Synthesis and activity of novel 16-dehydropregnenolone acetate derivatives as inhibitors of type 1 5α-reductase and on cancer cell line SK-LU-1

Article abstract of DOI:10.1016/j.bmc.2015.10.047

Testosterone (T) plays a crucial role in prostate growth. In androgen-dependent tissues T is reduced to dihydrotestosterone (DHT) because of the presence of the 5α-reductase enzyme. This androgen is more active than T, since it has a higher affinity for the androgen receptor (AR). When this mechanism is altered, androgen-dependent diseases, including prostate cancer, could result. The aim of this study was to synthesize several 16-dehydropregnenolone acetate derivatives containing a triazole ring at C-21 and a linear or alicyclic ester moiety at C-3 of the steroidal skeleton. These steroids were designed as potential inhibitors of the activity of both types (1 and 2) of 5α-reductase. The cytotoxic activity of these compounds was also evaluated on a panel of PC-3, MCF7, and SK-LU-1 human cancer cell lines. The results from this study showed that with the exception of steroids 20-oxo-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3β-yl-propionate and 20-oxo-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3β-yl-pentanoate, the compounds exhibit a lower inhibitory activity for both isoenzymes of 5α-reductase than finasteride. Furthermore the 3β-hydroxy-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-20-one and 20-oxo-21-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3β-yl-acetate derivatives display 80% cytotoxic activity on the SK-LU-1 cell line. These results also indicated that the triazole derivatives, which have a hydroxyl or acetoxy group at C-3, could have an anticancer effect, whereas the derivatives with a alicyclic ester group at C-3 do not show biological activity.

Full text of DOI:10.1016/j.bmc.2015.10.047

Accepted Manuscript
Synthesis and activity of novel 16-dehydropregnenolone acetate derivatives as
inhibitors of type 1 5α-reductase and on cancer cell line SK-LU-1
Aylin Viviana Silva-Ortiz, Eugene Bratoeff, Teresa Ramírez-Apan, Yvonne
Heuze, Araceli Sánchez, Juan Soriano, Marisa Cabeza
PII:
S0968-0896(15)30123-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.10.047
BMC 12648
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
26 August 2015
30 October 2015
31 October 2015
Please cite this article as: Silva-Ortiz, A.V., Bratoeff, E., Ramírez-Apan, T., Heuze, Y., Sánchez, A., Soriano, J.,
Cabeza, M., Synthesis and activity of novel 16-dehydropregnenolone acetate derivatives as inhibitors of type 1
5α-reductase and on cancer cell line SK-LU-1, Bioorganic & Medicinal Chemistry (2015), doi: http://dx.doi.org/
1
0.1016/j.bmc.2015.10.047
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1
Synthesis and activity of novel 16-dehydropregnenolone acetate derivatives as
inhibitors of type 1 5α-reductase and on cancer cell line SK-LU-1
a
†
b
c
Aylin Viviana Silva-Ortiz , Eugene Bratoeff , Teresa Ramírez-Apan , Yvonne Heuze ,
c
d
c,
Araceli Sánchez , Juan Soriano , Marisa Cabeza *.
a
Departamento de Farmacia, Facultad de Química, Universidad Nacional Autónoma de
México, Av. Universidad 3000, Copilco Universidad, Coyoacán, 04510 Ciudad de México,
Distrito Federal, México D.F.
b
Intituto de Quimica, Universidad Nacional Autónoma de México, Av. Universidad 3000,
Copilco Universidad, Coyoacán, 04510 Ciudad de México, Distrito Federal, México D.F.
c
Departamento de Sistemas Biológicos y de Producción Agrícola y Animal, Universidad
Autónoma Metropolitana-Xochimilco, Calzada del Hueso 1100. Col. Villa Quietud, 04960
Ciudad de México. México, D. F.
d
Departamento de Patología, Hospital General de México, Dr. Balmis 148, México, D. F.
ASO: aylinsilva@hotmail.es
TRA: mtrapan@unam.mx
ASM: ara_sm89@live.com.mx
EB: eugene@unam.mx
YH: ymheuze@gmail.com
JS: juansorianor@hotmail.com
MC: marisa@correo.xoc.uam.mx
*
Address correspondence to:
Marisa Cabeza Ph.D.
Departamento de Sistemas Biológicos
Universidad Autónoma Metropolitana-Xochimilco
Calzada Del Hueso No. 1100
México, D.F., C.P. 04960, México
Phone: [011-52-55] 5483-72-60
Fax: [011-52-55] 5483-72-60
E-mail: marisa@correo.xoc.uam.mx

2
Abstract
Testosterone (T) plays a crucial role in prostate growth. In androgen-dependent
tissues T is reduced to dihydrotestosterone (DHT) because of the presence of the 5α-
reductase enzyme. This androgen is more active than T, since it has a higher affinity for the
androgen receptor (AR). When this mechanism is altered, androgen-dependent diseases,
including prostate cancer, could result.
The aim of this study was to synthesize several 16-dehydropregnenolone acetate
derivatives containing a triazole ring at C-21 and a linear or alicyclic ester moiety at C-3 of
the steroidal skeleton. These steroids were designed as potential inhibitors of the activity of
both types (1 and 2) of 5α-reductase. The cytotoxic activity of these compounds was also
evaluated on a panel of PC-3, MCF7, and SK-LU-1 human cancer cell lines.
The results from this study showed that with the exception of steroids 20-oxo-21-
(
1H-1,2,4-triazole-1-yl)pregna-5,16-dien-3β-yl-propionate
and
20-oxo-21-(1H-1,2,4-
triazole-1-yl)pregna-5,16-dien-3β-yl-pentanoate, the compounds exhibit a lower inhibitory
activity for both isoenzymes of 5α-reductase than finasteride. Furthermore the 3β-hydroxy-
2
1-(1H-1,2,4-triazole-1-yl)pregna-5,16-dien-20-one and 20-oxo-21-(1H-1,2,4-triazole-1-
yl)pregna-5,16-dien-3β-yl-acetate derivatives display 80% cytotoxic activity on the SK-
LU-1 cell line.
These results also indicated that the triazole derivatives, which have a hydroxyl or
acetoxy group at C-3, could have an anticancer effect, whereas the derivatives with a
alicyclic ester group at C-3 do not show biological activity.

