New 11β-aryl-substituted steroids exhibit both progestational and antiprogestational activity

Article abstract of DOI:10.1016/S0039-128X(98)00060-9

The syntheses of three 11β-aryl-19-norpregna-4,9-dien-3-one derivatives with 17-spirolactone and 17β-hydroxy-17α-cyanoethyl substitutions are described. The progesterone agonist/antagonist activities of the new compounds are investigated using a recently

Full text of DOI:10.1016/S0039-128X(98)00060-9

New 11␤-aryl-substituted steroids
exhibit both progestational and
antiprogestational activity
Pemmaraju N. Rao,* Zhiqiang Wang,* James W. Cessac,* Rachel S. Rosenberg,†
David J. A. Jenkins,† and Eleftherios P. Diamandis‡
*Department of Organic Chemistry, Southwest Foundation for Biomedical Research, San Antonio,
Texas, USA; †Department of Nutritional Sciences, University of Toronto, Toronto, Ontario,
Canada; and ‡Department of Pathology and Laboratory Medicine, Mount Sinai Hospital, Toronto,
Ontario, Canada
The syntheses of three 11␤-aryl-19-norpregna-4,9-dien-3-one derivatives with 17-spirolactone and 17␤-hydroxy-
17␣-cyanoethyl substitutions are described. The progesterone agonist/antagonist activities of the new compounds
are investigated using a recently developed tissue culture system that relies on the progesterone agonist
up-regulation of the prostate-specific antigen (PSA) gene in female breast tumor cell lines. Two of the newly
synthesized compounds exhibit mixed agonistic/antagonistic progestational activity. (Steroids 63:523–530,
1998) © 1998 by Elsevier Science Inc.
Keywords: Steroids; synthesis; CDB-2914; progestin; antiprogestin; prostate-specific antigen; steroid hormone receptors; PSA
gene regulation
a 17␣-acetoxy-17␤-acetyl group exhibits activity similar to
mifepristone.7 In view of the significance of C-17 in reduc-
ing antiglucocorticoid activity, introduction of other sub-
stituents at C-17 position of 11␤-aryl substituted 19-
norsteroids may be useful in producing an antiprogestin
with reduced antiglucocorticoid activity. Modifications in-
corporating hydrophobic 17␣-substituents such as 17␣-
ethyl (compound 4), and 17␣-(1Ј-pentynyl, compound 5)
have given rise to in vivo antiprogestational activity supe-
rior to that of mifepristone.2 Philibert et al.8 have demon-
strated that a spirotetrahydrofuran at C-17 (compounds 6
and 7) causes marked increase in the ratio of antiprogesta-
tional/antiglucocorticoid activity. The corresponding 17-
spirolactone with a 6␤-methyl substituent was synthesized
by van den Heuvel and Groen9 and exhibited an improved
dissociation between progesterone receptor binding and glu-
cocorticoid receptor binding relative to mifepristone. How-
ever, due to a decreased progesterone receptor binding relative
to mifepristone, this compound was not tested for antiproges-
tational activity. We felt it was desirable to synthesize 17-
spirolactone derivatives without any substituents at the C-6
position and to evaluate their antiprogestational activity. Con-
veniently, the synthetic scheme chosen for these compounds
(Figure 2) allowed a facile conversion of intermediate 14b to
the 17␤-hydroxy-17␣-cyanoethyl derivative 16. This com-
pound is structurally similar to a 17␤-hydroxy-17␣-
cyanomethyl antiprogestational derivative synthesized by
Cook et al.7 In the present communication, we describe the
synthesis and biologic data of these derivatives.
Introduction
The discovery of the first competitive progesterone antagonist
mifepristone (Figure 1, RU-486, 1) has initiated an intense
search for more potent and more selective antiprogestins.1,2
Various attempts to dissociate the antiprogestin from the anti-
glucocorticoid activity of mifepristone and other analogs have
not been very successful, and the design and synthesis of new
compounds having antiprogestational activity devoid of anti-
glucocorticoid are highly desirable, both in terms of clinical
applications and basic endocrine research.3,4
Steroid receptors are closely related structurally and in
their mechanism of action. Slight modifications of the ste-
roidal structure have been found to induce important affinity
and specificity variations for the corresponding receptors.
