Synthesis and biological activity of two pregnane derivatives with a triazole or imidazole ring at C-21

Article abstract of DOI:10.1016/j.jsbmb.2016.02.013

Pregnane derivatives are studied as agents for the treatment of different hormone-dependent diseases. The biological importance of these steroids is based on their potential use against cancer. In this study, we report the synthesis, characterization and biological activity of two pregnane derivatives with a triazole (3β-hydroxy-21-(1H-1,2,4-triazol-1-yl)pregna-5,16-dien-20-one; T-OH) or imidazole (3β-hydroxy-21-(1H-imidazol-1-yl)pregna-5,16-dien-20-one; I-OH) moieties at C-21. These derivatives were synthesized from 16-dehydropregnenolone acetate. The activity on cell proliferation of the compounds was measured on three human cancer cells lines: prostate cancer (PC-3), breast cancer (MCF7) and lung cancer (SK-LU-1). The cytotoxic and antiproliferative effects of T-OH and I-OH were assessed by using SBR and XTT methods, respectively. The gene expressions were evaluated by real time PCR. In addition, results were complemented by docking studies and transactivation assays using an expression vector to progesterone and androgen receptor. Results show that the two compounds inhibited the three cell lines proliferation in a dose-dependent manner. Compound I-OH downregulated the gene expression of the cyclins D1 and E1 in PC-3 and MFC7 cells; however, effect upon Ki-67, EAG1, BIM or survivin genes was not observed. Docking studies show poor interaction with the steroid receptors. Nevertheless, the transactivation assays show a weak antagonist effect of I-OH on progesterone receptor but not androgenic or antiandrogenic actions. In conclusion, the synthesized compounds inhibited cell proliferation as well as genes key to cell cycle of PC-3 and MCF7 cell lines. Therefore, these compounds could be considered a good starting point for the development of novel therapeutic alternatives to treat cancer.

Full text of DOI:10.1016/j.jsbmb.2016.02.013

Journal of Steroid Biochemistry & Molecular Biology 159 (2016) 8–18
Contents lists available at ScienceDirect
Journal of Steroid Biochemistry & Molecular Biology
journal homepage: www.elsevier.com/locate/jsbmb
Synthesis and biological activity of two pregnane derivatives with a
triazole or imidazole ring at C-21
a
a,1
c
Aylin Viviana Silva-Ortiz , Eugene Bratoeff , María Teresa Ramírez-Apan ,
b
b
b
Rocío García-Becerra , David Ordaz-Rosado , Nancy Noyola-Martínez ,
Rafael Castillo-Bocanegra , David Barrera *
Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de México, Mexico, D.F. 04510, Mexico
Departamento de Biología de la Reproducción, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Avenida Vasco de Quiroga No. 15, Col.
a
b,
a
b
Belisario Domínguez, Sección XVI, México, D.F. 14080, Mexico
c
Instituto de Química, Universidad Nacional Autónoma de México, México, D.F. 04510, Mexico
A R T I C L E I N F O
A B S T R A C T
Article history:
Pregnane derivatives are studied as agents for the treatment of different hormone-dependent diseases.
The biological importance of these steroids is based on their potential use against cancer. In this study, we
report the synthesis, characterization and biological activity of two pregnane derivatives with a triazole
Received 3 November 2015
Received in revised form 22 January 2016
Accepted 11 February 2016
Available online 23 February 2016
(
(
3
b
-hydroxy-21-(1H-1,2,4-triazol-1-yl)pregna-5,16-dien-20-one; T–OH) or imidazole (3
b-hydroxy-21-
1H-imidazol-1-yl)pregna-5,16-dien-20-one; I–OH) moieties at C-21. These derivatives were synthesized
from 16-dehydropregnenolone acetate. The activity on cell proliferation of the compounds was measured
on three human cancer cells lines: prostate cancer (PC-3), breast cancer (MCF7) and lung cancer (SK-LU-
Keywords:
Pregnane derivatives
Prostate cancer
Breast cancer
Lung cancer
Cyclin D1
1). The cytotoxic and antiproliferative effects of T–OH and I–OH were assessed by using SBR and XTT
methods, respectively. The gene expressions were evaluated by real time PCR. In addition, results were
complemented by docking studies and transactivation assays using an expression vector to progesterone
and androgen receptor.
Cyclin E1
Results show that the two compounds inhibited the three cell lines proliferation in a dose-dependent
manner. Compound I–OH downregulated the gene expression of the cyclins D1 and E1 in PC-3 and
MFC7 cells; however, effect upon Ki-67, EAG1, BIM or survivin genes was not observed. Docking studies
show poor interaction with the steroid receptors. Nevertheless, the transactivation assays show a weak
antagonist effect of I–OH on progesterone receptor but not androgenic or antiandrogenic actions.
In conclusion, the synthesized compounds inhibited cell proliferation as well as genes key to cell cycle
of PC-3 and MCF7 cell lines. Therefore, these compounds could be considered a good starting point for the
development of novel therapeutic alternatives to treat cancer.
ã 2016 Elsevier Ltd. All rights reserved.
1. Introduction
feature a tetracyclic nucleus formed by three ciclohexanes and one
cyclopentane. They differ by the oxidation state, the chains and
It is well known that the steroids play a pivotal role in several
processes, such as the differentiation, development, growth, as
well as physiological and reproductive functions in the human
body. Steroid hormones are molecules that share as structural
functional groups attached to this four-ring structure which
change their chemical functions and biologic specificity [1].
Generally, many of the cellular effects of steroids are mediated
via the classical genomic mechanism, where the steroids are taken
from circulation to cells and bind to specific receptors, two
hormone-receptor complexes dimerize and are translocated from
cytoplasm to nucleus to recognize the hormone-response ele-
ments and induce changes in the transcriptional expression of
target genes [2]. In addition, several non-genomic actions have
been described. In this respect, ion channels, membrane-associat-
ed steroid receptors, enzyme-linked receptors and cytoplasmic
proteins activate intracellular signaling cascades that in turn
activate second messengers [3]. In fact, the steroid effects may
Abbreviations: AR, androgen receptor; PR, progesterone receptor; VDR, vitamin
D receptor; P
4
, progesterone.
*
Corresponding author at: Departamento de Biología de la Reproducción,
Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Avenida Vasco
de Quiroga No. 15, Col. Belisario Domínguez, Sección XVI, Tlalpan, 14080 México, D.
F., México.
E-mail addresses: barrera1912@gmail.com, vidadav@hotmail.com (D. Barrera).
Deceased.
1
http://dx.doi.org/10.1016/j.jsbmb.2016.02.013
0
960-0760/ã 2016 Elsevier Ltd. All rights reserved.

