Molecular interactions of progesterone derivatives with 5α-reductase types 1 and 2 and androgen receptors

Article abstract of DOI:10.1016/j.steroids.2010.03.006

The aim of this study was to ascertain the inhibitory effect of several progesterone derivatives for 5α-reductase types 1 and 2 isozymes and to determine the binding to the androgen receptor. The 3,20-dioxopregna-4-ene-17α-yl acetate 4 containing an acetoxy group in C-17 and steroid 17α-hydroxypregn-4-ene-3,20-dione 5 having a hydroxyl group in the same position inhibited both isozymes. On the other hand, 17α-hydroxy-4,5-epoxypregnan-3,20-dione 6 with an epoxy function at C-4, inhibited only the type 1 enzyme. Steroid 4-chloro-17α-hydroxypregn-4-ene-3,20-dione 7a and 4-bromo-17α-hydroxypregn-4-ene-3,20-dione 7b having the C-4 conjugated system and a chlorine or a bromine atom at C-4 respectively, inhibited both types of 5α-reductase. These results indicate that an increase in the electronegativity of ring A produces a major inhibitory activity for 5α-reductase type 1; however this increase was not observed for type 2 enzyme. When the free hydroxyl group of 7a or 7b was esterified, compounds 3,20-dioxo-4-chloropregn-4-ene-17α yl-4-ethylbenzoate 8a and 3,20-dioxo-4-bromopregn-4-ene-17α yl-4-ethylbenzoate 8b were obtained; these steroids inhibited only the 5α-reductase type 2 enzyme. Finasteride and steroids 4, 5, 7b, 8a showed a comparable in vivo pharmacological activity, however the IC₅₀ values of these compounds were higher as compared to that of finasteride. These results indicated also that steroids 4, 5, 7a, and 7b bind to the androgen receptor whereas compounds 6, 8a and 8b failed to do so. The overall data from this study showed that steroids 5 and 7b bind to the AR and decreased of the growth of prostate and seminal vesicles. Moreover, 4 decreased also the growth of seminal vesicles.

Full text of DOI:10.1016/j.steroids.2010.03.006

Steroids 75 (2010) 499–505
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Molecular interactions of progesterone derivatives with 5␣-reductase types 1
and 2 and androgen receptors
Eugene Bratoeffa, Perla Garcíaa, Yvonne Heuzeb, Juan Sorianoc, Adriana Mejíab, Ana María Labastidab,
Norma Valenciab, Marisa Cabezab,∗
a Department of Pharmacy, Faculty of Chemistry, National University of Mexico City, Mexico, D.F., Mexico
b Department of Biological Systems and Animal Production Metropolitan University-Xochimilco, Mexico, D.F., Mexico
c Department of Pathology of the General Hospital of Mexico (SS), Mexico, D.F., Mexico
a r t i c l e i n f o
a b s t r a c t
Article history:
The aim of this study was to ascertain the inhibitory effect of several progesterone derivatives for 5␣-
reductase types 1 and 2 isozymes and to determine the binding to the androgen receptor.
The 3,20-dioxopregna-4-ene-17␣-yl acetate 4 containing an acetoxy group in C-17 and steroid 17␣-
hydroxypregn-4-ene-3,20-dione 5 having a hydroxyl group in the same position inhibited both isozymes.
On the other hand, 17␣-hydroxy-4,5-epoxypregnan-3,20-dione 6 with an epoxy function at C-4, inhibited
only the type 1 enzyme. Steroid 4-chloro-17␣-hydroxypregn-4-ene-3,20-dione 7a and 4-bromo-17␣-
hydroxypregn-4-ene-3,20-dione 7b having the C-4 conjugated system and a chlorine or a bromine atom at
C-4 respectively, inhibited both types of 5␣-reductase. These results indicate that an increase in the elec-
tronegativity of ring A produces a major inhibitory activity for 5␣-reductase type 1; however this increase
was not observed for type 2 enzyme. When the free hydroxyl group of 7a or 7b was esterified, compounds
3,20-dioxo-4-chloropregn-4-ene-17␣ yl-4-ethylbenzoate 8a and 3,20-dioxo-4-bromopregn-4-ene-17␣
yl-4-ethylbenzoate 8b were obtained; these steroids inhibited only the 5␣-reductase type 2 enzyme.
Finasteride and steroids 4, 5, 7b, 8a showed a comparable in vivo pharmacological activity, however
the IC50 values of these compounds were higher as compared to that of finasteride.
Received 12 January 2010
Received in revised form 19 March 2010
Accepted 22 March 2010
Available online 30 March 2010
Keywords:
Progesterone derivatives
5␣-Reductase types 1 and 2
Androgen receptors
Prostate
Seminal vesicles
Molecular interactions
These results indicated also that steroids 4, 5, 7a, and 7b bind to the androgen receptor whereas
compounds 6, 8a and 8b failed to do so.