The most prominent structural feature of mifepristone is the
4-(dimethylamino)phenyl group in the 11␤-position of a
19-nor steroid. Without this substituent, the molecule would
be expected to act as a progestin. Replacement of the
4-(dimethylamino)phenyl group with a 4-acetylphenyl
(ZK112993, 2)5 leads to equal or more potent antipro-
gestins, and minor changes at the C-17 position dramati-
cally produces antiprogestins with reduced antiglucocorti-
coid activity.4,6 For example, CDB-2914 (compound 3) with
Address reprint requests to Pemmaraju N. Rao, Department of Organic
Chemistry, Southwest Foundation for Biomedical Research, P.O. Box
760549, San Antonio, Texas 78245-0549.
Received March 13, 1998; accepted June 5, 1998.
Steroids 63:523–530, 1998
© 1998 by Elsevier Science Inc. All rights reserved.
655 Avenue of the Americas, New York, NY 10010
0039-128X/98/$19.00
PII S0039-128X(98)00060-9

Papers
Figure 1. Antiprogestins.
Experimental
deuterochloroform either at 300 MHz using a General Elec-
tric QE-300 spectrometer or at 90 MHz using a Varian
EM-390 spectrometer. Infrared spectra were recorded on a
Perkin–Elmer model 1600 FTIR instrument equipped with a
diffuse reflectance accessory using a KBr matrix. Mass
Chemistry
Melting points were determined on a Thomas–Hoover ap-
paratus and are uncorrected. H NMR were determined in
1
Figure 2. Synthesis of 11␤-Aryl steroids.
524 Steroids, 1998, vol. 63, October

New 11␤-Aryl substituted steroids: Rao et al.
spectral analyses (EI) were conducted by Dr. Susan Wein-
traub of the University of Texas Health Science Center at
San Antonio using a Finnigan-MAT model 4615 mass spec-
trometer. Combustion analyses were performed by Midwest
MicroLabs Ltd., Indianapolis Indiana. “Flash column” chro-
matography was performed on 32–64 ␮M silica gel ob-
tained from Scientific Absorbents Inc., Atlanta Georgia.
TLC Analyses were carried out on silica gel GF (Analtech)
glass plates (2.5 ϫ 10 cm with 250 ␮M layer and pres-
cored).
Most chemicals and solvents were analytical grade and
used without further purification. 4-Bromoacetophenone
ethylene ketal was prepared according to the procedure of
Detty et al.10
reaction mixture for 30 min via a 6-inch needle inserted
through a rubber septum and a slight vacuum applied to the
top of the condenser. The reaction was extracted with ethyl
acetate (3 times). The organic fractions were washed with
10% NH4Cl solution (2 times) and brine (1 time), com-
bined, dried over Na2SO4, filtered, and concentrated in
vacuo. Flash chromatography (hexanes/ethyl acetate 2:1) of
the residue followed by trituration with ether/hexanes gave
the pure adduct (12a, 8.17 g, 85.37%). m.p. ϭ 190–191°C.
1H NMR (300 MHz) ␦ 0.466 (s, 18-CH3), 1.637 (s, CH3 of
11␤-substituent), 3.742–4.061 (m, -OCH2CH2O-), 4.310 (d,
J ϭ 4.8 Hz, 11␣CH), 7.189 (d, J ϭ 8.4 Hz, aromatic 2Ј-H
and 6Ј-H), 7.350 (d, J ϭ 8.4 Hz, aromatic 3Ј-H and 5Ј-H)
ppm. IR (cmϪ1) 3520, 2943, 1739, 1609, 1506. MS (m/z)
Mϩ ϭ 494. Analysis calculated for C30H38O6: C, 72.85; H,
7.74. Found: C, 73.03; H, 7.87.
3,3-Ethylenedioxy-5␣,10␣-epoxyestr-9,11-en-17-one
(11)
3,3-Ethylenedioxy-5␣-hydroxy-11␤-[4-(N,N-
dimethylamino)phenyl]-estr-9-en-17-one (12b)
Hydrogen peroxide (30%, 21.3 mL, 208 mmol) was added
to a mixture of hexafluoroacetone trihydrate (17 mL, 121.93
mmol) in CH2Cl2 (320 mL) cooled to 0°C in an ice bath.
