Androgenic and anti-androgenic effects of progesterone derivatives with different halogens as substituents at the C-6 position

Article abstract of DOI:10.1016/S0039-128X(99)00018-5

The pharmacological activities of four pregnane derivatives:17α- hydroxy-16β-methylpregna-4,6-diene-3,20-dione (7), 17α-acetoxy-16β- methylpregna-4,6-diene-3,20-dione (8), 17α-acetoxy-6-bromo-16β- methylpregna-4,6-diene-3,20-dione (10), and 17α-acetoxy-6-chloro-16β- methylpregna-4,6-diene-3,20-dione (11), were determined. The derivatives were evaluated on gonadectomized male hamster flank organs and seminal vesicles. The results indicate that topical applications of testosterone (T) on the flank organs increased the diameter of the pigmented spot. Similarly, the same phenomenon occurred on the glands treated with compound 11, whereas compound 10 decreased the size of the spot significantly. In this study, we determined the effects of several new steroids on the conversion of T to DHT in flank organs and seminal vesicles. The results show that compound 10 inhibited T conversion to DHT, but compound 11, at a dose of 200 μg, stimulated T conversion in both flank organs and seminal vesicles. However, when 2 mg of compound 11 was applied, it inhibited the conversion of T to DHT, suggesting that this compound also represses gonadotropin release. The difference between compounds 10 and 11 involves the electronegativity of the halogen at the C-6 position of the progesterone skeleton. These data clearly indicate that by decreasing the electronegativity of the halogen at C-6 (compound 10), 5α-reductase is inhibited in both tissues and at different pHs. On the other hand, when the electronegativity of the halogen atom was increased (11), there was a much lower inhibitory effect on the conversion of T to DHT.

Full text of DOI:10.1016/S0039-128X(99)00018-5

Steroids 64 (1999) 413–421
Androgenic and anti-androgenic effects of progesterone derivatives with
different halogens as substituents at the C-6 position
M. Cabeza*, E. Guti e´ rrez, R. Miranda, I. Heuze, E. Bratoeff, G. Flores, E. Ram ´ı rez
Departments of Biological Systems and Agricultural-Animal Production, Metropolitan University-Xochimilco and Faculty of Chemistry,
National University of Mexico, Mexico D.F., Mexico
Received 4 December 1997; accepted 14 December 1998
Abstract
The pharmacological activities of four pregnane derivatives:17␣-hydroxy-16␤-methylpregna-4,6-diene-3,20-dione (7), 17␣-acetoxy-
1
6␤-methylpregna-4,6-diene-3,20-dione (8), 17␣-acetoxy-6-bromo-16␤-methylpregna-4,6-diene-3,20-dione (10), and 17␣-acetoxy-6-
chloro-16␤-methylpregna-4,6-diene-3,20-dione (11), were determined. The derivatives were evaluated on gonadectomized male hamster
flank organs and seminal vesicles. The results indicate that topical applications of testosterone (T) on the flank organs increased the diameter
of the pigmented spot. Similarly, the same phenomenon occurred on the glands treated with compound 11, whereas compound 10 decreased
the size of the spot significantly. In this study, we determined the effects of several new steroids on the conversion of T to DHT in flank
organs and seminal vesicles. The results show that compound 10 inhibited T conversion to DHT, but compound 11, at a dose of 200 ␮g,
stimulated T conversion in both flank organs and seminal vesicles. However, when 2 mg of compound 11 was applied, it inhibited the
conversion of T to DHT, suggesting that this compound also represses gonadotropin release. The difference between compounds 10 and 11
involves the electronegativity of the halogen at the C-6 position of the progesterone skeleton. These data clearly indicate that by decreasing
the electronegativity of the halogen at C-6 (compound 10), 5␣-reductase is inhibited in both tissues and at different pHs. On the other hand,
when the electronegativity of the halogen atom was increased (11), there was a much lower inhibitory effect on the conversion of T to DHT.
©
1999 Elsevier Science Inc. All rights reserved.
Keywords: Flank organs; Seminal vesicles; 5␣-Reduction; T-Conversion; Halogen substituents at C-6; Progesterone derivatives
1
. Introduction
5␣-reduction in androgen-responsive tissues, such as the
prostate and seminal vesicles [2]. This compound, however,
increases the weight of these glands in castrated rats. Ex-
amples of steroidal molecules with high anti-androgenic
effects are cyproterone acetate [5], which is an antagonist of
androgen receptors, and finasteride, which selectively inhib-
its 5␣-reductase isozyme type 2 [6].
