Synthesis and biological evaluation of partially fluorinated antiprogestins and mesoprogestins

Article abstract of DOI:10.1016/j.steroids.2012.09.010

A series of antiprogestins have been synthesized by partially fluorinating the steroid molecule in positions relevant for receptor binding. By introducing fluorine at the exo-methylene of the 17 spirofuran ring, we obtained partial agonists (mesoprogestins) with significant applications for antiproliferative and antiovulatory treatment strategies in gynecological therapy such as uterine fibroids, endometriosis and heavy menstrual bleeding. Compared to the standard drug RU486, our synthesized compounds exhibited considerable dissociation between antiprogestational and antiglucocorticoid PR receptors. Furthermore, our studies have shown that pure antiprogestins can be generated by partially fluorinating the 17 propenyl and propynl group or by substituting the 4′ acetyl phenyl group in the 11 position using trifluromethyl group.

Full text of DOI:10.1016/j.steroids.2012.09.010

Steroids 78 (2013) 255–267
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and biological evaluation of partially fluorinated antiprogestins and
mesoprogestins
a
a
a
a
b
Klaus Nickisch a, , Walter Elger , James Cessac , Narkunan Kesavaram , Baishakhi Das , Robert Garfield ,
⇑
Shao-Qing Shi b, Olga Amelkina c, Reinhard Meister d
a Evestra, Inc., San Antonio, TX, USA
b St. Joseph’s Hospital and Medical Center, Phoenix, AZ, USA
c Leibniz Institute for Zoo and Wildlife Research, IZW, Berlin, Germany
d Beuth University of Applied Science, Berlin, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of antiprogestins have been synthesized by partially fluorinating the steroid molecule in posi-
tions relevant for receptor binding. By introducing fluorine at the exo-methylene of the 17 spirofuran
ring, we obtained partial agonists (mesoprogestins) with significant applications for antiproliferative
and antiovulatory treatment strategies in gynecological therapy such as uterine fibroids, endometriosis
and heavy menstrual bleeding. Compared to the standard drug RU486, our synthesized compounds
exhibited considerable dissociation between antiprogestational and antiglucocorticoid PR receptors. Fur-
thermore, our studies have shown that pure antiprogestins can be generated by partially fluorinating the
17 propenyl and propynl group or by substituting the 40 acetyl phenyl group in the 11 position using tri-
fluromethyl group.
Received 23 July 2012
Received in revised form 11 September
2012
Accepted 25 September 2012
Available online 21 November 2012
Keywords:
Mesoprogestins
Fluorine
Ó 2012 Elsevier Inc. All rights reserved.
Antiproliferation
1. Introduction
metriosis and fibroids [3,4]. The beneficial implications of both PR
antagonists and partial agonists (mesoprogestins) have resulted in
1.1. SARs considerations for choice of compounds (PRMs) to be
synthesized
efforts to better understand the structure–activity relationships
[5,6] and have led to the synthesis of new antiprogestins depicted
in Fig. 1.
Progesterone antagonists of the RU486 (Mifepristone) family
comprise pharmacodynamically different compounds. These phar-
macodynamic differences result from compound-specific interac-
tions with nuclear receptors apart from the PR (progesterone
receptor) for example the GR (glucocorticoid receptor). Further-
more, the respective compounds also differ in terms of their bio-
logical effects at the PR, due to the relative amount of PR-
antagonistic and PR-agonistic properties as well as the mechanism
of type I and type II antagonists [1]. Up until now, progesterone
receptor antagonists (PRMs) have been clinically developed by dif-
ferent strategies ever since Mifepristone (RU486) was first discov-
ered by Teutsch et al. [2]. The first approach deals with ‘‘PR-
antagonists’’ for the purpose of investigating different types of fer-
tility control and cancer indications, such as post-coital contracep-
tion, therapeutic pregnancy termination (Mifepristone, CDB-2914),
and breast cancer (Onapristone, ZK-230211). The second approach
requires a partial agonistic profile as was first observed with Aso-
prisnil for the treatment of gynecological indications, such as endo-
SARs lead to the understanding that the substitution pattern at
17 position of the steroid establishes binding to the PR and/or GR
receptor. The 17-alpha propinyl group of Mifepristone leads to
equal strong binding to both receptors therefore limiting its long
term application. Substitution of the 17-propinyl group with a 3-
hydroxypropyl, (Z)-3-hydroxyprop-1-enyl group [7],
a 17,17-
spiro-oxazole group [8], a 17-acetoxyprogesterone side chain [9],
or the 17,17 spiro ether group [10] and most recently, ZK-
230211 [11] a perfluoralkyl steroid leads to potent antiprogestins
with significantly reduced antiglucocorticoid activity.
In contrast substitution at the 11 position influences the ratio of
agonistic to antagonistic activity of the steroid molecule. Mesopro-
gestins were first observed with Asoprisnil [3] and later with other
derivatives [13,14]. Partial agonistic profile can be explained by
crystal structure investigations of the progesterone receptor bind-
ing domain complexed with Asoprisinil and the nuclear receptor
corepressor and SMRT [12].
The concept of partial fluorination was applied on different
moieties in the 11 and 17 positions of the steroid molecule in order
to accomplish two objectives, the first aim was to acquire antipro-
gestins with reduced binding to the GR receptor and our second
⇑
Corresponding author. Tel.: +1 210 673 3300; fax: +1 210 673 3322.
E-mail address: knickisch@evestra.com (K. Nickisch).
0039-128X/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2012.09.010

256
K. Nickisch et al. / Steroids 78 (2013) 255–267
N
O
N
O
HO
N
OH
OMe
OH
Me
OH
OMe
O
Mifepristone
Onapristone
Asoprisnil
O
O
O
OMe
N
O
N
O
O
CF
F
F
O
OH
3
O
OAc
OAc
O
ORG-33628
CDB-2914
CDB-4124
ZK-230211
Fig. 1. Significant RU486 derivatives.
R3
R1
OH
R2
O
CF2
O
O
1a(EC301) R1 = Ac, R2
2a(EC307) R3 = Ac
2b(EC310) R3 = NMe2
=
CF3
O
O
2c(EC311) R3
2e(EC313) R3
=
=
2d(EC312) R3 =
C2F5
F
1b(EC306) R1
=
, R2
CF3
=
N
F
1c(EC308) R1 = Ac , R2
=
O
F
O
C2F5
1d(EC316) R1
=
, R2 =
CF3
Fig. 2. Compound design.
O
O
O
O
O
OH
O
OH
Br
CF3
CF3
CF3
LDA, -78oC
50% H2SO4
MeOH, H2O
O
O
O
4
O
OH
O
OH
3
1a (EC301)
Fig. 3. Synthesis of compound 1a (EC301).
aim was to synthesize pure antiprogestins and mesoprogestins
(refer to Fig. 2). The paper covers basic molecular studies pertain-
ing to partially fluorinated steroids and a preliminary first qualita-
tive in vivo assay which permits a corresponding distinction
(Fig. 3).
SERMs varies in tissues and is sharply different compared to low
doses of estrogen. By analogy to SERMs at the progesterone recep-
tor, PRMs or SPRMs (selective progesterone receptor modulators) can
induce a unique blend of PR-agonistic (e.g., kind of secretory trans-
formation) and PR-antagonistic changes (e.g., thick-walled spiral
arteries) in the human endometrium [17]. This has lead to ongoing
studies to modulate PR-agonistic PRMs by improving the ratio of
PR-agonist over PR-antagonistic effects. For example, one signifi-
cant compound that does exhibit higher PR agonist over PR antag-
onistic response is RU486. RU486 is not a pure antagonist at the PR
[1,3,18] and has shown remarkable PR-agonistic activity in the gui-
nea pig model. The PR-agonistic activity due to RU486 may explain
lower labor conditioning effects when combined with a prosta-
glandin; and lower labor inducing properties when administered
without a prostaglandin as compared to pure PR antagonist
Onapristone [19].