Solid Na2HPO4 (17.2 g, 121 mmol) was added and the
mixture stirred mechanically at 0°C for 20 min. A solution
of the 3-ketal 10 (31.84 g, 101.3 mmol) in methylene
chloride (320 mL) was then added dropwise over a period of
15 min. The reaction mixture was then stirred at 0–4°C for
18 h. The reaction was extracted with CH2Cl2 (3 times), and
the organic fractions were washed successively with 10%
Na2SO3 solution (2 times), saturated sodium bicarbonate
solution (2 times), and brine (1 time). The organic layers
were combined, dried over anhydrous Na2SO4, filtered, and
evaporated in vacuo. Crystallization of the residue from
diisopropyl ether (2 times) afforded the pure 5␣,10␣-
epoxide 11 (20 g, 59.76%). m.p. ϭ 153–155°C (158°C11).
1H NMR (300 MHz) ␦ 0.810 (s, 18-CH3), 3.8–3.9 (m,
-OCH2CH2O-), 5.972–6.005 (m, 11␣ CH). IR (cmϪ1) 2938,
2876, 2842, and 1736.
Following the same procedure used in the preparation of
12a, a solution of 4-bromo-N,N-dimethylaniline (26.5 g,
132.4 mmol) in dry THF (100 mL) was added to activated
magnesium (3.2 g, 132.4 mmol) in dry THF (100 mL). After
the reaction had initiated, the mixture was stirred at ambient
temperature for 1.5 h. Solid copper (I) chloride (1.3 g, 13.2
mmol) was added and the mixture stirred at room temper-
ature for an additional 30 min. The reaction was then cooled
to Ϫ30°C and a THF (100 mL) solution of the ␣-epoxide
(11, 7.0 g, 21.18 mmol) was added dropwise. The reaction
was then stirred at Ϫ30 ϳ Ϫ20°C for 3 h. The reaction was
quenched and worked up in an identical manner as carried
out for the preparation of compound 12a. Flash chromatog-
raphy (hexanes/ethyl acetate 1:1) followed by trituration
with ether gave the pure adduct 12b (8.17 g, 85.37%) as a
light green solid. A small portion of this material was
recrystallized from ethyl acetate/petroleum ether for char-
acterization. m.p. ϭ 176–177°C (176°C). 13 1H NMR (300
MHz) ␦ 0.518 (s, 18-CH3), 2.909 (s, NMe2), 3.905–4.025
(m, -OCH2CH2O-), 4.245 (d, J ϭ 6.9 Hz, 11␣ CH), 6.642
(d, J ϭ 8.7 Hz, aromatic 3Ј-H and 5Ј-H), 7.065 (d, J ϭ 8.7
Hz, aromatic 2Ј-H and 6Ј-H) ppm. IR (cmϪ1) 3510, 2946,
1737, 1612, 1519. MS (m/z) Mϩ ϭ 451. Analysis calculated
for C28H37NO4: C, 74.47; H, 8.26; N, 3.10. Found: C,
74.67; H, 8.24; N, 3.23.
3,3-Ethylenedioxy-5␣-hydroxy-11␤-[4-(2-methyl-1,3-
dioxolan-2-yl)phenyl]-estr-9-en-17-one (12a)12
Under anhydrous conditions, magnesium turnings (3.2 g,
132.4 mmol) were weighed into a 1-L round bottom four-
neck flask equipped with a stirring shaft, thermometer,
addition funnel, and a reflux condenser. The system was
flushed with dry argon, and a few crystals of iodine and 2
drops of 1,2-dibromoethane were added. The contents of the
flask were then agitated for a few minutes to activate the
magnesium surface. Dry THF (100 mL) was added followed
by a slow (30 min.) dropwise addition of a solution of
4-bromoacetophenone ethylene ketal (32.2 g, 132.4
mmol)10 in dry THF (100 mL). Most of the magnesium had
