New-D-homoandrost-4,6-diene derivatives as potent progesterone receptor antagonist

Article abstract of DOI:10.1016/j.steroids.2009.11.001

The aim of this study was to synthesize three different D-homoandrostadiene derivatives (2-4) and study their biological activity. We carried out in vivo and in vitro experiments using female cycling mice, which were synchronized for estrus with luteinizi

Full text of DOI:10.1016/j.steroids.2009.11.001

Steroids 75 (2010) 101–108
Contents lists available at ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
New-D-homoandrost-4,6-diene derivatives as potent progesterone receptor
antagonist
Marisa Cabezaa,∗, Mario García-Lorenzanab, Montserrat Garcésa,
Ivonne Heuzea, Nayeli Teranc, Eugene Bratoeffc
a Department of Biological Systems and Animal Production Metropolitan University-Xochimilco, Mexico D. F., Mexico
b Department of Biology of Reproduction, Metropolitan University-Iztapalapa, Mexico D. F., Mexico
c Department of Pharmacy, Faculty of Chemistry, National University of Mexico City, 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 synthesize three different D-homoandrostadiene derivatives (2–4) and study
their biological activity. We carried out in vivo and in vitro experiments using female cycling mice, which
were synchronized for estrus with luteinizing hormone-releasing hormone (LHRH) and injected with
the steroidal compounds. It was also determined the binding of these compounds to the progesterone
receptors (PR). Since these steroids have a new D-homoandrostandienone skeleton in their molecular
structure, it was of interest also to study their binding to the androgen receptors (AR).
After LHRH treatment, the mice of the control group showed the presence of 14 4 corpus lutea in the
ovary whereas the animals treated with steroids 2–4, with RBAs of 100%, exhibited 11 7, 12 2, and
10 4 respectively. As a result of this study, it is evident that these steroids did not inhibit the ovulation
in these animals.
Received 15 September 2009
Received in revised form 14 October 2009
Accepted 5 November 2009
Available online 12 November 2009
Keywords:
Antagonist of the progesterone receptors
Endometriosis
New D-homoandrost-4,6-diene derivatives
Uterine histology
The uterus of the control group, showed the typical progestational activity with an enlarged endome-
trial thickness with a secretory activity. However, the endometrium of the mice treated with steroids
2–4 did not show an enlargement of the endometrium and no secretory activity could be detected. This
fact indicates that compounds 2–4 had antagonistic activity in this tissue.
Androgen receptors
The overall data show that steroids 2–4 are antagonists of the PR. However, they do not bind to the AR.
These results also demonstrate that 2–4 have an antiprogestational activity in vivo, but do not decrease
the number of corpus lutea in the ovary of mice treated with LHRH.
© 2009 Elsevier Inc. All rights reserved.
1. Introduction
the treatment of hormone-related pathological conditions such as
breast cancer, endometriosis and uterine leiomyomas. Mifepris-
Blocking progesterone receptor (PR) function by using PR
antagonists should allow the modulation of various reproduc-
tive processes. On this basis antiprogestins were developed which
disrupt the normal progesterone-induced signal transduction path-
way by competitive binding to PR. Therefore, antiprogestins
have considerable potential as therapeutic drugs for numerous
gynecological, obstetrical and oncological afflictions as well as con-
traceptives since these compounds can block ovulation [1] and
prevent implantation [2].
tone 1 (RU 486) was the first steroid of this class to show high
affinity for binding to the progesterone receptor (PR) [3,4] and
was recommended for the treatment of endometriosis [5]. How-
ever, mifepristone induces the luteolysis in pregnant mice [6] and
inhibits gonadotropin activity in women [7]. These are not desir-
able effects for the treatment of sterility produced by endometriosis
since the ovulation is inhibited. Mifepristone also produces side
effects that include hot flushes and transient increases in liver
transaminases [5].