1.2. Pharmacodynamic concept
SERMs (specific estrogen receptor modulators) are known to gen-
erate a tissue-dependent mosaic of ER-agonistic and/or antagonis-
tic effects [15,16]. The blend of estrogenic and antiestrogenic
effects is advantageous for the use of SERMs (Tamoxifen and
Raloxifen) for prevention and treatment of estrogen-dependent
mammary gland tumors in human. Moreover, SERMs have the
capability to partially abolish the negative effects of endogenous
and exogenous estrogens because the blend of effects induced by

K. Nickisch et al. / Steroids 78 (2013) 255–267
257
From a clinical perspective, ‘‘pure’’ antagonists may lead to
2.1.2. 11b-(40-Acetylphenyl)-17b-hydroxy-17-(3,3,3-trifluoro-1-
propynyl)-estra-4,9-diene-3-one (1a, EC301)
superior PRMs for the induction of labor. While on the other hand,
mixed PR-agonist/antagonists, in particular those with prominent
PR-agonistic properties, appear to be an improved option for
chronic uses in women with uterine disorders, or as an oral contra-
ceptive. As previously stated, Asoprisnil the first observed com-
pound with prominent PR agonistic effects [3,4], was used in a
treatment study for women with fibroid disease and resulted in
preventing endometrial antiproliferative effects in all subjects. In
separate studies, women treated with Asoprisnil showed inhibition
of menstruation irrespective of ongoing ovarian hormone secretion
and ovulation, which demonstrates that the endometrium is the
site of this antiproliferative effect [20]. More detailed studies re-
vealed an arrest of endometrial mitotic activity [17], stressing
the fact that the arrest of menstrual bleedings was only seen at
antiovulatory doses of RU486. These two examples of using Aso-
prisnil and RU486 in clinical applications beyond pregnancy high-
light the need to build up antiprogestins which can be modulated
by shifting the pharmacodynamic ratio to produce desired agonis-
tic and/or antagonistic effects.
A solution of 3,3-ethylenedioxy-5a,17b-dihydroxy-17-(3,3,3-
trifluoro-1-propynyl)-11b-{40-[10,10-(ethylenedioxy)-ethyl]phenyl}-
estr-9-ene (4) (3.5 g, 6 mmol) in methanol (35 mL) at 0 °C was
treated dropwise with 50% sulfuric acid (2.2 mL) and was allowed
to stir at room temperature for 2 h. The reaction mixture was care-
fully quenched with sodium bicarbonate solution (15 mL) and ex-
tracted with dichloromethane (3 ꢀ 20 mL). The combined organic
layers were washed with water and brine, dried over sodium sul-
fate and evaporated in vacuo to afford the crude product. Purifica-
tion was carried out on a silica gel column using 25% ethyl acetate
in hexane to afford 1a, EC301 (2.5 g, 87%).
1H NMR (d, 300 MHz) 0.52 (s, 3H), 1.3–2.9 (m, 17H), 2.58 (s, 3H),
4.0 (bs, 1H), 4.46 (d, J = 7.1 Hz, 1H), 5.81 (s, 1H), 7.26 (d, J = 8.3 Hz,
2H), 7.89 (d, J = 8.4 Hz, 2H).
13C NMR (75 MHz) 13.6, 23.4, 25.8, 26.4, 27.3, 31.0, 36.5, 38.3,
39.071, 39.169, 40.6, 47.5, 50.1, 73.5 (q, J = 52 Hz), 79.3 (d,
J = 1 Hz), 90.8 (q, J = 6.3 Hz), 114.3 (q, J = 256 Hz), 123.3, 126.8,
127.2, 128.8, 130.3, 134.9, 144.0, 150.4, 156.5, 197.9, 199.6.
2.1.3. 3,3-Ethylenedioxy-5a-hydroxy-17b-cyano-17a-
2. Experimental
trimethylsilyloxy-11b-{40-[10,10-(ethylenedioxy)-20202-
trifluoroethyl]phenyl}-estr-9-ene (6)
2.1. Chemistry
Under argon, a few crystals of iodine were added to magnesium
turnings (0.28 g, 11.5 mmol) in dry THF (11 mL) and the mixture
was stirred until it turned colorless. A solution of 40-bromo-triflu-
oroacetophenone ethylene glycol ketal [19] (3.32 g, 11.2 mmol)
in dry THF (20 mL) was added and the reaction was refluxed for
1 h. The resulting Grignard solution was cooled in an ice bath
and solid CuCl (0.33 g, 3.3 mmol) was added. After stirring at 0 °C
Nuclear magnetic resonance spectra were recorded on a Bruker
ARX (300 MHz) spectrometer as deuterochloroform (CDCl3) solu-
tions using tetramethylsilane (TMS) as an internal standard
(d = 0) unless noted otherwise. ‘Flash column’ chromatography
was performed on 32–64 lM silica gel obtained from EM Science,
Gibbstown, NJ. Thin-layer chromatography (TLC) analyses were
for 30 min, a solution of 3,3-ethylenedioxy-5
a,10a-epoxy-17b-cy-
carried out on silica gel GF (Analtech) glass plates (2.5 cm ꢀ 10 cm
ano-17 -trimethylsilyloxyestra-9(11)-ene (5) (2.45 g, 5.7 mmol) in
a
with 250 lM layer and pre-scored). Most chemicals and solvents
dry THF (20 mL) was added and the mixture stirred at 0 °C for 1 h
and at room temperature overnight. The reaction mixture was
quenched with saturated ammonium chloride solution and ex-
tracted with ethyl acetate (3ꢀ). The combined organic layers were
washed once with saturated ammonium chloride solution, dried
over anhydrous sodium sulfate and concentrated in vacuo. The res-
idue was purified on a silica column using 20% ethyl acetate in hex-
anes to afford 6 (2.8 g, 75%).
were analytical grade and used without further purification. Com-
mercial reagents were purchased from Aldrich Chemical Company
(Milwaukee, WI). All experiments were carried out in oven dried
glassware under inert atmosphere using dry nitrogen or argon gas.
2.1.1. 3,3-Ethylenedioxy-5a,17b-dihydroxy-17-(3,3,3-trifluoro-1-
propynyl)-11b-{40-[10,10-ethylenedioxy)-ethyl]phenyl}-estr-9-ene (4)
n-Butyl lithium (55 mL, 2.5 N, 137.5 mmol) was added dropwise
to a solution of di-isopropylamine (21.6 mL, 154 mmol) in THF
(40 mL) at ꢁ78 °C under argon and the resulting LDA solution was
stirred at ꢁ78 °C for 30 min. Separately, a solution of 2-bromo-
3,3,3-trifluoropropene (12 g, 68.5 mmol) in THF (80 mL) was made,
cooled to ꢁ78 °C and above prepared LDA was slowly added over a
period of 20 min. After stirring for 15 min, a solution of 3,3-ethyl-
1H NMR (d, 300 MHz) 0.245 (s, 9H), 0.49 (s, 3H), 3.9–4.3 (m, 8H),
4.39 (d, J = 6 Hz, 1H), 4.44 (s, 1H), 7.24 (d, J = 9 Hz, 2H), 7.49 (d,
J = 9 Hz).
2.1.4. 3,3-Ethylenedioxy-5a
-hydroxy-11b-{40-[10,10-(ethylenedioxy)-
20202-trifluoroethyl]phenyl}-estr-9-en-17-one (7)
enedioxy-5
a
-hydroxy-11b-{40-[10,10-(ethylenedioxy)-ethyl]phenyl}-
A solution of the Grignard adduct (6) (4.0 g, 6.2 mmol) in THF
(15 mL) was treated dropwise with a solution of tetrabutylammo-
nium fluoride in THF (1 M, 15.5 mL, 15.5 mmol) and the reaction
mixture was stirred at room temperature for 1 h. The reaction
was quenched with water (50 mL) and extracted with ethyl acetate
(2ꢀ). The combined organic fractions were washed with brine,
dried over sodium sulfate and concentrated under reduced pres-
sure. The residue was dissolved in 20 mL of 10% aqueous methanol
and 10 mL of CH2Cl2 and cooled in an ice bath. A solution of sodium
hydroxide (2 M, 10 mL) was added and the reaction mixture stirred
at 0 °C for 2 h. The reaction was diluted with water and extracted
with methylene chloride (2ꢀ) and the combined organic fractions
were washed with water, dried over sodium sulfate and concen-
trated in vacuo. The residue was purified on a silica gel column
using 30% ethyl acetate in hexanes followed by trituration with
diisopropyl ether to give 7 (2.8 g, 82%).
estra-9-ene-17-one (3) (6 g, 12.1 mmol) [21] in THF (80 mL) was
added dropwise and stirred at ꢁ78 °C for 1 h and slowly allowed
to warm to room temperature over 15 h. Reaction mixture was
quenched with saturated aqueous ammonium chloride solution
(50 mL) and extracted with ethyl acetate (3 ꢀ 100 mL). The com-
bined organic layers were washed with water and brine, dried over
sodium sulfate, and evaporated in vacuo to afford the crude product.