reacted after stirring 1.5 h at room temperature. Solid cop-
per (I) chloride (1.3 g, 13.2 mmol) was added, and the
reaction mixture was stirred an additional 30 min at room
temperature. The reaction was then cooled to Ϫ30°C, and a
THF (100 mL) solution of the ␣-epoxide (11, 7.0 g, 21.18
mmol) was added dropwise. The reaction was then stirred at
Ϫ30 ϳ Ϫ15°C for 3 h. Ammonium chloride solution (33 g
in 150 mL water) was added dropwise to quench the reac-
tion. To oxidize Cu (I) to Cu (II), air was drawn through the
3,3-Ethylenedioxy-11␤-[4-(2-methyl-1,3-dioxolan-2-
yl)phenyl]spiro[estr-9-en-17␤,2Ј-oxirane] 13a
Under nitrogen, 60% sodium hydride (0.45 g, 11.3 mmol) in
mineral oil was weighed into a 100 mL three neck round
bottom flask equipped with a magnetic stir bar, a reflux
condenser, glass stopper, and a rubber septum. The sodium
hydride suspension was washed free of mineral oil using
petroleum ether (3 times). Dry DMSO (21 mL, 296 mmol)
was introduced via syringe and the mixture was stirred and
heated at 70°C for 45 min. The reaction was cooled to room
temperature, diluted with dry THF (35 mL), and further
cooled to Ϫ5°C. A solution of trimethylsulfonium iodide
(2.1 g, 10.35 mmol) in dry DMSO (15 mL) was added and
the reaction stirred at Ϫ5 ϳ 0°C for 35 min. A solution of
Steroids, 1998, vol. 63, October 525

Papers
the Grignard adduct 12a (1.2 g, 2.43 mmol) in dry THF (12
mL) was introduced and the reaction mixture stirred at 0°C
for 3 h. The reaction was poured into cold water and
extracted with CH2Cl2 (3 times). The organic fractions were
washed with brine (2 times), combined, dried over Na2SO4,
filtered and concentrated in vacuo. Crystallization of the
residue from ether gave the pure 17-oxirane 13a (1.0 g,
8.1 Hz, aromatic 2Ј-H and 6Ј-H), 7.350 (d, J ϭ 8.1 Hz,
aromatic 3Ј-H and 5Ј-H) ppm. IR (cmϪ1) 3470, 2940, 2242,
1609, 1505. MS (m/z) Mϩ ϭ 549. Analysis calculated for
C33H43NO6 ⅐ 4/5 H2O: C, 70.26; H, 7.97; N, 2.48. Found: C,
70.08; H, 7.67; N, 2.43.
3,3-Ethylenedioxy-5␣,17␤-dihydroxy-11␤-[4-(N,N-
dimethylamino)phenyl]-19-nor-17␣-pregn-9-ene-21-
carbonitrile 14b
1
81.3%) as a white solid. m.p. ϭ 126–128°C. H NMR (90
MHz) ␦ 0.47 (s, 18- CH3), 1.62 (s, CH3 of 11␤-substituent),
2.59 and 2.94 (both d, J ϭ 6.3 Hz, 17␣-CH2), 3.73–4.13 (m,
-OCH2CH2O-), 4.2–4.37 (m, 11␣ H plus OH), 7.15 (d, J ϭ
9 Hz, aromatic 2Ј-H and 6Ј-H), 7.35 (d, J ϭ 9 Hz, aromatic
3Ј-H and 5Ј-H) ppm. IR (cmϪ1) 3533, 2928, 1679, 1602,
1505. MS (m/z) Mϩ ϭ 508. Analysis calculated for
C31H40O6: C, 73.20; H, 7.93. Found: C, 73.38; H, 7.88.
Following the same procedure used in the preparation of
14a, a mixture of 1.5 M cyclohexane solution of lithium
diisopropylamide mono(tetrahydrofuran) (20 mL, 30
mmol), THF (40 mL), and acetonitrile (2 mL, 30 mmol) was
reacted with a solution of the 17-oxirane 13b (2.0 g, 4.3
mmol) in THF (15 mL) at ambient temperature for 6 h. The
reaction was quenched and worked up in an identical man-
ner as carried out for the preparation of compound 14a.