3
Keywords
Synthesis of derivatives dehydropregnenolone, 5α-reductase 1 inhibitors, antiproliferative
activity on SKLU-1 cells

4
1
. Introduction
Steroids are transcription factors that regulate gene expression; these messengers are
able to cross the plasma membrane and bind to specific receptors. The complex comprising
the steroid receptor has a direct interaction with DNA, altering gene expression. The result
of this process induces growth and differentiation of its target tissues, so any alteration in
this mechanism can cause deregulation of the expression, resulting in the modification of
several signaling pathways and therefore of functions. [1-2]
It is known that in some metastatic tumors such as lung adenocarcinoma as well as
in cancer cell line SK-LU-1, gene CYP24A1 is highly expressed. [3] This gene is a member
of the CYP450 superfamily, which codes for 24 hydroxylase enzyme. [3] It is also
recognized that the expression of glyoxalase enzyme genes is regulated by testosterone (T)
and that altered expression of genes encoding this glyoxalase is present in various types of
cancerous tumors of the prostate. [4] Furthermore, androgens also induce the development
of benign prostatic hyperplasia (BPH). Both cancer and BPH have also been associated
with an increase of dihydrotestosterone (DHT) in this gland. [4] T is converted to DHT by
the action of 5α-reductase (5α-R) isoenzymes. There are three 5α-reductase isoenzymes in
prostate tissue. Type 1 5α-R (5α-R1) is expressed in the prostate epithelial cells and is
related to the development of prostate cancer. [5] Type 2 5α-R (5α-R2) is associated with
benign prostatic hyperplasia (BPH) [1], and type 3 5α-R (5α-R3) is found in the brain and
pancreas, and is related to hormone-refractory prostate cancer (HRPC) [6, 7] Each is
encoded by a different gene; these have been characterized in several species. [8]

5
Therefore the development of new inhibitors of enzymes involved in the synthetic
routes of steroidal hormones and antagonists of steroidal receptors could improve current
anticancer therapies. [9, 10]
The cytotoxic effect of dehydroepiandrosterone derivatives with a triazole moiety
and an aromatic ester at C-3 was demonstrated by our group. [9] These derivatives
displayed high cytotoxic activity in cell lines PC-3, MCF7 and SK-LU-1. We also
demonstrated that several progesterone derivatives inhibited the activity of both types (1
and 2) of 5α-R. These steroids also showed a high pharmacological activity inhibiting
prostate growth. [10]
Based on these results we decided to study the synthesis and biological activity of a
series of 21-(1H-1,2,4-triazole-1-yl)-5,16-pregnadiene-20-one-3β-yl derivatives (9a-j).
Their biological activity was evaluated by measuring their antiproliferative effect in a panel
of human cancer cell lines; PC-3 (prostate), MCF7 (breast) and SK-LU-1 (lung). In
addition we also evaluated their effect as inhibitors of the activity of 5α-R1 and 5α-R2 and
their binding capacity to the androgen receptor (AR). The pharmacological effect of these
pregnane derivatives was evaluated in an animal model.
In the cancer cell lines tested, ketoconazole 10 (Fig. 1) was used as a positive
control. This compound is an antimycotic drug and an agent that inhibits cytochrome P450
enzymes, which are required for the synthesis of androgens. [11]

6
Figure 1. 16-dehydropregnenole acetate (1), Ketoconazole (10), Finasteride (11)
and Mibolerone (12).
2
2
2
. Material and methods
.1. Chemical and radioactive materials
.1.1. Reagents
Solvents were purchased from commercial sources and used without further
purification. Melting points were determined on a Fisher Johns melting point apparatus and
are uncorrected. IR spectra were recorded on a Perkin-Elmer 200 spectrometer. The UV
1
13
lamp (254 nm) was from UVP. H and C NMR were taken on a Varian VRX-400
1
13
spectrometer operating at 400 ( H) and 100 ( C) MHz with TMS as internal standard (δ=0)
in CDCl (the abbreviations of signal patterns are as follows: s, singlet; d, doublet, t, triplet;
3
m, multiplet). High resolution mass spectra (HRMS) were obtained with a Thermo DFS
+
spectrometer by direct infusion and using FAB ionization mode.

7
3
3
(
1,2,6,7- H) Testosterone [ H] T specific activity: 95 Ci/mmol and Mibolerone
3
3
(
17a-methyl- H) [ H] MIB 10 specific activity 70–87 Ci/mmol were provided by Perkin
Elmer Life and Analytical Sciences (Boston, MA).
Radioinert T, 5α-DHT and MIB were supplied by Steraloids (Wilton, NH, USA).
Sigma Chemical Co. (St. Louis, MO) provided NADPH. Finasteride was obtained by
extraction from Proscar® (Merck, Sharp & Dohme). The tablets were crushed, extracted
with chloroform and the solvent was eliminated in vacuum; the crude product was purified
by silica gel 60 (63–200 µm) column chromatography (Sigma–Aldrich) with ethyl acetate
as eluant. The melting point of the isolated finasteride (252–254 °C) was identical to that
reported in the literature.
The ketoconazole used as a reference compound was purchased from Sigma Life Sciences.
2
.1.2. Synthesis of derivates of the 16-dehydropregnenolone acetate (8, 9a−j)
The synthetic pathway for the preparation of compounds 8 and 9a−j are outlined in
Fig. 2. These steroids were prepared from the commercially available 16-
dehydropregnenolone acetate (1). The synthesis of these novel compounds is described
below.
2
.1.2.1.
Synthesis of 16,17-epoxy-3-hydroxypregn-5-en-20-one 2. A solution
30 % (4
the steroid 1 (1 g, 2.82 mmol), sodium hydroxide 4 N (2 mL, 8 mmol) and H
2
O
2
mL, 135 mmol) in hot methanol (66 mL) was stirred at room temperature for 4 h. After this
time the methanol was evaporated and the product was washed with water, obtaining a
white solid.

8
-1
1
Yield 0.665 g (72%), m.p. 197–199 °C, IR (KBr) cm : 3457, 1692, 1642 and 1042. H
RMN (400 MHz, CDCl ) δ: 1.00 (s, H-18, 3H), 1.03 (s, H-19, 3H), 2.01 (s, H-21, 3H), 2.29
s, H-16, 1H), 3.47 (m, J = 3.4 Hz, H-3, 1H), 3.66 (s, OH, 1H), 5.32 (t, J = 5.3 Hz, H-6,
3
(
1
3
1
1
H). C RMN (100 MHz, CDCl
6), 71.16 (C-17), 71.75 (C-3), 121.13 (C-6), 141.27 (C-5), 205.07 (C-20). HRMS cal. for
330.2195, found 330.2187.
3
) δ: 15.38 (C-18), 19.30 (C-19), 27.51 (C-21), 60.55 (C-
C
21
H
30
O
3
Fig. 2. Reagents and conditions: i) H
DMF; iii) NaOH, C I(OAc) , MeOH; iv) SOCl
triazole, K CO , 80 °C, 5 h; vi) HCl, acetone; vii) CrCl
acetic anhiydride, Py; viii b-e) R-COOH, DCC, DMAP, CHCl
2
O
2
, NaOH 4N; ii) TBDMS, imidazole,
, Py, CH Cl ; v) 1,2,4-
, Ac. Acetic; viii a)
6
H
5
2
2
2
2
2
3
2
3
.