Antiprogestins, such as mifepristone 1 (Fig. 1), have poten-
tial use for both regular and emergency contraception and for
Previously, we synthesized several progesterone derivatives
containing a phenylacetyloxy substituent at C-17 which bind
selectively to the PR and showed an antiprogestin activity
[8]. These steroids inhibited the ovulation and interrupted the
endometrial maturation of estrous mice. Since these compounds
inhibited the ovulation it was of interest to synthesize similar
D-homoandrostadiene derivatives having a phenyl substituent at
C-16, methyl and phenylacetoxy functions at C-17 (compound 2)
or a fluorophenyl acetoxy group at C-17 (compounds 3 and 4) and
∗
Corresponding author at: Departamento de Sistemas Biológicos, Universidad
Autónoma Metropolitana-Xochimilco, Calzada del Hueso No. 1100, Col Villa Qui-
etud, 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 © 2009 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2009.11.001

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M. Cabeza et al. / Steroids 75 (2010) 101–108
Fig. 1. Structures of mifepristone and the novel steroids 2–4.
determine the antiprogestational effect in the endometrium with-
out blocking ovulation or to produce degenerative changes in the
ovary.
The aim of this research was to synthesize three different D-
homoandrostadiene derivatives (Fig. 2): 17␤-Methyl-16␤-phenyl-
17␣-phenylacetoxy-D-homoandrost-4,6-diene-3,17a-dione (2),
17␤-methyl-16-␤-phenyl-17␣-(2-fluorophenyl)acetoxy-D-homo-
2.2. Synthesis of steroidal derivatives
The synthesis of the new steroids 2–4 is briefly described below.
The preparation of the intermediates 6–11 is given in Ref. [10].
A mixture of the corresponding phenyl acetic acid (1.79 mmol),
p-toluenesulfonic acid (0.001 g, 0.0052 mmol) and trifluoroacetic
anhydride (0.19 g, 0.81 mmol) was stirred for 2 h at room tempera-
ture. Steroid 11 (0.2 g, 0.49 mmol) was added; the reaction mixture
was stirred for an additional 2 h at room temperature (nitrogen
atmosphere). It was neutralized with an aqueous sodium bicar-
bonate solution to pH 7 and diluted with chloroform (10 mL). The
organic phase was separated and dried over anhydrous sodium sul-
fate; the solvent was eliminated in vacuum. The crude product was
purified by silica gel column chromatography and recrystallized
from methanol.
androst-4,6-diene-3,17a-dione
(3)
and
17␤-methy-16␤-
phenyl-17␣-(3-fluorophenyl)acetoxy-D-homoandrost-4,6-diene-
17a-dione (4) and to study their activities as potent progesterone
receptor antagonists. It was also of interest to determine the bind-
ing of these steroids to the androgen receptor (AR). Furthermore
we also examined the effect of these compounds in the ovulation,
as well as their function as interrupters of endometrial maturation.
2. Materials and methods
2.2.1. 17ˇ-Methyl-16ˇ-phenyl-17˛-phenylacetoxy-D-
homoandrost-4,6-diene-3,17a-dione
(2)
2.1. Chemical and radioactive material
Yield 0.12 g, 0.19 mmol (39%) of pure product 2, m.p. 186–189 ◦C.
UV (nm): 283 (ε = 22,900). IR (KBr) cm−1: 1735, 1720, 1704, 1667,
1620, 870, 761, 700. 1H NMR (CDCl3) ı: 1.10 (3H, s, H-18), 1.17
(3H, s, H-19), 1.26 (3H, s, methyl at C-17), 5.4 (2H, s, COOCH2-Ph),
5.68 (1H, s, H-4), 6.13 (1H, d, J = 8.8 Hz, H-6), 6.26 (1H, q, J1 = 8.8 Hz,
J2 = 2 Hz, H-7), 7.23 (5H, m, phenyl at C-16), 7.35 (5H, m, phenyl of
ester). 13C NMR (CDCl3) ı: 16.2 (C-18), 17.6 (C-19), 22.9 (methyl
at C-17), 123.8 (C-4), 128.2 (C-6), 139.2 (C-7), 162.2 (C-5), 170.7
(ester carbonyl), 199.4 (C-3), 211.6 (C-17a). FAB-MS (m/z) calcd for
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 (ı = 0) in CDCl3 (the abbreviations
of signal patterns are as follows: s, singlet; d, doublet, t, triplet, m,
multiplet, q, quartet). Mass spectra were obtained with an HP5985-
B spectrometer. IR spectra were recorded on a PerkinElmer 200 s
spectrometer.