Purification via flash chromatography (ethyl acetate/hex-
anes 1:1) gave the 21-carbonitrile compound 14b (1.25 g,
57%) as a foam. 1H NMR (300 MHz) ␦ 0.506 (s, 18- CH3),
2.915 (s, NMe2), 3.9–4.1 (m, -OCH2CH2O-), 4.255 (d, J ϭ
6.9 Hz, 11␣ H), 6.655 (d, J ϭ 9 Hz, aromatic 3Ј-H and
5Ј-H), 7.047 (d, J ϭ 9 Hz, aromatic 2Ј-H and 6Ј-H) ppm. IR
3,3-Ethylenedioxy-11␤-[4-(N,N-
dimethylamino)phenyl]spiro[estr-9-en-17␤,2Ј-
oxirane] 13b
Following the same procedure used in the preparation of
13a, Grignard adduct 12b (4.0 g, 8.86 mmol) in THF (50
mL) was reacted with a mixture prepared from 60% sodium
hydride suspension (1.64 g, 41.02 mmol) in DMSO (50 mL)
and THF (120 mL) and trimethylsulfonium iodide (7.65 g,
37.74 mmol) in DMSO (50 mL). The reaction mixture was
stirred at 0°C for 3 h and worked up in an identical fashion
to provide after crystallization from ether the pure 17-
(cmϪ1) 3506, 2944, 2245, 1612, 1517. MS (m/z) Mϩ
ϭ
506. Analysis calculated for C31H42N2O4 ⅐ 1/4 EtOAc: C,
72.69; H, 8.39; N, 5.30. Found: C, 72.93; H, 7.47; N, 5.37.
1
oxirane 13b (3.4 g, 82.4%). m.p. ϭ 145–150°C (d). H
17␤-hydroxy-11␤-(4-acetylphenyl)-19-nor-17␣-
pregna-4,9-diene-21-carboxylic acid, ␥-lactone 15a
NMR (300 MHz) ␦ 0.522 (s, 18- CH3), 2.598 and 2.939
(both d, J ϭ 5.0 Hz, 17␣-CH2), 2.900 (s, NMe2), 3.904–
4.043 (m, -OCH2CH2O-), 4.192 (d, J ϭ 6 Hz, 11␣ H), 6.626
(d, J ϭ 9 Hz, aromatic 3Ј-H and 5Ј-H), 7.017 (d, J ϭ 9 Hz,
aromatic 2Ј-H and 6Ј-H) ppm. IR (cmϪ1) 3511, 2940, 1612,
1518. MS (m/z) Mϩ ϭ 465.
Under nitrogen, a mixture of the 21-carbonitrile 14a (0.8 g,
1.46 mmol) 45% potassium hydroxide (2 mL, 16 mmol),
and methanol (10 mL) was heated to reflux for 5 hours. The
reaction was cooled to room temperature, and water (7 mL)
and acetic acid (22 mL) were added. The reaction was then
heated to 50°C for 5 h. The solvents were removed in vacuo
and the residue extracted with CH2Cl2 (3 times). The or-
ganic fractions were washed with water (once) and brine
(once), combined, dried over sodium sulfate, filtered, and
concentrated in vacuo. Crystallization of the residue from
ethyl acetate gave the 21,17-carbolactone 15a (0.54 g,
83.5%) as a yellow solid. m.p. ϭ 251–252°C. 1H NMR (300
MHz) ␦ 0.585 (s, 18-CH3), 2.587 (s, 4Ј-acetyl), 4.488 (d,
J ϭ 7.2 Hz, 11␣ H), 5.810 (s, 4-CH), 7.268 (d, J ϭ 8.1 Hz,
aromatic 2Ј-H and 6Ј-H), 7.894 (d, J ϭ 8.1 Hz, aromatic
3Ј-H and 5Ј-H) ppm. IR (cmϪ1) 2945, 1771, 1671, 1654,
1601. MS (m/z) Mϩ ϭ 444. Analysis calculated for
C29H32O4 ⅐ 1/4 EtOAc: C, 77.23; H, 7.34. Found: C, 77.19;
H, 7.32. Analysis by HPLC (CH3CN/H2O, 1:1; Waters
Associates NovaPak C18; 1 mL/min; ␭ ϭ 260 nm) indicated
the product to be Ͼ 99% pure with a retention time of 3.74
min.
Analysis calculated for C29H39NO4: C, 74.80; H, 8.44;
N, 3.01. Found: C, 74.52; H, 8.54; N, 3.01.