A.V. Silva-Ortiz et al. / Journal of Steroid Biochemistry & Molecular Biology 159 (2016) 8–18
9
occur using one or both mechanisms in a tissue-dependent
manner.
200 spectrometer (Perkin-Elmer Life and Analytical Science,
1
13
Shelton CT, USA). H and C NMR were taken on a Varian VRX-
1
Indeed, the literature describes numerous examples of the
natural and synthetic compounds used for the treatment of both
sex hormone-dependent and independent cancer tumors [4–9].
However, the resistance to therapies impedes the successful
treatment. Consequently, the design of compounds with a specific
activity for each molecular target should be performed.
In this sense, the replacement of one or more carbon atoms in a
steroid molecule by nitrogen atoms affects the chemical properties
400 spectrometer (MR resources NC, USA) operating at 400 ( H)
13
and 100 ( C) MHz with tetramethylsilane (TMS) as internal
standard ( = 0) in CDCl (the abbreviations of signal patterns are as
d
3
follows: s, singlet; d, doublet, t, triplet; m, multiplet). High
resolution mass spectra (HRMS) were obtained with a Thermo DFS
+
spectrometer by direct infusion and using FAB ionization mode
(Thermo Fisher Scientific, San Jose, CA USA).
16-dehydropregnenolone acetate, progesterone (P
4
), RU486,
0
of the steroid and changes its biological activity. Since 1980 s,
5a
-dihydrotestosterone (5 -DHT), flutamide (FLUT) and sulfo-
a
azasteroids had received much attention for the development of
new compounds that are being used for the treatment of hormone-
dependent diseases such as adenocarcinomas, offering an impor-
tant strategy for tumor control [10]. Actually, many drugs used in
the treatment of a number of androgen-independent, androgen-,
estrogen-, and other steroid-dependent diseases, have in its
structure a heterocyclic group. Some interesting compounds in
the pharmaceutical market are for example aminoglutethimide
and its derivatives; esters of 4-pyridineacetic acid; bis-chloro-
phenyl-pyrimidine analogues; as well as imidazole and triazole
derivatives, such as ketoconazole, liarozole, fadrozole, CGS 18320
B, vorozole and CGS 20267 [11].
In particular, pharmacological studies have displayed that some
pregnane derivatives have a variety of bioactivities, such as
cytotoxicity and immunoregulation among others, whereas some
synthetic steroids have been previously described as regulators of
apoptosis or implicated in cellular proliferation [12–17]. In this
study, we synthesized two steroidal derivatives from 16-dehy-
rhodamine B (SRB) were purchased from Sigma-Aldrich (St. Louis,
MO, USA), Ketoconazol was purchased from Sigma Life Science (St.
Lois, MO, USA). Cell culture medium (DMEM and RPMI) was
obtained from Gibco-Invitrogen. TRIzol and oligonucleotides for
real time polymerase chain reaction (qPCR) were from Invitrogen
(CA, USA). The probes, capillaries, the TaqMan Master reagents, the
Transcriptor First Strand cDNA synthesis kit, the cell proliferation
assay (XTT) were purchased from Roche (Roche Applied Science,
3
IN, USA). The [H]chloramphenicol (specific activity 38.9Ci/mmol)
was purchased from DuPont NEN Research products (Perkin-
Elmer, Boston, MA) and radioactivity was determined in a Beckman
LS6500 scintillation system (Beckman Instruments, CA) using
Biodegradable Counting Scintillant (Amersham, CA) as counting
solution.
2.2. Synthesis of 16-Dehydropregnenolone derivates (T–OH and I–
OH)
dropregnenolone acetate with triazole (3
triazol-1-yl) pregna-5,16-dien-20-one; T–OH) or imidazole (3
b
-hydroxy-21-(1H-1,2,4-
The synthetic pathways for the preparation of T–OH and I–OH
are outlined in Fig. 2. These compounds were prepared from the
commercially available 16-dehydropregnenolone acetate (1) as
follows:
b-
hydroxy-21-(1H-imidazol-1-yl) pregna-5,16-dien-20-one; I–OH)
moieties at C-21 (Fig. 1). The compounds were tested in vitro to
determine the effect on cell growth, and upon gene expression of
proliferative and apoptotic proteins using a panel of human cancer
cell lines: PC-3 (prostate), MCF7 (breast) and SK-LU-1 (lung).
In order to rationalize and visualize at the molecular level the
important features of these pregnane derivatives and their
implication in ligand-protein interaction we used docking tools
to calculate the thermochemical parameters between T–OH and I–
OH versus progesterone, androgen and vitamin D receptors (PR, AR
and VDR, respectively). Additionally, transactivation assays were
performed with I–OH compound to determine its agonistic or
antagonistic actions.
2.2.1. 16
A solution of steroid 1 (1 g, 2.82 mmol), sodium hydroxide 4 N
(2 mL, 8 mmol) and H 30% (4 mL, 135 mmol) in hot methanol
a,17a-Epoxy-3b-hydroxypregn-5-en-20-one (2)
2 2
O
(66 mL) was stirred at room temperature for 4 h. After this time the
methanol was evaporated and the product was washed with water
and air dried to give a white solid.
ꢀ
ꢁ1
Yield 0.665 g (72%), m.p. 197–199 C, IR (KBr) cm : 3457, 1692,
1
3
1642 and 1042. H RMN (400 MHz, CDCl ) d: 1.00 (s, H-18, 3H), 1.03
(s, H-19, 3H), 2.01 (s, H-21, 3H), 3.47 (m, J = 3.4 Hz, H-3, 1H), 3.50 (s,
OH, 1H), 3.66 (s, H-16, 1H), 5.32 (t, J = 5.3 Hz, H-6, 1H). 13C RMN
(
100 MHz, CDCl
(C-16), 71.16 (C-17), 71.75 (C-3), 121.13 (C-6), 141.27 (C-5), 205.07
C-20). HRMS cal. for C21 330.2195, found 330.2187.
3
) d: 15.38 (C-18), 19.30 (C-19), 27.51 (C-21), 60.55
2. Materials and methods
(
30 3
H O
2.1. Reagents
2
.2.2. 3
b-(Tetrahydro-2H-pyran-2-yloxy)-16a,17a-epoxypregn-5-
Reagents and solvents were purchased from commercial
en-20-one (3)
sources. Melting points (uncorrected) were determined on a
Fisher Johns melting point apparatus (Fisher Johns, Mexico City,
Mexico). Infrared spectra (IR) were recorded on a Perkin-Elmer
A solution of steroid 2 (1 g, 2.42 mmol), 3,4-dihydro-2H-pyran
(30 mL) and p-toluensulfonic acid monohydrate (0.100 mg,
0.058 mmol) in dichloromethane (40 mL), was stirred at room
N
N
N
N
N
O
O
H
H
H
H
H
H
HO
HO
T-OH
I-OH
Fig. 1. Molecular structures of T–OH and I–OH.