The overall data from this study showed that steroids 5 and 7b bind to the AR and decreased of the
growth of prostate and seminal vesicles. Moreover, 4 decreased also the growth of seminal vesicles.
© 2010 Elsevier Inc. All rights reserved.
1. Introduction
The 5␣-reductase enzyme (EC 1.3.99.5) converts T to 5␣-DHT
in androgen dependent tissues. The activity of this enzyme in
Androgens play a crucial role in the development, maintenance,
and regulation of male phenotype and reproductive biology [1].
Testosterone 1 (T) and dihydrotestosterone 2 (DHT) (Fig. 1) are
androgens principally produced by the testicles; these hormones
are uptaken from the blood by the target tissues, which possess the
androgen receptor (AR). The binding of these hydrophobic ligands
induces the AR to assume a configuration that leads to transcription
activation (or inhibition) and allows transmission of extra cellular
signals into intracellular responses by targeting response elements
and recruiting cofactors [2].
androgen dependent tissues has long been known. Two types of
5␣-reductase had been identified: 1 and 2, each encoded by differ-
ent genes, which have been characterized in several species [3,4].
5␣-Reductase type 2 isozyme plays a major role in prostate cancer
and benign prostatic hyperplasia as it is predominantly expressed
in this tissue. However, some evidence indicates that type 1 is
expressed in the prostate epithelial cells while the type 2 is mainly
located in the stromal compartment [5,6]. 5␣-Reductase type 1
is also located in the liver and skin and acts in a neutral or basic
medium, whereas type 2 is active in acidic pH [3,4].
Previously our group had reported the synthesis and
biological activity of several pregnane derivatives [7,8];
however the mechanism of action of these compounds
had not been studied. The purpose of this research
∗
Corresponding author at: Departamento de Sistemas Biológicos, Universidad
was
3,20-dione
6, 4-chloro-17␣-hydroxypregn-4-ene-3,
to
synthesize
5, 17␣-hydroxy-4,5-epoxypregnan-3,20-dione
20-dione 7a,
steroids
17␣-hydroxypregn-4-ene-
Autónoma Metropolitana-Xochimilco, Calzada del Hueso no. 1100, México, D.F C.P
04960, Mexico. Tel.: +52 55 5483 72 60; fax: +52 55 5483 72 60.
E-mail address: marisa@correo.xoc.uam.mx (M. Cabeza).
0039-128X/$ – see front matter © 2010 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2010.03.006

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E. Bratoeff et al. / Steroids 75 (2010) 499–505
2.2.1. 17˛-Hydroxypregn-4-ene-3,20-dione 5
A solution of 17␣-acetoxyprogesterone 4 (commercially avail-
able) (1 g, 2.69 mmol), methanol (60 mL) and 10% aqueous sodium
hydroxide solution (20 mL) was allowed to reflux for 1 h. Part of the
methanol (25 mL) was eliminated by distillation from the reaction
mixture and ice (30 g) was added. The precipitated crude product
was isolated by filtration; it was recrystallized from methanol. Yield
735 mg, 2.23 mmol (83.3%) of pure product 5, mp 201–204 ◦C. UV
(nm) 240 (ε = 12,500). IR (KBr) cm−1: 3428, 3029, 1708, 1665, 1208.
1HNMR (CDCl3) ı: 0.73 (3H, s, H-18), 1.1 (3H, s, H-19), 2.2 (3H, s, H-
21), 4.4 (1H, s, hydroxyl proton), 5.8 (1H, s, H-4). 13CNMR (CDCl3)
ı: 15.2 (C-18), 16.5 (C-19), 27.6 (C-21), 124.2 (C-4), 171.1 (C-5),
199.7 (C-3), 211.8 (C-20). FAB-MS (m/z) calc for C21H30O3 331.4712
(M+H), found 331.4213.
Fig. 1. The structure of testosterone, dihydrotestosterone and mibolerone (MIB).
4-bromo-17␣-hydroxypregn-4-ene-3, 20-dione 7b, 3, 20-
dioxo-4-chloropregn-4-ene 17␣ yl-4-ethylbenzoate 8a and
3,20-dioxo-4-bromopregn-4-ene 17␣ yl-4-ethylbenzoate 8b from
the commercially available 3, 20-dioxopregn-4-ene-17␣ yl acetate
4 (Fig. 2) similar to those reported earlier [7,8], and to study
the relationship of their structure with the inhibitory activity of
5␣-reductase types 1 and 2 as well as its interaction with the AR.
Furthermore, it was of interest to study their pharmacological
effect on the weight of prostate and seminal vesicles.