It is well known that in some classes of steroids, blocking
known sites of metabolic inactivation, such as by C-6 hy-
droxylation, leads to substantial increases in androgenicity
[7]. Changing the electronegativity of the C-6 substituent
could also potentially produce a profound effect on the
molecular interactions of cells. Since hydroxylation at C-6
of some androgen molecules enhances their androgenicity
in target tissues, increases or decreases in the electronega-
tivity at C-6 may also produce different significant effects.
Therefore, the purpose of this study was to determine the
pharmacological effects of the C-6 halogen atom on the
pregnane derivatives synthesized. The tested compounds
Researchers are still interested in the synthesis of an
ideal anti-androgenic molecule that can offer useful treat-
ment for androgen-mediated diseases, such as prostate can-
cer, acne, seborrhea, androgenic alopecia, precocious pu-
berty, and benign prostatic hyperplasia [1]. Using this idea
as a starting point, many researchers have synthesized a
variety of steroids showing higher androgenic activity [2–4]
than testosterone, as well as several new compounds with
anti-androgenic activity. An example of a compound with
high androgenic activity is 7␣-methyl-19-norethisterone
(MENT), a steroid more potent than T in the suppression of
serum gonadotropin levels, but one which can not undergo
*
Corresponding author. Biological System Department, Metropolitan
University-X, Calzada del Hueso #1100, 04860 M e´ xico, D.F. Mexico. Tel:
ϩ52-5-724-52-60; fax: ϩ52-5-724-52-37.
E-mail address: marisa@cueyatl.uam.mx (M. Cabeza)
0
039-128X/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved.
PII: S0039-128X(99)00018-5

4
14
M. Cabeza et al. / Steroids 64 (1999) 413–421
were the following: 17␣-hydroxy-16␤-methylpregna-4,6-
diene-3,20-dione (7); 17␣-acetoxy-16␤-methylpregna-4,6-
diene-3,20-dione (8); 17␣-acetoxy-6-bromo-16␤-methyl-
pregna-4,6-diene-3,20-dione (10), and 17␣-acetoxy-6-
chloro-16␤-methylpregna-4,6-diene-3,20-dione (11). These
compounds were synthesized from commercially available
2.2. Synthesis of compounds 7, 8, 10, and 11
The syntheses of 17␣-hydroxy-16␤-methylpregna-4,6-
diene-3,20-dione (7), 17␣-acetoxy-16␤-methylpregna-4,6-
diene-3,20-dione (8), 17␣-acetoxy-6-bromo-16␤-methyl-
pregna-4,6-diene-3,20-dione (10), and 17␣-acetoxy-6-
chloro-16␤-methylpregna-4,6-diene-3,20-dione (11) were
performed as previously described in the literature [17–25].
1
6-dehydropregnenolone acetate.
Analysis of the biologic potency of steroids consists of
two potentially independent processes: one involves meta-
bolic change in the steroid molecule produced by enzymes
2
.3. Pharmacological evaluation of compounds 7, 8, 10,
and 11
[8,9] and the other involves steroid action at the target organ
[10], where the compound binds to its cognate receptors
[11].
2
.3.1. Animals and tissues
Adult male Syrian Golden hamsters (150–200 g) were
To test the androgenic effects of the synthesized com-
obtained from Metropolitan University-Xochimilco of Mex-
ico. The animals were kept in a room with controlled tem-
perature (22°C) and light-dark periods of 12 h. Food and
water were provided ad libitum.
Gonadectomies were performed under light ether anes-
thesia. Animals were sacrificed by ether anesthesia. The
flank organs and seminal vesicles were immediately re-
moved, blotted, and weighed prior to their use in different
experimental procedures. Tissues used in the metabolic ex-
periments were homogenized with a tissue homogenizer
pounds, we used the flank organ model [12], and the
seminal vesicles of gonadectomized male hamster [13].
Flank organs are two pigmented nodules located in the
dorsal skin surface of hamsters. In female hamsters, the
diameter of the pigmented spot measures 2 mm, whereas
in males it is 8 mm. In males, these nodules shrink after
castration until they resemble the nodules of the female
[
12]. However, daily injections or topical applications of
testosterone (T) or 5␣-dihydrotestosterone (DHT) re-
stores their original size [13,14]. These glands can me-
tabolize T to DHT in both intact and gonadectomized
animals. Anti-androgenic drugs [15,16] have been eval-
uated using the flank organ model.