9
2
.1.2.2.
Synthesis of 3-{[tert-butyl(dimethyl)silyl]oxy}-16,17-epoxypregn-5-
en-20-one 3. To a solution of steroid 2 (1 g, 3.02 mmol), t-butyl dimethylsilylbchloride
0.75 g, 5.51 mmol) and imidazole (0.488g, 7.17 mmol) in dry DMF (15 mL) were added.
(
The solution was stirred at room temperature for 2 h. When the reaction was finished, the
DFM was evaporated at vacuum and the product was washed with water, obtaining a white
solid.
-1
Yield 1.17 g (87%), m.p. 118–120 °C, IR (KBr) cm : 2935, 1704, 1642, 1698 and 1083.
1
H RMN (400 MHz, CDCl
3
) δ: -0.051 (s, CH
3
of protector group, 6H), 0,048 (s, CH of
3
protector group, 9H), 0.88 (s, H-18, 3H), 1.00 (s, H-19, 3H), 2.02 (s, H-21, 3H), 2.25 (s, H-
1
3
1
6, 1H), 3.47 (m, J = 3.4 Hz, H-3, 1H), 5.29 (1H, d, J = 5.2 Hz, H-6),. C RMN (100 MHz,
CDCl ) δ: -4.33 (CH of protector group) 15.40 (C-18), 19.32 (C-19), 27.70 (C-21), 26.08
of protector group), 60.57 (C-16), 71.11 (C-17), 72.55 (C-3), 120.43 (C-6), 141.93
C-5), 205.08 (C-20). HRMS cal. for C27 Si 444.3060, found 444.3052.
Synthesis of 3β-{[tert-butyl(dimethyl)silyl]oxy}-20,20-dimethoxy-16α,
7α-epoxypregn-5-en-21-ol 4. To a solution of steroid 3 (1 g, 2.2 mmol), (diacetoxyiodo)
3
3
(
(
CH
3
H
44
O
3
2
.1.2.3.
1
benzene (1.2 g, 3.72 mmol) and sodium hydroxide (1 g, 25 mmol) in methanol (21 mL) and
dichloromethane (10 mL) were added, and stirred at room temperature overnight. The
solvent was evaporated under reduced pressure. Finally the purification was made with a
basic aluminum column chromatography using mixture of hexane and ethyl acetate (95:5).
-1
Yield 0.959 g (84 %), m.p. 215-218 °C, IR (KBr) cm : 3597, 2930, 1666, 1070 and 1033.
1
H RMN (400 MHz, CDCl
3
) δ: -0.051 (s, CH
3
of protector group, 6H), 0.048 (s, CH of
3
protector group, 9H), 0.88 (s, H-18, 3H), 1.01 (s, H-19, 3H), 2.25 (s, H-16, 1H), 3.29 (s,

10
1
3
OCH
3
, 6H), 3.44 (m, J = 3.6, H-3, 1H), 3.69 (s, OH, 1H), 5.30 (d, J = 5.3 Hz, H-6, 1H). C
RMN (100 MHz, CDCl
3
) δ: -4.33 (CH
3
of protector group), 15.30 (C-18), 19.66 (C-19),
), 62.73 (C-21), 69.78 (C-16), 72.50 (C-3),
2
1
5
2
6.08 (CH
3
of protector group), 49.51 (OCH
3
01.99 (C-20), 120.74 (C-6), 141.86 (C-5). HRMS cal. for C29
06.3423.
H
50
5
O Si 506.3428, found
.1.2.4.
Synthesis
of
3-{[tert-butyl(dimethyl)si1yl]oxy}-21-chloro-20,20-
dimethoxy-16,17-epoxypregn-5-ene 5. To a cold solution of steroid 4 (1 g, 1.97 mmol)
in a mixture of dry dicholoromethane (8 mL) and pyridine (1 mL) thionyl chloride (0.25
mL) was added dropwise under nitrogen atmosphere and stirred at room temperature for 30
min. Chloroform (100 mL) was added and it was washed two times with 10% aqueous
hydrochloric acid, aqueous sodium bicarbonate and water. It was dried over sodium sulfate
and the solvent was evaporated in vacuum. The crude product was recrystallized from
methanol.
-1
1
Yield 0.830 g (80%), m.p. 150–152 °C, IR (KBr) cm : 2930, 1668, 1088 and 1033. H
RMN (400 MHz, CDCl ) δ: -0.051 (s, CH of protector group, 6H), 0.048 (s, CH of
protector group, 9H), 0.88 (s, H-18, 3H), 1.00 (s, H-19, 3H), 2.24 (s, H-16, 1H), 3.24 (s,
OCH , 6H), 3.46 (m, J = 3.4 Hz, H-3, 1H), 4.01 (s, 2H, H-21), 5.28 (d, J = 5.2 Hz, H-6,
3
3
3
3
1
3
1
H). C RMN (100 MHz, CDCl
C-19), 26.08 (CH of protector group), 49.87 (OCH
C-3), 101.96 (C-20), 121.18 (C-6), 142.08 (C-5). HRMS cal. for C29
3
) δ: -4.33 (CH
3
of protector group), 15.99 (C-18), 18.83
), 59.57 (C-21), 69.50 (C-16), 72.96
Si 524.3089,
(
(
3
3
H49ClO
4
found 524.3093.
2
1
.1.2.5.
Synthesis
of
3-{[tert-butyl(dimethyl)silyl]oxy)}-20,20-dimethoxy-
6,17-epoxy-21-(1H-1,2,4-triazol-1-yl)pregn-5-ene 6. A mixture of 1,2,4-triazole

11
(
0.069 g, 1.0 mmol), K
2
CO
3
(0.263 g, 1.90 mmol) and the corresponding compound 5 (1 g,
atmosphere for 4
1
.90 mmol) dissolved in dry DMF (5 mL) was heated at 90 °C under N
2
h. Cold water was added and the resulting solid was filtered. The compound was purified
by a column chromatography with florisil.
-1
1
Yield 0.753 g (71%), m.p. 188–190 °C, IR (KBr) cm : 2929, 1616, 1086 and 1058. H
RMN (400 MHz, CDCl ) δ: -0.051 (s, CH of protector group, 6H), 0.048 (s, CH of
protector group, 9H), 0.88 (s, H-18, 3H), 1.00 (s, H-19, 3H), 2.30 (s, H-16, 1H), 3.24 (s,
OCH , 6H,), 3.48 (m, J = 3.3 Hz, H-3, 1H), 4.01 (s, 2H, H-21), 5.29 (d, J = 5.3 Hz, H-6,
H,), 7.47 (s, H-Het., 1H), 7.84 (s, H-Het., 1H) . C RMN (100 MHz, CDCl
CH of protector group),15.20 (C-18), 18.03 (C-19), 26.08 (CH of protector group), 49.40
OCH ), 60.14 (C-21), 68.99 (C-16), 71.99 (C-17), 72.65 (C-3), 101.77 (C-20), 121.08 (C-
), 121.60 (C-Het.), 141.44 (C-Het.), 142.25 (C-5). HRMS cal. for C31 Si 557.3649,
found 557.3645
3
3
3
3
1
3
1
3
) δ: -4.84
(
(
3
3
3
6
H
51
N
3
O
4
2
5
.1.2.6.
Synthesis of 16,17-epoxy-3-hydroxy-21-(1H-1,2,4-triazol-1-yl)pregn-
-en-20-one 7. This compound was prepared from compound 6 (0.5 g, 0.89 mmol). The
hydrolysis was carried out in acetone (1.5 mL) with 36% chlorhydric acid (1.3 mL), at
room temperature 1h. The solvent was evaporated under reduced pressure and the crude
product was recrystallized from methanol.
-1
Yield 0.275 g (77%), m.p. 155–157 °C, IR (KBr) cm : 3346, 2928, 1724, 1378, 1274 and
1
1
3
051. H RMN (400 MHz, CDCl
3
) δ: 0.90 (s, H-18, 3H), 1.20 (s, H-19, 3H), 3.24 (m, J =
.5, H-3, 1H), 3.51 (s, OH, 1H), 3.68 (s, H-16, 1H), 4.18 (s, 2H, H-21), 5.33 (d, J = 5.3 Hz,
1
3
H-6, 1H), 7.51 (s, H-Het, 1H), 7.70 (s, H-Het, 1H). C RMN (100 MHz, CDCl ) δ: 15.32
3

