In vivo and in vitro effect of androstene derivatives as 5α-reductase type 1 enzyme inhibitors

Article abstract of DOI:10.3109/14756366.2012.729827

The aim of these studies was to synthesize twelve ester derivatives of dehydroepiandrosterone with therapeutic potential. The effect of 1-12 was demonstrated in the flank organs of gonadectomized hamsters treated with testosterone and the synthesized ster

Full text of DOI:10.3109/14756366.2012.729827

Journal of Enzyme Inhibition and Medicinal Chemistry, 2013; 28(6): 1247–1254
© 2013 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.729827
RESEARCH ARTICLE
In vivo and in vitro effect of androstene derivatives as
5α-reductase type 1 enzyme inhibitors
Eugene Bratoeff1, Araceli Sánchez2, Yazmín Arellano1, Yvonne Heuze2, Juan Soriano3,
and Marisa Cabeza2
1Department of Pharmacy, Faculty of Chemistry, National University of Mexico City, Mexico, D. F., Mexico,
2Department of Biological Systems and Animal Production Metropolitan University-Xochimilco, Mexico, D. F.,
Mexico, and 3Department of Pathology of the General Hospital of Mexico (SS), Mexico, D. F., Mexico
Abstract
The aim of these studies was to synthesize twelve ester derivatives of dehydroepiandrosterone with therapeutic
potential. The effect of 1–12 was demonstrated in the flank organs of gonadectomized hamsters treated with
testosterone and the synthesized steroids. In vitro studies were carried out determining the IC50 values for the
inhibition of the activity of 5α-reductase type 1 and 2, which are present in rat liver and human prostate respectively.
The binding of 1–12 to the androgen receptors (AR) was determined using rat’s prostate cytosol. Steroids 1–12
containing different substituents in the phenyl group of the ester moiety in C-3 reduced the flank organs and
inhibited the activity of 5α-R type 1; however only steroids 1 and 2 inhibited 5α-R type 2. 1–12 did not bind to the
AR. The modification of one atom of the substituents in the phenyl group of the ester moiety in C-3 changed their
biological potency (IC50).
Keywords: 5α-reductase, prostate cancer, flank organs, dehydroepiandrosterone derivatives
Introduction
is 8 mm. ese nodules shrink after castration until they
e 5α-reductase enzyme (EC 1.3.99.5) converts
∆4-3-ketosteroids to 5α-3-ketosteroids in androgen
dependent tissues. Two types of 5α-reductase enzymes
(5α-R) had been identified, 1 and 2, each encoded by a
different gene, which have been characterized in several
species1. 5α-R type 2 plays a major role in prostate cancer
and benign prostatic hyperplasia as it is predominantly
expressed in this tissue. However, some evidence indi-
cates that type 1 is expressed in the prostate epithelial
cells while the type 2 is mainly located in the stromal
compartment2. 5α-R 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 (1).
resemble the nodules of the female animals. However, it
had been previously reported that daily injections or top-
ical applications of testosterone 1 (T) or 2 (DHT) restore
their original size3. ese glands can metabolize T to
DHT in both intact and gonadectomized hamster since
the 5α-R is present in this tissue4. Hamster flank organs
also have an androgen receptor5. ese glands had been
used as a model to test antiandrogenic drugs as well as
5α-inhibitory compounds as finasteride and others6.
Some dehydroepiandrosterone derivatives had
previously been reported as inhibitors of 5α-R enzyme
and antagonists of the AR7 which produced a decrease
of prostate cancer tumors8 and antagonized the effects of
DHT as well as androst-5-ene-3β-17α−diol (Adiol) on the
growth of culture LNCaP cells9.