3,3-Ethylenedioxy-5␣,17␤-dihydroxy-11␤-[4-(2-
methyl-1,3-dioxolan-2-yl)phenyl]-19-nor- 17␣-pregn-
9-ene-21-carbonitrile 14a
Under nitrogen, a 1.5-M cyclohexane solution of lithium
diisopropylamide mono(tetrahydrofuran) (10 mL, 15 mmol)
was added to dry THF (40 mL), and the mixture was cooled
to Ϫ30°C. Dry acetonitrile (1.0 mL, 16 mmol) was added,
and the mixture was stirred at Ϫ30°C for 30 min. A solution
of the 17-oxirane 4a (1.36 g, 2.67 mmol) in dry THF (15
mL) was added dropwise over a period of 10 min and the
reaction was then allowed to warm to ambient temperature
and stirred for 3 h. The reaction mixture was then poured
into saturated ammonium chloride solution and extracted
with ethyl acetate (3 times). The organic fractions were
washed with brine (once), combined, dried over sodium
sulfate, filtered, and concentrated in vacuo. The residue was
purified via flash chromatography (hexanes/ethyl acetate
1:1) followed by crystallization from ethyl acetate/hexanes
to give the 21-carbonitrile 14a (0.8 g, 54.4%) as a white
17␤-Hydroxy-11␤-[4-(N,N-dimethylamino)phenyl]-
19-nor-17␣-pregna-4,9-diene-21-carboxylic acid,
␥-lactone 15b
1
solid. m.p. ϭ 191–193°C. H NMR (300 MHz) ␦ 0.451 (s,
Following the same procedure used in the preparation of
15a, the 21-carbonitrile 14b (0.6 g, 1.18 mmol) was reacted
with 45% potassium hydroxide (2 mL, 16 mmol) in meth-
18- CH3), 1.641 (s, CH3 of 11␤-substituent), 3.7–4.1 (m,
-OCH2CH2O-), 4.328 (d, J ϭ 7.5 Hz, 11␣ H), 7.170 (d, J ϭ
526 Steroids, 1998, vol. 63, October

New 11␤-Aryl substituted steroids: Rao et al.
Figure 3. Previously synthesized 11␤-aryl-substituted steroids with agonist or mixed agonist/antagonist progestational activity.
anol (10 mL) at reflux for 4.5 h followed by the addition of
acetic acid (22 mL) and water (7 mL) and reaction at 60°C
for 5 h. The reaction was worked up in an identical manner
as carried out for the preparation of compound 15a. Purifi-
cation via Flash chromatography (ethyl acetate/hexanes 3:2)
gave the 21,17-carbonitrile compound 15b (0.37 g, 70.2%)
as a yellow foam.
37°C and 5% CO2 in RPMI 1640 medium (Gibco BRL,
Gaithersburg MD), supplemented with 10% fetal calf serum
(FCS), 0.2 I.U. bovine insulin per mL, and 29 g/L glu-
tamine. Cells were grown until confluency in the flask, then
detached by trypsin-EDTA treatment to be split or subcul-
tured in 48-well microtiter plates for experimentation. The
subculture medium was the same as above except charcoal
stripped FCS was used instead of the regular FCS, as it is
devoid of any steroid hormones. Cell clumps were mini-
mized by passing cells and media through an 18-gauge
syringe. Cells were grown as monolayers until confluency,
1H NMR (300 MHz) ␦ 0.645 (s, 18- CH3), 2.921 (s,
NMe2), 4.367 (d, J ϭ 5.7 Hz, 11␣ H), 5.773 (s, 4-CH),
6.659 (d, J ϭ 9 Hz, aromatic 3Ј-H and 5Ј-H), 6.982 (d, J ϭ
9 Hz, aromatic 2Ј-H and 6Ј-H) ppm. IR (cmϪ1) 2945, 1775,
1660, 1612, 1518. MS (m/z) Mϩ ϭ 445. Analysis calculated
for C29H35NO3: C, 78.17; H, 7.92; N, 3.14. Found: C,
78.07; H, 7.81; N, 3.14. Analysis by HPLC (CH3CN/H2O,
1:1; Waters Associates NovaPak C18; 2 mL/min; ␭ ϭ 260
nm) indicated the product to be Ͼ 99% pure with a retention
time of 7.1 min.