12
(
C-18), 19.56 (C-19), 62.26 (C-21), 71.74 (C-3), 121.04 (C-6), 121.2 (C-Het.), 131.2 (C-
Het.), 141.24 (C-5), 206,08 (C-20). HRMS cal. for C23 397.2365, found 397.2369.
H
31
N
3
O
3
2
.1.2.7.
Synthesis of 3-hydroxy-21-(1H-1,2,4-triazol-1-yl)pregna-5,16-dien-20-
one 8. A solution of steroid 7 (0.5 g, 1.25 mmol) and chromous chloride (0.4 g, 3.25 mmol)
in acetic acid (20 mL) was stirred at room temperature for 30 min. The mixture was diluted
with cold water (150 mL) and the precipitate was filtered and dried. The product was
purified by silica gel column chromatography using a mixture of hexane and ethyl acetate
(
9:1). From this compound, compounds (9a–j) were derived.
-
1
Yield 0.370 g (77%), m.p. 120–122, IR (KBr) cm : 3355, 2927, 1709, 1670, 1276 and
1
1
1
1
1
1
051. H RMN (CDCl
3
) δ: 0.90 (s, H-18, 3H), 1.20 (s, H-19, 3H), 3.25 (m, J = 3.5 Hz, H-3,
H), 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,
13
H),7.50 (s, H-Het., 1H), 7.70 (s, H-Het., 1H). C RMN (100 MHz, CDCl
8), 20.01 (C-19), 62.15 (C-21), 71.78 (C-3), 121.04 (C-6), 141.23 (C-5), 141.38 (C-16),
41.47 (C-Het.), 144.83 (C-Het.), 196.66 (C-20). HRMS cal. For C23 381.2416,
3
) δ: 15.32 (C-
H
31
N
3
O
3
found 381.2420.
.1.2.8. Preparation of 20-oxo-21-(1H-1,2,4-triazol-1-yl)pregna-5,16-dien-3β-yl
2
acetate 9a. The synthesis of this compound was conducted from a solution of steroid 8 (0.1
g, 2.62 mmol), pyridine (0.4 mL) and acetic anhydride (0.4 mL). The resulting solution was
stirred at room temperature for 1 h. A solution of water and salt was added and the
precipitated was filtered. The compound was washed with water 3 times.
-
1
1
Yield 0.10 g (90%), m.p. 119–121 °C, IR (KBr) cm : 2927, 1731, 1270 and 1087. H RMN
(
40 MHz, CDCl ) δ: 1.15 (s, H-18, 3H), 1.30 (s, H-19, 3H), 2.03 (t, ester-H-2′, 3H), 3.25
3

13
(
m, J = 3.2 Hz, H-3, 1H), 4.19 (s, 2H, H-21), 5.34 (d, J = 5.3 Hz, H-6, 1H), 6.74 (s, J = 6.7,
1
3
C-16, 1H), 8.01 (s, H-Het., 1H), 8.30 (s, H-Het., 1H). C RMN (100 MHz, CDCl
3
) δ:
5.30 (C-18), 19.40 (C-19), 21.3 (C-2′ of ester), 65.50 (C-21), 73.61 (C-3), 121.97 (C-6),
29.62 (C-16), 140.02 (C-Het.), 141.24 (C-5), 148.32 (C-Het.), 156.57 (C-17), 173.46
423.2522, found 423.2525.
.1.2.9. General procedure for obtention of 20-oxo-21-(1H-1,2,4-triazol-1-
1
1
(
ester carbonyl), 201.02 (C-20). HRMS cal. for C25
H
33
N
3
O
3
2
yl)pregna-5,16-dien-3β-ylpropionate (9b) to 20-oxo-21-(1H-1,2,4-triazol-1-yl)pregna-
,16-dien-3β-ylcycloheptanecarboxylate (9j).
To a solution of steroid 8 (0,1 g, 0.262 mmol), DCC (0.110 g, 0.524 mmol) and DMAP
0.064 g, 0.524 mmol) in chloroform (3 mL) the corresponding acid (0.6 mmol) was stirred
5
(
at room temperature for 2 h. Ethyl acetate (8 mL) was added and the precipitated
dicyclohexyl urea was filtered. The organic phase was washed three times with 10%
aqueous hydrochloric acid, 5% aqueous sodium bicarbonate, water and was dried over
anhydrous sodium sulfate. The crude ester was recrystallized from chloroform-methanol.
2
.1.2.9.1. 20-oxo-21-(1H-1,2,4-triazol-1-yl)pregna-5,16-dien-3β-yl-propionate 9b. Yield
-
1
1
0
.097 g (85%), m.p. 119–121 °C, IR (KBr) cm : 2927, 1728, 1571, 1270 and 1087. H
) δ: 0.90 (s, H-18, 3H), 1.03 (s, H-19, 3H), 1.3 (t, H-3′ of ester,
H), 2.1 (q, H-2′ of ester, 2H), 3.25 (m, J = 3.2, H-3, 1H), 4.19 (s, 2H, H-21), 5.34 (d, J =
.3 Hz, H-6, 1H), 6.74 (s, J = 6.7 Hz, C-16, 1H), 8.02 (s, H-Het., 1H), 8.30 (s, H-Het., 1H).
RMN (400 MHz, CDCl
3
3
5
1
3
C RMN (100 MHz, CDCl
), 121.97 (C-6), 129.62 (C-16), 140.02 (C-Het.), 141.24 (C-5), 148.32 (C-Het.), 156.57
437.2678,
3
) δ: 8.30 (C-3′ of ester), 10.3 (C-18), 12.03 (C-19), 73.61 (C-
3
(
C-17), 173.48 (ester carbonyl), 201.02 (C-20). HRMS cal. for C26
H
35
N
3
O
3
found 437.2673.