Hamster flank organs are two pigmented spots formed
by a pilosebaceous complex, located in the dorsal skin
surface of hamsters. In female hamsters the diameter of
the pigmented spot measures 2 mm, whereas in males it
Previously, our group had reported7 that the ester
moiety in C-3 of the dehydroepiandrosterone esters
Address for Correspondence: Marisa Cabeza, Departamento de Sistemas Biológicos, Universidad Autónoma Metropolitana-Xochimilco,
Calzada del Hueso No. 1100, México, D.F., C.P. 04960, México, Tel.: [011-52-55] 5483-72-60, Fax: [011-52-55] 5483-72-60,
E-mail: marisa@correo.xoc.uam.mx.
(Received 11 July 2012; revised 07 September 2012; accepted 11 September 2012)
1247

1248 E. Bratoeff et al.
increases the 5α-R inhibitor activity as well as the antian-
drogenic effect. ese compounds contain an aliphatic
ester moiety (propionyloxy, butyroyloxi and valeroy-
loxy) at C-3 position of the steroidal skeleton. In view of
the fact that the aliphatic ester derivatives showed a high
pharmacologicalactivity, inthisstudywedecidedtosyn-
thesized similar compounds having an aromatic ester
(benzoic and p-substituted benzoic acids) and compare
the activity of the aromatic ester derivatives with that of
the previously publicized aliphatic esters. In this paper
we report the synthesis and the biological evaluation of
the following compounds: 17-oxaandrost-5-ene-3β-yl-
benzoate 1; 17-oxaandrost-5-ene-3β-yl-p-fluoroben-
Synthesis of the steroidal derivatives
General procedure for the preparation of esters
To
a solution of 3β-hydroxyandrost-5-ene-17-one
(1000 mg, 3.5 mmol), DCC (2147 mg, 10.5 mmol) and
DMAP (848 mg, 7.0 mmol) in chloroform (20 mL) was
added the corresponding acid (14 mmol). e resulting
solution was stirred at room temperature for 2h. Hexane
(15 mL) was added and the precipitated dicyclohexylurea
was filtered. e organic phase was washed with 10%
aqueous hydrochloric acid, 5% aqueous sodium bicar-
bonate and water. It was dried over anhydrous sodium
sulfate, and the solvent was removed in vacuum. e
crude ester was recrystallized from ethyl acetate.
17-oxaandrost-5-ene-3β-yl- benzoate (1). Yield
82% of pure product, mp 261–262°C. IR (KBr) cm–1: 2960,
1735, 1711, 1253, 1272. 1H-NMR (CDCl3) δ: 0.9 (3H,
s, H-19), 1.1 (3H, s, H-18), 4.8 (1H, m, H-3), 5.4 (1H, d,
J = 4 Hz, H-6), 7.4 (2H, t, J = 8 Hz, H-3′ and H-5′), 7.5
(1H, t, J = 8 Hz, H-4′), 8.0 (2H, d, J = 8 Hz, H-2′ and H-6′).
13C-NMR (CDCl3) δ: 13.5 (C-18), 19.4 (C-19), 74.3 (C-3),
121.9 (C-6), 128.6 (C-3′ and C-5′), 129.5 (C-2′ and C-6′),
130.7 (C-1′), 132.7 (C-4′), 139.9 (C-5), 168.1 (ester car-
bonyl), 221 (C-17). HRMS calc for C26H32O3 392.3287
found 392.5436. e spectral data for compounds 2–6 are
given in the Info File.
zoate
zoate
2;
17-oxaandrost-5-ene-3β-yl-p-chloroben-
17-oxaandrost-5-ene-3β-yl-p-bromoben-
3;
zoate 4; 17-oxaandrost-5-ene-3β-yl-p-iodobenzoate
5; 17-oxaandrost-5-ene-3β−yl-p-methylbenzoate 6;
17-chloro-16-formylandrost-5,16-diene-3β-yl-ben-
zoate 7; 17-chloro-16-formylandrost-5,16-diene-3β-yl-
p-fluorobenzoate 8; 17-chloro-16-formylandrost-
5,16-diene-3β-yl-p-chlorobenzoate 9; 17-chloro-
16-formylandros-5,16-diene-3β-yl-p-bromobenzoate
10;
17-chloro-16-formylandrost-5,16-diene-3β-yl-p-
iodobenzoate 11; 17-chloro-16-formylandrost-5,16-
diene-3β-yl-p-methylbenzoate 12 with therapeutic
potential as antiandrogens.