Seminal vesicles are male accessory organs that are also
androgen-dependent. These organs are capable of reducing
T to DHT in both intact and gonadectomized animals [8].
(model 985–370; variable speed 5000–30 000 rpm; Biospec
Products, INC).
2
.3.2. Flank organ test
The flank organ test was performed as previously re-
ported by Vermorken et al. [16]. The effect of steroids on
the flank organs of male hamsters, which were gonadecto-
mized 8 days before the experiments, was determined on six
groups of four animals/experiment, selected at random. The
animals were kept in a room with controlled temperature
2
. Experimental
(22°C) and light-dark periods of 12 h. Food and water were
provided ad libitum.
2
.1. Chemicals and radioactive material
2
.3.3. Hormone treatment of flank organs
Both flank organs of the animal were shaven prior to the
Solvents were laboratory grade or better. Melting points
were determined on a Fisher Johns melting point apparatus
and are uncorrected. Ultraviolet spectra were determined on
a Perkin Elmer 202 spectrometer. IR spectra were recorded
on a Perkin Elmer 549B spectrometer. NMR spectra were
determined on a Varian VXR-300 Spectrometer using deu-
teriochloroform as a solvent and tetramethylsilane as the
internal standard. High resolution mass spectra were re-
corded on a HP 5985-B apparatus.
experiments. Steroids were dissolved in 40 ␮l of ethyl
acetate and applied topically daily on the hamster’s right
flank organ [12,26]. Applications were made with an Edp 2
pipette (model E2 100, distributed by Rainin Instrument
Co). After 15 days of steroid treatment, the diameter of the
pigmented organ was determined. The left flank organ of
each animal received 40 ␮l of ethyl acetate and served as
the control.
3
3
(1,2,6,7- H) testosterone [ H]T, specific activity: 85–105
Topical applications on the right flank organs were as
follows:
Ci/mmol, was provided by New England Nuclear Co. (Bos-
ton, Mass.) and radio-inert T was kindly provided by Syntex
of Cuernavaca (Mexico). D(alpha)-glucose, which had a
melting point of 146°C, was purchased from Merck (Mex-
ico). Sigma Chemical Co. (St. Louis, MO.) supplied cypro-
terone acetate (CA), 5␣-dihydrotestosterone (DHT), and
GROUP 1: n ϭ 4200 ␮g of testosterone (T) [12,26]
GROUP 2: n ϭ 4200 ␮g of cyproterone acetate [26]
GROUP 3: n ϭ 4200 ␮g of steroid 7
GROUP 4: n ϭ 4200 ␮g of steroid 8
GROUP 5: n ϭ 4200 ␮g of steroid 10
ϩ
NADPH .
GROUP 6: n ϭ 4200 ␮g of steroid 11

M. Cabeza et al. / Steroids 64 (1999) 413–421
415
Another group of four animals served as the untreated
control to which solvent was applied only on the right flank
organ. Two separate experiments were performed for each
group of steroid-treated animals. The results were analyzed
using one-way analysis of variance with EPISTAT soft-
ware.
rificed by ether anesthesia, and the seminal vesicles were
dissected out and weighed on a balance.
Treatments for the animals were as follows:
GROUP 1: n ϭ 4200 ␮g of testosterone (T) [12,26]
GROUP 2: n ϭ 4200 ␮g of Tϩ200 ␮g cyproterone
acetate
n ϭ 4200 ␮g of Tϩ500 ␮g of cyproterone acetate [26]
n ϭ 4200 ␮g of Tϩ1 mg of cyproterone acetate
n ϭ 4200 ␮g of Tϩ2 mg of cyproterone acetate
GROUP 3: n ϭ 4200 ␮g of Tϩ200 ␮g compound 10
n ϭ 4200 ␮g of Tϩ500 ␮g of compound 10
n ϭ 4200 ␮g of Tϩ1 mg compound 10
2
.3.4. In vitro metabolic studies in flank organs
Homogenates from male hamster flank organs (around
.3 mg protein) were prepared from intact adult male ani-
4
mals using Krebs—Ringer phosphate buffer solution, pH 8.
