-way ANOVA test followed by multiple compar-
isons test.
2.4.3. Crystal violet assay
Cell proliferation was assessed using the CVS assay. In this method,
the Crystal Violet dye binds to DNA of eukaryotic cells [23]. The
amount of dye bound permits the estimation of all adherent cells, ir-
respective of their viability [24]. We used the method described by
Saotome et al. [23] with some modifications. The assay was performed
3. Results and discussion
3.1. Chemistry
Due to the potential as anticancer agents and other biological activities,
3

D. Soto-Castro et al.
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Scheme 1. Synthesis of β-aminoalcohol derivatives 5 and 6 from cholesterol 1 and diosgenin 2.
Fig. 1. 1H NMR spectra of a) cholesterol 5,6-epoxides, b) aminolysis reaction and c) 6β-phenylamino-cholestan-3β,5α-diol (5).
the synthesis of β- aminoalcohols is an important topic. Hence, with the aim
to obtain a steroidal drug with potential anticancer activity that could have
a good compromise among pharmacokinetic behavior, cellular uptake,
target specificity, and safety we started the synthesis with cholesterol 1 [29]
and diosgenin 2 [30]. The synthesis was achieved in two steps, 5,6-epoxide
formation and subsequent aminolysis (Scheme 1).
The oxidation of cholesterol and diosgenin with m-chloroperbenzoic
acid (m-CPBA) [19] at Δ5, gave the corresponding 5,6-epoxides as a
mixture of α:β isomers in a ratio of 5:1 in both cases. The mixture of
5,6α-epoxy-5α-cholestan-3β-ol (3a) and 5,6β-epoxy-5β-cholestan-3β-ol
(3b)) was obtained in 98% yield, and diosgenin epoxy derivatives 4a
and 4b in 96% yield. The epoxides ratio was determined from the 1H
4

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Fig. 2. Percentage of proliferation of MCF-7 cells exposed to β-aminoalcohol 5 evaluated 48 h later by MTT and CVS assays. The results are the average of nine independent assays and are
expressed as percentages with respect to untreated cells.
Fig. 3. Percentage of proliferation of MCF-7 cells exposed to β-aminoalcohol 6 evaluated 48 h later by MTT and CVS assays. The results are the average of nine independent assays and are
expressed as percentages with respect to untreated cells.
NMR spectrum (supporting information S1.3) based on the coupling
constant values and the chemical shift which permit differentiation of
the diastereoisomeric epoxides. Thus, H-6 in compounds 3b and 4b
show J = 2.0 Hz in agreement the value reported for 5,6β-epoxides
(< 2.0 Hz), while compounds 3a and 4a show J = 4.4 Hz characteristic
of 5,6α-epoxides (> 3.0 Hz). Moreover, H-19 is observed at 1.03 and
1.06 in 3a and 4a respectively while H-6 in 5β,6β-epoxides appear at
higher frequencies [21,31]. The mixture of 5,6-epoxides was used
without further purification in the aminolysis reaction, taking ad-
vantage of the preferred attack to the α-epoxide [12]. This was carried
with aniline and catalyzed by SZ to give the corresponding steroidal β-
aminoalcohol. A systematic variation of reaction conditions was made,
and 1:4 epoxide:aniline ratio, 6 h of reaction at 120 °C, 50% w/w of SZ
and solvent free were stablished as optimized conditions (Supporting
information Table S1). Under these conditions the β-aminoalcohol of
cholesterol (6β-phenylamino-cholestan-3β,5α-diol, 5) and diosgenin
((25R)-6β-phenylaminospirostan-3β,5α-diol, 6) were obtained in yields
of 97 and 68%, respectively.
On the basis of the 1H NMR data it was possible to determine the
chemoselectivity in the ring opening reaction, since only the signals for
the 5,6α-epoxide disappeared whereas those of the 5,6β-epoxide re-
mained unchanged. In Fig. 1 it is observed that the ratio of integrals of
H-6 from the α-epoxide and β-epoxides changes from 5/1 (at the be-
ginning of the reaction) to those corresponding to the aminolysis pro-
duct. This chemoselectivity is attributed to preferred attack of the
amine to the oxirane from the beta face leading to the trans diaxial 6β-
amino-5α-alcohol where the hydroxyl group at C-5 is also trans to the C-
19 methyl group. The energetically unfavorable C-5β hydroxy group
(cis-fused) is not formed in accordance with Paillasse et al. [32].
β-Aminoalcohols 5 and 6 were spectroscopically characterized by
1H, PENDANT 13C NMR, and HETCOR experiments, which allowed
unambiguous signal assignment complemented with FTIR-ATR and
HRMS (supporting information S2). The most characteristic shifts in 1
H
NMR that evidenced the formation of desired products 5 (6) are the
triplets, at 7.15 (7.14) and 6.66 (6.67) ppm, and doublet at 6.58 (6.56)
ppm, associated to the aromatic ring, together with the shifting of H-3,
5