General procedure for the preparation of 17-chloro-16-
formylandrost-5,16-dien-3β-yl benzoates
Material and methods
A solution of the corresponding ester (500 mg, 1–1.3
mmol) in chloroform (5 mL) was added dropwise to a cold
and stirred solution of phosphorus oxychloride (4.2mL,
45.3 mmol) dissolved in dimethylformamide (4.2 mL, 55
mmol). e mixture was allowed to reflux under N2 for 5 h
at 40°C. It was combined with 100 mL of a saturated cold
solution of sodium bicarbonate and extracted with chlo-
roform (3 × 125 mL); it was dried over anhydrous sodium
sulfate and the solvent was removed to in vacuum. e
yellow crude product was purified by flash column chro-
matography [FCC, silica gel 60 hexane:ethyl acetate 98:2].
17-chloro-16-formylandrost-5,16-dien-3β-yl ben-
zoate (7). Yield 43% of pure product, mp 188–190°C. IR
Chemical and radioactive materials
Solvents were laboratory grade or better. Melting points
were determined on a Fisher-Johns melting point appa-
1
ratus and are uncorrected. H-NMR and 13C-NMR were
taken on Varian Gemini 200 and VRX-300, respectively.
Chemical shifts are given in ppm relative to that of Me4Si
(δ= 0) in CDCl3 (the abbreviations of signal patterns are
as follows: s, singlet; d, doublet, t, triplet, m, multiplet).
Mass spectra were obtained with a HP5985-B spectrom-
eter. IR spectra were recorded on a Perkin-Elmer 200s
spectrometer.
(1, 2, 6, 7-3H) Testosterone [3H] T specific activity:
95 Ci/mmol and Mibolerone (17α-methyl-3H) [3H]
MIB specific activity 70–87 Ci/mmol were provided
by Perkin Elmer Life and Analytical Sciences. (Boston,
MA). Radioinert T, 5α-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). e tablets were
crushed, extracted with chloroform and the solvent was
eliminated in vacuum; the crude product was purified
by silica gel column chromatography. e melting point
of the isolated finasteride (252–254°C) was identical to
that reported in the literature. Bio-gel Hydroxyapatite
(HAP) was provided by Bio-Rad Laboratories,
(CA, USA).
1
(KBr) cm–1: 2860, 1708, 1711, 1664, 1272, 1253. H-NMR
(CDCl3) δ: 1.0 (3H, s, H-18), 1.1 (3H, s, H-19), 4.9 (1H, m,
H-3), 5.4 (1H, d, J = 5.2 Hz, H-6), 7.4 (2H, t, J = 7.6 Hz,
H-3′ and H-5′), 7.5 (1H, t, J = 7.6 Hz, H-4′), 8.0 (2H, d,
J = 7.2 Hz, H-2′ and H-6′), 9.9 (1H, s, formyl group). 13C-
NMR (CDCl3) δ: 15.3 (C-19), 19.4 (C-18), 74.4 (C-3), 122.1
(C-6), 128.4 (C-3′ and C-5′), 129.7 (C-2′ and C-6′), 130.8
(C-1′), 132.9 (C-4′), 136.6 (C-16), 140.1 (C-5), 162.5 (C-17),
166.1 (ester carbonyl),188.3 (formyl group). HRMS calc
for C27H31ClO3 438.2452 found 438.7835. e spectral data
for compounds 8–12 are given in the Info file.
Biological activity of the steroidal compounds
e human prostate of a man 53 years old, who died
from diabetes and renal insufficiency was introduced
Journal of Enzyme Inhibition and Medicinal Chemistry

In vivo and in vitro effect of androstene derivatives 1249
to a solution of NaCl 150 mM and stored at –70ºC. is
gland was obtained from the Department of Pathology,
e General Hospital (SS) in Mexico City. Frozen human
prostate was thawed on ice and minced with scissors.