Purification was performed on a silica gel column using 25% ethyl
acetate in hexane to afford 4 (5.0 g, 70%).
1H NMR (d, 300 MHz) 0.45 (s, 3H), 1.63 (s, 3H), 1.1–2.5 (m, 19H),
3.7–4.1 (m, 8H), 4.34 (d, J = 6.3 Hz, 1H), 4.44 (s, 1H), 7.17 (d,
J = 8.2 Hz, 2H), 7.34 (d, J = 8.2 Hz).
13C NMR (75 MHz) 13.5, 23.4, 23.9, 24.1, 27.5, 35.1, 38.3, 38.7,
39.21, 39.25, 47.37, 47.49, 50.1, 59.54, 64.15, 64.53, 64.6, 64.76,
70.1, 74.1 (q, J = 52 Hz), 80.0 (d, J = 1.1 Hz), 90.5 (q, J = 6.5 Hz),
108.7, 108.9, 114 (q, J = 255 Hz), 125.2, 127.0, 133.2, 135.1, 140.6,
146.2.
1H NMR (d, 300 MHz) 0.45 (s, 3H), 3.96–4.39 (m, 9H), 7.24 (d,
J = 9 Hz, 2H), 7.48 (d, J = 9 Hz).

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K. Nickisch et al. / Steroids 78 (2013) 255–267
2.1.5. 17b-Hydroxy-17
a
-pentafluoroethyl-11b-{40-[10,10-(ethylenedioxy)-
colorless. A solution of 2-(4-bromophenyl)-2-methyl-1,3-dioxo-
lane (2.1 g, 8.5 mmol) in THF (5 mL) was introduced over a period
of 5 min and allowed to reflux for 1 h. Reaction mixture was cooled
under ice and solid CuCl (150 mg, 1.5 mmol) was added to it and
continued to stir at 0 °C for 30 min. Finally a solution of 3,3-ethyl-
20202-trifluoroethyl]phenyl}-estrr-4,9-dien-3-one (1b, EC306)
Pentafluoroiodoethane (2.5 g, 10 mmol) was condensed into a
solution of the 17-ketone (7) (0.49 g, 0.9 mmol) in dry toluene
(18 mL) at ꢁ78 °C. A solution of methyl lithium lithium bromide
in ether (1.5 M, 6 mL, 9 mmol) was added and the reaction mixture
was stirred at ꢁ78 °C for 1 h and at 0 °C for another hour. The reac-
tion was quenched with saturated ammonium chloride solution
and extracted with ethyl acetate. The combined organic fractions
were washed with water, brine, dried over sodium sulfated and
concentrated in vacuo. The residue was taken up in methanol
(10 mL), cooled to 0 °C and 50% sulfuric acid (0.4 mL) was added.
The reaction was stirred at room temperature for 1 h, neutralized,
extracted with ethyl acetate, dried over sodium sulfate and con-
centrated in vacuo. The residue was purified over silica gel using
30% ethyl acetate in hexanes followed by crystallization from
methanol to give 1b, EC306 (0.38 g, 70%).
enedioxy-5a,10a-epoxy-17b-hydroxy-17-(2,3,3-trifluoroprop-2-
enyl)-estr-9(11)-ene (10) (730 mg, 1.7 mmol) in THF (5 mL) was
added into the cuprate solution and allowed to stir for 2 h at
0 °C. The reaction was quenched with aqueous ammonium chlo-
ride solution (30 mL) and extracted with ethyl acetate
(3 ꢀ 25 mL). The combined organic layers were washed further
with water and brine, dried over sodium sulfate and evaporated
in vacuo to afford the crude product. Purification was carried out
on a silica gel column using 25% ethyl acetate in hexane to afford
11 (810 mg, 80%).
1H NMR (d, 300 MHz) 0.48 (s, 3H), 0.8–2.7 (m, 24H), 3.6–4.6 (m,
10H), 6.79 (d, J = 8.8 Hz, 1H), 7.18 (d, J = 8.2 Hz, 2H), 7.30–7.40 (m, 3H).
1H NMR (d, 300 MHz) 0.58 (s, 3H), 4.05–4.29 (m, 4H), 4.47 (d,
J = 9 Hz, 1H), 5.79 (s, 1H), 7.20 (d, J = 7.5 Hz, 2H), 7.52 (d, J = 7.5 Hz).
2.1.9. 11b-(40-Acetylphenyl)-17b-hydroxy-17-(2,3,3-trifluoroprop-2-
enyl)-estra-4,9-diene-3-one (1c, EC308)
2.1.6. 3,3-Ethylenedioxy-17b-hydroxy-17-(2,3,3-trifluoroprop-2-
enyl)-5(10),9(11)-estradiene (9)
Compound 1c was prepared by following exactly the procedure
outlined for compound 1a where the hydrolysis of 11 (1.5 g) was
carried out using 50% sulfuric acid in methanol to give after work-
up and purification 1c, EC308 (1.1 g).
A solution of 1,1,1,2-tetrafluoroethane (820 mg, 8 mmol) in ether
(10 mL) was cooled to ꢁ78 °C as a 2.5 M solution of n-BuLi (2.4 mL,
6.1 mmol) was introduced over a period of 10 min and allowed to stir
for 1 h at ꢁ78 °C. A solution of spiro-20-(10-oxacyc1opropane)-17(S)-
[3,3-(ethylenedioxy)-5(10),9(11)-estradiene] 8 (1 g, 3.04 mmol) in
ether (7 mL) was introduced, followed by boron trifluoride etherate
(0.38 mL, 3.04 mmol) dropwise. The reaction mixture was stirred at
ꢁ78 °C for 1 h and allowed to warm to room temperature over a per-
iod of 1 h. The reaction was quenched with sodium bicarbonate solu-
tion (20 mL) and extracted with ethyl acetate (3 ꢀ 15 mL). The
combined organic layer was washed further with water and brine,
dried over sodium sulfate and evaporated in vacuo to afford the crude
product. Purification was carried out on a silica gel column using 20%
ethyl acetate in hexane to afford 9 (430 mg, 35%).
1H NMR (d, 300 MHz) 0.56 (s, 3H), 1.0–2.8 (m, 22H), 4.48 (d,
J = 6.7 Hz, 1H), 5.80 (s, 1H), 7.29 (d, J = 8.4 Hz, 2H), 7.88 (d,
J = 8.4 Hz, 2H).
13C NMR (75 MHz) 15.2, 22.7, 23.5, 25.8, 26.4, 27.4, 30.9, 33.5 (d,
J = 18.7 Hz), 34.3, 36.6, 37.1, 39.3, 40.5, 46.7, 50.0, 82.8 (d,
J = 2.7 Hz), 123.4, 125–130 (m), 127.0, 128.6, 130.2, 134.9, 143.9,
150.0, 154.7 (ddd, J = 47, 272, 286 Hz), 155.9, 197.4, 198.9.
2.1.10. 3,3-Ethylenedioxy-17b-hydroxy-17-(1,1,2,2,2-
pentafluoroethyl)-estra-5(10),9(11)-diene (13)
Pentafluoroiodoethane (4 g, 16 mmol) was condensed into a
solution of 3,3-ethylenedioxyestra-5(10),9(11)-diene-17-one (12)
(2.5 g, 8 mmol) in toluene (32 mL) at ꢁ78 °C, and stirred for
10 min. Methyl lithium lithium bromide solution in ether (1.5 M,
9.8 mL) was introduced dropwise and continued to stir at ꢁ78 °C
for 1 h. Reaction mixture was warmed to 0 °C and stirred at that
temperature for 1 h before quenching with saturated ammonium
chloride solution. Extracted with ethyl acetate, the combined or-
ganic layer was washed once with brine, dried over sodium sulfate,
concentrated under reduced pressure to obtain 13 (3.0 g, 85%),
which was used for epoxidation without further purification.