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Steroidsxxx(xxxx)xxx–xxx
Fig. 4. Morphological changes of MCF-7 cells treated with DMSO or different concentrations of 5 for 48 h. Cell morphology was observed under inverted phase-contrast microscope at
magnification 40×.
from 3.87 (3.89) to 4.07 (4.02) ppm, and H-6, from 2.89 (2.89) to 3.35
(3.31) ppm, mainly. The HR-ESI spectra from 5 shows a peak at
496.4154 associated to M+H+ at 524.3739 and 546.3542 for 6 which
correspond to M+H+ and M+Na+, respectively.
high temperatures [33] and the use of lithium perchlorate [12] or BF3
[22] which are not environmentally friendly. Additionally, the se-
lectivity in the aminolysis attained using catalysis by SZ is high and
avoids purification steps.
The presented methodology to obtain steroidal β-aminoalcohols
represents an alternative to avoid long reaction times (up to 80 h) very
6

D. Soto-Castro et al.
Steroidsxxx(xxxx)xxx–xxx
Fig. 5. Morphological changes of MCF-7 cells treated with DMSO or different concentrations of 6 for 48 h. Cell morphology was observed under inverted phase-contrast microscope at
magnification 40×.
3.2. Biological evaluation
For compound 5, the cells treated with 0.45, 0.9 and 1.81 µM showed
activity. According to the results by CVS assay, a proliferation between
29 and 49% was obtained, in other words the mean of dead cells are of
60%. While by MTT assay only a 20% of active metabolic cells are
maintained, this can be interpreted as an inhibition of 80% (Fig. 2).
Regardless of the method, the results of the statistical analysis suggest
To determine the effect on cell proliferation of the two novel ster-
oidal β-aminoalcohols, MCF-7 cells were chosen as cancer cell line. To
validate the accuracy of the method used to assess the effects of com-
pounds 5 and 6, we performed MTT and CVS tests for both compounds.
7

D. Soto-Castro et al.
Steroidsxxx(xxxx)xxx–xxx
that cytotoxic effects are observed in MCF-7 cells at 0.45, 0.9 and
1.81 µM.
of cell size and apoptotic body formation in a dose dependent way, in
the range of concentrations tested.
On the other hand, for compound 6, the mean percent proliferation
was statistically equal for the same concentration assessed by both
methods (Fig. 3). However, concentrations of 0.12, 0.25, 0.51 and
1.025 µM showed significant difference with respect to the control by
the MTT assay, showing that a mean of 40% of active metabolic cells
are maintained. For CVS staining only concentrations of 0.25, 0.51 and
1.025 µM exhibited significant difference with respect to the control,
with a mean of 60% of proliferation. In these sense, many researchers
agree that the CVS method recognizes only live cells but Chiba et al.
[34] postulated that it also considers dead cells and underestimates the
cytotoxicity of a compound. In particular, studies carried out by Śliwka
et al. [35] suggest that MTT test was less reliable than CVS for the
compounds investigated. For that reason, the test method to study the
mechanism of action of the compound must be chosen with caution.
Finally, between the examined compounds, we can see that both
compounds (5 and 6) showed inhibitory effect on proliferation against
MCF-7 cells, either by CVS or MTT assays. Although MTT assay could be
overestimating the values, derivative 5 from cholesterol, at concentra-
tions of 0.45, 09 and 1.81 µM, is more active than compound 6.
Acknowledgements
The authors thank CONACyT for financial support and scholarships
to Roberto Lara Contreras and María Teresa Hernández-Huerta. We
acknowledge Ma. T. Cortez for NMR spectra and G. Cuéllar Rivera for
HRMS.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.steroids.2017.08.008.
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