Unless specified, the following procedures were carried
out at 4ºC.
e 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.
Human prostate tissue was homogenized in two
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 homogenizer Ultra-Turrax IKA,
T18 basic. (Wilmington, NC). Homogenates were centri-
fuged at 1500g for 60 min in a SW 60 Ti rotor (Beckman
instruments, Palo Alto, CA). e pellets were separated,
suspended in medium A and kept at –70ºC. e suspen-
sion, 5 mg of protein/mL for human prostates, deter-
mined by the Bradford method10 was used as source of
5α-R type 2 isozyme.
Tissues were homogenized in one volume of buffer
TEMD plus protease inhibitors in an ice bath with a tis-
sue homogenizer. Homogenates were centrifuged13 in a
SW 60 Ti rotor (Beckman Instruments, Palo Alto, CA).
e 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 µL) were determined by the Bradford
method10.
In vitro experiments
Animals and tissues
Characterization of 5α-R type 1
For the in vivo experiments, adult male golden ham-
sters 2.5 month old (150–200 g) were obtained from the
Metropolitan University Xochimilco, Mexico. Hamster’s
gonadectomies were performed under pentobarbital
anesthesia and the castrated hamsters were kept in a
room with controlled temperature (22ºC) and light–
dark periods of 12 h. Food and water were provided ad
libitum. After 36 days maintaining these conditions, the
hamsters were sacrificed with CO2. is protocol was
approved by the Institutional Care and Use Committee
of the Metropolitan University of Mexico (UAM). e
experiment with the gonadectomized animals was car-
ried out on 8 groups of 4 animals/experiment.
Determination of protein concentration-dependent
5α-R type 1 activities
In order to determine the optimum concentration of pro-
tein to obtain the maximum activity of 5α-R enzyme type
1, different concentrations of solubilized microsomes
(30, 60 and 90 µg of protein) were used as a source of this
enzyme. A mixture containing a final volume of 1 mL,
1 mM DTT sodium phosphate buffer 20 mM, at pH 6.5,
2 nM of [1,2,6,7 3H] T, and 2 mM NADPH11 was prepared.
e reaction in duplicate was started when the enzymatic
fraction was added to the mixture and this was incubated
at 37°C for 60 min. e reaction was stopped by mixing
with 1 mL of dichloromethane. Incubation without tissue
was used as a control.
With the aim to prepare microsomes as source of 5α-R
type 1 isozyme, two livers from male adult rats, 8 months
old were extirpated. e 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 buf-
fer A using a tissue mill IKA A11 basic. Unless specified,
the following procedures were carried out at 4ºC. e tis-
sue was homogenized and the suspension centrifuged at
10,000g for 30 min (Beckman L70K ultracentrifuge); the
pellet was discarded. e supernatant liquid was filtered
through a strainer and centrifuged at 100,000g for 60min.
e microsomal pellet was washed by resuspension in
five volumes of buffer A with a homogenaizer and the
proteins were determined by the Bradford method10. e
suspension was centrifuged at 100,000g and the pellet
suspended in buffer A to a concentration of 10–20 mg of
potein/mL; the microsomal solution was stored at –80°C
prior to the preparation of the solubilized steroid 5α-R
type 1. e solubilization of 5α-R type 1 isozyme from rat
liver microomes was carried out according to Levy et al.11.
In order to determine the binding of steroids 1–12 to
the AR, the prostate of adult rats 8 months old weighing
500 g was extirpated12. In this study we used also rats
because the prostate gland is bigger and there is no dif-
ference in the binding activity between rats and hamsters
cytosol.
Determination of pH dependence of 5α-R activity
To measure the pH dependent 5α-R type 1 activities, the
assays were carried out in a similar way that we described
above but using 20 mM potassium phosphate buffers
ranging from pH 6–8 and different concentrations of
unlabeled T.