1H NMR (d, 300 MHz) 0.93 (s, 3H), 1.2–2.8 (m, 18H), 3.98 (s, 4H),
5.6 (bs, 1H).
1H NMR (d, 300 MHz) 0.90 (s, 3H), 1.0–2.7 (m, 20H), 3.99 (s, 4H),
5.50–5.60 (bs, 1H).
13C NMR (75 MHz) 14.4, 23.6, 24.6, 27.6, 31.2, 31.3, 32.8, 33.7
(dd, J = 2.8, 18.6 Hz), 34.7, 39.4, 41.3, 45.3, 46.2, 46.8, 64.36,
64.49, 82 (m), 108.2, 117.7, 126.1, 125–130 (m), 130.2, 136.5,
154.8 (ddd, J = 285, 271, 47.2 Hz).
2.1.7. 3,3-Ethylenedioxy-5a,10a-epoxy-17b-hydroxy-17-(2,3,3-
trifluoroprop-2-enyl)-estr-9(11)-ene (10)
Hydrogen peroxide (0.18 mL, 30%, 1.6 mmol) was added to an
ice-cold solution of hexafluoroacetone trihydrate (350 mg,
1.6 mmol) in dichloromethane (3 mL). Solid Na2HPO4 (180 mg,
1.3 mmol) was introduced and the reaction mixture was stirred
for 1 h at 0 °C. An ice-cold solution of 3,3-ethylenedioxy-17b-hy-
droxy-17-(2,3,3-trifluoroprop-2-enyl)-5(10),9(11)-estradiene (9)
(410 mg, 1 mmol) in dichloromethane (3 mL) was added and the
mixture was stirred at 0 °C for 3 h, then at 5 °C for 15 h. The reac-
tion mixture was diluted with dichloromethane (15 mL) and
washed with 10% sodium sulfite solution (15 mL), water, dried
over sodium sulfate and concentrated under vacuum to obtain
the mixture of crude epoxides. Separation of the isomeric epoxides
was carried out on a silica gel column using 20% ethyl acetate in
2.1.11. 3,3-Ethylenedioxy-5a,10a-epoxy-17b-hydroxy-17-(1,1,2,2,2-
pentafluoroethyl)-estra-9(11)-ene (14)
Following the procedure outlined for the synthesis of com-
pound 10, the epoxidation of 13 (3.0 g) was carried out using
hydrogen peroxide and hexafluoroacetone in dichloromethane
and gave after workup and purification 14 (1.8 g).
1H NMR (d, 300 MHz) 0.92 (s, 3H), 1.1–2.8 (m, 18H), 3.70–4.00
(m, 4H), 6.0 (bs, 1H).
13C NMR (75 MHz) 15.8, 22.8, 25.1, 25.2, 28.1, 31.8, 34.4, 35.4
(dd, J = 3.6, 8.1 Hz), 38.4, 40.3, 48.8, 49.0, 49.1, 60.1, 61.7, 64.1,
64.4, 84.2 (dd, J = 25.1, 21.5 Hz), 107.1, 117 (m), 122 (m), 126.86,
126.89, 135.1.
hexane to afford 10 (240 mg, 56%) of pure a-isomer.
1H NMR (d, 300 MHz) 0.9 (s, 3H), 1.0–2.8 (m, 20H), 3.8–4.0 (m,
4H), 5.90–6.10 (m, 1H).
2.1.12. 3,3-Ethylenedioxy-5a,17b-dihydroxy-17-(1,1,2,2,2-
2.1.8. 3,3-Ethylenedioxy-5
a
,17b-dihydroxy-17-(2,3,3-trifluoroprop-2-
pentafluoroethyl)-11b-[40-(tert-butyldimethylsilyloxymethyl)phenyl]-
estr-9-ene (15)
Following the procedure outlined for the synthesis of com-
pound 11, the Grignard reaction of 14 (230 mg) was carried out
enyl)-11b-{40-[10,10-(ethylenedioxy)-ethyl]phenyl}-estr-9-ene (11)
Magnesium turnings (220 mg, 9 mmol) in THF (10 mL) contain-
ing a crystal of iodine was heated to reflux for 10 min to become

K. Nickisch et al. / Steroids 78 (2013) 255–267
259
using 4-(t-butyldimethylsilyloxymethyl) bromobenzene and mag-
nesium and gave after workup and purification the required prod-
uct 15 (340 mg).
in hexanes followed by trituration with diisopropyl ether to give
1d EC316 (0.9 g).
1H NMR (d, 300 MHz) 0.58 (s, 3H), 4.52 (d, J = 9 Hz, 1H), 5.82 (s,
1H), 7.40 (d, J = 6 Hz, 2H), 8.01 (d, J = 6 Hz).
1H NMR (d, 300 MHz) 0.08 (s, 6H), 0.53 (s, 3H), 0.93 (s, 9H), 1.2–
2.5 (m, 18H), 3.8–4.1 (m, 4H), 4.4 (bd, 1H), 4.70 (s, 2H), 7.0–7.2 (m,
4H).
2.1.17. 3,3-Ethylenedioxy-21,21-difluoro-17,23-epoxy-19,24-dinor-
17a-chola-5(10),9(11),20-triene (20)
2.1.13. 3,3-Ethylenedioxy-5
pentafluoroethyl)-11b-[40-(hydroxymethyl) phenyl]-estr-9-ene (16)
solution of 3,3-ethylenedioxy-5 ,17b-dihydroxy-17-
a,17b-dihydroxy-17-(1,1,2,2,2-
To a solution of diisopropylamine (0.95 mL, 6.8 mmol) in THF
(10 mL) at ꢁ78 °C, n-BuLi (2.7 mL, 2.5 M, 6.8 mmol) was added
and stirred for 30 min. A solution of diethyl difluoromethylphosph-
onate (1.1 mL, 6.8 mmol) in THF (10 mL) was added and stirred for
1 h at ꢁ78 °C. Finally, a solution of 3,3-ethylenedioxy-40,50-dihy-
A
a
(1,1,2,2,2-pentafluoroethyl)-11b-[40-(tert-butyldimethylsilyloxym-
ethyl)phenyl]-estr-9-ene (15) (340 mg) in THF (3 mL) was treated
with TBAF (1.0 M, 1.3 mL) and stirred at r.t. for 2 h. Solvents were
removed under reduced pressure and directly purified on a silica
gel column using 60% ethyl acetate in hexane to afford 16
(210 mg, 75%).
drospiro[estra-5(10),9(11)-diene-17b,20(30H)-furan]-30-one
[1]
(19, 1.0 g, 2.7 mmol) in THF (10 mL) was added dropwise, stirred
for 30 min at ꢁ78 °C, slowly warmed to room temperature over a
period of 1 h and allowed to reflux for 15 h. Reaction mixture
was quenched with water (30 mL) and extracted with ethyl acetate
(3 ꢀ 25 mL). The combined organic layers were washed once with
brine, dried over sodium sulfate and evaporated in vacuo to afford
the crude product. Purification was carried out on a silica gel col-
umn using 10% ethyl acetate in hexane to afford 400 mg (37%) of
the required product (20).
2.1.14. 3,3-Ethylenedioxy-5
pentafluoroethyl)-11b-(40-formylphenyl)-estr-9-ene (17)
suspension of 3,3-ethylenedioxy-5 ,17b-dihydroxy-17-
(1,1,2,2,2-pentafluoroethyl)-11b-[40-(hydroxymethyl)
phenyl]-
a,17b-dihydroxy-17-(1,1,2,2,2-
A
a
estr-9-ene (17) (210 mg), NMO (66 mg) and powdered 4 Å molec-
ular sieve (190 mg) in dichloromethane (8 mL) was stirred at r.t., as
TPAP (7 mg) was introduced as solid at once and stirred for 2 h.
Pure product was obtained by directly passing the reaction mixture
through a silica gel column using 30% ethyl acetate in hexane to af-
ford 200 mg of 17.
1H NMR (d, 300 MHz) 0.83 (s, 3H), 1.1–2.9 (m, 20H), 3.60–3.95
(m, 2H), 3.96 (s, 4H), 5.49 (bs, 1H).