10
A.V. Silva-Ortiz et al. / Journal of Steroid Biochemistry & Molecular Biology 159 (2016) 8–18
Fig. 2. Synthesis of two 16-dehydropregnenolone acetate derivates.
0
temperature for 1 h. Then, the solvent was removed with the
rotaevaporator and the product was recrystallized from methanol.
17), 97.13 (C-20), 101.95 (C-1 of protector group), 121.22 (C-6),
141.29 (C-5). HRMS cal. for C28
44
H O
6
476.3138, found 476.3134.
ꢀ
ꢁ1
Yield 0.88 g (71%), m.p. 114–116 C, IR (KBr) cm : 2935, 1703,
1
1
438 and 1024. H RMN (400 MHz, CDCl
3
)
d: 1.00 (s, H-19, 3H), 1.02
2.2.4. 21-Choloro-20,20-dimethoxy-3
b
-(tetrahydro-2H-pyran-2-
(
s, H-19, 3H), 2.01 (s, H-21, 3H), 3.47 (m, J = 3.4 Hz, H-3, 1H), 3.66 (s,
yloxy)-16 ,17
a
a
-epoxypregn-5-ene (5)
0
0
H-16, 1H), 3.89 (d, H-5 of protector group, 2H), 4.69 (t, H-1 of
To a cold solution of steroid 4 (0.5 g, 1.01 mmol) in a mixture of
dry dichloromethane (4 mL) and pyridine (0.5 mL) was added
dropwise thionyl chloride (0.15 mL) under a nitrogen atmosphere,
and the mixture was stirred at room temperature for 20 min.
Chloroform (50 mL) was added to stop the reaction and then it was
evaporated in vacuum. The crude product was recrystallized from
methanol.
Yield 0.418 g (80%), m.p. 101–103 C, IR (KBr) cm : 2932, 1670,
3
1065 and 1039. H RMN (400 MHz, CDCl ) d: 1.02 (s, H-18, 3H), 1.23
(s, H-19, 3H), 3.25 (s, OCH3, 6H), 3.43 (m, J = 3.4, H-3,1H), 3.48 (s, H-
protector group, 1H), 5.31 (1H, d, J = 5.3 Hz, H-6). 13C RMN
(
100 MHz, CDCl
3
)
d
: 15.37 (C-18), 19.28 (C-19), 27.65 (C-21),
0.63 (C-5 of protector group), 62,96 (C-16), 75,99 (C-17), 76.06 (C-
),120.92 (C-6),141.38 (C-5), 205.06 (C-20). HRMS cal. for C26
14.2770, found 414.2774.
6
3
4
38 4
H O
ꢀ
ꢁ1
2
1
.2.3. 20,20-Dimethoxy-3
,17 -epoxypregn-5-en-21-ol (4)
A solution of steroid 3 (1 g, 2.11 mmol), (diacetoxyiodo) benzene
1.2 g, 3.72 mmol) and sodium hydroxide (1 g, 25 mmol) in
b-(tetrahydro-2H-pyran-2-yloxy)-
1
6
a
a
0
(
21, 2H), 3.67 (s, H-16,1H), 3.89 (d, H-5 of protector group, 2H), 4.69
0
13
methanol (21 mL) and dichloromethane (10 mL) was stirred at
room temperature for 3 h. The solvent was evaporated under
reduced pressure, and the residue was purified by a column
chromatography packed with basic aluminum oxide eluted with a
mixture of hexane and acetone (95:5).
(t, H-1 of protector group,1H), 5.32 (d, J = 5.3 Hz, H-6,1H). C RMN
(100 MHz, CDCl
3
)
d
: 15.26 (C-18), 19.50 (C-19), 50.45 (OCH
3
), 59.51
0
(C-21), 68.30 (C-16), 76.08 (C-3), 60.63 (C-5 of protector group),
97.13 (C-20), 101.95 (C-1 of protector group), 121.22 (C-6), 141.29
(C-5). HRMS cal. for C28
0
5
H43ClO 494.2799, found 494.2793.
ꢀ
ꢁ1
Yield 0.751 g (65%), m.p. 139–141 C, IR (KBr) cm : 3504, 2937,
1
1
083 and 1030. H RMN (400 MHz, CDCl
3
)
d
: 1.00 (s, H-18, 3H), 1.24
2.2.5. 20,20-Dimethoxy-16
yloxy)-21-(1H-1,2,4-triazol-1-yl) pregn-5-ene (6a)
mixture of 1,2,4-triazole (0.031 g, 0.443 mmol),
a,17
a-epoxy-3
b
-(tetrahydro-2H-pyran-2-
(
s, H-19, 3H), 3.25 (s, OCH
3
, 6H), 3.43 (m, J = 3.4, H-3, 1H), 3.44 (s,
0
OH, 1H), 3.48 (s, H-21, 2H), 3.67 (s, H-16, 1H), 3.89 (d, H-5 of
protector group, 2H), 4.69 (t, H-1 of protector group, 1H), 5.32 (d,
A
K
2
CO
3
0
(0.123 g, 0.89 mmol) and compound 5 (0.1 g, 0.202 mmol) was
dissolved in dry DMF (5 mL) and the solution was heated at 90 C
under nitrogen for 3 h. Cold water was added and the resulting
1
3
ꢀ
J = 5.3 Hz, H-6,1H). C RMN (100 MHz, CDCl
C-19), (CH
3
)
d
: 15.27 (C-18), 19.41
0
(
3
of protector group), 50.49 (OCH
3
), 60.63 (C-5 of
protector group), 62.69 (C-16), 63.02 (C-21), 76.08 (C-3), 76.02 (C-

A.V. Silva-Ortiz et al. / Journal of Steroid Biochemistry & Molecular Biology 159 (2016) 8–18
11
solid was filtered. The compound was purified by a column
chromatography with florisil.
Yield 0.078 g (73%), m.p. 124–125 C, IR (KBr) cm : 2934, 1379,
2.3. Cell culture
ꢀ
ꢁ1
PC-3, MCF7, SK-LU-1 and HeLa human cell lines were supplied
by National Cancer Institute (NCI), USA. These lines were
maintained at 37 C in a humidified atmosphere with 5% CO
95% air. Experimental procedures were performed with different
supplemented medium as described in each section.