Determination of 5α-R type 1 activity
e activity of the 5α-R type 1 isozyme was determined
by following the conversion of T to DHT at pH 7.5. 2nM of
3
[1,2,6,7 H] T, and different concentrations of unlabeled
T (5 × 10–7–6.3 × 10–6 M) or DHT (5 × 10–7–2.7 × 10–5M) and
2 mM NADPH11 was prepared. e reaction in duplicate
was started when 5α-R type 1 isozyme fraction (60 µg
protein in a volume of 7.5 µL) was added to the mixture
and incubated at 37°C for 60 min.
Determination of 5α-R type 2 activity
e activity of the isozyme 5α-R type 2 was assayed as
previously described14,15
.
e radioactive zone that had identical chromato-
graphic behavior as the standard T (Rf value of 0.56) corre-
sponds to 70% of the accounted radioactivity in the plate.
e radioactivity contained in the zone corresponding
© 2013 Informa UK, Ltd.

1250 E. Bratoeff et al.
to DHT standard (Rf value of 0.67) of the experimental
chromatogram was identified as the transformed DHT
and corresponds to 20–27% of the total radioactivity
accounted in the plate. is result was considered to be
100% of the activity of 5α-R (types 1 or 2) for the develop-
ment of inhibition plots. Unmodified [3H]T was identi-
fied (Rf value of 0.56) from control incubations which did
not contain tissue and had identical chromatographic
behavior as the non labeled standard (identified by UV
lamp, 254 nm). e 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.
e 5α-R type 1 activity was calculated from the per-
centage of the labeled DHT, formed, taking into consid-
eration the recovery, blank values, specific activity of [3H]
T and ratio of added [3H] T to unlabeled T or DHT. e
5α-R type 2 activity was calculated taking into consider-
ation the recovery, blank values and the specific activity
of [3H] T.
In vivo experiments
For the daily subcutaneous injections, 2 mg/kg of the
steroids 1–12 were dissolved in 200 µL of sesame oil and
administered for 6 days to gonadectomized hamsters,
together with 1 mg/kg of testosterone. ree groups of
gonadectomized hamsters were kept as control; one
was injected with 200 µL 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. e flank organs diameter
size of each hamster was measured using a vernier. Two
separate experiments were performed for each group of
steroid treated animals. e results were analyzed using
one-way analysis of variance and Dunnett’s Method to
compare means, with JMP IN 5.1 software.
Results
Synthesis of the steroidal derivatives 1–12
e steroidal derivatives 1–12 were synthesized using
dehydroepiandrosterone as the starting material. is
compound was esterified with the corresponding acid in
the presence of dicyclohexylcarbodiimide and dimeth-
ylaminopyridine. e resulting esters 1–6 were treated
with phosphorus pentachloride in dimethylformamide
(Vilsmeyer-Haack reaction) to form the final compounds,
the esters of 17-chloro-16-formylandrost-5,16-diene
moiety 7–12.
Km and Vmax values were derived from Linewear–Burk
plots, in which the regression lines were computed by
the least square method. e statistical significance
of the means was determined by Student’s t test in all
experiments.
e efficiency of T 5α-R type 1 was estimated accord-
ing to Weisser and Krieg report16.
Determination of 50% of the inhibitory concentration of
steroids 1–12 in 5α-R type 1 and 2 activities
In vitro experiments
Protein concentration-dependent 5α-R type 1 activities
When different concentrations of protein obtained from
solubilized microsomes of rat’s liver were used, 5α-R
enzyme type 1 shows different activity. e optimum
concentration of protein was of 60 µg.