The radioligands: promegestone (17␣-methyl-3H) [3H] R5020
(synthetic progestin with high affinity for the PR [9]) specific
activity of 87 Ci/mmol; mibolerone (17␣-methyl-3H) [3H] MIB
(synthetic androgen with high affinity by the AR) specific activ-
ity of 70–87 Ci/mmol, were provided by PerkinElmer Life Sciences,
Inc. (Boston, MA). Radio inert mibolerone and R5020, were sup-
plied by Steraloids (Wilton, NH, USA) and PerkinElmer Life Sciences,
Inc. (Boston, MA), respectively. dl-Dithiothreitol and protease
inhibitors were purchased from Sigma–Aldrich (St. Louis, MO,
USA). Activated charcoal (acid washed with hydrochloric acid) and
Dextran (Mr-70,000) were supplied by Sigma–Aldrich (St. Louis,
MO).
C35H38O4 523.5736 (M+H), found 523.5654.
2.2.2. 17ˇ-Methyl-16ˇ-phenyl-17˛-(2-fluorophenyl)
acetoxy-D-homoandrost-4,6-diene-3,17a-dione (3)
Yield 0.1 g, 0.19 mmol (37.4%) of pure product 3 m.p. 192–194 ◦C
UV (nm) 284 (ε = 22,700). IR (KBr) cm−1: 1730, 1712, 1662, 1618,
877. 1H NMR (CDCl3) ı: 1.11 (3H, s, H-18), 1.15 (3H, s, H-19), 1.22
(3H, s, methyl at C-17), 5.4 (2H, COOCH2-Ph), 5.67 (1H, s, H-4), 6.13
(1H, d, J = 8.8 Hz, H-6), 6.27 (1H, q, J1 = 8.8 Hz, J2 = 2 Hz, H-7), 7.04
(5H, m, phenyl at C-16), 7.26 (4H, m, phenyl of ester). 13C NMR
(CDCl3) ı: 16.4 (C-18), 18.0 (C-19), 23.1 (methyl at C-17), 127.4
(C-4), 128.1 (C-6), 128.4 (C-para, aromatic, 1C), 129.1 (C-ortho,

M. Cabeza et al. / Steroids 75 (2010) 101–108
103
Fig. 2. Conditions and reagents: the preparation of compounds 5–11 is given in Ref. [10]. (i) (CF3CO)2O, PTSA, RCOOH, r.t., 3 h.
aromatic, 1C), 129.5 (C-meta aromatic, 2C), 130.2 (C-F aromatic,
2.4. Receptor binding assays
1C), 138.5 (C-7), 162.9 (C-5), 170.2 (ester carbonyl), 199.9 (C-3),
211.8 (C-17a). FAB-MS (m/z) calcd for C35H37FO4 541.3412 (M+H),
found 541.3465.
In this study we determined the binding of compounds: 2–4
to the progesterone receptors. Since these steroids have a new D-
homoandrostandienone skeleton in their molecular structure, it
was of interest also to determine their binding to the androgen
receptors.
2.2.3. 17ˇ-Methyl-16ˇ-phenyl-17˛-(3-fluorophenyl)
acetoxy-D-homoandrost-4,6-diene-3,17a-dione (4)
Yield 0.1 g, 0.19 mmol (37.4%) of pure product 4 m.p. 178–180 ◦C
UV (nm): 285 (ε = 22,500). IR (KBr) cm−1: 1725, 1711, 1665, 1620.
1H NMR (CDCl3) ı: 1.11 (3H, s, H-18), 1.14 (3H, s, H-19), 1.23 (3H,
s, methyl at C-17), 5.4 (2H, s, COOCH2-Ph), 5.70 (1H, s, H-4), 6.15
(1H, d, J = 8.8 Hz, H-6), 6.25 (1H, q, J1 = 8.8 Hz, J2 = 2 Hz H-7), 6.91
(5H, m, phenyl at C-16), 7.37 (4H, m, phenyl of ester). 13C NMR
(CDCL3) ı: 16.22 (C-18), 17.73 (C-19), 22.85 (methyl at C-17), 123.85
(C-4), 125.26 (C-6), 127.18 (C-para, aromatic, 1C), 128.12 (C-ortho,
aromatic, 2 C), 128.75 (C-meta, aromatic, 1C), 130.11 (C-F, aromatic,
1C), 138.10 (C-7), 163.90 (C-5), 172.21 (ester carbonyl) 199.41 (C-3),
211.45 (C-17a). FAB-MS (m/z) calcd for C35H37FO4 541.3513 (M+H),
found 541.3526.