Table 1. Progestational/Androgenic Activity
BT-474 cells
T-47D cells
% Agonist PSA, % Agonist
Compound
PSA, ng/La Activityb ng/La Activityb
17␤-Hydroxy-11␤-[4-(N,N-dimethylamino)phenyl]-
19-nor-17␣-pregna-4,9-diene-21-carbonitrile 16
Under nitrogen, a solution of the 21-carbonitrile 14b (0.3
g, 0.59 mmol) in acetic acid (14 mL), THF (6 mL), and
water (6 mL) was heated to 60°C for 5 h. The solvents
were removed in vacuo and the residue extracted with
CH2Cl2 (3 times). The organic fractions were washed
with saturated sodium bicarbonate solution (2 times) and
brine (two times), combined, dried over sodium sulfate,
filtered, and concentrated in vacuo. Crystallization of the
residue from ether gave product 16 (0.178 g, 67.6%) as a
7
0.8
0.6
45
58
8
9
5
1
yellow solid. m.p. ϭ 241–242°C. H NMR (300 MHz) ␦
0.580 (s, 18-CH3), 2.916 (s, NMe2), 4.365 (d, J ϭ 6.6 Hz,
11 ␣ H), 5.760 (s, 4-CH), 6.659 (d, J ϭ 8.7 Hz, aromatic
3Ј-H and 5Ј-H), 7.002 (d, J ϭ 8.7 Hz, aromatic 2Ј-H and
6Ј-H) ppm. IR (cmϪ1) 3362, 2950, 2242, 1641, 1600,
1518. MS (m/z) Mϩ ϭ 444. Analysis calculated for
C29H36N2O2: C, 78.34; H, 8.16; N, 6.30. Found: C,
78.32; H, 8.24; N, 6.31. Analysis by HPLC (CH3CN/
H2O, 1:1; Waters Associates NovaPak C18; 2 mL/min; ␭
ϭ 260 nm) indicated the product to be 97.6% pure with
a retention time of 2.75 min.
Ͻ1
336
118
Ͻ0.1
40
0
0
373
190
54
27
Assessment of biological activity
14
Growth and maintenance of cells
aThe amount of PSA present in the tissue culture is proportional
to the progestational/androgenic activity of these compounds.
All experiments were done in duplicate and results were repro-
ducible within Ϯ10%.
The breast cancer cell lines T47-D and BT-474 were ob-
tained from the American Type Culture Collection (ATCC),
Rockville, MD 20852. Both cell lines were cultured accord-
ing to the instructions of the ATCC.
bComparison to norgestrel, a progestin, whose activity was ar-
bitrarily set at 100%.
T47-D and BT-474 cells were initially grown in flasks at
Steroids, 1998, vol. 63, October 527

Papers
Table 2. Antiprogestational/Antiandorgenic Activity of Compounds, as Tested in a Tissue Culture System
% Blocking of
Norgestrel
Activityb,c
% Blocking of
Norgestimate
Activityb,c
% Blocking of
Dihydrotestosterone
Activityb,d
Concentration
Ma
Compound
10Ϫ8/10Ϫ7
10Ϫ10/10Ϫ9
96
100
–
86
80
100
10Ϫ8/10Ϫ7
10Ϫ10/10Ϫ9
40
27
0
–
0
0
10Ϫ8/10Ϫ7
10Ϫ10/10Ϫ9
100
93
100
–
91
64
10Ϫ8/10Ϫ7
10Ϫ10/10Ϫ9
84
50
100
–
87
54
aDenotes concentration of stimulant/blocker under the conditions of the experiments.
bAll experiments were repeated twice and results were reproducible within Ϯ10%.
cIndicates antiprogestational activity. For details, see “Methods.”
dIndicates antiandrogen activity. For details, see “Methods.”
with approximately 2 ϫ 105 cells per well and each well
contained 1 mL of medium.
Harvesting of supernatant
Supernatant was separated from the cells through aspiration,
using plastic pipettes. Supernatant was immediately trans-
ferred into labeled Eppendorf tubes, and either immediately
used for PSA assay or stored at Ϫ20°C until analysis.
Agonistic activity
BT-474 and T47-D breast cancer cells were grown to con-
fluency in 48-well and 24-well plates, respectively, under
the conditions described, and stimulated with compounds
CDB-2914, 15a, 15b, or 16 at final concentration of 10Ϫ7
and 10Ϫ9 M. All compounds were diluted with pure anhy-
drous ethyl alcohol. Positive controls were norgestrel (a
progestin), testosterone (an androgen) and norgestimate (a
progestin devoid of androgenic activity) (Sigma Chemical
Co., St. Louis, MO 63178). Alcohol was the negative con-
trol. Mifepristone (Roussel-UCLAF, Romainville, France)
was used as a known antiprogestin with weak progestational
activity13 Tissue culture supernatants were collected after 7
days (see below).