2.2.2. 17˛-Hydroxy-4,5epoxypregnan-3,20-dione 6
To a solution of 5 (0.5 g, 1.52 mmol) in dichloromethane (5 mL)
and methanol (6 mL) was added 10% aqueous solution of sodium
hydroxide (0.6 mL) and 30% hydrogen peroxide (0.8 mL). The mix-
ture was stirred for 24 h at room temperature. The methanol was
eliminated in vacuum. Upon cooling, the desired compound 6 pre-
cipitated as colorless crystals. Yield 376 mg, 1.14 mmol (73%), mp
153–155 ◦C. IR (KBr) cm−1: 3480, 2980, 1705, 1264, 890. 1HNMR
(CDCl3) ı: 0.78 (sH, s, H-18), 1.1 (3H, s, H-19), 2.2 (3H, s, H-21), 3.6
(1H, s, H-4), 4.8 (1H, s, hydroxyl proton), 13CNMR (CDCl3) ı: 15.4
(C-18), 16.8 (C-19), 26.3 (C-21), 62.9 (C-4), 70.4 (C-5), 198.0 (C-
3), 203.6 (C-20). FAB-MS (m/z) calc for C21H30O4 347.4712 (M+H),
found 347.5132.
2. Materials and methods
2.1. Chemical and radioactive materials
Solvents were laboratory grade or better. Melting points were
determined on a Fisher Johns melting point apparatus and are
uncorrected. 1H-NMR and 13C-NMR were taken on Varian gem-
ini 200 and VRX-300 respectively. Chemical shifts are given in ppm
relative to that of Me4Si (( = 0) in CDCl3 (the abbreviations of signal
patterns are as follows: s, singlet; d, doublet, t, triplet, m, multi-
plet). Mass spectra were obtained with a HP5985-B spectrometer.
IR spectra were recorded on a Perkin-Elmer 200s spectrometer.
(1,2,6,7-3H) Testosterone [3H] T specific activity: 95 Ci/mmol
2.2.3. Preparation of compounds 8a–8b
A
solution containing chloroform (3 mL), 4-ethylbenzoic
acid (290 mg; 1.98 mmol) trifluoroacetic anhydride (0.6 mL), p-
toluenesulfonic acid (100 mg) and compound 7a or 7b (1.5 mmol)
(the preparation of these compounds is described in Ref. [7])
was stirred at room temperature for 3 h. An aqueous saturated
sodium bicarbonate solution (5 mL) was added and the mixture was
stirred at room temperature for 30 min. It was extracted 3 times
with chloroform (5 mL), the combined organic phase was dried
with anhydrous sodium sulfate and the solvent was eliminated
in vacuum. The crude reaction product was purified by thin layer
chromatography (solvent system: hexane–ethyl acetate = 4:1).
and Mibolerone (17␣-methyl-3H) [3H] MIB
3 (Fig. 1) spe-
cific activity 70–87 Ci/mmol were provided by Perkin Elmer
Life and Analytical Sciences (Boston, MA). Radioinert T, 5␣-
dihydrotestosterone and MIB were supplied by Steraloids (Wilton,
NH, USA). Sigma Chemical Co. (ST. Louis, MO) provided NADPH
and Lubrol PX. 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 column chromatography.
2.2.4. 3, 20-Dioxo-4-chloropregn-4-ene-17˛ yl-4-ethylbenzoate
8a
2.2. Synthesis of the steroidal derivatives
Yield 89 mg, 0.127 mmol (25%), mp 225–228 ◦C, UV (nm) 251
(ε = 12,200). IR (KBr) cm−1: 3020, 1720, 1675, 800. 1HNM (CDCl3)
ı: 0.9 (3H, s, H-18), 1.2 (3H, s, H-19), 1.3 (3H, t, CH3 of the ethyl side
chain), 2.1 (3H, s, H-21), 2.8 (2H, q, CH2 of the ethyl side chain),
The synthesis of the new steroidal derivatives 5, 6, 8a, 8b is
briefly described below. The preparation of the intermediates 7a
and 7b is given in Ref. [7].
Fig. 2. Reaction scheme for the synthesis of the final compounds 8a and 8b.

E. Bratoeff et al. / Steroids 75 (2010) 499–505
501
7.5 (4H, m, aromatic protons). 13CNMR (CDCl3) ı: 15.0 (C-18), 16.9
2.3.2. Solubilization of 5˛-reductase type 1 from rat liver
microsomes
(C-19), 25.2 (C-21), 29.3 (CH2 of the ethyl side chain), 127.7 (C-4),
128.4 (2C-metha aromatic ring), 130.0 (2C-ortho aromatic ring),
150.9 (1C-para-aromatic ring), 190.9 (C-3), 204.5 (C-20). FAB-MS
(m/z) calc for C30H38O4Cl, 498.3462 (M+H), found 498.4658.
The solubilization of 5␣-reductase type 1 isozyme from rat liver
microsomes was carried out according to Levy et al. [12]. Briefly, the
microsomes were suspended in five volumes of buffer B (100 mM
sodium citrate, 100 mM potassium chloride, 1.0 mM dithiothreitol
(DTT). 0.1% nonaethylene glycol monododecyl ether (Lubrol PX),
20% glycerol, and 0.1 mM NADPH adjusted to pH 7.5). The blend was
mixed and the solution incubated on ice for 30 min and then cen-
trifuged at 100,000 × g for 60 min. Aliquots from the supernatant
liquid, which contained greater than 80% of the microsomal activity
were stored at −80 ◦C.