2.4.1. Androgen receptor (AR)
Rat prostates were homogenized with a tissue homogenizer
(Teckmar, Cincinnati, OH), in one volume of buffer TEMD at pH 8
and protease inhibitors (2 mM PMSF, 10 ␮g/mL antipain, 5 mM leu-
peptin [11]) in ice bath with a tissue homogenizer. Homogenates
were centrifuged at 140,000 × g for 60 min [12] 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 (4 mg of protein in 200 ␮L) were deter-
mined by the Bradford method [13].
For competitive studies, tubes containing 1 nM of [3H] MIB plus
a range of increasing concentrations (1 × 10−10 to 4 × 10−7 M) of
cold MIB or steroids 2–4 in ethanol or chloroform, or in the 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
[14]; the solvent was completely eliminated.
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 18 h at 4 ◦C in the tubes as previously
described [11]. After incubation 0.27 mL of saturated ammonium
sulfate in TEMD buffer (35%) was added [15]. The mixture was fur-
ther incubated for 1 h with occasional shaking for the precipitation
of the [3H] MIB-complex. The precipitate was collected by centrifu-
gation at 10,000 × g for 10 min and the pellet was redissolved in
0.5 mL of TEMD and mixed with 0.5 mL of 0.1% dextran-coated 1%
2.3. Biological activity of the progesterone derivatives
The biological activity of 2–4 (Fig. 2) was determined in in
vivo and in vitro experiments using CD1 female mice and adult
New Zealand white female rabbits. The animals (mice 20–25 g
and rabbits 4 kg) were obtained from the Metropolitan University-
Xochimilco of Mexico. At the end of the in vivo experiment, the mice
were sacrificed by cervical dislocation. In order to obtain the cytosol
from the uteri of rabbits, the animals were sacrificed with CO2. This
protocol was approved by the Institutional Care and Use Committee
of the Metropolitan University of Mexico. The uteri of the rabbits
were removed, blotted, weighed in ice bath and soaked in cold
TEMD (40 mM Tris–HCl, 3 mM EDTA and 20 mM sodium molyb-
date, dithiothreitol 0.5 mM, 10% glycerol at pH 7.4) and maintained
in ice bath prior to their use.

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M. Cabeza et al. / Steroids 75 (2010) 101–108
charcoal in TEMD buffer. The mixture was incubated for 10 min at
4 ◦C. To prepare the dextran-coated charcoal mixture, the dextran
was agitated for 30 min before adding the charcoal to the mix-
ture. The mixture was agitated for two more hours. The tubes were
vortexed and immediately centrifuged at 800 × g for 10 min to pel-
let the charcoal; aliquots (600 ␮L) were taken and submitted for
radioactive counting using Ultima Gold (Packard) as counting solu-
tion. The radioactivity was determined in a scintillation counter
(Packard tri-carb 2100 TR). The IC50 of each compound was calcu-
lated according to the plots of concentration versus percentage of
binding.
of the test compounds; then they were grouped, with four mice
per treatment. In the morning of the 9th day following luteinizing
hormone-releasing hormone treatment, the mice of three differ-
ent groups, were injected with compounds 2–4 with RBAs of 100%
(0.22 mg/kg in 100 ␮L of sesame oil) once daily subcutaneously for
four consecutive days. One vehicle-treated group was maintained
as a control. The animals were sacrificed in the morning follow-
ing the last treatment. The ovaries and the uteri of mice of each
group were removed and prepared for histological examination.
Tissues were fixed with buffered formaldehyde (10%) [18], pro-
cessed with conventional histological techniques and included in
Paraplast (Oxford Labware, St. Louis, MO 63103, USA). The ovary
from each group of mice was cut sagittally, whereas the uteri were
cut transversally; 5 ␮m serial sections of the included tissues were
obtained. These sections were stained using hematoxylin–eosin
[19] and analyzed under a clear field light microscope (Axioskope II,
Carl Zeiss) and image analyzer (Axiovision 4.5, Carl Zeiss). The cor-
pus lutea present in the ovary of each animal/group were counted.