In order to calculate the IC50 values (the concentration
of steroids 1–12 or finasteride required to inhibit 50%
of the activity 5α-R types 1 and 2 enzyme, six series of
tubes containing increasing concentrations of these ste-
roids (10–11–10–3 M solubilized in DMSO) were incubated
in duplicate: For type 2 isozyme assay, pH of 6.5; 2 mM
[1,2,6,7-3H]T, 2 mM NADPH and 500 µg of protein from
the enzymatic fraction obtained from human prostate
were used14,15. For 5α-R type 1 assay, we used a pH of 7.5;
2 mM NADPH, 2 nM [1,2,6,7-3H]T and 6.31 µM of non
labeled T. e reaction was started by adding 60 µg of
soluble 5α-R type 1 isozyme.
pH Dependent 5α-R type 1 activities
5α-R type 1 present in the solubilized microsomes from
rat’s liver, showed the conversion of T to DHT in the
presence of NADPH. Furthermore, the pH profile of this
enzyme was observed by using different concentrations
of T. In the presence of 6.31 µM of T, 5α-R type 1 shows
maximal conversion to DHT at pH 7.5. When smaller or
higher concentrations of T were used, a lower activity of
5α-R type 1 at pH 7.5 was observed. e kinetic param-
eters were also calculated and these data are shown in
Table 1. e higher Vmax value was determined at pH 7.5;
however, this enzyme shows major affinity for T at pH 6.5
(lower value of Km = 2.97) see Table 1.
e concentration of finasteride and compounds 1–12
required for inhibiting 5α-R type 1 and 2 activities by 50%
(IC50) were determined from the inhibition plots using
SigmaPlot Software.
Androgen receptor competitive binding assay
For AR competitive binding studies, tubes containing
1 nM of [3H] MIB plus a range of increasing concentra-
tions (1 × 10–10–4 × 10–7 M) of unlabeled MIB or steroids
1–12 in ethanol or acetone, or in the absence of competi-
tor were prepared. e [3H] MIB-AR complex was sepa-
rated by the hydroxyapatite (HAP) method17.
In addition, the efficiency of 5α-R type 1 for the con-
version of T to DHT was calculated. is efficiency value
(Vmax/Km)16 was obtained from the rate between maximal
velocities of production of DHT (Vmax), using different
pHs and the Michaelis constants (Km).
Vmax/Km rate values showed that 5α-R type 1 was effec-
e IC50 value of each compound was calculated
according to the plots of concentration versus percent-
age of binding using SigmaPlot Software.
tive at pHs of 6.5, 7.5 and 8. However this efficiency was
higher at pH 7.5 (40.16 value) as compared to that at pH
6.5 and 8 (37.03 and 18.78 values, respectively).
Journal of Enzyme Inhibition and Medicinal Chemistry

In vivo and in vitro effect of androstene derivatives 1251
Table 1. Effect of the testosterone concentration on 5α-reductase type 1 activity at different pHs. Solubilized microsomes from rat liver were
used as source of 5α-reductase type 1.
5α-Reductase
type 1 efficiency
Vmax
pmol/mL/h
Concentration nM
Km [µM]
T
38.2
1.87
96.4
2.37
196
500
1250
2.28
1670
2.91
2300
1.53
4000
1.33
pH 6.5% of
Conversion to
DHT
2.44
2.86
pmol/mL/h
T
0.71
500
2.29
1250
3.32
4.68
1670
3.88
14.3
2300
4.1
28.5
4000
3.5
48.59
5180
3.66
33.19
6100
3.75
53.2
6308
3.48
37.03
100
2.97
pH 7.5% of
Conversion to
DHT
4.01
pmol/mL/h
20.05
0.667
41.5
2.33
64.79
1.17
94.3
2.04
140
189.5
1.83
200
219.5
2.80
40.16
18.78
1111.1
526
27.6
28
pH 8% of
Conversion to
DHT
1.94
1.89
pmol/mL/h
3.33
29.13
19.53
46.92
77.6
94.79 125.2
176.62
On the other hand, labeled T in the presence of increas-
ing concentrations of unlabeled T showed a higher con-
version to labeled DHT (higher 5α-R activity) Vmax = 111.1
pmol/mg of protein/h (Km = 27.6) as compared to that
of the experiment where increasing concentrations of
DHT and labeled T were used (Vmax = 0.597 pmol/mg of
protein/h and Km 1.17); as a result of this, the efficiency of
the enzyme 5α-R was higher in the absence of DHT.