2.2.5. 3, 20-Dioxo-4-bromopregn-4-ene-17˛ yl-4-ethylbenzoate
8b
Yield 82 mg. 0.151 mmol (30%), mp 239–241 ◦C. UV (nm) 255
(ε = 12,650). IR (KBr) cm−1: 3035, 1725, 1680, 639. 1HNMR (CDCl3)
ı: 0.8 (3H, s, H-18), 1.2 (3H, s, H-19), 1.4 (3H, t, CH3 of the ethyl side
chain), 2.2 (3H, s, H-19), 2.7 (2H, q, CH2 of the ethyl side chain),
7.4 (4H, m, aromatic protons). 13CNMR (CDCl3) ı: 14.5 (CH3 of the
ethyl side chain), 15.3 (C-18), 16.9 (C-19), 25.6 (C-21), 29.3 (CH2
of the ethyl side chain), 126.6 (C-4), 128.2 (2C-metha aromatic
ring), 130.6 (2C-ortho aromatic ring), 150.8 (1C-para aromatic ring),
166.4 (ester carbonyl), 191.5 (C-3), 206.5 (C-20). FAB-MS calc for
In order to determine the binding of steroids 4, 5, 6, 7a, 7b, 8a and
8b to the androgen receptors, the prostate of adult rats 8 months
old weighing 500 g was extirpated. In this study we used also rats
because the prostate gland is bigger and there is no difference in
the binding activity between rats and hamsters cytosol.
The prostate of the rats was blotted, weighed and soaked in cold
TEMD (40 mM Tris–HCl, 3 mM EDTA and 20 mM sodium molybdate,
dithiotreitol 0.5 mM, 10% glycerol at pH 8) prior to their use. Unless
specified, all procedures were carried out in an ice bath. Tissues
were homogenized with a tissue homogenizer.
C30H38O4 Br 542.4361 (M+H) found 542.4913.
2.3. Biological activity of the steroidal compounds
The prostate of a man 48 years old, who died from renal lithiasis
was extirpated and the tissue was immediately chilled in ice-cold
150 mM NaCl and stored at −20 ◦C. Frozen human prostate was
thawed on ice and minced with a tissue mill IKA A11 basic. Unless
specified, the following procedures were carried out at 4 ◦C.
Human prostate tissue was homogenized in 2 volumes of buffer
A (20 mM sodium phosphate, pH 6.5 containing 0.32 M sucrose,
0.1 mM dithiothreitol Sigma–Aldrich, Inc.) with a tissue homoge-
nizer Ultra-Turrax IKA, T18 basic. (Wilmington, NC). Homogenates
were centrifuged at 1500 × g for 60 min [9,10] in a SW 60 Ti rotor
(Beckman instruments, Palo Alto, CA). The pellets were separated,
suspended in medium A and kept at −70 ◦C. The suspension, 5 mg
of protein/mL for human prostates, determined by the Bradford
method [11] was used as source of 5␣-reductase type 2 isozyme.
Tissues were homogenized in one volume of buffer TEMD plus
protease inhibitors (2 mM PMSF, 10 ␮g/mL antipain, 5 mM leu-
peptin [13]) in an ice bath with a tissue homogenizer. Homogenates
were centrifuged at 140,000 × g for 60 min [14] 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.
Prostatic cytosol proteins (6 mg of protein in 200 ␮L) were deter-
mined by the Bradford method [11].
2.3.3. In vitro experiments
2.3.3.1. Determination of 5˛-reductase type 1 activity. The activity
of the 5␣-reductase type 1 isozyme was determined by following
the conversion of T to DHT. A mixture containing a final volume of
1 mL, 1 mM DTT sodium phosphate buffer 40 mM, at pH 8.0, 2 nM of
[1,2,6,7-3H] T, 108 nM unlabeled T and 2 mM NADPH [12]. The reac-
tion in duplicate was started when it was added to the enzymatic
fraction (61 ␮g protein in a volume of 7.5 ␮L) incubated at 37 ◦C
for 60 min and stopped by mixing with 1 mL of dichloromethane;
this was considered as the end point. Incubation without tissue was
used as a control.
2.3.1. Animals and tissues
For the in vivo experiments, adult male golden hamsters 2.5
months old (150–200 g) were obtained from the Metropolitan
University in Xochimilco, Mexico. Hamster gonadectomies were
performed under pentobarbital anesthesia and the castrated ham-
sters were kept in a room with controlled temperature (22 ◦C) and
light–dark periods of 12 h. Food and water were provided ad libi-
tum. After 36 days maintaining these conditions, the hamsters were
sacrificed with CO2. This protocol was approved by the Institutional
Care and Use Committee of the Metropolitan University of Mex-
ico (UAM). The experiment with the gonadectomized animals was
carried out on 8 groups of 4 animals/experiment.