14
2
.1.2.9.2. 20-oxo-21-(1H-1,2,4-triazol-1-yl)pregna-5,16-dien-3β-yl-butirate 9c. Yield
-
1
1
0
.095 g (80%), m.p. 202–205 °C, IR (KBr) cm : 2927, 1732, 1575, 1270 and 1087. H
) δ: 0.90 (t, ester-H-4′, 3H), 1.03 (s, H-18, 3H), 1.20 (s, H-19, 3H),
.20 (q, ester-H-3′, 2H), 3.25 (m, J = 3.2 Hz, H-3, 1H), 4.20 (s, 2H, H-21), 5.34 (d, J = 5.3
RMN (400 MHz, CDCl
3
2
1
3
Hz, H-6, 1H), 6.74 (s, J = 6.7 Hz, C-16, 1H), 7.51 (s, H-Het., 1H), 7.84 (s, H-Het., 1H). C
RMN (100 MHz, CDCl ) δ: 10.1 (C-3′ of ester), 12.32 (C-18), 15.20 (C-19), 18.6 (C-4′ of
ester), 67.01 (C-21), 73.61 (C-3), 121.97 (C-6), 129.62 (C-16), 140.02 (C-Het.), 142. 2 (C-
), 148.32 (C-Het.), 156.57 (C-17), 173.48 (ester carbonyl), 201.02 (C-20). HRMS cal. for
3
5
C
27
H
37
N
3
O
3
451.2835, found 451.2830.
2
.1.2.9.3.
20-oxo-21-(1H-1,2,4-triazol-1-yl)pregna-5,16-dien-3β-yl-pentanoate 9d.
-1
Yield 0.095 g (78%), m.p. 195–197 °C, IR (KBr) cm : 2927, 1731, 1570, 1275 and 1087.
1
H RMN (400 MHz, CDCl ) δ: 0.98 (t, ester-H-5′, 3H), 1.03 (s, H-18, 3H), 1.20 (s, H-19,
3
3
5
H), 2.4 (q, ester-H-4′, 2H), 3.25 (m, J = 3.2 Hz, H-3, 1H), 4.20 (s, 2H, H-21), 5.34 (d, J =
.3 Hz, H-6, 1H), 6.74 (s, J = 6.7 Hz, C-16, 1H), 7.51 (s, H-Het., 1H), 7.82 (s, H-Het., 1H).
1
3
C RMN (100 MHz, CDCl ) δ: 9.82 (C-5′ of ester), 10.1 (C-4′ of ester), 10.31 (C-18),
3
1
1
5.4 (C-19), 67.31 (C-21), 73.59 (C-3), 121.97 (C-6), 129.62 (C-16), 140.02 (C-Het.),
42.25 (C-5), 148.32 (C-Het.), 156.57 (C-17), 173.46 (ester carbonyl), 200.02 (C-20).
HRMS cal. for C28
H
39
N
3
O
3
465.2991 found 465.2993.
,
2
.1.2.9.4.
20-oxo-21-(1H-1,2,4-triazol-1-yl)pregna-5,16-dien-3β-yl-hexanoate
9e.
-1
Yield 0.098 g, (73%), m.p. 197–199 °C, IR (KBr) cm : 2927, 1725, 1571, 1270 and 1087.
1
H RMN (400 MHz, CDCl ) δ: 1.0 (t, ester-H-6′, 3H), 1.23 (s, H-18, 3H), 1.30 (s, H-19,
3
3
5
H), 2.32 (q, ester-H-5′, 2H), 3.25 (m, J = 3.2 Hz, H-3, 1H), 4.20 (s, 2H, H-21), 5.33 (d, J =
.3 Hz, H-6, 1H), 6.74 (s, J = 6.7 Hz, C-16, 1H), 7.51 (s, H-Het., 1H), 7.80 (s, H-Het., 1H).

15
1
3
C RMN (100 MHz, CDCl
3.62 (C-3), 121.97 (C-6), 129.62 (C-16), 140.02 (C-Het.), 142.31(C-5), 148.32 (C-Het.),
56.57 (C-17), 173.46 (ester carbonyl), 200.02 (C-20). HRMS cal. for C29
3
) δ: 9.80 (C-6′), 12.30 (C-18), 15.42 (C-19), 67.30 (C-21),
7
1
H
41
N
3
O
3
4
79.3148 for
,
found 479.3141.
2
.1.2.9.5.
20-oxo-21-(1H-1,2,4-triazol-1-yl)pregna-5,16-dien-3β-yl-
-1
cyclopropanecarboxylate 9f. Yield 0.059 g, (50%), m.p. 131–134 °C, IR (KBr) cm :
1
2
1
2
928, 1722, 1625, 1575, 1270 and 1172. H RMN (400 MHz, CDCl
3
) δ: 0.90 (s, H-18, 3H),
.31 (s, H-19, 3H), 1.83 (m, ester-H-1′, 1H), 3.45 (m, J = 3.5 Hz, H-3, 1H), 4.90 (s, 2H, H-
1), 5.35 (d, J = 5.3 Hz, H-6, 1H), 6.74 (s, J = 6.7 Hz, C-16, 1H), 7.70 (s, H-Het., 1H), 8.01
1
3
(
(
s, H-Het., 1H). C RMN (100 MHz, CDCl
3
) δ: 13.6 (C-1′ of ester), 14.53 (C-18), 15.23
C-19), 63.2 (C-21), 73.55 (C-3), 121.03 (C-6), 121.1 (C-16), 141.52 (C-5), 144.04 (C-
Het.), 152.15 (C-Het.), 157.13 (C-17), 174.44 (ester carbonyl), 190.89 (C-20). HRMS cal.
449.2678 found 449.2672.
for C27
H
35
N
3
O
3
,
2
.1.2.9.6.
20-oxo-21-(1H-1,2,4-triazol-1-yl)pregna-5,16-dien-3β-yl-
-
1
cyclobutanecarboxylate 9g. Yield 0.068 g, (56%), m.p. 136–138 °C, IR (KBr) cm : 2930,
1
1
723, 1625, 1575, 1270 and 1167. H RMN (400 MHz, CDCl
3
) δ: 0.98 (s, H-18, 3H), 1.01
(
s, H-19, 3H), 2.30 (m, ester-H-1′, 1H), 3.45 (m, J = 3.4 Hz, H-3, 1H), 4.90 (s, 2H, H-21),
.36 (d, J = 5.3 Hz, H-6, 1H), 6.74 (m, J = 6.7 Hz, C-16, 1H), 7.80 (s, H-Het, 1H), 8.2 (s,
5
1
3
H-Het., 1H). C RMN (100 MHz, CDCl ) δ: 12.18 (C-18), 15.86 (C-19), 30.10 (C1′ of
3
ester), 65.51 (C-21), 73.55 (C-3), 121.93 (C-6), 121.96 (C-16), 141.53 (C-5), 143.98 (C-
Het.), 152.15 (C-Het.), 157.21 (C-17.), 174.96 (ester carbonyl), 190.92 (C-20). HRMS cal.
for C28
H
37
N
3
O
3
463.2835 found 463.2837.
,