1
1275 and 1026. H RMN (400 MHz, CDCl
3
)
d: 0.87 (s, H-18, 3H), 0.98
ꢀ
(
2
s, H-19, 3H), 3.28 (s, OCH
3
, 6H), 3.46 (m, J = 3.4, H-3,1H), 3.48 (s, H-
2
-
0
1, 2H), 3.68 (s, H-16,1H), 3.90 (d, H-5 of protector group, 2H), 4.70
0
(
t, H-1 of protector group, 1H), 5.32 (d, J = 5.3 Hz, H-6, 1H), 7.98 (s,
13
3
H-Het.,1H), 8.27 (s, H-Het.,1H). C RMN (100 MHz, CDCl ) d: 15.20
(
(
C-18), 19.43 (C-19), 49.66 (C-16), 50.47 (OCH
C-5 of protector group), 76.04 (C-3), 97.13 (C-20), 101.93 (C-1 of
3
), 58.52 (C-21), 62.68
2.4. Cell cytotoxic assay with SRB
0
0
protector group), 121.16 (C-6), 121.23 (C-Het.), 141.31 (C-5), 141.48
C-Het.). HRMS cal. for C30 527.3359, found 527.3355.
The cytotoxicity of 16-dehydropregnenolone acetate and I–OH
was determined using the protein-binding dye SRB in microculture
assay at a single concentration for the studied compounds and
compared with Ketoconazole and T–OH previously reported [16].
The compounds were screened in vitro on PC-3, MCF7 and SK-
LU-1, these cells were cultured in RPMI-1640 medium supple-
(
45 3 5
H N O
2
.2.6. 3
one (T–OH)
Compound 6a (0.1 g, 0.19 mmol) was hydrolyzed and the
b-Hydroxy-21-(1H-1,2,4-triazol-1-yl) pregna-5,16-dien-20-
epoxide group at C-16 was eliminated at the same time with a
reaction carried out in acetic acid (4 mL), chromous chloride
mented with 10% fetal bovine serum, 2 mM
L
-glutamine, 100 units/
mL penicillin G sodium, 100 g/mL streptomycin sulfate, 0.25
m
mg/
1
(
0.08 g, 0.65 mmol) and two drops of 36% hydrochloric acid at room
temperature for 20 min the mixture was diluted with cold water
150 mL) and the precipitate was filtered and dried. The product
mL amphotericin B (Gibco ), and 1% of nonessential amino acids
1
(Gibco ). The viability of these cells exceeds 95% as determined
(
with trypan blue.
was purified by silica gel column chromatography using a mixture
Cells were removed from the tissue culture flask and diluted
of hexane and acetone (9:1).
with fresh media. From this cell suspension, 100 mL containing
ꢀ
ꢁ1
Yield 0.370 g (77%), m.p. 120–122 C, IR (KBr) cm : 3355, 2927,
5000 cells per well, were seeded in 96-wells tissue culture plates
1
ꢀ
1
1
(
709, 1670, 1276 and 1051. H RMN (CDCl
.20 (s, H-19, 3H), 3.25 (m, J = 3.5 Hz, H-3, 1H), 3.52 (s, OH, 1H), 4.18
s, 2H, H-21), 5.34 (d, J = 5.3 Hz, H-6, 1H), 6.74 (s, H-16, 1H), 7.50 (s,
3
)
d
: 0.90 (s, H-18, 3H),
(Costar) and the material was incubated at 37 C for 24 h.
Furthermore, another plate was prepared with culture medium
only and it was incubated for 1 h. Next, 100 mL of a solution of the
compounds obtained by diluting the stocks were added to each
well.
13
H-Het., 1H), 7.70 (s, H-Het.,1H). C RMN (100 MHz, CDCl
3
)
d
: 15.32
C-18), 20.01 (C-19), 62.15 (C-21), 71.78 (C-3), 121.04 (C-6), 141.23
C-5), 141.38 (C-16), 141.47 (C-Het.), 144.83 (C-Het.), 196.66 (C-20).
381.2416, found 381.2409.
(
(
Cells were exposed for 48 h to the compounds at concentration
HRMS cal. for C23
H
31
N
3
O
2
of 50
m
M. Then, the cells were fixed by the addition of cold 50%
ꢀ
aqueous trichloroacetic acid and the plates were incubated at 4 C
2
(
.2.7. 21-(1H-Imidazol-1-yl)-20,20-dimethoxy-16
tetrahydro-2H-pyran-2-yl)-pregn-5-ene (6b)
This compound was obtained by the procedure described in the
section 2.2.5 using 1H-imidazole (0.04 g, 0.61 mmol) and Cs CO
0.40 g, 1.21 mmol).
Yield 0.083 g (78%), m.p. 90–92 C, IR (KBr) cm : 2935, 1377,
a
,17
a
-epoxy-3
b
-
2
for 1 h, washed with tap H O and air-dried. The trichloroacetic-
acid-fixed cells were stained by the addition of 0.4% SRB as
described [15]. Free SRB solution was removed by washing with 1%
aqueous acetic acid. The samples were air dried, and the bound dye
2
3
(
was solubilized with 100 mL (10 mM) of unbuffered Tris base. The
ꢀ
ꢁ1
plates were placed on a vortex for 5 min, and the absorption was
determined at 515 nm using an ELISA plates reader (Bio-Tek,
Winooski, VT, USA). Cell growth inhibition was calculated
according to the following expression:
1
1
(
1
258 and 1028. H RMN (400 MHz, CDCl
s, H-19, 3H), 3.28 (s, OCH , 6H), 3.44 (m, J = 3.4, H-3,1H), 3.67 (s, H-
6,1H), 3.90 (d, H-5 of protector group, 2H), 4.04 (s, H-21, 2H), 4.71
3
) d: 1.00 (s, H-18, 3H), 1.28
3
0
0
(
t, H-1 of protector group, 1H), 5.35 (d, J = 5.3 Hz, H-6, 1H), 7.12 (d,
13
100 ꢁ ðsample absortionÞ
H-Het., 1H), 7.71 (d, H-Het., 1H), 8.27 (s, H-Het., 1H). C RMN
cell growth inhibitionð%Þ ¼
ꢂ 100
vehicle absortion
(
100 MHz, CDCl
3
)
d
: 15.30 (C-18), 19.90 (C-19), 49.52 (C-16), 50.00
0
(
OCH
3
), 55.5 (C-21), 63.02 (C-5 of protector group), 76.10 (C-3),
7.09 (C-17), 101.97 (C-1 of protector group), 115.01 (C-20), 121.23
Ketoconazole was used as control for this assay. This compound
is an antimycotic drug that has previously been described as an
agent that inhibits cytochrome P450 enzymes and has shown
cytotoxic activity in vitro studies [18–20].