2.3.3.2. Determination of 5˛-reductase type 2 activity. The activity
of the isozyme 5␣-reductase type 2 was assayed as previously
described [9,10]. The reaction mixture contained a final volume
of 1 mL, 1 mM DTT, sodium phosphate buffer 40 mM, at pH 6.5 for
human prostates, 2 mM, NADPH, 2 nM [1,2,6,7-3H]T. The reaction
in duplicate was started when it was added to the enzymatic frac-
tion (500 ␮g protein in a volume of 80 ␮L) incubated at 37 ◦C for
60 min [10] and stopped by mixing with 1 mL of dichloromethane;
this was considered as the end point. Incubation without tissue was
used as a control.
With the purpose to prepare microsomes as source of 5␣-
reductase type 1 isozyme, two livers from male adult rats, 8 months
old were extirpated. The rats were fasted overnight to decrease
glycogen levels before the removal of the livers.
Rat livers were minced (15–30 g) in one volume of buffer A using
a tissue mill IKA A11 basic. Unless specified, the following proce-
dures were carried out at 4 ◦C. The tissue was homogenized and
the suspension centrifuged at 10,000 × g for 30 min (Beckman L70K
ultracentrifuge); the pellet was discarded. The supernatant liquid
was filtered through a strainer and centrifuged at 100,000 × g for
60 min. The microsomal pellet was washed by resuspension in five
volumes of buffer A with a homogenizer and the proteins were
determined by the Bradford method [11]. The suspension was cen-
trifuged at 100,000 × g and the pellet suspended in buffer A to a
concentration of 10–20 mg of protein/mL; the microsomal solu-
tion was stored at −80 ◦C prior to the preparation of the solubilized
steroid 5␣-reductase type 1 [12].
2.3.3.3. Extraction and purification of DHT formed from T (5˛-
reductase types
1 and 2 activities). The mixtures (incubation
medium/dichloromethane) were agitated on a vortex for one
minute and the dichloromethane phase was separated and placed
in another tube. This procedure was repeated 4 more times. The
dichloromethane extract was evaporated to dryness under a nitro-
gen stream and suspended in 50 mL of methanol that was spotted
on HPTLC Keiselgel 60 F254 plates. T and DHT were used as car-
riers and were applied in different lanes on both lateral sides of
the plates (T, T + DHT and DHT). The plates were developed in
chloroform–acetone = 9:1 and were air-dried; the chromatography

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E. Bratoeff et al. / Steroids 75 (2010) 499–505
a range of increasing concentrations (1 × 10−10 to 4 × 10−7 M) of
cold MIB 3 (Fig. 1) or: 4, 5, 6, 7a, 7b, 8a and 8b in ethanol or ace-
tone, or in absence of the competitor were prepared [14]. Incubates
also contained 200 nM triamcinolone, in ethanol (Sigma) to prevent
interaction of MIB with glucocorticoid receptors and progesterone
receptors, the solvent was evaporated.
was repeated 2 more times. The steroid carriers were detected
using phosphomolibdic acid reagent (DHT) and T with an UV lamp
(254 nm). After the plates were segmented in areas of one cm
each, they were cut off and the strips soaked in 5 mL of Ultima
Gold (Packard). The radioactivity was determined in a scintillation
counter (Packard tri-carb 2100 TR). The radioactivity content in the
segment corresponding to T and DHT carriers was identified. The
radioactivity that has identical chromatographic behavior as the
DHT standard was considered as the DHT transformation. Control
incubations, chromatography separations and identifications, were
carried out in the same manner as described above except that the
tubes did not contain tissue. The DHT transformation yields were
calculated from the strips, taken into account the entire radioactiv-
ity in the plate.
The 5␣-reductase type 1 activity was calculated from the per-
centage of the labeled DHT, formed, taking into consideration the
recovery, blank values, specific activity of [3H] T or and ratio of
added [3H] T to unlabeled T. The 5␣-reductase type 2 activity was
calculated taking into consideration the recovery, blank values and
the specific activity of [3H] T.
The activity of both types of 5␣-reductase enzymes was
assessed incubating the enzymatic fractions with labeled T. The
dichloromethane extracts from human prostate membranes or rat
liver 5␣-reductase soluble fraction from microsomes were sub-
jected to TLC analysis. The radioactive zone that had identical
chromatographic behavior as the standard T (Rf value of 0.56) cor-
responds to 70% of the accounted radioactivity in the plate. The
radioactivity contained in the zone corresponding to DHT standard
(Rf value of 0.67) of the experimental chromatogram was identi-
fied as the transformed DHT and corresponds to 20–27% of the total
radioactivity accounted in the plate. This result was considered to
be 100% of the activity of 5(-reductase (types 1 or 2) for the develop-
ment of inhibition plots. Unmodified [3H]T was identified (Rf value
of 0.56) from control incubations which did not contain tissue and
had identical chromatographic behavior as the non labeled stan-
dard (identified by UV lamp, 254 nm). The radioactivity contained
in the zone corresponding to DHT standard (Rf value of 0.67) of the
control chromatogram was 1% of the total radioactivity accounted
in the plate and was considered as an error; it was subtracted from
the experimental chromatograms.