16
2
.1.2.9.7.
20-oxo-21-(1H-1,2,4-triazol-1-yl)pregna-5,16-dien-3β-yl-
-1
cyclopentanecarboxylate 9h. Yield 0.073 g, (58%), m.p. 118–120 °C, IR (KBr) cm :
1
2
1
2
8
928, 1724, 1625, 1574, 1270 and 1168. H RMN (400 MHz, CDCl
3
) δ: 0.96 (s, H-18, 3H),
.20 (s, H-19, 3H), 2.31 (m, ester-H-1′, 1H), 3.48 (m, J = 3.4 Hz, H-3, 1H), 4.90 (s, 2H, H-
1), 5.36 (d, J = 5.3 Hz, H-6, 1H), 6.74 (m, J = 6.7 Hz, C-16, 1H), 7.70 (s, H-Het., 1H),
1
3
.01 (s, H-Het., 1H). C RMN (100 MHz, CDCl ) δ: 15.87 (C-18), 19.25 (C-19), 30.10
3
(
(
C1′ of ester), 65.10 (C-21), 74.45 (C-3), 121.92 (C-6), 140.01 (C-16), 141.23 (C-5), 144.0
C-Het.), 152.17 (C-Het.), 157.29 (C-17), 176.37 (ester carbonyl), 190.96 (C-20). HRMS
cal. for C29
.1.2.9.8.
H
39
N
3
O
3
477.2991
,
found 477.2997.
2
20-oxo-21-(1H-1,2,4-triazol-1-yl)pregna-5,16-dien-3β-yl-
-1
cyclohexanecarboxylate 9i. Yield 0.071 g, (55%), m.p. 115–118 °C, IR (KBr) cm : 2930,
1
1
724, 1627, 1585, 1275 and 1166. H RMN (400 MHz, CDCl
3
) δ: 0.96 (s, H-18, 3H), 1.31
(
s, H-19, 3H), 2.30 (m, ester-H-1′, 1H), 3.78 (m, J = 3.7 Hz, H-3, 1H), 4.90 (s, 2H, H-21),
.36 (d, J = 5.3 Hz, H-6, 1H), 6.74 (m, J = 6.7 Hz, C-16, 1H), 7.70 (s, H-aromatic, 1H),
5
8
3
1
1
3
.01 (s, H-aromatic, 1H). C RMN (100 MHz, CDCl ) δ: 15.80 (C-18), 19.25 (C-19),
3
0.10 (C1′ of ester), 65.50 (C-21), 73.52 (C-3), 121.92 (C-6), 140.10 (C-16), 141.50 (C-5),
43.92 (C-Het.), 152.20 (C-Het.), 175.57 (ester carbonyl), 199.37 (C-20). HRMS cal. for
C
30
H
41
N
3
O
3
491.3148 for
,
found 491.3144.
2
.1.2.9.9.
20-oxo-21-(1H-1,2,4-triazol-1-yl)pregna-5,16-dien-3β-yl-
-1
cycloheptanecarboxylate 9j. Yield 0.068 g, (51%), m.p. 197–199 °C, IR (KBr) cm :
1
2
1
2
932, 1726, 1629, 1583, 1270 and 1160. H RMN (400 MHz, CDCl
3
) δ: 0.90 (s, H-18, 3H),
.33 (s, H-19, 3H), 2.30 (m, ester-H-1′, 1H), 3.48 (m, J = 3.4 Hz, H-3, 1H), 4.90 (s, 2H, H-
1), 5.35 (d, J = 5.3 Hz, H-6, 1H), 6.73 (m, J = 6.7 Hz, C-16, 1H), 7.70 (s, H-aromatic, 1H),

17
1
3
7
3
1
.98 (s, H-aromatic, 1H). C RMN (100 MHz, CDCl ) δ: 15.87 (C-18), 19.31 (C-19),
3
0.71 (C1′ of ester), 65.50 (C-21), 72.05 (C-3), 121.07 (C-6), 140.30 (C-16), 141.50 (C-5),
43.94 (C-Het.), 152.20 (C-Het.), 176.58 (ester carbonyl), 191.05 (C-20). HRMS cal. for
C
31
H
43
N
3
3
O 505.3304, found 505.3310.
2
2
.2. Animal and human tissues
.2.1 Type 1 5-R isoenzyme isolated from rat liver
Two adult (8-month old) rats were obtained by the Metropolitan University in
Xochimilco from Animal Care Facility, Mexico. All procedures with animals were
approved by the Institutional Care and Use Committee of UAM.
The adult rats weighing 500 g had been fasted overnight to decrease glycogen levels
before their livers were extirpated for use as a source of 5-R1 [12]. To prepare
microsomes, the livers (30 g) were minced in one volume of buffer A (20 mM sodium
phosphate, pH 6.5, containing 0.32 M sucrose, 0.1 mM DTT (Sigma-Aldrich, Mexico City,
Mexico) with an IKA−A11 basic tissue mill (IKA Laboratory Equipment, Mexico City,
Mexico). Unless otherwise specified, the following procedures were carried out at 4 °C.
The tissue was homogenized and the suspension was centrifuged (13,200 rpm; 20
min; 0 °C) (Beckman L70 K ultracentrifuge). The pellet was discarded. The supernatant
was filtered through a nylon mesh filter (pore size 11 µm, distributed by OEM-Membrane
Solution, Dallas TX) and centrifuged again (10,000 rpm; 60 min, 4 °C); the microsomal
pellet was resuspended in five volumes of buffer A with a homogenizer. The protein
amount was measured by the Bradford method. [13]

18
The suspension was re-centrifuged (13,200 rpm; 20 min, 4 °C) and the pellet was
resuspended in buffer A to give a final concentration of 20 mg protein/mL. The microsomal
suspension was stored at −70 °C prior to the preparation of the sample steroid.
2
.2.2. Type 2 5R isoenzyme isolated from human prostate
Four hours after a 53-year-old patient had died of a heart attack, his normal prostate
was extirpated in the Pathology Department of the General Hospital in Mexico City. The
Ethical Committee of the General Hospital in Mexico City approved this protocol.
The tissue was rinsed and immediately chilled in ice-cold 150 mM NaCl and stored
at −20 ºC. The frozen human prostate was thawed on ice, rinsed, and minced in buffer A
(
20 mM sodium phosphate, pH 6.5, containing 0.32 M sucrose, 0.1 mM DTT; Sigma-
Aldrich, Mexico City, Mexico) with an IKA® A11 basic tissue mill (IKA Laboratory
Equipment, Mexico City, Mexico). Unless otherwise specified, the following procedures
were carried out at 4 ºC.
Human prostate was used in this experiment because this tissue is an abundant
source of 5α-R2, but not of 5α-R1; the AR study in this model is very complex.
The human prostate tissue was homogenized in two volumes of buffer A with a
tissue homogenizer Ultra-Turrax IKA, T18 basic (Wilmington, NC). The homogenates
were centrifuged (1500×g; 60 min) [10] in a SW 60 Ti rotor (Beckman Instruments, Palo
Alto, CA). The pellets were resuspended in buffer A, and stored at −70 °C. This suspension