0
9
(
C-6), 141.29 (C-Het.), 141.31 (C-5), 141.89 (C-Het.), 151.55 (C-Het.).
HRMS cal. for C31 526.3407, found 526.3411.
46 2 5
H N O
2
(
.2.8. 3
I–OH)
The procedure to prepare this compound is the same as that
b
-Hydroxy-21-(1H-imidazol-1-yl) pregna-5,16-dien-20-one
2.5. Cell proliferation assay with XTT method
The PC-3 cells were incubated in RPMI 1640, MCF7 in DMEM-
HG and the SK-LU-1 in DMEM-F12 medium, all supplemented and
with phenol red. The proliferation was determined using the
previously described in section 2.2.6.
ꢀ
ꢁ1
Yield 0.053 g (73%), m.p. 179–181 C, IR (KBr) cm : 3334, 2930,
1
1
1707,1668,1277 and 1051. H RMN (CDCl
3
)
d: 0.95(s, H-18, 3H),1.30
colorimetric XTT assay Kit (Roche ). The T–OH and I–OH
(
2
s, H-19, 3H), 3.52 (m, J = 3.5 Hz, H-3, 1H), 3.66 (s, OH, 1H), 4.25 (s,
proliferative effects were evaluated at different concentrations
and its inhibitory concentrations 20% and 50% (IC20 and IC50) were
determined.
H, H-21), 5.32 (d, J = 5.3 Hz, H-6, 1H), 6.74 (s, H-16, 1H), 7.04 (d, H-
13
Het., 1H), 7.35 (d, H-Het., 1H), 7.78 (s, H-Het., 1H). C RMN
100 MHz, CDCl : 15.30 (C-18),19.9 (C-19), 61.31 (C-21), 71.69 (C-
), 121.01 (C-6), 141.21 (C-5), 144.30 (C-16), 141.24 (C-Het.), 144.89
C-Het.), 151.55 (C-Het.), 154.40 (C-17), 206–09 (C-20). HRMS cal.
for C24 380.2464, found 380.2470.
Reagents and conditions: (i) H , NaOH 4N, 4 h; (ii) DHP, p-
toluensulfonic acid, 2 h; (iii) NaOH, C
(
3
(
3
)
d
For this assay, 1000 cells were seeded in 96-well tissue culture
plates by sextuplicate. After 24 h, culture medium was removed
and cells were incubated with T–OH or I–OH at different
ꢁ
10
ꢁ4
H
32
N
2
O
2
concentrations (1 ꢂ10 –1 ꢂ10 M). Ethanol (0.1% v/v) was used
ꢀ
2
O
2
as vehicle (V). The cells were incubated at 37 C for 4 h (control
H
5
I(OAc)
) a) 1,2,4-triazole, K
, CH
2
, MeOH, 3 h; (iv)
plate) or 4 days (evaluation plates) using the colorimetric XTTassay
according to manufacturer’s instructions. The absorbance at
492 nm was determined in a microplate reader (BioTek, Winooski,
6
ꢀ
SOCl
1
2
, Py, CH
2
Cl
2
, 20 min; (
v
2
CO
3
, 80 C, 5 h; b)
ꢀ
H-imidazole, K
2
CO
3
, 80 C, 5 h; (vi) HCl, CrCl
2
3
COOH, 20 min.

12
A.V. Silva-Ortiz et al. / Journal of Steroid Biochemistry & Molecular Biology 159 (2016) 8–18
VT, USA). The IC20 and IC50 values were calculated by means of a
scientific graphing software (Origin 5, OriginLab Corporation,
Northampton MA) using a non-linear regression analysis with
sigmoidal fitting based in a dose-response curve as described [21].
the antagonists RU486 and FLUT. The plates were incubated for
ꢀ
24 h at 37 C in 5% CO
The CAT activity was determined using 5
2
atmosphere. Ethanol was used as vehicle.
g of protein, 10 g of
m
m
5
3
butyryl coenzyme-A (Sigma), 2 ꢂ10 cpm of xylene-extracted [ H]
chloramphenicol in 0.25 M Tris–HCl, pH 8.0. The reporter plasmid
and expression vectors used to transfect HeLa cells were kindly
provided by Dr A. J. Cooney (Baylor College of Medicine).
2.6. Gene expression analysis by qPCR
The cancer cells were incubated in the presence of different I–
ꢁ
9
ꢁ4
OH concentrations (1 ꢂ10 –1 ꢂ10 M) or its vehicle.
2.8. Docking studies
Afterwards, medium was aspired and total RNA was extracted
using Trizol reagent and 1
transcriptor RT system (Roche, Germany).
The qPCR was carried out using the LightCycler Taqman Master
System and the LightCycler 2.0 Instrument from Roche (Roche
Diagnostics, Mannheim, Germany). The following protocol was
m
g was reverse transcribed using the
To characterize the molecular interactions between the
receptors and the synthetized compounds we performed a docking
study. Considering the structural origin of these compounds, the
progesterone receptor (PR: NR3C3) and androgen receptor (AR:
NR3C4) were used as templates for the docking analysis.
Additionally, vitamin D receptor (VDR: NR1I1) was evaluated.
The X-ray crystal structure of PR, AR and VDR of Petit-Topin, He
and Moras complexes were used, these were obtained from
Protein Data Bank (PDB) codes: 3D90, 1XQ3 and 1S19 respectively.
used: Taq DNA polymerase activation and DNA denaturation at
ꢀ
9
5 C for 10 min, proceeded by 45 amplification cycles, each cycle
ꢀ
ꢀ
ꢀ
consisting of 10 s at 95 C, 30 s at 60 C, and 1 s at 72 C.
The genes studied related with cell proliferation were cyclin D1
(
CCND1), cyclin E1 (CCNE1), Ki-67 and the potassium channel
PR (A/B chains) was co-crystalized with 13-
ethynyl-17 -hydroxygon-4-en-3-one [24], AR was co-crystalized
with (17 )-17-hydroxy-17-methylestra-4,9,11-trien-3-one [25],
and the VDR was co-crystalized with calcipotriol at the active site
[26].
b-ethyl-17-alpha-
Ether-à-go-go (EAG1), while for apoptosis were BIM and survivin.
Additionally, in order to establish a possible binding of the
compounds to the VDR, the expression of CYP24A1 gene was
b
b
ꢁ
9
evaluated with different concentrations of I–OH (1 ꢂ10
–
ꢁ
6
ꢁ8
1
ꢂ10 M), in absence and presence of calcitriol (1 ꢂ10 M).