Aliquots of 200 ␮L of prostate cytosol were added and incu-
bated in the presence of 300 ␮L of TEMD buffer containing protease
inhibitors (duplicate) for 24 h at 4 ◦C in the tubes as previously
described. After incubation 0.27 mL saturated ammonium sulfate
in TEMD buffer (35%) was added [10]. The mixture was further
incubated for 1 h with occasional shaking to facilitate the precip-
itation of the [3H] MIB-complex. The precipitate was collected by
centrifugation at 10,000 × g, 10 min and the pellet was redissolved
in 0.5 mL of TEMD and mixed with 0.5 of 0.1% dextran-coated 1%
charcoal in TEMD buffer. The mixture was incubated for 40 min at
4 ◦C. To prepare the dextran-coated charcoal mixture, the dextran
was agitated for 30 min before adding the charcoal to the mixture.
The tubes were agitated on a vortex and immediately centrifuged
at 800 × g for 10 min to pellet the charcoal; aliquots (600 ␮L) were
taken and submitted for radioactive counting. The IC50 value of each
compound was calculated according to the plots of concentration
versus percentage of binding.
2.3.4. In vivo experiments
For the daily subcutaneous injections, 2 mg/kg of the steroids
4, 5, 6, 7a, 7b, 8a and 8b were dissolved in 200 ␮L of sesame oil
and administered for 6 days to gonadectomized animals, together
with 1 mg/kg of testosterone. Three groups of gonadectomized ani-
mals were kept as control; one was injected with 200 ␮L of sesame
oil, the second with 1 mg/kg of testosterone and the third with T
plus 1 mg/kg of finasteride for 6 days. After the treatment, the ani-
mals were sacrificed with CO2. The prostate and seminal vesicles
of each animal were dissected and weighed. Two separate exper-
iments were performed for each group of steroid treated animals.
The results were analyzed using one-way analysis of variance and
Dunnett’s Method to compare means, with JMP IN 5.1 software.
3. Results
3.1. Synthesis of the steroidal derivatives 8a and 8b
2.3.3.4. Determination of 50% of the inhibitory concentration of
steroids 4, 5, 6, 7a, 7b, 8a and 8b in 5˛-reductase type 1 and 2
activities. In order to calculate the IC50 values (the concentration of
steroids 4, 5, 6, 7a, 7b, 8a and 8b or finasteride required to inhibit
50% of the activity 5␣-reductase types 1 and 2 enzyme, six series of
tubes containing increasing concentrations of these steroids (10−11
to 10−3 M) were incubated in duplicate, in the presence of: 1 mM
of dithiothreitol, 40 mM sodium phosphate buffer pH of 6.5; 2 mM
NADPH, 2 nM[1,2,6,7-3H]Tfor type2and 500 ␮g ofproteinfrom the
enzymatic fraction obtained from human prostate as we described
above. For 5␣-reductase type 1 assay, we used: 1 mM of dithio-
threitol, 40 mM sodium phosphate buffer pH of 8.0; 2 mM NADPH,
2 nM [1,2,6,7-3H]T and 108 nM of non labeled T. Increasing concen-
trations of the synthesized steroids (10−11 to 10−3 M) were added
also to the incubation mixture. The reaction was started by adding
60 ␮g of soluble 5␣-reductase type 1 isozyme and the final volume
of incubation was of 1 mL.
The intermediate compounds 7a and 7b were prepared from the
commercially available 17␣-progesterone acetate 4 [7] (Fig. 2). The
preparation of steroids 5, 6, 8a and 8b is described in the experi-
mental section of this paper. The final compounds 8a and 8b were
obtained by a relatively unconventional esterification of the free
alcohols 7a and 7b with 4-ethylbenzoic acid. The NMR, IR and
mass spectra of all intermediates and final compounds confirmed
unequivocally the correct structure.
3.2. Biological activity
3.2.1. In vitro experiments. Determination of the 50% inhibitory
concentration of the synthesized compounds in human prostate
The concentration of finasteride and compounds 4, 5, 6, 7a,
7b, 8a and 8b required for inhibiting 5␣-reductase type 1 and
2 activities by 50% (IC50) were determined from the inhibition
plots, using different concentrations of the tested steroids; the
results standard deviations are shown in Table 1. Finasteride
inhibited 5␣-reductase type 2 with a low value of IC50, whereas this
value was higher for type 1 isozyme. Steroid 4 inhibits both types of
5␣-reductase isozymes; similar results were found for 5, however,
this compound showed an IC50value higher for the type 1 enzyme
than compound 4. Steroid 6 did not inhibit the type 2 enzyme; it
The reactions were carried out in duplicate at 37 ◦C for 60 min;
1 mL of dichloromethane was added to stop the reaction. The
extraction and the chromatographic procedures were carried out as
described above. The radioactivity contained in the fraction corre-
sponding to DHT standard was determined as we described above.