19
had a final concentration of 5 mg protein/mL, as determined by the Bradford method, and
was used as source of 5-R2 isozyme.
2
.2.3. Rat prostate cytosol as source of AR
In order to evaluate the binding of steroids 8 and 9a−j to the androgen receptor, the
prostates of 50 adult rats (8 months old; 500 g) were removed, blotted, weighed and soaked
in cold TEMD (40 mM tris–HCl, 3 mM EDTA and 20 mM sodium molybdate,
dithiothreitol 0.5 mM, 10% glycerol at pH 8) before use. All procedures were carried out in
an ice bath at 0 °C.
The tissues were homogenized with a tissue homogenizer (Teckmar, Cincinnati,
OH) in one volume of buffer TEMD plus protease inhibitors (2 mM PMSF, 10 µg/mL
antipain, 5 mM leupeptin) [14] in an ice bath with a tissue homogenizer; subsequently the
homogenates were centrifuged (14,000 rpm, 60 min) [15] in a SW 60 Ti rotor (Beckman
Instruments, Palo Alto, CA). The cytosolic fraction obtained from the supernatant liquid of
the rat prostate homogenate described above was stored at −70 °C. The protein (6 mg of
protein in 200 µL) was determined by the Bradford Method.
In this study we used rats because the prostate gland is bigger than that of the
hamster and there is no difference in the binding activity of the AR between rat and hamster
cytosol. [15]
2
.2.4 Characteristics of cell lines

20
The following panel of cells was used in this study: PC-3 (human prostate cancer
cell line), MCF7 (human breast cancer cell line), and SK-LU-1 (human lung cancer cell
line), supplied by the National Cancer Institute (USA).
2
.2.5 In vivo experiments
For the experiments in vivo, 80 adult male golden hamsters (2.5 months old; 150–
2
00 g) were used. After gonadectomies had been performed on the hamsters under
isoflurane anesthesia, the castrated hamsters were allowed to recover for 30 days prior to
experimentation. The castrated hamsters and the remaining eight intact hamsters were
housed in a room with controlled temperature (22 °C) and light-dark periods of 12 h; the
hamsters were fed with food and water ad libitum. Thirty days post gonadectomy, the
hamsters were separated into nine groups consisting of four animals per group. The
hamsters were treated for six days, and thereafter sacrificed with CO
was carried out twice under the same conditions.
2
[10]. This experiment
2
2
.3 Biological activity of the new compounds
.3.1 In vitro experiments
The effect of steroids 8 and 9a–j, (Fig. 2) on the activity of 5α-R1 and 5α-R2 was
evaluated under the same in vitro conditions that we described previously [10]
We give a brief description of some of the details of the technique to evaluate the
5
0% inhibitory concentration (IC50) of 8 and 9a–j required to inhibit the activity of 5α-R
−
10
−4
isozymes. Different concentrations (1 × 10 to 10 M) of each of the steroid derivatives
were added to tubes containing the culture medium [10]. The transformation of T to DHT
in the presence of 8 and 9a–j was calculated from radioactive compounds present in each of

21
the lanes of the chromatographic plate, taking into account the entire radioactivity present
in the rest of the plate. These data were plotted using SigmaPlot software 12 inhibition
curves. [10].
2
.3.2 Competitive studies in AR
The binding capacity of steroids 8 and 9a–j to the AR was determined using the
same technique that was described previously by our group [9] and the IC50 of each
compound was calculated according to the plots of concentration versus percentage of
binding, using SigmaPlot 12.
2
2
.3.3 In vivo experiments
.3.3.1 Weight of the prostate, seminal vesicles and diameter of the pigmented spot
For six consecutive days, each of the steroid derivatives 8, 9a–e (2 mg/kg body
weight (BW)) dissolved in 200 µL of sesame oil, together with 1 mg/kg (BW) of
testosterone, was administered by subcutaneous (SC) injection to a group of
gonadectomized hamsters (four animals per derivative). Three groups of gonadectomized
animals were kept as control; the first group was injected SC with 200 µL sesame oil, the
second group with 1 mg /kg (BW) of testosterone, and the third group with 1 mg T plus 1
mg/kg (BW) of finasteride also prepared in sesame oil. Additionally, one group of four
intact hamsters was used as the intact control. After treatment, the animals were sacrificed
with CO . The diameters of the flank organs were measured using a vernier caliper; the
2
prostate and seminal vesicles of each hamster were dissected and weighed. Two separate
experiments were performed for each group of steroid treated hamsters. The results were

22
analyzed by using one-way analysis of variance and Dunnett’s method to compare means,
using JMP IN 5.1 software (JMP, Statistical Discovery, Cary, NC, USA).
2
.3.4 Cytotoxicity assay
Cytotoxic activity of steroids 8 and 9a–j on three different cell lines; PC-3, MCF7,
and SK-LU-1, was evaluated as reported previously. [16, 17]
The percentage of cell growth inhibition was calculated according to the following
expression:
1
00 – (simpleabsorption)
X 100
vehicle absorption)
Cell growthinhibition (%)=
(
3
3
. Results
.1. Chemistry
In this synthesis the strategy for the preparation of 8 and 9a−j (Fig. 2) was the following:
we used the commercially available 16-dehydropregnenolone acetate (1) treated with
hydrogen peroxide and sodium hydroxide 4N to form the 16β, 17β-epoxy compound 2. The
protection in C-3 with tert-butyldimethylsilyl chloride and imidazole in DMF afforded the
compound 3. The oxidation of 3 with (diacetoxyiodo) benzene, sodium hydroxide and
methanol generated the compound with the alcohol in C-21 4. This compound treated with
thionyl chloride yielded the chlorinated compound 5. 6 was prepared using potassium
carbonate and 1,2,4-triazole in DMF and warmed at 80 °C for 5 h. Compound 6 upon
treatment with hydrochloric acid in acetone afforded the compound 7. The 16β,17β-epoxy
elimination of 7 was performed with chromium chloride (II) in acetic acid giving

23
compound 8. Finally compounds 9b−j were obtained from 8 by using the Steglish
esterification method. [18] Their esters were obtained with a high and regular yields (51-
9
0%). The synthesis of 9a was done with acetic anhydride and pyridine (77% yield). All
compounds were isolated and the melting points showed a maximum of two degrees
1
difference. The desired compounds and intermediates were characterized by IR, H NMR,
1
3
C NMR and mass spectrometry.

24
Table 1. Biological activity of novel derived compounds on human cancer cell line growth inhibition at 50 µM, IC50 value for 5α-R
isoenzymes and AR binding assay.
Structure
Compound
R
PC-3
MCF7
SK-LU-1
5α-R1
IC50 nM
5α-R2
IC50 µM
RBA
AR
%
Ketoconazole
Finasteride
10
11
8
-
-
-
88.48±3.9 95.16±4.8 73.39±1.6
ND
630
NA
ND
0.0085
NA
ND
NA
NA
NA
NA
NA
61.3±0.3
62.1±0.8
88.9±5.5
9
a
-CH3
56.2±1.3
59.4±2.3
74.8±1.5
NA
100
NA
9
9
9
9
b
c
d
e
-C H5
NA
5.9±2.2
25.6±2.7
38.5±0.9
14.4±0.2
NA
8.8±0.7
15.6±1.8
20.2±2.8
6.4±1.7
NA
88
NA
320
NA
NA
NA
NA
118
NA
NA
NA
NA
NA
2
-C H7
3
9.2±1.6
38.2±2.2
16.9±1.8
NA
-C H9
4
3
-C H
5
11
100x10
NA
9
f
9
9
g
h
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
9
i
j
3
9
98x10