Expression levels were calculated after normalization to the
housekeeping gene -actin (internal control). Probes and primers
The geometry optimizations of these compounds were
submitted by Molecular Mechanics using the MMFF94x force-
field as implemented in the Spartan 10 program (Wave-function
Inc. Irvine, CA), this optimized the structure conformers and the
minimum energy of each compound were filtered. The torsional
root and branches of the ligands were chosen utilizing MGLTools
1.5.4. Docking calculations were performed with AutoDock 4.2 [27]
and Glide 5.7 softwares [28]. To perform Glide docking the ligands
and the receptors were prepared with Maestro v9.8. [29].
A three-dimensional grid box of size 70Å ꢂ 70Å ꢂ 70 Å, with
0.375 Å spacing and centered at the amino acid GLN725 in PR,
GLN711 in the AR and SER278 in VDR was performed to establish
the possible interaction of the compounds with each receptor.
The parameters used in the docking study were: number of GA
runs (25), the population (150), the energy evaluations
(2,500,000), and the maximum number of top individuals that
automatically survive (27,000). The best binding mode of each
molecule was selected based on the lowest free binding energy. All
molecular graphics figures were prepared with PyMOL [30].
b
were designed with the Universal Probe Library Assay Design
Center from Roche, and they are reported in Table 1.
Data obtained from qPCR were analyzed using the statistical
software Sigma Plot 12.0. The representative differences for dose-
response were determined by One-Way ANOVA followed by
appropriate post-hoc test (Holm-Sidak method for pair-wise
comparisons). Data were expressed as the mean ꢃ standard
deviation (S.D.) and the differences were considered statistically
significant at P ꢄ 0.01.
2
.7. Plasmid and transfection in HeLa cell line
HeLa cells were plated the day before transfections, at 6 well
plates in DMEM-HG medium without red phenol, supplemented
with 5% stripped FBS, 100 U/mL penicillin and 100 g/mL
streptomycin [22]. Transfections were performed in triplicate
using PolyFect
protocol provided by the manufacturer. The cells were co-
transfected with 1.0 g of the expression vector pLEN-hPRB or
pSVhAR.BHEXE containing the coding sequence of the PR-B and AR,
respectively; and 0.5 g of reporter plasmid PRE-E1b-CAT which
m
TM
(QIAGEN Inc., Valencia, CA) following the
3. Results
m
3.1. Chemistry
m
contains an oligonucleotide with a progesterone/androgen re-
sponse element upstream of the adenovirus E1bTATA box fused to
the chloramphenicol acetyltransferase (CAT) gene [23]. Twenty-
four hours later, the cells were treated with different concen-
The synthetic pathways for the preparation of T–OH and I–OH
are outline in Fig. 2. Commercially available 16-dehydropregne-
nolone acetate (1) was treated with hydrogen peroxide and sodium
hydroxide 4 N to form the 16b,17b-epoxy compound 2 with a 71%
4
trations of I–OH alone, or in combination with P and 5a-DHT, or
yield. The hydroxyl group at C-3 was protected with 3,4-dihydro-
Table 1
Probes and primers used to the qPCR.
Gen/accession number
L-primer
R-primer
Probe numbera
CCND1/NM_053056.2
CCNE1/NM_001238.1
Ki-67/X65550.1
EAG1/AF078741.1
BIM/AY305716.1
gaagatcgtcgccacctg
ggccaaaatcgacaggac
ggtgtgcagaaaatccaaaga
cctggaggtgatccaagatg
gctgtggaggctgaatcc
gcccagtgtttcttctgctt
catcatggccatcaaaacaa
ccaaccgcgagaagatga
gacctcctcctcgcscttct
gggtctgcacagactgcat
actgtccctatgacttctggttg
ccaaacacgtctccttttcc
tcggctgcttggtaattattc
aaccggacgaatgcttttta
gcagctcgactggagtgac
ccagaggcgtacagggatag
67
36
73
49
63
11
88
64
Survivin/AB154416.1
CYP24A1/NM_000782.3
b-actin/NM_001101.3
a
From the universal probe library (Roche).

A.V. Silva-Ortiz et al. / Journal of Steroid Biochemistry & Molecular Biology 159 (2016) 8–18
13
Fig. 3. Effects of T–OH and I–OH on proliferation of three different cancer cells lines. PC-3 (&), MCF7 (*) and SK-LU-1 (~) were incubated with different concentrations of T–
OH (A) and I–OH (B) during 4 days. Cell growth assays by the XTT colorimetric method were performed. Results are the mean ꢃ S.D. of sextuplicate and represented at least
three different experiments. The vehicle (V) data were normalized to 100% for each experiment. *P ꢄ 0.01 vs. V.
2
H-pyran and p-toluenesulfonic acid monohydrate in dichloro-
order to inhibition by T–OH and I–OH was: PC-3 > MCF7 > SK-LU-1.
Nevertheless, PC-3 cells were more sensitive to I–OH antiprolifer-
ative effect compared with T–OH, whereas in MCF7 and SKLU-
1 cell lines, an equipotential effect was observed.
methane to provide compound 3 with a 65% yield.
The oxidation of 3 with (diacetoxyiodo) benzene and sodium
hydroxide in methanol generated the alcohol in C-21 thus
affording compound 4 with a regular yield of 65%.
Treatment of
4
with thionyl chloride gave chlorinated
3.3. Effects of I–OH on different genes involved in proliferation or
compound 5 (80%). Similarly, it has been reported others methods
to perform this same reaction [16,31].
apoptosis
Compound 6a with a triazole ring at C-21, and 6b, with an
imidazole ring at C-21, were prepared in 73% and 78% respectively
Since I–OH had better antiproliferative effect than T–OH gene
expression studies were performed with I–OH for CCND1, CCNE1,
Ki-67, EAG1, BIM, and survivin on three cell lines.
As depicted in Fig. 4, the gene expression of CCND1 and
CCNE1 decreased in a concentration-dependent manner both in
PC-3 and MCF7 line cells whereas Ki-67, EAG1, BIM and survivin
gene expression was not affected significantly by I–OH (data not
shown). In SK-LU-1 cell line a significant response on these genes
was not observed (data not shown).
from 5 potassium carbonate or cesium carbonate and 1,2,4-trizole
ꢀ
or imidazole, respectively in DMF 80 C for 5 h. The 16
b
,17b
-epoxy
elimination and C-3 hydrolysis were carried out with chromium
chloride (II), hydrochloric acid and acetic acid affording T-OH and
I-OH with 77% and 73% yield, respectively.
All synthesized compounds were isolated and the melting
points showed a maximum of two degree difference. The desired
1
compounds and intermediates were characterized by IR, H NMR,
13
C NMR and mass spectrometry.