2.3.3.5. Androgen receptor competitive binding assay. For AR com-
petitive binding studies, tubes containing 1 nM of [3H] MIB plus

E. Bratoeff et al. / Steroids 75 (2010) 499–505
503
Table 1
Table 2
Effect of different compounds as inhibitors of the activity of 5␣-reductase type 1
Binding of the synthesized compounds to the androgen receptors.
and type 2.
STEROID STRUCTURE
IC50 (nM)
RBA
100%
0.15%
0.48%
–
2.3%
0.23%
–
Steroid structure
5␣-reductase Type 1
IC50 (␮M)
5␣-reductase Type 2
IC50 (␮M)
MIBOLERONE
fx9
fx10
fx11
fx12
fx13
fx14
fx15
1.9
1200
400
NA
82
800
NA
fx1
fx2
fx3
fx4
fx5
fx6
fx7
fx8
58.9 3.3
31.7 5.0
62.6 4.8
50.1 9.0
0.50 0.08
0.32 0.075
NA
0.0085 0.0002
39 8.7
37 9.0
NA
42 6.2
40 4.2
65 10
NA
–
The source of the androgen receptor was the cytosol obtained from homogenates
from rat prostate.
NA
100 8.0
5␣-reductase type 1 was obtained from the solubilized fraction of rat liver micro-
somes, whereas that 5␣-reductase type 2 was obtained from the pellet fraction of
homogenates of human prostate centrifuged at 1500 × g. The homogenates were
incubated with labeled and unlabeled T and increasing concentration of the syn-
thesized compounds. The results indicate the concentration of the novel steroids
necessary for inhibit the 50% of the activity of both enzyme types. NA non-active
compound.
The IC50 and relative binding affinity (RBA) values were determined using
non-labeled MIB and the synthesized steroids. The IC50 value represents the con-
centration of the steroid that inhibits 50% of labeled MIB binding to the AR and was
determined as described in the experimental section. NA, not binding.
3
IC of [ H] Mibolerone
50
RBA =
× 100.
IC of synthesized steroid
50
ment with vehicle alone did not change this condition, whereas
s.c. injections of 1 mg/kg of T for 6 days significantly increased
(P < 0.05) the weight of these glands in castrated male hamsters
(Fig. 4). When T (1 mg/kg) plus finasteride (1 mg/kg) or compounds
5, 7b, 8a (2 mg/kg) were injected together, the weight of the
prostate and seminal vesicles decreased significantly P < 0.05 as
compared to that of T-treated animals (Fig. 4); compound 4 sig-
nificantly decreased the weight of the seminal vesicles, whereas
the weight of prostate did not show a significant difference with
those treated with T. However steroids 7a and 8b exhibited a ten-
dency to decrease the weight of the prostate and seminal vesicles,
but the difference with the T-treated hamsters was not significant
(P > 0.05). Compound 5 showed the lowest weight of the prostate
gland thus exhibiting the highest antiandrogenic effect, whereas 6
did not significantly reduce the weight of prostate in the treated
hamsters, P > 0.05 (Fig. 4).
showed an IC50 value for type 1 enzyme slightly lower as compa-
rable to that of finasteride On the other hand compounds 7a and
7b showed lower IC50 values for type 1 than for type 2 enzyme; 8a
as well as 8b did not show an inhibitory activity for 5␣-reductase
type 1, however both steroids inhibited the type 2 isozyme.
3.3. Binding of the synthesized compounds to the androgen
receptor
The binding of steroids 4, 5, 6, 7a, 7b, 8a and 8b to the AR was
determined from the IC50 and relative binding affinity (RBA) val-
ues for each compound. The IC50 value was calculated according to
the plots of concentration of non-labeled MIB or synthesized com-
pounds versus percentage of binding. MIB 3 (Figs. 1 and 3) showed
an IC50 of 1.9 nM, whereas compounds 6, 8a and 18b did not inhibit
labeled MIB binding to the androgen receptor. However 4, 5, 7a and
7b, bind to the androgen receptor with RBA values of 0.15, 0.48, 2.3
and 0.23% respectively Table 2 and Fig. 3.
3.4. In vivo experiments
3.4.1. Effect of the synthesized steroids on the weight of the
prostate and seminal vesicles
After castration, the weight of the hamster prostate and seminal
vesicles decreased (P < 0.05) compared to the normal glands. Treat-
Fig. 4. Weight of prostate and seminal vesicles ( standard deviations) obtained
from groups of castrated hamster (4 animals/each) receiving s.c. different treatments
for 6 days. The control animals (C) were treated with vehicle only. The pharmaco-
logical experiment was carried out in duplicate. The treatment with T increased the
weight of the prostate and seminal vesicles compared with the glands from animals
treated with the vehicle alone. The weight of the prostate and seminal vesicles from
animals, decreased with the treatment with T plus F or steroids 4, 5, 6, 7a, 7b, and
8a, compared with the glands from animals treated with T alone.