However, the 5α-R type 1 showed a higher affinity
(minor Km = 0.39 and Vmax = 169.49) when labeled T, unla-
beled T [1 µM] and an increasing concentrations of DHT
were present in the incubating medium. In this case, the
efficiency value of 5α-R indicated by the rate of Vmax/Km
was higher as compared to that of the experiment where
T in minor concentration or T (in minor concentration)
+DHT were present in the incubating medium. e affin-
ity of the 5α-R type 1 was also evaluated in the presence
of labeled T, unlabeled T [1.5 µM] and an increasing con-
centration of DHT; in this test, higher values of Km = 0.79
µM and Vmax = 188.67 were observed. is trial showed
that the efficiency rate value was lower than that found
for 1 µM of T.
inhibited 5α-R type 2 with a low value of IC50 (0.0085 µM),
whereas this value was higher for type 1 isozyme (0.63 µM).
Steroids 1 and 2 were the only one from the synthesized
compounds that inhibited the enzyme 5α-R type 2, with
IC50 values of 100 and 0.00358 µM, respectively (Figure 1).
Steroids 1–12 inhibited the enzyme 5α-R type 1 activ-
ity; however, compounds 3 and 6 showed a higher IC50
value (0.9 and 5 µM, respectively; lower activity) as com-
pared to that of the remaining steroids. On the other hand
compound 11 and Finasteride showed a lower IC50 value
(0.55 0.02 and 0.63 0.005 µM, respectively; a higher
inhibitory activity).
Binding of the synthesized compounds to the
androgen receptor
e binding of steroids 1–12 to the AR was determined
from the IC50 and the relative binding affinity (RBA) val-
ues for each compound. e IC50 value was calculated
according to the plots of concentration of the non labeled
MIB or the synthesized compounds versus percentage
of binding to MIB (Figure 1). As can be seen in Figure 1,
none of the studied compounds bound to the AR.
e data from this experiment indicated very clearly
that the affinity of 5α-R enzyme type 1 with DHT
increases in the presence of 1 µM of T, (small Km value
of 0.39) as compared to that using 1.5 µM of T giving a
larger Km value (0.79) thus showing a reduced affinity.
Nevertheless the presence of the product (DHT) did not
inhibit the efficiency of the conversion of T to DHT at
these concentrations (1 and 1.5 µM).
In vivo experiments
Flank organ test
After castration, the diameter of the pigmented spot of
the male hamster flank organs decreased (p < 0.005) as
compared to that of the normal glands. Treatment with
vehicle alone did not change this condition, whereas
s.c. injections of 1mg/kg of T for 6 days significantly
increased (p < 0.005) the diameter of the pigmented spot
in castrated male hamsters flank organs (Figure 2). e
diameter of the pigmented spot significantly decreased
(p < 0.005) when testosterone (T) and finasteride (1 mg/
kg) or steroids 1–12 (2 mg/kg) were injected together,
as compared to that of testosterone-treated animals
(Figure 1). Compound 12 showed the highest pharma-
cological activity (small diameter of the pigmented spot
1.0 mm).
Biological activity of the novel compounds
Determination of the 50% inhibitory concentration of the
synthesized compounds in human prostate
e concentration of finasteride and compounds 1–12
required for inhibiting 5α-R 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 Figure 1. Finasteride
© 2013 Informa UK, Ltd.

1252 E. Bratoeff et al.
Figure 1. Steroidal structures and effect of different dehydroepiandrosterone derivatives on the activity of 5α-R enzyme type 1 and 2 (5α-R):
1–12. e IC50 values indicate the required concentration of the novel steroids for the inhibition of 50% of the activity of 5α-R1 and 5α-R2.
is figure shows also the relative binding affinity (RBA) of these derivatives to the androgen receptor (AR). e following abbreviations were
used: NA, non active compound.
type 1 source was of 60 µg, thus at this protein concentra-
tion the active sites of the enzyme were saturated by the
substrate and coenzyme.
e kinetic data for 5α-R type 1 enzyme indicated
that Vmax was of 1.1 nmol/mL/h at pH 7.5. is result
correlates well with the previously reported data for this
enzyme18 and is corroborated by the efficiency rate value
of the reaction at this pH. On the other hand, it had been
previously demonstrated19 that a different activity of the
enzymes is observed when a range of pHs are used. is
fact had been explained on the ground that some elec-
trostatic changes were effected in the molecules involved
in the reaction19. is could be in agreement with the
variation in the activity of the 5α-R type 1 observed in
this study.