PSA assay
PSA protein was measured with a highly sensitive immun-
ofluorometric procedure that can measure PSA at levels of
1 ng/L or higher (up to 10,000 ng/L) with a precision of
Ͻ10%. This assay uses a mouse anti-PSA capture antibody
coated to polystyrene microtiter wells, a biotinylated mouse
monoclonal detection antibody, and alkaline phosphatase-
labeled streptavidin. The details and evaluation of this assay
are described elsewhere.14
Calculations
Antagonistic activity
The agonistic activity of the various steroids tested was
expressed both as ng/L of PSA in the tissue culture super-
natant at 7 days stimulation as well as a percentage of
norgestrel’s agonistic activity. The antagonistic activity was
expressed as a percentage of blocking of either norgestrel
(antiprogestational/antiandrogenic), norgestimate (antipro-
gestational) or dihydrotestosterone (antiandrogenic) activ-
ity. For this, we evaluated the PSA concentration at 7 days
in the tissue culture supernatant without any blocker (0%
blocking) or in the presence of the blocker (blocking from
0–100%).
T47-D cells were grown to confluency under standard con-
ditions in 48-well microtiter plates. The cells were incu-
bated in 1 mL media with the candidate antagonists CDB-
2914, 15a, 15b, or 16 for 1 h at final concentrations of 10Ϫ7
or 10Ϫ9 M. Norgestrel or dihydrotestosterone were then
added at final concentrations of 10Ϫ8 or 10Ϫ10 M, or norg-
estimate at 10Ϫ8 M. Controls did not contain the antagonist
(only stimulating steroid) or the stimulating steroid (only
antagonist). Alcohol was the negative control. Incubation
time was 7 days.
528 Steroids, 1998, vol. 63, October

New 11␤-Aryl substituted steroids: Rao et al.
The evidence presented here indicates the importance of
D-ring substituents in determining the balance of agonist/
antagonist activity. This is consistent with the findings of
Cook et al.19 that a derivative of CDB-2914 lacking the
17␣-acetoxy group was found to have mixed agonist/antag-
onist progestational activity and its 16␣-ethyl-derivative
was found to be a pure agonist. A similar reversal of
antiprogestational activity has been observed upon 18-
methylation of an 11␤-[4-(methylthio)phenyl]-derivative of
CDB-2914.20 At the present time, the data are insufficient
for a definitive structure activity relationship with regard to
agonist/antagonist activity, but it is evident that modifica-
tions of or near C-17 substituents play a key role in deter-
mining agonist/antagonist progestational activity of these
compounds.
Results and discussion
Chemistry
The syntheses of the key 17-oxirane intermediates 4a-b
(Figure 2) was carried out following the general strategy
of Cook et al..7 The Regioselective 5,10-epoxidation of
3,3-ethylenedioxyestra-5(10),9(11)-dien-17-one16
1
was
achieved using hexafluoroacetone hydroperoxide generated
in situ from hexafluoroacetone trihydrate and hydrogen
peroxide following the general procedure of Teutsch et al.17
From the resulting epoxide mixture, the pure 5␣,10␣-isomer
11 could be isolated in 60% yield. The copper (I) catalyzed
addition of the Grignard reagent prepared from the ap-
propriate aryl bromide (4-bromodimethylaniline and
4-bromoacetophenone ketal10) gave the corresponding
Grignard adducts 12a-b in 85% yield. Reaction of 12a-b
with dimethylsulphonium methylide gave the 17-oxirane
intermediates 13a-b in 81–82% yield. Condensation of the
17-oxirane intermediates 13a-b with in situ generated ace-
tonitrile anion according to Faraj et al.18 afforded the 17␣-
cyanoethyl-17␤-hydroxy intermediates 14a-b in 54–57%
yields. A one step alkaline hydrolysis-acid treatment of
compounds 14a-b then gave the desired 17,21-carbolactone
products 15a-b in 70–84% yields. Acid hydrolysis of in-
termediate 14b gave the 17␣-cyanoethyl derivative 16.
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In Table 1 we present the structures of compounds RU-
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agonistic activity with T47-D and BT-474 cells. At 10Ϫ7
M
concentration, the most potent agonistic activity (progestin
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with compound CDB-2914, while compound 15a has very
little or no agonistic activity. The ability of these com-
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