13C NMR (75 MHz) 13.8, 23.9, 24.6, 27.6, 28.4, 31.2, 31.4, 33.02,
36.9, 38.6, 41.3, 46.79, 46.82, 64.4, 64.5, 65.1, 93.6 (t, J = 3.9 Hz),
95.1 (dd, J = 17.9, 19.5 Hz), 108.1, 117.6, 126.1, 130.3, 136.5,
150.9 (dd, J = 282, 280 Hz).
1H NMR (d, 300 MHz) 0.51 (s, 3H), 1.0–2.8 (m, 18H), 3.8–4.1 (m,
4H), 4.2–4.4 (m, 2H), 7.41 (d, J = 8.1 Hz, 2H), 7.78 (d, J = 8.3 Hz, 2H),
9.96 (s, 1H).
2.1.18. 3,3-Ethylenedioxy-21,21-difluoro-5a,10a;17,23-bisepoxy-
19,24-dinor-17 -chola-9(11),20-diene (21)
a
2.1.15. 17b-Hydroxy-17
hydroxy-20202-trifluoroethyl] phenyl}-estra-4,9-dien-3-one (18)
solution of the benzaldehyde substrate (17) (1.7 g,
a
-(1,1,2,2,2-pentafluoroethyl)-11b-{40-[10-
Hydrogen peroxide (0.18 mL, 30%, 1.6 mmol) was added to an
ice-cold solution of hexafluoroacetone trihydrate (350 mg,
1.6 mmol) in dichloromethane (3 mL). Solid Na2HPO4 (180 mg,
1.3 mmol) was introduced and the reaction mixture was stirred
for 1 h at 0 °C. An ice-cold solution of (20) (400 mg, 1 mmol) in
dichloromethane (3 mL) was added and the mixture was stirred
at 0 °C for 3 h then at 5 °C for 15 h. The reaction mixture was di-
luted with dichloromethane (15 mL) and washed with 10% sodium
sulfite solution (15 mL), water, dried over sodium sulfate and con-
centrated under vacuum to obtain the mixture of crude epoxides.
Separation of isomeric epoxides was carried out on a silica gel col-
umn using 10% ethyl acetate in hexane to afford 230 mg (55%) of
A
3.05 mmol) in dry THF (6 mL) at 0 °C was treated with trifluoro-
methyl trimethylsilane (1.8 mL, 12.2 mmol) followed by a few
drops of tetrabutylammonium fluoride in THF (1 M). The reaction
was stirred from 0 °C to room temperature for 1 h, and then cooled
back to 0 °C. A 10% H2SO4 solution (10 mL) was carefully added and
the reaction was stirred overnight at room temperature. The reac-
tion was neutralized with bicarbonate solution and extracted with
dichloromethane. The combined organic extracts were dried over
sodium sulfate and concentrated in vacuo. The residue was dis-
solved in THF (10 mL), treated with tetrabutylammonium fluoride
in THF (1 M, 6 mL, 6 mmol) and stirred at room temperature for
1 h. The reaction was quenched with the addition of saturated
ammonium chloride solution and extracted with ethyl acetate.
After concentration in vacuo, the residue was purified over silica
gel using 30% ethyl acetate in hexanes to give 18 (1.0 g, 58%).
1H NMR (d, 300 MHz) 0.58 (s, 3H), 4.49 (d, J = 9 Hz, 1H), 5.01 (m,
1H), 5.67 (s, 1H), 7.22 (d, J = 9 Hz, 2H), 7.38 (d, J = 9 Hz).
pure a-isomer (21).
1H NMR (d, 300 MHz) 0.85 (s, 3H), 1.1–2.9 (m, 20H), 3.6–4.0 (m,
6H), 5.8–6.0 (m, 1H).
2.1.19. 3,3-Ethylenedioxy-21,21-difluoro-5a
-hydroxy-11b-{40-[10,10-
(ethylenedioxy)-ethyl]phenyl-} 17,23-epoxy-19,24-dinor-17a-chola-
9(10),20-diene (22a)
Magnesium turnings (85 mg, 3.5 mmol) in THF (5 mL) contain-
ing a crystal of iodine was heated to reflux for 10 min to become
colorless. A solution of 2-(4-bromophenyl)-2-methyl-1,3-dioxo-
lane (835 mg, 3.5 mmol) in THF (5 mL) was added to the reaction
mixture and was allowed to reflux for 1 h. Reaction mixture was
cooled under ice and solid CuCl (100 mg, 1.0 mmol) was added to
it and continued to stir at 0 °C for 30 min. Finally a solution of
(21) (480 mg, 1.15 mmol) in THF (5 mL) was added into the cup-
rate solution and allowed to stir for 2 h at 0 °C, quenched with sat-
urated aqueous ammonium chloride solution (30 mL) and
extracted with ethyl acetate (3 ꢀ 25 mL). The combined organic
layers were washed further with water and brine, dried over so-
dium sulfate and evaporated in vacuo to afford the crude product.
Purification was carried out on a silica gel column using 25% ethyl
acetate in hexane to afford 350 mg (52%) of the required product
(22a).
2.1.16. 17b-Hydroxy-17a
-(1,1,2,2,2-pentafluoroethyl)-11b-40-(20202-
trifluoracetyl)phenyl-estra-4,9-dien-3-one (1d, EC316)
A solution of the alcohol (18) (1 g, 1.77 mmol) in dichlorometh-
ane (15 mL) was cooled to 0 °C and treated successively with tri-
ethylamine (2.5 mL, 18 mmol) and a solution of pyridine sulfur
trioxide complex (2.3 g, 14.4 mmol) in DMSO (8.6 mL). The reac-
tion was allowed to warm to room temperature and stirred over-
night. The reaction was cooled to 0 °C and quenched with the
addition of saturated ammonium chloride (30 mL). The pH of the
mixture was adjusted to ꢂ4.0 with 10% H2SO4 and the mixture ex-
tracted with methylene chloride. The organic fractions were
washed with saturated sodium bicarbonate solution, water and
brine, combined, dried over sodium sulfate and concentrated in va-
cuo. The residue was purified over silica gel using 30% ethyl acetate

260
K. Nickisch et al. / Steroids 78 (2013) 255–267
1H NMR (d, 300 MHz) 0.45 (s, 3H), 1.0–2.9 (m, 24H), 3.6–4.1 (m,
1H NMR (d, 300 MHz) 0.58 (s, 3H), 1.0–2.9 (m, 19H), 2.14 (s, 3H),
3.18 (d, J = 30 Hz, 1H), 3.7–4.0 (m, 3H), 4.33 (d, J = 7.0 Hz, 1H), 5.06
(d, J = 1.3 Hz, 1H), 5.37 (s, 1H), 5.77 (s, 1H), 7.11 (d, J = 8.4 Hz, 2H),
7.39 (d, J = 8.3 Hz, 2H).
10H), 4.20 (d, J = 7.4 Hz, 1H), 4.39 (s, 1H), 6.75 (d, J = 8.5 Hz, 1H),
7.12 (d, J = 8.2 Hz, 2H), 7.26–7.32 (m, 2H).
13C NMR (75 MHz) 14.3, 23.2, 23.9, 24.1, 27.4, 28.2, 32.57, 32.60,
35.0, 38.2, 39.0, 39.4, 40.8, 47.3, 48.0 (t, J = 1.9 Hz), 49.48, 49.52,
64.04, 64.3, 64.4, 64.5, 64.6, 65.2, 70.2, 93.8 (t, J = 3.9 Hz), 94.4
(dd, J = 17.1, 19.8 Hz), 108.7, 108.8, 114.9, 125.1, 126.6, 126.9,
133.9, 134.3, 140.3, 146.5, 150.8 (t, J = 282 Hz), 156.1.
2.1.25. 3,3-Ethylenedioxy-21,21-difluoro-5
bromophenyl)-17,23-epoxy-19,24-dinor-17
a
-hydroxy-11b-(40-
a-chola-9(10),20-diene
(22f)
Following the procedure outlined for the synthesis of com-
pound (22a), the Grignard reagent prepared from 1,4-dibromoben-
zene was reacted with compound (21) and CuCl in THF to give after
workup the required product (22f).