Figure 2. Diameter size standard deviations of the pigmented
spot from gonadectomized hamster’s flank organs receiving
different treatments with T plus the novel steroids for 6 days.
Control gonadectomized hamsters were treated with vehicle
only. e following abbreviations were used: Testosterone (T),
Finasteride (F). *Significant differences with the T-treatment were
observed.
e kinetic results obtained in this experiment indi-
cated also that when DHT was added to the incubated
medium, the conversion of T to DHT was not inhibited at
concentrations of 1 and 1.5 µM of T. On the contrary, the
presence of 1 µM of T induced an increase of the affinity
of 5α-R type 1 for its product, DHT (lower Km value) and
an increase in Vmax was observed. On the other hand, the
efficiency rate value for this enzyme was higher when
1 µM of T plus increasing concentrations of DHT were
used, as compared to the experiment containing 1.5 µM
of T plus increasing concentrations of DHT. e result of
Discussion
Since the optimal concentration of the protein obtained
from the solubilized microsomes of rat’s liver as 5α-R
Journal of Enzyme Inhibition and Medicinal Chemistry

In vivo and in vitro effect of androstene derivatives 1253
these experiments is similar to that previously reported
by our group for 5α-R type 220.
derivatives described in this paper showed an opposite
phenomenon (the aromatic steroids exhibited a higher
5α-R type 1 enzyme inhibitory activity as compared to
their aliphatic homologues).
Furthermore, these studies demonstrated also that the
treatment with steroids 1–12 applied to the hamsters for
6 days in the dose used did not produce any toxicological
effect.
e overall data suggested that the decrease of T
observed in elderly man could produce higher affinity
of 5α-R for its product DHT. As a consequence of this,
an increase of the affinity for both isozymes21 could pro-
duce a higher synthesis of DHT in the target organs. is
metabolic imbalance could induce a pathological illness
such as prostate cancer, where an increase of 5α-R type 1
activity had been noticed21.
On the other hand, it is important to consider that
high levels of intracellular DHT result in cellular prolif-
eration and delay cellular differentiation22. In this con-
text previously it had been reported23 that in advanced
prostatic cancer the 5α-R type 1 enzyme is expressed
to a higher degree than the type 2. Furthermore, these
authors demonstrated also that in the tissue located
around cancer cells, the gene expression of 5α-R type 1
is increased as compared with that found in benign pros-
tatic hypertrophy23.
Future studies are planned in order to test the effect
of these compounds in PC-3 line cells that exhibit some
5α-R activity.
Declaration of interest
We thank CONACYT for its support for the project No
165049. is research did not have any real or per-
ceived conflicts of interest and disclosure arising from
intellectual, personal or financial circumstances of the
research.
On the ground of the findings given above, the effect
of steroids 1–12 described in this study as inhibitors of
5α-R type 1 could have a therapeutic potential for pros-
tate cancer, since they are specific for the inhibition of
5α-R type 1 enzyme, with the exception of 1 and 2 that
also inhibited type 2 isozyme. ese compounds have
also the advantage that they did not bind to the AR and
this phenomenon decreases the adverse effects and
improves also their therapeutic potential. However one
of the important goals is to decrease the IC50 value of
the5 α-R types 1 and 2 inhibitors; these studies demon-
strated that the change of the nature of the substituent in
phenyl group of the ester moiety in C-3 of dehydroepi-
androsterone skeleton is sufficient to change the inhibi-
tory potency of synthesized compounds for the5α-R
enzymes.
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