Tissue preparations were incubated in duplicate with 85 nM
3
ϩ
n ϭ 4200 ␮g of Tϩ2 mg compound 10
[
H]T in the presence of 1 mM NADPH [12], 11 ␮M of
GROUP 4: n ϭ 4200 ␮g of Tϩ200 ␮g compound 11
n ϭ 4200 ␮g of Tϩ500 ␮g of compound 11
n ϭ 4200 ␮g of Tϩ1 mg of compound 11
unlabeled cyproterone acetate, and compound 10 or com-
pound 11 in a Dubnoff metabolic incubator at 37.5°C for 60
min with O /CO (95/5) as the gas phase. The final incu-
2
2
n ϭ 4200 ␮g of Tϩ2 mg compound 11
bation volume was 1 ml. Incubations without tissues were
used as controls. Incubation was terminated by addition of
Another group of four animals, which were injected with
only vehicle, served as the untreated control. Two separate
experiments were performed for each group of steroid-
treated animals. The results were analyzed using one-way
analysis of variance with EPISTAT software.
3
ethyl acetate, and [ H]steroids were extracted (4X) using 3
volumes of ethyl acetate. The organic extract was washed
with hexane in order to remove the remaining lipids. The
protein content of the homogenates was determined by
Bradford’s dye-binding method [27] using bovine serum
albumin (BSA) as the standard.
2
.3.7. In vitro metabolic studies with seminal vesicles
Homogenates from male hamster seminal vesicles
Isolation and purity assessment of radioactive dihy-
drotestosterone (DHT) by the reverse isotope dilution tech-
nique was performed. The isolated compound was purified
with steroid carriers (T and DHT) in two different thin-layer
chromatographic systems, (chloroform-acetone, 9:1; ben-
zene-ethyl acetate, 2:1) as well as by successive recrystal-
lizations to obtain constant specific activity. The radioactive
conversion product was identified on chromatographic
plates by autoradiography, while nonradioactive steroid car-
riers were detected using phosphomolybdic acid reagent and
an ultraviolet lamp (240 nm). Radioactivity was determined
in a Beckman LS 6000TA liquid scintillation spectrometer,
using Ultima Gold (Packard, Downers Grove, IL) as the
(
around 22 mg protein) were prepared from intact adult
male animals, using Krebs—Ringer phosphate buffer solu-
tion, pH 6. Tissue preparations were incubated in duplicate
3
ϩ
with 85 nM [ H]T in the presence of 1 mM NADPH [12],
1 ␮M unlabeled cyproterone acetate, compound 10, or
1
compound 11 in a Dubnoff metabolic incubator at 37.5°C
for 60 min with O /CO (95/5) as the gas phase. The final
incubation volume was 1 ml. Incubations without tissue
were used as controls. Incubation was terminated by addi-
tion of ethyl acetate, and [ H]steroids were extracted (4X)
using 3 volumes of ethyl acetate. The organic extract was
washed with hexane in order to remove the remaining lipids.
Isolation and purity assessment of radioactive DHT by
the reverse isotope dilution technique was performed as
described above for flank organs.
2
2
3
3
counting solution. The counting efficiency of [ H] was 67%.
The losses of radioactivity during the procedure were cal-
culated in agree at the results obtained from control exper-
iment without tissue. The formation of DHT was calculated
and expressed as fmol/mg protein/h and as percentage.
3
. Results
2
.3.5. Seminal vesicles test
The effect of steroids on seminal vesicles from castrated
The following is a brief description of the preparation of
the active and inactive compounds:
7␣-hydroxy-16␤-methylpregna-4,6-diene-3,20-dione 7;
7␣-acetoxy-16␤-methylpregna-4,6-diene-3,20-dione 8; 17␣-
male hamsters was determined in four groups of four ani-
mals/experiment selected at random and gonadectomized 8
days prior to experimentation. The animals were kept in a
room with controlled temperature (22°C) and light-dark
periods of 12 h. Food and water were provided ad libitum.