The results were analyzed using one-way analysis of variance and
Dunnett’s Method to compare means, with JMP IN 5.1 software.
Micrographs of the specimen were taken with an AxioCamMRc5
(Carl Zeiss).
2.4.2. Progesterone receptor (PR)
Uteri were isolated from mature rabbits treated for 7 days with
10 ␮g/animal/estradiol valerate. The uteri from the rabbits were
minced and homogenized in equal volume of TEMD buffer plus pro-
tease inhibitors (2 mM PMSF, 10 ␮g/mL antipain, 5 mM leupeptin
[9]; 40 mM Tris–HCl, 3 mM EDTA and 20 mM sodium molybdate,
dithiothreitol 0.5 mM, 10% glycerol at pH 7.4) [8]. The homogenate
was centrifuged at 140,000 × g for 1 h at 0 ◦C in a SW 60 Ti rotor
(Beckman Instruments, Palo Alto, CA).
The cytosolic fraction obtained from the supernatant liquid of
the rabbit uteri homogenate described above, was stored at −70 ◦C.
Uteri cytosol proteins (4 mg of protein in 200 ␮L) were determined
by the Bradford method [13].
3. Results
For competitive studies, tubes containing 0.4 nM of [3H] R5020
[15]; plus a range of increasing concentrations (1 × 10−10 to
4 × 10−6 M) of cold R5020, Mifepristone (RU486) or steroids 2–4
(1 × 10−10 to 4 × 10−6 M) in ethanol or chloroform, or in the absence
of the competitor were prepared. In all tubes the solvent was
eliminated in vacuum. We determined also the binding of the
antiprogestin Mifepristone (RU486) to the PR using different con-
centrations of this steroid (1 × 10−10 to 4 × 10−6 M) and 0.4 nM of
[3H] R5020 for the competitive binding.
3.1. Synthesis of steroidal derivatives 2–4
Compounds 5–11 were prepared from the commercially avail-
able 16-dehydropregnenolone acetate 5 [10] (Fig. 2). The final
products 2–4 were obtained by esterification of the free alcohol
11 with the corresponding acid. The NMR, UV, IR and mass spectra
of all intermediates and final products confirmed unequivocally the
structure of these compounds.
Aliquots of 200 ␮L of uteri cytosol (4 mg of protein) were added
and incubated in the presence of 300 ␮L of TEMD buffer contain-
ing protease inhibitors in the tubes (duplicate) for 18 h at 4 ◦C
as previously described [11]. After incubation 0.21 mL saturated
ammonium sulfate in TEMD buffer (30%) was added [8,16,17]. The
mixture was further incubated for 1 h with occasional shaking for
precipitation of the [3H] R5020-complex. The precipitate was col-
lected by centrifugation at 10,000 × g for 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 incu-
bated for an additional 10 min at 4 ◦C. The tubes were vortexed
and immediately centrifuged at 800 × g for 10 min to pellet the
charcoal; aliquots (600 ␮L) were taken and the radioactive count-
ing was determined. The IC50 of each compound was calculated
according to the plots of concentration versus percentage of bind-
ing.
3.2. Relative binding affinities of the homoandrostadiene
derivatives to the PR
We evaluated three new steroidal compounds: 2 without fluo-
rine atom in the ester side chain, 3 having a fluorine atom in ortho
position in the side chain and 4 with a fluorine atom in meta posi-
tion in the side chain for the inhibition of [3H] R5020 binding to
the progesterone receptor. The “IC50 values” for the displacement
of [3H] R5020 binding to the progesterone receptor and RBAs are
shown in Table 1. Progesterone and R5020 as well as steroids 2–4
have similar IC50 values; these steroids bind to the progesterone
receptor with RBAs of 100%.
On the other hand RU486 also binds to the PR with an IC50
value of 1.39 nM and a RBA value of 28.7% (Table 1). This com-
pound has an affinity for the PR as previously had been reported
[7].