25
3
.2. Effect of steroids 8 and 9a−j as inhibitors of the activity of 5α-R isozymes
The in vitro effect of steroids 8, 9a−j and finasteride (11 in Fig. 1) required for
inhibiting 5α-R1 and 5α-R2 activity for 50% (IC50 value) are shown in Table 1.
These results indicate that 9b and 9d with IC50 values for 5α-R1 of 88 nM and 320
nM respectively (Table 1) have higher inhibitory activity (lower IC50 values) than
finasteride (IC50=630). However, the rest of these compounds do not show this inhibitory
activity by 5α-R1.
The data in Table 1 also show that 9a and 9f inhibited the activity of 5α-R2 with
IC50 values of 100 and 118 µM respectively. These IC50 values are higher than that of
finasteride (0.0085 µM), which is the steroid we used as a reference control. Thus 9a and 9f
are less potent than finasteride for inhibiting the activity of 5αR-2.
3
.3. Competitive studies
These studies show that 8 and 9a-j did not displace the tritium-labeled MIB (12 in
Fig. 1) of AR in the trial. However, the unlabeled MIB used as reference control shifted the
labeled MIB from AR, giving an IC50 value of binding to the AR of 1 nM. MIB is a very
potent agonist of AR; this is the reason we used this steroid as a reference control.
3
.4. Cytotoxic activity on cancer cell lines
The percentage of antiproliferative effect produced by steroids 8, 9a−j on the
growth of human cancer cell lines PC-3 (prostate), MCF7 (breast) and SK-LU-1 (lung) is
shown in Table 1. Ketoconazole was used in this study as a reference standard because it is
known that this compound has an antiproliferative effect in cancer cell lines.
The data in Table 1 also show that compounds 8 and 9a have lower antiproliferative
activity on PC-3 and MCF7 (61.3%, 56.2%, 62.1% and 59.4% respectively) than in SK-

26
LU-1 cells. Furthermore these compounds have a high inhibitory activity on this last cell
line (88.9% and 74.8% respectively). However steroids 9b-9j displayed a lower cytotoxic
activity than ketoconazole; this low cytotoxic activity could be due to its low solubility in
DMSO, which could prevent availability in the cell culture medium.
3
.5. Pharmacological results
After castration, the weight of the hamster prostate and seminal vesicles decreased
(
p < 0.05) compared to the normal glands, as did the diameter of the pigmented spot.
Treatment with vehicle alone did not change these conditions. However, injections of 1
mg/kg of T for 6 days significantly increased (p ≤0.05) the diameter of the pigmented spot
as well as the weight of the prostate and seminal vesicles (Table 2). When T (1 mg/kg) and
finasteride were injected together, the diameter of the flank organs and the weight of the
prostate and seminal vesicles decreased significantly (p ≤ 0.05) compared to that of T-
treated animals (Table 2). This reduction of the weight of these glands and diameter of the
pigmented spot was comparable to that produced by finasteride 11 (Table 2).
The results of this experiment (Table 2) show the effect of steroids 8, 9a−j on the
size of the pigmented spot of the flank organs as well as the weight of prostate and seminal
vesicles. The Dunnett test, which compares the mean obtained in the pharmacological
experiments, indicated that no significant differences exist between the response obtained
with T-treatment and T plus 8, 9a−j.

27
Table 2. Diameter of the pigmented spot of the flank organs; weight of the prostate and
seminal vesicles glands ± standard deviation from castrated hamsters receiving different
treatments for 6 days.
Treatment
DIAMETER OF
THE PIGMENTED
SPOT
WEIGHT OF
PROSTATE
WEIGHT OF
SEMINAL
VESICLES
(
mg)±standard
error
(
mm)±standard
error
(mg)±standard
error
Control
T
2.03±.63
5.65± 1.1
3.57±0.9
3.05 1.5
2.9±0.9
50.4±9.9
98 ±32.6
69.6± 21.2
89.0 ±16
87.65±21.8
89.55±21.1
81.3±57
96.11±12.5
200.6 ±55
161.4± 44.5
163.33± 41.4
216.075±67.2
157.08±32.6
138.9±45
T+F
T + 8
T + 9a
T + 9b
T + 9c
T + 9d
T + 9e
T + 9f
T + 9g
T + 9h
T + 9i
T + 9j
2.79±1.4
2.71±0.7
3.71±1.0
2.8±0.4
82.30±21.9
86.9±19.5
107.48±22.8
100±40
180.03±39.5
200±56.7
2.88±0.8
2.14±2.5
2.8±.1.0
3±0.3
180.3±39.5
130±58
97±30
120±25
88±10
150±15
4.45±0.4
78.2±18.5
203.98±47.8
The control animals were treated with vehicle only

28
4
. Discussion
In this study we demonstrated that derivatives 8 and 9a-j were less potent for
inhibition of the activity of 5α-R2 than other 16-pregnenolone acetate and 16-pregnandiene
acetate derivatives previously studied. [19] This low activity in vitro (8 and 9a-j) fully
explains the lack of pharmacological effect of these steroids observed in these experiments.
However 9b and 9d were more potent for inhibition of 5αR1 than the previously
reported derivatives. [19] Compounds 9b and 9d have ester groups with an even number of
carbon atoms in their structure, which may favor union with 5α-R1. This is important
because some types of prostate tumor cells, such as LnCaP, proliferate due to the presence
of 5α-R1 specifically, so these compounds may have therapeutic potential for this type of
condition. [5]
The structure of the two isozymes of 5α-R has not been elucidated because they are
membrane proteins, which makes them very difficult to purify in their functional form. [1]
For this reason its crystal structure is unknown and docking and SAR studies remain in the
theoretical stage. [9, 10, 19]
Data obtained in this study demonstrated that steroid 8 with a trizole moiety at C-21
showed high cytotoxic activity on the cell line SK-LU-1 (lung cancer). In a previous study
it has been reported that DHEA derivatives to which triazole groups were added at C-17
were able to inhibit the growth of the same three cell lines used in this study. [9] But these
derivatives of DHEA [9] also had antiproliferative action on cell lines PC-3 and MCF7.
The triazole group added to the synthesized pregnane derivatives increased their solubility
in the culture medium of cells studied. [20] In addition, the ester group attached at C-3 of

29
these derivatives enhances the value of log P and therefore its permeability through the cell
membrane. When the antiproloferative effect produced by steroid derivatives with
imidazole or a pyrazole ring is compared with triazole it is observed that the
antiproliferative effect increases triazole ring. [21] Other 21-triazolyl derivatives of
pregnenolone have displayed a potent antitumor activity. For example the compound 21-
{
4-[(4-methoxyphenoxy)methyl]-1H-1,2,3-triazol-1-yl}-3-hydroxypregn-5-en-20-one
synthesized by the group of Banday, which was very active to inhibit the growth of DU-
1
45 and PC-3 cell lines [22].
These data indicate that the hydroxylated derivative of pregnenolone at C-3 and
with a triazole group at C-21 synthesized and studied in this work displays antiproliferative
specificity on the SK-LU-1 cells, which could be considered an advantage for its
therapeutic use. This activity could be attributed to the presence of a free OH group at C-3
(
8), which forms hydrogen bonds with different molecules in the cells.