1
1
acetoxy-6-bromo-16␤-methylpregna-4,6-diene-3,20-dione 10,
and 17a-acetoxy-6-chloro-16␤-methylpregna-4,6-diene-3,20-
dione 11. This sequence of reactions is shown in Figure 1. The
starting material used for this synthesis was the commercially
available 16-dehydropregnenolone acetate 1. In the first step of
this synthesis, the C-16 double bond was epoxidized [22] with
hydrogen peroxide in basic medium, thus giving the desired
epoxy compound 2 (in this strong alkaline medium, the ace-
2
.3.6. Hormone treatment of seminal vesicles
Daily subcutaneous (s.c.) injections of the steroids at
different doses dissolved in 0.5 ml castor oil were admin-
istered for 3 days. After treatments, the animals were sac-

4
16
M. Cabeza et al. / Steroids 64 (1999) 413–421
Fig. 1. Synthesis of compounds 7, 8, 10, and 11.
toxy group at C-3 was hydrolyzed). Subsequent acetylation of
with acetic anhydride and pyridine afforded the desired
acetoxy compound 3.
In view of the fact that the epoxy moiety at C-16, 17
would be opened with methylmagnesium chloride in a sub-
sequent step, it was necessary to protect the C-20 carbonyl
group and thus avoid a possible reduction. With compound
3, this protection was carried out using ethylene glycol and
trimethyl orthoformate and p-toluenesulfonic acid as a cat-
alyst [23]. This reaction afforded the dioxolane derivative 4
in good yield.
2
In the subsequent step, the oxiran ring in 4 was opened

M. Cabeza et al. / Steroids 64 (1999) 413–421
417
Fig. 2. Effect of different compounds on diameter size. Significant differences were observed between the diameter of glands from castrated control animals
vehicle-treated, C) and testosterone-treated (T) animals (P Ͻ 0.05). Cyproterone acetate (CA) did not significantly change the diameter of the pigmented
spot of the glands (P Ͼ 0.05), and the same was observed for compounds 7 and 8. However, compound 10 significantly decreased the diameter of the spot
P Ͻ 0.05), and 11 showed a tendency to increased it.
(
(
with methylmagnesium chloride. The intermediate 5 was
obtained with the C-16␤-methyl group [17]. Treatment of 5
with acetone, acetic acid and p-toluenesulfonic acid af-
forded 6 with the original C-20 carbonyl group [24]. Oxi-
dation of the C-3 hydroxyl group in 6 was carried out
according to Shapiro’s method [25]. This reaction afforded
the pregna-4,6-diene-3,20-dione moiety 7 in high yield.
Acetylation of the C-17 hydroxyl group in 7 with acetic acid
and trifluoroacetic anhydride [26] yielded the desired ace-
toxy ester 8. Subsequent epoxydation of the C-6 double
bond with m-chloroperbenzoic acid [25] afforded the 6a,7a-
epoxy derivative 9.
When compound 9 was treated with anhydrous hydrochlo-
ric or hydrobromic acid in acetic acid [26], the first step in the
reaction resulted in the formation of the corresponding halo-
hydrines (C-6 halo, C-7 hydroxy compound). Both crude halo-
hydrines, without further purification, were heated in the same
reaction medium to dehydrate the hydroxyl group and thus
give the desired final compounds 10 and 11. The spectral data
of these compounds confirm the proposed structures. Addi-
tional information on the synthesis of these compounds can be
obtained from the literature [18].
that of the uncastrated animals. Topical applications of
vehicle alone did not change this condition. However, treat-
ment with testosterone (T) restored the diameter of the spot
(Fig. 2). Cyproterone acetate as well as compounds 7 and 8
did not have a significant (P Ͼ 0.5) effect on the diameter
of the pigmented spot (Fig. 2).
3
.1.2. Compound 10
This compound significantly decreased (P Ͻ 0.05) the di-
ameter of the pigmented spot of hamster flank organs from
castrated animals (Fig. 2). This suggests that compound 10
inhibits the conversion of testosterone (T) to DHT in this
tissue. This was also demonstrated by the inhibition of the
3
conversion of T( H) to DHT by the same compound at pH 8
(P Ͻ 0.05) as shown in Fig. 3. The purity of radioactive DHT
in all experiments was established by successive recrystalliza-
tions to constant specific activity (Table 1).
The overall data of these experiments indicate that com-
pound 10 significantly inhibits (P Ͻ 0.05) the conversion of
T to DHT in castrated male hamster flank organs.