3.4. Molecular docking
3.2. Cytotoxic and metabolic effect
The biological importance of pregnane derivatives lies on their
4
structural similarities with P , and, at the same time, with some
In this study, using SRB method was determined the cytotoxic
androgen derivatives, giving them progestogenic and androgenic
effect of 16-dehyropregnenolone acetate and I–OH at 50 mM
concentration and compared with Ketoconazole and T–OH
previously reported [16]. Similarly to Ketoconazole, I–OH showed
a high cytotoxic activity in the three cancer cells lines: PC-3,
MCF7 and SK-LU-1 compared with the precursor that had a weak
effect. Likewise, I–OH was more effective than T–OH in PC-3 and
MCF7 cells (data not shown).
Interestingly, using XTT method the compounds inhibited in a
concentration-dependent manner the cell cancer growth (Fig. 3).
As can be seen in Table 2, the IC20 and IC50 were calculated and the
Table 2
Inhibitory concentrations (IC20 and IC50) values of T–OH and I–OH on a panel cancer
cell lines studied.
Compound
IC
PC-3
(mol/L)
MCF7
(mol/L)
SK-LU-1 (mol/L)
ꢁ7
ꢁ5
ꢁ4
ꢁ5
ꢁ4
T–OH
I–OH
20
50
20
3.1 ꢂ10
1.7 ꢂ10
4.3 ꢂ10
4.1 ꢂ10
1.1 ꢂ10
ꢁ
5
ꢁ4
3.6 ꢂ10
2.3 ꢂ10
ꢁ
9
6
ꢁ4
4.1 ꢂ10
1.9 ꢂ10
1.3 ꢂ10
2.0 ꢂ10ꢁ
ꢁ5
1.8 ꢂ 10
ꢁ4
5
0

14
A.V. Silva-Ortiz et al. / Journal of Steroid Biochemistry & Molecular Biology 159 (2016) 8–18
Fig. 4. Regulation of CCND1 and CCNE1 gene expression by I–OH. The PC-3 (A, B) and MCF7 (C, D) cells were incubated with different concentrations of I–OH. The mRNA was
extracted and the qPCR was performed. Data are expressed as mean ꢃ S.D. of three independent experiments per triplicate. Values were normalized by
b-actin mRNA used as
housekeeping gene. Data were normalized to 1 for vehicle-treated cells (V). *P ꢄ 0.01 vs. V.
properties. In order to rationalize and visualize at molecular level
the importance of the structural features of T–OH and I–OH and
their implication in ligand-protein interaction, both compounds
were docked into PR, AR and VDR model. The VDR was included
because the synthesized compounds in this work also share a
geometry, hydrogen bonding, binding free energy and structural
chemical similitudes with the calcitriol (1,25-dihydroxyvitamin
Interestingly, the analyses using VDR demonstrated that both
compounds were docked into the VDR ligand binding domain, with
similar interactions that the calcitriol. The interactions observed
are based in hydrogen bonds with TYR 143, HIS 305 and HIS
397 residues. Strong interactions are considered those with less
than 4 angström (Å) between atoms, suggesting an interaction
with the VDR in live systems.
D
3
), the hormonally active form of vitamin D [32].
In Table 3 is presented the lowest binding energy and cluster
3.5. Transfection studies on HeLa cell line
size conformation using AutoDock and Glide Score of the
compounds and the natural ligands of each receptor.
To address the question as to whether I–OH elicits agonistic or
As expected, the binding energies for P
4
, 5a
-DHT and calcitriol
antagonistic actions comparable to those previously observed for
showed highly affinity for their specific receptors as previously was
reported [33,34] but not for the others compounds. Likewise, the
Glide was a more effective tool to visualize and compare the
binding energies between the natural ligands with its receptor.
Generally, T–OH and I–OH into PR, AR and VDR had similar
4
P and 5a-DHT or RU486 and FLUT respectively, we used in vitro
reporter gene assays. In general, results showed that I–OH did not
stimulate reporter gene transcription, which discarded the
progestagenic (Fig. 6A) and androgenic activity by I–OH (data
4
not shown). However, I–OH partially inhibited the P effect on the
energy but minor clusters size compared with P
4
, 5a
-DHT and
transcriptional activity indicated an antiprogestagenic effect
calcitriol, respectively. The Fig. 5 shows the proposed binding
models for these binding energies. The results suggested that the
compounds induce conformational changes on PR and these
(Fig. 6B) whereas no effect of I–OH was observed upon reporter
gene activation induced by 5a-DHT (data not shown). These
results indicated that I–OH did not act as antiandrogenic
compound.
4
modifications possibly block P interactions with the GLN725
amino acid in the B chain [25]. On the other hand, none compound
binds suitably in the binding pocket of the AR.
Unfortunately, we do not have VDR plasmid, therefore gene
CYP24A1 expression was evaluated which is a gene highly induced
Table 3
Binding energies and cluster size of T–OH and I–OH in the PR, AR and VDR model.
PR
AR
VDR
Compound
Glide score
AutoDock
Cluster Glide score
AutoDock
Cluster Glide score
AutoDock
Cluster size
D
G
bind (Kcal/mol)
D
G
bind (Kcal/mol) size
D
Gbind (Kcal/mol)
D
G
bind (Kcal/mol) size
DG
bind (Kcal/mol)
D
G
bind (Kcal/mol)
a
a
a
P
5
4
ꢁ12.2
ꢁ5.8
ꢁ5.2
ꢁ7.3
ꢁ7.4
ꢁ9.42
ꢁ6.86
ꢁ6.25
ꢁ8.45
ꢁ7.47
17
4
3
7
6
ꢁ5.8
ꢁ10.8
ꢁ5.1
ꢁ6.94
ꢁ8.99
5
21
3
9
8
ꢁ8.1
ꢁ7.5
ꢁ9.28
ꢁ8.68
11
11
17
8
a
a
a
a
a
-DHT
a
a
a
a
Calcitriol
T–OH
I–OH
ꢁ6.21
ꢁ6.37
ꢁ13.3
ꢁ9.8
ꢁ8.5
ꢁ9.91
a
ꢁ5.4
ꢁ7.4
ꢁ10.63
a
ꢁ8.23
ꢁ9.61
7
a
Lowest energy conformation for each compound.