Having demonstrated in this study that the novel steroids bind
to the PR, we also evaluated their binding to the AR [12]. It was
shown that the tested steroids did not bind to the AR since none of
the steroids inhibited the [3H] mibolerone binding to the AR present
in the cytosol obtained from castrated rats. Cold (not radioactive)
mibolerone inhibited the binding to the AR at concentration of
6 nM.
2.5. In vivo experiments
2.5.1. Determination of the effect of 2–4 on the number of corpus
lutea present in the ovary and on the endometrial thickness with a
secretory activity from treated mice
The animals were kept in a room with controlled temperature
(22 ◦C) and light–dark periods of 12 h. Food and water were pro-
vided ad libitum.
3.3. In vivo experiments
In order to know the number of corpus lutea present in
the ovary we used random cycling mature females CD1 Mice
(20–25 g) which were synchronized for estrus with 2 ␮g of luteiniz-
ing hormone-releasing hormone (Sigma) (in phosphate-buffered
saline containing 0.1% of bovine serum albumin) administered
subcutaneously per mouse at 9:00 h and again at 16:00 h [8]. Ani-
mals were allowed to rest for 8 days before the administration
3.3.1. Effect of 2–4 in the number of corpus lutea in the ovary of
mice
After 13 days following luteinizing hormone-releasing hormone
treatment, the mice of the control group showed the presence
of 14 4 corpus lutea, whereas the animals treated with steroids
2–4 with RBAs of 100%, had 11 7, 12 2, and 10 4 respectively

M. Cabeza et al. / Steroids 75 (2010) 101–108
105
Table 1
Relative binding affinities (RBAs) of the novel steroidal compounds 2–4 to the progesterone receptor.
Structures
IC50 (nM)
RBA (%)
0.37 0.065
100
0.36 0.045
100
1.39 0.19
28.7
0.37 0.011
100
0.37 0.089
100
0.37 0.036
100
IC50 standard deviations and RBA values of the novel steroidal compounds to the progesterone receptor obtained from rabbits’ uteri, previously treated
with estrogens. Each experiment was carried out in two different times by duplicate. The RBA was calculated according to the following equation: RBA =
(IC50 of [3H] promegestone/IC50 of inhibitor) × 100.
(Fig. 3). It is evident from this study that the novel steroids 2–4 did
not significantly inhibit the ovulation. On the other hand, no degen-
erative change was observed in the ovary of the mice treated with
steroids 2–4.
activity and an increase of the luminal folding of the endometrium
were also observed, both indicating a progestational activity. How-
ever, the endometrium of the mice treated with steroids 2–4 (with
RBAs of 100%), showed a reverse progesterone-induced transfor-
mation effect, with no enlargement of the endometrial thickness.
Furthermore, we also observed a large reduction of the secretory
activityandluminal foldingin the endometrium ofthe treated mice,
thus indicating that compounds 2–4 have antagonistic effect for
this tissue. On the other hand, we also observed a reduction of the
diameter of the uteri of the treated mice compared to the con-
3.3.2. Mice uterine transformations
After 13 days following luteinizing hormone-releasing hormone
treatment, the uterus specimens of the mice of the control group,
showed an enlargement of the endometrial thickness, which is
characteristic of the progestational phase. An intense secretory

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M. Cabeza et al. / Steroids 75 (2010) 101–108
Progesterone, promegestone as well as steroids 2–4 showed
similar IC50 values and RBAs of 100%. These data indicated that the
studied compounds had high affinity for the PR. Steroids 2–4 also
showed a higher affinity for the PR than RU486.
Since the binding assay showed that the concentration of the
unlabeled promegestone necessary for the displacement of 50% of
the labeled promegestone (0.4 nM) used for the binding to PR was
0.4 nM, this fact indicates a high reliability for this assay. The com-
petition studies previously reported by Palmer et al. [16] showed an
IC50 value for the unlabeled progesterone of 4 nM, when they used
labeled promegestone in a concentration of 0.4 nM. The difference
between Palmer’s and our studies is that we could precipitate the
PR with ammonium sulfate [8]; this procedure permits the forma-
tion of a purified fraction of the PR [17] and better competition
analysis.