3
.1.3. Compound 11
When the animals were treated with T and 11 the diam-
3
.1. Effect of the synthesized compounds on flank organs
and seminal vesicles of castrated male hamsters
eter of the pigmented spot of the glands increased compared
to that of the control (Fig. 2). Since the development of the
pigmented spots in male hamster flank organs depends on
the conversion of T to DHT, which is mediated by 5␣-
reductase enzyme [12,13], it was important to ascertain the
3
.1.1. Flank organs
After castration, the diameter of the pigmented spot on
the glands decreased significantly (P Ͻ 0.05) compared to

4
18
M. Cabeza et al. / Steroids 64 (1999) 413–421
3
3
Fig. 3. Effect of different synthetic steroids on in vitro conversion of [ H]T to [ H]DHT in castrated male hamster flank organs. A significant difference is
observed between control (C) and testosterone (T) plus 10 treatments, and control and T plus 11 treatments (P Ͻ 0.05). Values are given as fmol/mg of
protein/h Ϯ standard deviations.
3
effect of compound 11 on the conversion of [ H]T to
[
0
When T and cyproterone acetate were injected together,
the weight of the vesicles did not significantly diminish at
any of the doses used (P Ͼ 0.05); however, a tendency was
observed showing a decrease of the weight of the glands
(Table 2).
3
H]DHT. This compound significantly increased (P Ͻ
.05) the conversion of T to DHT by 150 fold as shown in
Figure 3. The purity of radioactive DHT was determined by
successive recrystallizations to constant specific activity
(
Table 1). It was observed that compound 11 at pH 8
Steroid 10 in the range of 0.5–2 mg decreased the
weight of the glands significantly (P Ͻ 0.05) compared to
the T treated animals (Table 2). Since the weight of the
seminal vesicles does not depend on the 5␣-reduced
androgens [6,7], it was important to determine the effect
of compound 10 on the conversion of T to DHT in this
tissue. The results obtained from two separate experi-
ments performed in duplicate demonstrated a difference
between the conversion of [ H]T to [ H]DHT in the
control, T, and T plus cyproterone acetate-treated ani-
mals, indicating that cyproterone acetate is a substrate for
conversion at pH 6. These data showed a significant
difference when T plus compound 10 were used (Fig. 4).
Steroid 10 completely inhibited completely the conver-
sion of T to DHT at pH 6 in seminal vesicle homogenates
from castrated male hamsters treated with a dose of 200
enhanced the conversion of T to DHT (Fig. 3).
3
.1.4. Seminal vesicles
After castration, the weight of the male hamster seminal
vesicles significantly decreased (P Ͻ 0.05) compared to that
of the normal glands. Treatment with vehicle alone did not
change this condition (Table 2), whereas s.c. injections of
3
3
2
00 mg of testosterone (T) for 3 days significantly increased
(P Ͻ 0.05) the weight of the seminal vesicles in castrated
male hamsters.
Table 1
The radioactive compound was obtained from extracts of flank organs
from castrated male hamsters pretreated with topical applications of
Tϩ10 or Tϩ11
3
Treatment
Specific activity [ H]DHT (dpm/mg)
␮
g (Fig. 4).
Tϩ10
Cr 109
The protein content in seminal vesicles under different
1
Cr 128
treatments is shown in Table 2. These data indicate that
protein synthesis is enhanced when the weight of the sem-
inal vesicles decreases.
2
ML 120
2
Tϩ11
Cr 613
1
When compound 11 was evaluated in the same man-
ner, it produced a decrease in the weight of the seminal
vesicles in a dose-dependent manner (Table 2), suggest-
ing another mechanism of action different from that of
steroid 10.
Cr 525
2
ML 600
2
3
Glands were incubated previously with [ H]T as described in the exper-
imental section.
Cr, crystallization number; ML mother liquor number.

M. Cabeza et al. / Steroids 64 (1999) 413–421
419
Table 2
conversion is higher at pH 6 as compared to that of the
less electronegative substituent bromine. These results
suggest that 5␣-reduction is dependent on the electro-
negativity of the C-6 substituent. However, this phenom-
enon does not explain the contrary effect observed when
the doses of compound 11 are increased to 2 mg. It is
possible that this compound inhibits the hypothalamic-
pituitary axis and consequently results in decreased T to
DHT conversion.
Weight and protein content of seminal vesicles from animals receiving
different s.c. treatments and doses
Treatment
mg)
Weight of seminal
vesicles (mg)
Protein content
(␮g/mg of tissue)
(
Control
196.0 Ϯ 29.0
19.5 Ϯ 0.050
7.6 Ϯ 0.002
T
0
.2
690.0 Ϯ 74.0
The overall data from these experiments show that
compound 10 inhibits the conversion of T to DHT,
whereas compound 11 increases DHT formation at low
doses. However, at high doses, steroid 11 completely
inhibits DHT formation, thus suggesting an additional
mechanism of action.