2.1.20. 21,21-Difluoro-11b-(40-acetyl)phenyl-17,23-epoxy-19,24-
dinor-17a-chola-4,9,20-triene (2a, EC307)
A solution of 22a (350 mg, 0.6 mmol) in methanol (5 mL) at 0 °C
was treated with 50% sulfuric acid (0.35 mL) and allowed to stir at
room temperature for 2 h. The reaction mixture was carefully
quenched with sodium bicarbonate solution (5 mL) and extracted
with dichloromethane (3 ꢀ 15 mL). The combined organic layers
were washed further with water and brine, dried over sodium sul-
fate and evaporated in vacuo to afford the crude product. Purifica-
tion was carried out on a silica gel column using 25% ethyl acetate
in hexane to afford 250 mg (88%) of the required product (2a,
EC307).
1H NMR (d, 300 MHz) 0.50 (s, 3H), 1.0–2.9 (m, 21H), 3.7–4.0 (m,
7H), 4.17 (d, J = 7 Hz, 1H), 4.33 (s, 1H), 7.06 (d, J = 8.3 Hz, 2H), 7.36
(d, J = 8.5 Hz, 2H).
2.1.26. 21,21-Difluoro-11b-(40-bromophenyl)-17,23-epoxy-19,24-
dinor-17a-chola-4,9,20-triene (2f)
Following the procedure outlined for the synthesis of com-
pound (2a), the Grignard adduct (22f) was hydrolyzed in 50% sul-
furic acid to give after workup the required product (2f).
1H NMR (d, 300 MHz) 0.55 (s, 3H), 1.0–2.9 (m, 19H), 3.7–4.0 (m,
3H), 4.27 (d, J = 7.1 Hz, 1H), 5.77 (s, 1H), 7.03 (d, J = 8.1 Hz, 2H),
7.39 (d, J = 8.5 Hz, 2H).
1H NMR (d, 300 MHz) 0.52 (s, 3H), 1.1–2.9 (m, 19H), 2.57 (s, 3H),
3.70–4.0 (m, 2H), 4.37 (d, J = 7.2 Hz), 5.77 (s, 1H), 7.25 (d, J = 8.1 Hz,
2H), 7.87 (d, J = 8.1 Hz, 2H).
13C NMR (75 MHz) 14.6, 23.6, 25.9, 26.6, 27.5, 28.4, 31.05, 32.7,
32.8, 36.8, 39.1, 40.79, 40.84, 48.4, 49.67, 49.72, 65.4, 93.6 (t,
J = 3.9 Hz), 94.7 (dd, J = 17, 20 Hz), 123.4, 127.3, 128.8, 130.0,
135.1, 144.4, 150.6, 151.0 (t, J = 282 Hz), 156.2, 197.6, 199.2.
13C NMR (75 MHz) 14.7, 23.6, 25.9, 27.5, 28.4, 29.2, 31.0, 32.8 (d,
J = 3 Hz), 36.8, 39.0, 40.2, 40.8, 48.4, 49.72, 49.77, 53.5, 65.4, 93.7 (t,
J = 4 Hz), 94.73 (dd, J = 17, 20 Hz), 119.7, 123.4, 128.8, 129.9, 131.8,
143.8, 144.6, 151.0 (t, J = 281 Hz), 156.2, 199.2.
2.1.21. 3,3-Ethylenedioxy-21,21-difluoro-5
a
-hydroxy-11b-[40-
2.1.27. 21,21-Difluoro-11b-[40-(30-pyridyl)phenyl]-17,23-epoxy-
(dimethylamino)-phenyl]-17,23-epoxy-19,24-dinor-17
a
-chola-
19,24-dinor-17a-chola-4,9,20-triene (2d, EC312)
9(10),20-diene (22b)
One hundred milligram (0.2 mmol) of (2f), 3-pyridinylboronic
acid (60 mg, 0.5 mmol), potassium carbonate (40 mg, 0.3 mmol),
bis(triphenylphosphine)palladium(II) chloride (7 mg, 0.01 mmol)
and triphenylarsine (7 mg, 0.02 mmol) were dissolved in a mixture
of dioxane (5 mL) and water (1 mL) under a nitrogen atmosphere.
The reaction mixture was stirred for 3 h at 100 °C and then cooled
to room temperature. Water (15 mL) was added and the mixture
was extracted twice with EtOAc (3 ꢀ 10 mL). The combined organ-
ic layers were washed with brine, dried (MgSO4) and evaporated to
dryness. Purification by column chromatography using 50% ethyl
acetate in hexane afforded 75 mg (75%) of the required product
(2d, EC312).
Following the procedure outlined for the synthesis of com-
pound (22a), the Grignard reagent prepared from 4-bromo-
dimethylaniline was reacted with compound (21) and CuCl in
THF to give after workup the required product (22b).
1H NMR (d, 300 MHz) 0.54 (s, 3H), 1.10–2.85 (m, 21H), 2.91 (s,
6H), 3.7–4.2 (m, 7H), 4.33 (s, 1H), 6.64 (d, J = 8.7 Hz, 2H), 7.02 (d,
J = 8.6 Hz, 2H).
2.1.22. 21,21-Difluoro-11b-[40-(dimethylamino)phenyl]-17,23-epoxy-
19,24-dinor-17a-chola-4,9,20-triene (2b, EC310)
Following the procedure outlined for the synthesis of com-
pound (2a), the Grignard adduct (22b) was hydrolyzed in 50% sul-
furic acid to give after workup the required product (2b, EC310).
1H NMR (d, 300 MHz) 0.62 (s, 3H), 1.0–2.9 (m, 19H), 2.93 (s, 6H),
3.7–4.0 (m, 2H), 4.27 (d, J = 6.9 Hz, 1H), 5.76 (s, 1H), 6.66 (d,
J = 8.8 Hz, 2H), 6.99 (d, J = 8.7 Hz, 2H).
1H NMR (d, 300 MHz) 0.58 (s, 3H), 1.2–2.9 (m, 19H), 3.7–4.0 (m,
2H), 4.39 (d, J = 7.1 Hz), 5.77 (s, 1H), 7.1–7.4 (m, 3H), 7.50 (d,
J = 8.3 Hz, 2H), 7.8–7.9 (m, 1H), 8.5–8.6 (m, 1H), 8.82 (d,
J = 1.7 Hz, 1H).
13C NMR (75 MHz) 14.6, 23.6, 24.7, 25.9, 27.5, 28.4, 31.1, 32.7 (d,
J = 2.7 Hz), 36.7, 36.8, 39.0, 40.4, 40.7, 48.4 (t, J = 1.9 Hz), 49.7, 49.8,
65.3, 93.7 (t, J = 3.9 Hz), 94.7 (dd, J = 17, 20 Hz), 123.3, 123.6, 127.3,
127.8, 129.7, 134.2, 135.3, 136.1, 144.8, 145.0, 148.1, 148.3, 150.9
(t, J = 282 Hz), 156.3, 199.3.
2.1.23. 3,3-Ethylenedioxy-21,21-difluoro-5a
-hydroxy-11b-(40-
isopropenylphenyl)-17,23-epoxy-19,24-dinor-17a-chola-9(10),20-
diene (22c)
Following the procedure outlined for the synthesis of com-
pound (22a), the Grignard reagent prepared from 4-bromoisop-
ropenylbenzene was reacted with compound (21) and CuCl in
THF to give after workup the required product (22c).
1H NMR (d, 300 MHz) 0.52 (s, 3H), 1.0–3.0 (m, 21H), 2.14 (s, 3H),
3.6–4.0 (m, 6H), 4.23 (d, J = 7.1 Hz, 1H), 4.34 (s, 1H), 5.04 (d,
J = 1.2 Hz, 1H), 5.38 (s, 1H), 7.14 (d, J = 8.3 Hz, 2H), 7.36 (d,
J = 8.3 Hz, 2H).
2.1.28. 21,21-Difluoro-11b-[40-(30-furanyl)phenyl]-17,23-epoxy-
19,24-dinor-17a-chola-4,9,20-triene (2e, EC313)
Following the procedure outlined for the synthesis of com-
pound (2d), (2f), 3-furanylboronic acid, potassium carbonate,
bis(triphenylphosphine)palladium (II) chloride and triphenylarsine
were reacted in a mixture of dioxane and water to give, after puri-
fication, the required product (2e).