Fig. 3. Binding specificity of the steroids 4, 5, 7a, 7b and MIB to the AR. Gonadec-
tomized rat’s cytosol aliquots were incubated for 18 h at 0 ◦C in presence of 1 nM of
[3H] mibolerone and increasing concentrations of unlabeled steroids.
Steroids 6, 8a and 8b did not bind to the AR.
Significant differences (P < 0.05) with on the weight of prostate and seminal vesicles
from the animals treated with T.
T, testosterone, F, finasteride.

504
E. Bratoeff et al. / Steroids 75 (2010) 499–505
4. Discussion
mibolerone. However this binding could be sufficient to produce
an antagonistic effect.
In this paper we report the synthesis and biological activ-
ity of several steroidal compounds (4, 5, 6, 7a, 7b, 8a and
8b) based on the pregnane skeleton. Compounds 4 and 5 hav-
The hepatic metabolism of 4-chloro-17␣-methyl-17␤-hydroxy-
1,4-androstadien-3-one, which has a similar structure in ring A as
compared to steroid 7a, had been previously reported. This halo-
gen derivative produced chlorine metabolites which were present
in the urine of treated horses [17]. On this basis, we could hypoth-
esize that 7a and 7b could produce halogen metabolites in the
liver, which are excreted in the urine. In this way 7a and 7b
could convert in compound 5 and show higher biological activ-
ity.
In a similar manner as described above, our results indicate
that steroid 4 binds lightly to the AR and this could be suffi-
cient to produce an antagonistic activity (it showed a decrease
in the weight of seminal vesicles). However compound 4 could
also be hydrolyzed by hepatic estearases [18] to produce com-
pound 5 which binds also to the AR. This binding could give
antagonistic effect that causes a decrease of the weight of seminal
vesicles.
It is interesting to observe that compounds 4, 5, 7a and
7b, which form a complex with the androgen receptor showed
a triple mechanism of action, since they inhibited both types
of 5␣-reductase enzyme and also bind to the androgen recep-
tor.
The results from this study indicate that steroids 5, 7b and 8a are
active compounds for the inhibition of the growth of the prostate
gland and the seminal vesicles, whereas steroid 4 decreased only
the weight of seminal vesicles.
ing an acetoxy and
a hydroxyl group at C-17 respectively
inhibited both types 1 and 2 of 5␣-reductase isozymes. How-
ever when the double bond in C-4 was replaced with an
epoxy function (compound 6), it inhibited only the type
1
isozyme.
The steroidal derivatives 7a and 7b having the C-4 conjugated
system and a chlorine or a bromine atom at C-4 (compounds 7a and
7b respectively) inhibited both types of 5␣-reductase isozymes.
Both steroidal derivatives showed a lower IC50 values (higher
inhibitory activity) as compared to that of compound 6 (having
a 4, 5 epoxy function). Apparently an increase of the electroneg-
ativity of ring A (7a and 7b) enhanced the inhibitory activity for
the type 1 enzyme as compared to steroid 6. However these com-
pounds (7a and 7b) showed a lower inhibitory activity for the type 2
isozyme as compared to finasteride and compounds 4 and 5. When
the free hydroxyl group in compounds 7a and 7b was esterified
(steroids 8a and 8b), these compounds did not inhibit the type 1
5␣-reductase enzyme and showed a very low inhibitory activity
for the type 2 isozyme. Apparently this reduction of the polarity of
the molecule decreased the steroid-5␣ reductase enzyme associ-
ation and as a result of this a much lower inhibitory activity was
observed.
The results from this study indicated very clearly that 5␣-
reductase types
1 and 2 isozymes have different molecular
interaction with the tested compounds. Apparently the differ-
ent protein structures of typ1 and type 2 isozymes could be
responsible for the different innhibitory activity of the tested
steroids.
Acknowledgements
We would like to thank CONACYT for its support for the project
No. 54853.
Previously it had been reported that the two types of 5␣-
reductase isozymes share 50% of amino acid sequence identity
and have similar substrate specificity; however, the two isozymes
show different sensibility to inhibitors [3]. On the other hand amino
acid sequence identities with 5␣-reductase isozymes from human
and rat is in the magnitude of 60% between type 1 isozymes and
77% between type 2 isozymes respectively. When the two classes
of 5␣-reductase isozymes are compared, they average 47% iden-
tity, thus indicating that the rat and human type 1 isozymes are
true homologues, as are the type 2 isozymes [3]. Thus it is possi-
ble to compare the IC50 values for steroids 4, 5, 6, 7a, 7b, 8a and
8b between type 1 (obtained from rat) and type 2 (from human)
isozymes.
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