TϩCA
0.2
0.5
1.0
2.0
546.0 Ϯ 40.0
378.9 Ϯ 47.0
414.7 Ϯ 37.0
408.8 Ϯ 149
10.8 Ϯ 0.007
12.0 Ϯ 0.001
13.0 Ϯ 0.002
15.6 Ϯ 0.001
Tϩ10
0.2
0.5
1.0
2.0
706.0 Ϯ 30.0
270.0 Ϯ 72.0
326.8 Ϯ 58.0
233.2 Ϯ 26.0
10.3 Ϯ 0.002
70.7 Ϯ 16.60
63.2 Ϯ 12.20
56.0 Ϯ 5.20
4
. Discussion
This paper describes the synthesis of four new steroidal
Tϩ11
0
0
1
2
.2
.5
.0
.0
427.1 Ϯ 35.0
419.4 Ϯ 18.7
219.0 Ϯ 50.0
120.0 Ϯ 19.6
10.0 Ϯ 0.002
8.8 Ϯ 0.025
15.6 Ϯ 0.020
34.0 Ϯ 0.003
compounds 7, 8, 10, and 11 (Fig. 1). Compounds 7 and 8 are
devoid of a halogen atom at C-6 and are biologically inac-
tive. Steroids 10 and 11 have a halogen atom at C-6, bro-
mine and chlorine, respectively. They are biologically ac-
tive, and apparently the difference in the electronegativity of
the substituent at C-6 imparts distinct biologic properties.
Compound 10 decreases the diameter of the pigmented spot
in flank organs (Fig. 2).
Significant differences were observed between the weights of cas-
trated controls (vehicle-treated) and testosterone-treated (T) animals.
Cyproterone acetate (CA) did not significantly change the weight (P Ͼ
0
.05) with any of the doses used; however compound 10 decreased
weight significantly (P Ͻ 0.05). Also, compound 11 decreased the
weight of vesicles; but in this case, the effect was dependent on the
doses used. Protein content change in an inverse way relative to the
seminal vesicles weight in all cases. Compounds 10 and 11 increased
protein content in seminal vesicles.
The development of the pigmented spot in male hamster
flank organs depends on DHT [28] and the conversion of T
to DHT in the flank organ is mediated by 5␣-reductase
[12,14,28]. The inhibitory effect of 10 on the diameter of the
spot can be explained by the inhibition of this enzyme at pH
8. The major difference between compounds 10 and 11 is
the electronegativity of the halogen substituent at C-6. Ev-
idence suggests that when the electronegativity of the halo-
gen is decreased, as with the bromine atom in compound 10,
The hexane extracts from castrated male hamster sem-
inal vesicles receiving different treatments were sub-
jected to TLC analysis. The zone corresponding to the
DHT standard (Rf values 0.33) of each experimental
chromatogram was eluted and the radioactivity deter-
mined. The results obtained from two separate experi-
ments performed in duplicate demonstrated that signifi-
cant differences (P Ͻ 0.05) existed between the
conversion of [ H]T to [ H]DHT in control samples (T
and T plus compound 11) at pH 6 (Fig. 4). Compound 11
increased the conversion of T to DHT by three fold at the
same dose (Fig. 4), suggesting that 11 also enhanced the
conversion of T to DHT in this tissue at pH 6.
3
3
it inhibits the conversion of [ H]T to [ H]DHT in flank
organs. Contrary to this, when the halogen substituent has a
higher electronegativity, as with the chlorine atom in com-
3
pound 11, it increases the formation of [ H]DHT from
3
3
3
[ H]T.
It is a well-known fact that in humans and animal tissues
two isozymes of 5␣-reductase exist and have different activi-
ties at different pHs [29]. The fact that compound 10 inhibits
3
3
the conversion of [ H]T to [ H]DHT at pH 8 in flank organs
suggests that this steroid interacts with the type 1 isozyme,
which has optimal activity at basic conditions.
When the animals were injected with high doses of 11
(
2 mg for three days), the formation of DHT was com-
pletely inhibited. This confirms the fact that a dose-
dependent phenomenon, contrary to that observed above,
is evident in this case.
Taken together, these findings suggest that steroid 10 has
an anti-androgenic effect in the flank organ inhibiting the
conversion of T to DHT.