1H NMR (d, 300 MHz) 0.58 (s, 3H), 1.1–2.9 (m, 19H), 3.6–4.0 (m,
2H), 4.34 (d, J = 7.1 Hz, 1H), 5.77 (s, 1H), 6.67 (t, J = 0.9 Hz, 1H), 7.15
(d, J = 8.2 Hz, 2H), 7.40 (d, J = 8.2 Hz, 2H), 7.45 (t, J = 1.7 Hz, 1H),
7.70 (s, 1H).
2.1.24. 21,21-Difluoro-11b-(40-isopropenylphenyl)-17,23-epoxy-
19,24-dinor-17a-chola-4,9,20-triene (2c, EC311)
Following the procedure outlined for the synthesis of com-
pound (2a), the Grignard adduct (22c) was hydrolyzed in 50% sul-
furic acid to give after workup the required product (2c, EC311).
13C NMR (75 MHz) 14.6, 23.6, 25.8, 27.5, 28.3, 31.0, 32.7 (d,
J = 2.6 Hz), 36.8, 39.0, 40.4, 40.7, 48.4 (t, J = 1.9 Hz), 49.7, 49.8,

K. Nickisch et al. / Steroids 78 (2013) 255–267
261
65.3, 93.7 (t, J = 3.9 Hz), 94.7 (dd, J = 17, 19.7 Hz), 108.7, 123.1,
126.0, 126.1, 127.4, 129.5, 130.0, 138.4, 143.4, 143.7, 145.3, 150.1
(t, J = 281.5 Hz), 156.4, 199.3.
each well and the plate is incubated for 2 h at room temperature.
The plate is read on a fluorescence plate reader.
2.2.5. The guinea pig model for the assessment of PR-agonistic and
antagonistic properties of PRMs
2.2. Biological assays
The animal studies were performed in Dr. Robert Garfield Lab,
St. Joseph’s Hospital and Medical Center, Phoenix, AZ, USA, after
approval (no. 369) by the local ethical committee. Dunkin-Hartley
guinea pigs (400–500 g body weight) were purchased from Charles
River Laboratory. Animals were kept in an automatically climatized
(21 °C) and illuminated (12:12 light–dark cycle) facilities. Tap
water was available ad libitum from sipper tubes; the provided
pelleted food was fortified with vitamin C and supplemented with
fruits (oranges).
The studies were performed in cycling guinea pigs for the spe-
cific assessment of both PR-agonistic and PR-antagonistic proper-
ties and the interaction of corresponding properties. The studies
were performed in the second half of the guinea pig cycle which
is about 16 days long. The treatment was from cycle day 10–17
by daily s.c. injection of test compounds in 0.2 mL of the vehicle
(0.2 mL benzylbenzoate/castor oil, ratio 1:4, v/v). Control animals
were treated with 0.2 mL vehicle. The time of autopsy (day 18) is
24–48 h after the expected ovulation of the next cycle. This timing
permits the study of the effects of the tested compounds on the
ovulation and on the functional state of the old corpora lutea. Fresh
corpora lutea confirm normal ovulation.
2.2.1. Molecular studies
In vitro studies were performed by Invitrogen Corporation,
Madison. Nuclear receptor studies use beta-lactamase cDNA under
transcriptional control of an upstream activator sequence (UAS).
Details of the respective screenings are as follows.
Test compounds were supplied at 1000ꢀ of the desired starting
concentration (usually 100 nM) in 100% DMSO. The 1000ꢀ test
compounds were serially diluted (10 point ½-log increments) in
100% DMSO. An aliquot of the serial dilution is diluted 1:100 in As-
say Media to a 10ꢀ concentration. The 10ꢀ concentration is then
added to the assay plate where the addition of other assay reagents
dilutes the compounds to a final concentration of 1ꢀ in the assay
with a final DMSO.
2.2.2. PR-agonist screen
PR-UAS-bla HEK 293T cells are thawed and resuspended in as-
say media (DMEM phenol red free, 2% CD-treated FBS, 0.1 mM
NEAA, 1 mM sodium pyruvate, 100 U/mL/100 lg/mL Pen/Strep)
to a concentration of 468,750 cells/mL. Four microliters of a 10ꢀ
serial dilution of R5020 (control agonist starting concentration,
100 nM) or compounds are added to appropriate wells of a 384-
well TC-treated assay plate. Thirty-two microliters of cell suspen-
sion (15,000 cells) is added to each well. Four microliters of assay
media is added to all wells to bring the final assay volume to 40
The plate is incubated for 16–24 h at 37 °C/5% CO2 in a humidified
incubator. Eight microliters of 1 M substrate loading solution is
Typically, ‘‘pure’’ PR-antagonists lead to a proliferation and cor-
nification of the vaginal epithelium. An advanced stage of shedding
of the cornified layers of the vaginal epithelium prevails in ovulat-
ing controls at this stage of cycle (metestrus).
lL.
l
2.2.5.1. Assessment of unopposed estrogenic effects. Vaginal prolifer-
ation and cornification reflect the unopposed effects of the basal
ovarian estrogen secretion of the ovary-intact animals. Uterine
growth may occur, but is sometimes missing under such pure
antagonists. These indicators of estrogen dominance may occur de-
spite the presence of very high levels of progesterone in the circu-
lation, which is brought about by the maintenance of corpora lutea
(‘‘antiluteolytic effect’’ of pure PR-antagonists) [25].
added to each well and the plate is incubated for 2 h at room tem-
perature. The plate is read on a fluorescence plate reader.
2.2.3. PR-antagonist screen, activated by R5020
PR-UAS-bla HEK 293T cells are thawed and prepared as de-
scribed above for the agonist screen. Four microliters of a 10ꢀ se-
rial dilution of RU-486 (control antagonist starting concentration,
100 nM) or compounds are added to appropriate wells of a TC-
treated assay plate. Thirty-two microliters of cell suspension is
added to the wells and pre-incubated at 37 °C/5% CO2 in a humid-
ified incubator with compounds and control antagonist titration
for 30 min. Four microliters of 10ꢀ control agonist R5020 at the
pre-determined EC80 concentration is added to wells containing
the control antagonist or compounds. The plate is incubated for
16–24 h at 37 °C/5% CO2 in a humidified incubator. Eight microli-
2.2.5.2. Assessment of PR-agonistic properties. Corresponding prop-
erties lead to an interference with the proliferation and cornifica-
tion of the vaginal epithelium and induce
change, the mucification of vaginal epithelium.
a morphological
Marginal PR-agonistic effects: Reduction or inhibition of the vag-
inal cornification in presence of an ongoing proliferation of the ba-
sal squamous vaginal epithelium. Pronounced effects: absence of
cornification, reduction or complete inhibition of epithelial prolif-
eration. Simultaneously, a columnar epithelium of typical mucified
cells appears. This is seen as a marker of a PR-mediated agonistic
effect. The mucified epithelium may cover the surface of an abnor-
mally branching layer of ceratinized cells. Similarly, the upper
layer of squamous (non-cornifying) epithelium may be covered
by a layer of mucified cells. As ultimate inhibitory step in this
direction, a complete arrest of proliferation in the vaginal epithe-
lium may be seen. This may then consist of a single layer of colum-
nar mucified epithelial cells (see Figs. 8–10).
ters of 1 lM substrate loading solution is added to each well and
the plate is incubated for 2 h at room temperature. The plate is
read on a fluorescence plate reader.
2.2.4. GR antagonist screen activated by dexamethasone
GR-UAS-bla HEK 293T cells are thawed and resuspended in as-
say media (DMEM phenol red free, 2% CD-treated FBS, 0.1 mM
NEAA, 1 mM sodium pyruvate, 100 U/mL/100 lg/mL Pen/Strep)
to a concentration of 625,000 cells/mL. Four microliters of a 10ꢀ
serial dilution of RU486 (control antagonist starting concentration,
10 nM) or compounds are added to appropriate wells of a TC-trea-
ted assay plate. Thirty-two microliters of cell suspension is added
to the wells and pre-incubated at 37 °C/5% CO2 in a humidified
incubator with compounds and control antagonist titration for
30 min. Four microliters of 10ꢀ control agonist dexamethasone
at the pre-determined EC80 concentration is added to wells con-
taining the control antagonist or compounds. The plate is incu-
bated for 16–24 h at 37 °C/5% CO2 in a humidified incubator.