A.V. Silva-Ortiz et al. / Journal of Steroid Biochemistry & Molecular Biology 159 (2016) 8–18
15
Fig. 5. Molecular docking studies that show binding interactions of T–OH and I–OH with hormonal receptors. PR: A) P
4
, B) P
4
on chain A and chain B of PR, C) T–OH and D) I–
OH on PR in B chain; AR: E) 5
a-DHT, F) T–OH and G) I–OH; VDR: H) Calcitriol, I) T–OH, J) I–OH. A, D and G were the natural ligands used as controls to compare the energies
and the spatial distribution.
by calcitriol. However, I–OH was incapable to induce the mRNA of
this enzyme (data not shown).
process to be used as anticancer treatment, and the steroids are not
the exception [10–17,35–37]. Particularly, Bratoeff E, et al., showed
that a synthetic compound from dehydroepiandrosterone with an
ester moiety at C-3 and an imidazole group at C-17 (16-formyl-17-
4. Discussion
(
1H-imidazol-1-yl) androsta-5,16-diene-3
biological activity of -reductase type
proliferation of the human prostate cancer cell line; PC-3 [15].
b
-yl) inhibited both the
Over the last years, a large number of molecules have been
modified and synthetized as potential inhibitors of proliferative
5
a
1
as well as cell

16
A.V. Silva-Ortiz et al. / Journal of Steroid Biochemistry & Molecular Biology 159 (2016) 8–18
4
Fig. 6. Antiprogestagenic activity of I–OH. HeLa cells transfected were incubated in absence or presence of different concentrations of I–OH, P and RU486 (A). Cells were
ꢁ
8
treated with P
4
(1 ꢂ10 M) alone or plus increased concentration of I–OH or RU486 (B). The results are the mean ꢃ S.D. of the transactivation of PR-B of three independent
experiments per triplicate. *P ꢄ 0.01 vs. 0.
Similarly, Banday A. et al., reported a series of novel D-ring
substituted 1,2,3-triazolyl 20-keto pregnenane derivatives. Among
these compounds 21-{4-[(4-acetylphenoxy) methyl]-1H-1,2,3-
triazol-1-yl}-3-hydroxypregn-5-en-20-one was the most active,
especially against proliferation of DU-145 and PC-3 cell lines [17].
Recently, was reported the synthesis and biological activity of new
CCND1 complex, and hyperphosphorylation of the retinoblastoma
protein as well as affects the regulation of the progression through
the restriction point R at the end of the G1-phase to allow cells to
enter S-phase [43,44]. On the other hand, Ki-67, EAG1, BIM and
survivin genes were not significantly altered, which indicates that
these proteins are not involved in the antiproliferative effect by I–
OH. Collectively, this study proposes that I–OH affects directly the
cell cycle progression of both MCF7 and PC-3 cells. In the case of
SK-LU-1, none gene studied was significantly modified, thus others
molecular targets deserve further investigation to this cell type.
Lastly, to elucidate the potential interaction between T–OH and
I–OH with hormonal receptors we use molecular docking
simulations. Results suggest that these synthetic compounds bind
to the hormone binding pocket into PR and induce conformational
changes to interfere with natural agonist activity by competitive
binding and thus have antagonist action [45]. Indeed, the
experimental results from transfection assays with the PR in HeLa
cells showed weak antiprogestagenic properties by I–OH. The
functional significance of this is unknown but has been showed
16-dehydropregnenolone derivatives with a triazole moiety at C-
21 [16]. Herein, we compared two compounds that contain each
one a triazole and imidazole moiety at C-21, respectively. As
expected, these compounds showed antiproliferative effects on
PC-3, MCF-7 and SK-LU-1 cancer cell lines. Interestingly, I–OH
compound had higher antiproliferative activity than T–OH,
showing a preferential inhibition upon PC-3 cells. These results
show that the imidazole moiety into the molecular structure of the
steroid improved its biological activity.
In addition, we demonstrated that CCND1 and CCNE1 gene
were downregulated by I–OH treatment in PC-3 and MCF7 cells.
These cyclins are key components of the core cell cycle machinery;
CCND1 is a key positive regulator of the G1-S phase transition and
its overexpression is implicated in genesis and progression of some
neoplasms [38]. Likewise, it has been reported that upregulation
CCND1 might be related to the evolution of androgen-independent
disease in prostate cancer [39,40]. In the same way, the
CCNE1 protein is tightly related as an endpoint of several
regulatory pathways that are critical for growth control and
frequently are altered in cancer cells. CCNE1 in conjunction with its
kinase subunit Cdk2 regulates essential processes at the G1–S-
phase boundary of the cell cycle. CCNE1 gene is undetectable in
G0 and G1 phases of the cell cycle, but it rises sharply during of
time that precedes each entry into S phase [41,42].
4
that P and some synthetic derivatives had a proliferative action in
some cancers and inducing the gene CCND1 expression. In fact, in
the progression from normal to malignant breast cells, the
expression of the two PR isoforms (A and B) is altered, and more
aggressive breast cancers are associated with a predominance of
4
either one of isoforms [46]. Contrary to P , some synthetic
pregnane derivates abolished the hyper-proliferative process and
downregulated the cyclins expressions. It is possible that T–OH
and I–OH act as antagonist of PR blocking cyclins activation during
cancer, which deserves further study.
Conversely, the transfection assays with the AR, I–OH did not
On the basis of these findings, we suggest that I–OH avoids the
accumulation and activity of cyclin dependent kinase 4 (Cdk4)/
have androgenic or antiandrogenic effect compared with 5
or FLUT respectively. These results demonstrated that effect of I–
a-DHT

A.V. Silva-Ortiz et al. / Journal of Steroid Biochemistry & Molecular Biology 159 (2016) 8–18
17
OH is AR-independent pathway and this corresponds with the
Appendix A. Supplementary data
effect observed in the PC-3 cells that is lacking of AR and was the
most sensitive cell line for this treatment.
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
jsbmb.2016.02.013.
Considering, that the synthesized compounds also share
structural and chemical similitudes with the calcitriol, a hormone
with antiproliferative effects, that use VDR signaling to affect
target genes in multiple cancer cells. Additionally, we complete
docking study using the VDR. Although the cluster size is lower,
these values indicated a possible interaction with the pocket
domain into VDR. Nonetheless, the experimental result showed
that I–OH was not able to induce the mRNA expression of CYP24A1,
a gene highly inducible by calcitriol via VDR, which discards totally,
that the antiproliferative effect uses the VDR pathway. Taken
together, these data are consistent with previous computer-based
quantitative structure-activity relationship studies of ligand-
receptor interactions [33,34].
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compounds can also be attributed to the nature of the azole groups
[
6] D.Q. Li, Z.B. Wang, J. Bai, J. Zhao, Y. Wang, K. Hu, Y.H. Du, Effects of mifepristone
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[
10,12,47]. These groups are highly soluble in water and are
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Acknowledgments
This work was supported by the National Council of Science and
Technology,CONACyT Mexico (grant number 165049; 154215) and
DGAPA project IN 211312 (UNAM), thank for their generous
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