As mentioned above, compound 11 at doses of 200 ␮g
increased the conversion of T to DHT at pH 6 by three-
fold in a similar manner to that observed at pH 8. These
data indicate that when the electronegativity of the C-6
substituent is increased (chlorine), the rate of T to DHT
As mentioned above, compound 11 shows a tendency to
increase the diameter of the pigmented spot (Fig. 2). This
result is similar to that found by Burdick and Hill [30] on
hamster flank organs treated with chlormadinone acetate, a
derivative of 17␣-acetoxy progesterone, which has a chlo-

4
20
M. Cabeza et al. / Steroids 64 (1999) 413–421
3
3
Fig. 4. Effect of different synthetic steroids on in vitro conversion of [ H]T to [ H]DHT in castrated male hamster seminal vesicles homogenates. Compound
1 stimulated significantly T conversion (P Ͻ 0.05), whereas compound 10 inhibited it completely until undetectable levels. Values are given in fmol/mg
of protein/h Ϯ standard deviations. Control (C), testosterone (T), cyproterone acetate (CA).
1
rine atom at C-6. Steroid 11 increases the conversion of T to
DHT (Fig. 3) producing androgen activity. This evidence
demonstrates that 11 stimulating T conversion.
Compound 10 in the range of 0.5 to 2 mg decreases the
weight of the seminal vesicles significantly (P Ͻ 0.05)
compared to T-treated animals; however, this effect is not
observed at doses of 200 ␮g (Table 2). This compound also
inhibits the conversion of T to DHT in seminal vesicle
homogenates from castrated males treated with doses of 200
completely inhibited the formation of DHT, thus indicating
that an effect is present contrary to that observed before
(Table 2). This inhibitory effect depends on the dose used
and suggests that steroid 11 could potentially inhibit the
hypothalamic-pituitary axis. These results could also ex-
plain the fact that chlormadinone acetate, at high doses (20
mg/rat), decreases the weight of rat seminal vesicles and
shows an inhibitory effect on gonadotropin secretion [31,
32].
␮
g (Fig. 4). The results suggest that compound 10 inhibits
The effect observed with 200 ␮g of compounds 10 and
11 on the conversion of T to DHT in seminal vesicles from
castrated male hamster follows a similar pattern to that
observed in flank organs (Figs. 3 and 4). These data confirm
the hypothesis that the high electronegativity of the halogen
atom at C-6 increases the conversion of T to DHT at both
pHs.
Protein synthesis in seminal vesicles under different
treatments was determined by Bradford’s method and is
shown in Table 2. These data indicate that when a signifi-
cant decrease (P Ͻ 0.05) in the weight of the seminal
vesicles has taken place, protein synthesis is increased. On
the other hand, the conversion of T to DHT enhances pro-
tein synthesis. These proteins could be enzymes, membrane
proteins, or cytosol receptors.
The overall data indicate that the biologic properties of
compounds 10 and 11 are determined by the electronega-
tivity of the halogen atom at C-6. These data show very
clearly that a decrease in the electronegativity of the halo-
gen atom (compound 10) significantly inhibits (P Ͻ 0.05)
the conversion of T to DHT. When the electronegativity of
the halogen atom is increased (compound 11), a significant
the 5␣-reductase isozyme type 2 at pH 6 [6]; however, it
does not change the weight of the seminal vesicles at the
same doses. From these data, we can infer that T and not
DHT is actually responsible for the weight increase of the
seminal vesicles, as has also been suggested by others [6,7].
With compound 10, the real anti-androgenic effect is ob-
served in the dose range of 0.5 to 2 mg (Table 2). This effect
was similar to that observed when the animals were treated
with cyproterone acetate; nevertheless, steroid 10 showed
higher biologic activity at lower doses.
Compound 11 significantly decreased (P Ͻ 0.05) the
weight of the seminal vesicles in a dose dependent man-
ner (Table 2), suggesting another mechanism of action
different from that of compound 10. Furthermore, while
the conversion of T to DHT in the presence of 11 was not
inhibited at doses of 200 ␮g, we observed a decrease in
the weight of the seminal vesicles. This effect could
originate from an increase in the androgens produced by
the tissue that could inhibit gonadotropin production by
the pituitary gland.
However, at a dose of 2 mg/day for 3 days, compound 11

M. Cabeza et al. / Steroids 64 (1999) 413–421
421
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