On the other hand the competition study reported by Chan-
drasekhar and Amstrong [20] showed a RBA value of Mifepristone
(RU486), for the PR, of 187.5%. However, in our method, which
uses ammonium sulfate for the precipitation of the PR from primed
estrogen rabbit uterus, we found a RBA value for RU486 of 28.7%.
This difference in the RBA value could be explained on the ground
that Chandrasekhar and Amstrong’s method did not use purified
fraction of the PR. These authors used labeled promegestone in a
concentration of 10 nM; however they needed 180 nM of unlabeled
promegestone for the displacement of 50% of the labeled promege-
stone (10 nM). These data demonstrated that the use ammonium
sulfate for the precipitation of PR is an advantage. Gestrinone with a
RBA value of 75% [21] is another steroidal antiprogestin, which had
Fig. 3. Number of corpus lutea present in the ovary of mice, after 8 days of treatment
with 2 ␮g of LHRH. The mice were treated daily for 4 days with vehicle (controls)
or with the novel steroids. No significant differences were observed between the
control and treated groups.
trol group. The pictures of the histological sections are shown in
Figs. 4 and 5.
4. Discussion
In this paper, we showed that steroids 2–4 bind to the PR present
in the cytosol from estrogen-primed rabbits. However, these com-
pounds did not bind to the AR existing in the cytosol of rat prostate.
Fig. 4. These photomicrographs show the histology of the mice’s uteri. Important changes can be observed. In the center are shown transverse sections of control and
experimental groups (2–4) (H–E, bar 1000 ␮m). In photomicrographs 2-4 can be observed an evident reduction of the uterus’ diameter and lumen (L), when compared to
the control group. Framed regions were enlarged to show endometrial glands (arrow). Experimental groups exhibit differences as compare to the control group (H–E, bar
200 ␮m).

M. Cabeza et al. / Steroids 75 (2010) 101–108
107
Fig. 5. These photomicrographs show the histology of the mice’s uteri. Experimental groups (photomicrographs 2–4) show a reduction in the number and activity of
endometrial glands as manifested by the size and dilatation when compared to the control; an abundant secretion can be observed, arrows (H–E bar 40 ␮m).
been used for the treatment of endometriosis. This steroid demon-
strated a weak agonistic activity and a marked pituitary inhibitory
activity.
advantage over the previously reported results [8]. The histological
analysis of the ovary in the treated animals indicated that 2–4 did
not produce any degenerative process in the ovary.
The fact that 2–4 did not bind to the AR could be advantageous
for its use in the control of fertility (menses-induction and abortion)
[7,22], in hormone dependent tumors (e.g. breast cancer) and the
control of cell growth and differentiation (endometriosis [5,21])
because they will not produce an androgenic effect.
The results from this study indicate very clearly that these
steroids showed a high binding affinity as well as high antagonis-
tic activity for the PR present in the estrogen-primed rabbit uterus.
In view of the fact that they had high potency in vivo, they could
probably be used for the treatment of endometriosis without sup-
pressing the ovulation, an important improvement as compared to
the presently used compounds.
The overall data obtained from this study indicate that the
PR antagonists: 2–4 (D-homoandrostadienone-derivatives) did not
exhibit androgenic effects, since they did not bind to the AR.
It has been shown by several authors [7,23–28] that endocrine
bioassays using the uterine transformations assay, as parameter for
the determination of progestational activity is a reliable method for
the in vivo evaluation. This assay gives more real information than
those carried out in vitro and can be used as a definite test. Previ-
ously, it had been demonstrated that progesterone antagonists such
as mifepristone have reverse progesterone-induced transformation
in estrogen-primed rabbit uterus [25] and later this compound was
successfully used for the treatment of endometriosis [21,29].
The novel steroids, 2–4, inhibited progesterone-stimulated
uterine transformations, in the mice treated with luteinizing
hormone-releasing hormone. Therefore, these compounds could be
considered as antagonists for the PR present in the mouse uterus.
These results indicated also that 2–4, produced a similar effect in
the mice uterus as previously reported for mifepristone in the rab-
bit uterus [24]. On the other hand 2–4 failed to inhibit the ovulation.
The effect of the antiprogestin mifepristone to inhibit the ovulation
has been reported in the past [29].
Acknowledgement
This study was supported by a Grant from CONACYT (Project
number 54853).
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