2.2.5.3. Effects on ovulation. Antiovulatory effects, absence of fresh
CL on cycle day 19, may result from both PR-agonistic and PR-
antagonistic properties.
2.2.5.4. Effects on old corpora lutea. The complete abolition of luteal
regression is a reliable indicator of ‘‘pure’’ PR-antagonism; (undis-
turbed) degeneration of CL and no formation of new CL indicate a
potential mesoprogestin.
Eight microliters of 1 lM substrate loading solution is added to

262
K. Nickisch et al. / Steroids 78 (2013) 255–267
2.3. Evaluation of uterine weight changes and bio-statistical analysis
with 2 equiv. of LDA generated the required 3,3,3-trif-
luoropropynyllithium at ꢁ78 °C which was then added to 3 to ob-
tain 4. Acid hydrolysis of 4 affords compound 1a (EC301, Fig. 4).
As shown in Fig. 5, reaction of intermediate 5 [22] with the
Effects on uterine weights of hormonal active substances were
compared to vehicle controls. Whereas a significant fold-change
of weight could be demonstrated for RU486, EC306, and EC308,
the uterine weights observed for all other substances were in the
range of the vehicle control. Data were log-transformed for analy-
sis. Effects are expressed as fold-change on the original scale. Mul-
tiple comparisons to the vehicle group were based on an ANOVA
and the Bonferroni–Holm adjustment [26] controlling the experi-
ment-wise error level <5%.
Grignard reagent generated from the ketal of a,a,a-trifluoro-4-bro-
moacetophenone gave the adduct 6. Fluoride ion removal of the
TMS ether along with base hydrolysis of the intermediate cyanohy-
drin gave intermediate 7. Acid hydrolysis of 7 affords compound 1b
(EC306).
Opening of epoxide 8 [23] with trifluorovinyllithium (generated
from 1,1,1,2-tetrafluoroethane and n-BuLi at ꢁ78 °C) in presence of
boron trifluoride etherate afforded 9. Subsequent epoxidation, con-
jugate Grignard addition, and hydrolysis yielded 1c (EC308)
(Fig. 6).
2.4. Screening data
As shown in Fig. 7, addition of pentafluoroethyl lithium to com-
pound 12 was carried out to give compound 13. Subsequent epox-
idation and aryl Grignard addition yielded 15. Removal of silyl
protecting group using TBAF, followed by oxidation using TPAP/
NMO resulted in the required benzaldehyde 17. Nucleophilic triflu-
oromethylation using trifluoromethyl trimethylsilane followed by
removal of the TMS protecting group gave 18. Oxidation using pyr-
idine sulfur trioxide yielded 1d (EC316).
In a screening study, substances were compared to a vehicle
control. This experiment should help distinguish between sub-
stances that do increase uterine weight and those that do not.
The data show heterogenous variances; therefore, we decided to
perform all analyses on log-scale of weight, thereby achieving
more homogenous variances. Axes in plots show log-scale. Sample
sizes are in a range of 3–11, with the largest size used for the vehi-
cle control.
The intermediate 17-spirodihydrofuran-30(20H)-one 19 was
synthesized following the procedure of Jiang et al. [24]. Subsequent
Wittig–Horner reaction with the reagent prepared from dif-
luoromethyldiphenylphosphine oxide gave the exocyclic-difluoro-
methylene intermediate 20. Subsequent epoxidation, conjugate
Grignard addition, and hydrolysis gave the targeted compounds
2a–2c (EC307, EC310, and EC311). Palladium-mediated Suzuki
3. Results and discussion
3.1. Chemistry
Compound 1a was synthesized according to Fig. 4 starting from
intermediate 3 [21]. Treatment of 2-bromo-3,3,3-trifluoropropene
O
O
O
F3C
O
CN
CN
F3C
OTMS
OTMS
TBAF
2 M NaOH
Br
Mg, CuCl
O
O
O
5
6
O
O
OH
O
O
O
O
1) CF3CF2I
OH
CF2CF3
O
F3C
F3C
MeLi.LiBr
-78 oC
2) 50% H2SO4
MeOH
O
O
O
OH
1b (EC306)
7
Fig. 4. Synthesis of compound 1b (EC306).
F
F
F
F
F
F
O
OH
OH
Li
F
F
F
H2O2
(CF3)2CO.3H2O
BF3.Et2O
O
O
O
O
8
10
9
O
O
O
O
O
O
F
F
F
F
F
F
O
O
OH
OH
50% H2SO4
MeOH, H2O
Br
O
Mg, CuCl
O
11
O
OH
1c (EC308)
Fig. 5. Synthesis of compound 1c (EC308).

K. Nickisch et al. / Steroids 78 (2013) 255–267
263
OH
C2F5
OH
O
C2F5
H2O2, CH2Cl2
C2F5I
MeLi.LiBr
-78 oC
O
O
O
O
(CF3)2CO.3H2O
14
13
O
12
O
O
OH
OTBDMS
OTBDMS
OH
OH
C2F5
C2F5
TBAF
THF
Br
Mg, CuCl, THF
TPAP/NMO
CH2Cl2
O
O
16
O
OH
15
O
OH
O
O
O
OH
F3C
F3C
OH
OH
OH
C2F5
C2F5
C2F5
1) Me3SiCF3, TBAF
THF
2) H2SO4 (10%)
Pyr.SO3
DMSO
CH2Cl2, Et3N
O
O
3) TBAF/THF
1d (EC316)
17
O
OH
18
Fig. 6. Synthesis of compound 1d (EC316).
O
O
O
O
CF2
CF2
1) F2CHP(O)(OEt)2
THF, -78oC
2) Reflux, 15 h
H2O2, CH2Cl2
O
O
O
O
(CF3)2CO.3H2O
19
20
21
O
O
O
R
R
O
O
O
CF2
CF2
R
MgBr
50% H2SO4
MeOH, H2O
O
CuCl, THF
H3C
O
O
OH
O
2a (EC307)R = Ac
22a R =
2b (EC310)R = NMe2
2c (EC311)R =
2f R = Br
22b R = NMe2
22c R =
22f R = Br
R
O
Br
O
O
O
R-B(OH)2
CF2
CF2
Ph3As, (Ph3P)2PdCl2
K2CO3, heat
2f
2d (EC312)R =
2e (EC313)R =
N
O
Fig. 7. Syntheses of compounds 2a, 2b, 2c, 2d and 2e (EC307, EC310, EC311, EC312, and EC313).
coupling of compound 2f with the appropriate boronic acid yielded
2d (EC312) and 2e (EC313).
performed. RU486 served as standard substance for the antigluco-
corticoid and antiprogestational activity and R5020 was used as
standard for the progestational activity. Data are reported in rela-
tive % compared to standard (see Table 1).
3.2. Biological assays
All compounds with the 17-difluoro exomethylene tetrahydro
furan moiety (307, 310, 311, 312, 313) showed a very high activity
in the antiprogestational test exceeding the activity of RU486 up to
nearly 4-fold. In addition, all these compounds exhibit a rather low
3.2.1. In vitro studies
For the determination of the dissociation between antiglucocor-
ticoid and antiprogestational activity transactivation studies were

264
K. Nickisch et al. / Steroids 78 (2013) 255–267
Table 1
RU486. The dissociation between antiprogestational and antigluco-
corticoid binding was also less pronounced.
Progesterone and glucocorticoid receptor profiling.
CMPD Invitrogen receptor profiling
Progesterone
Glucocorticoid
3.2.2. In vivo characterization
3.2.2.1. PR-Ranking. Ranking of PRM-compounds concerning their
ratio of PR-antagonistic and PR-agonistic properties was done by
an orienting testing using a single very high dose (10.0 mg/ani-
mal/day s.c. days 10–17, and autopsy day 18) in cycling guinea
pigs: Table 2 summarizes the results of the test compound.
The evaluation of a single high dose of the test compounds led
in all cases to distinct and compound-specific results, reflecting the
ratio of PR-agonistic and antagonistic in the plateau of the dose–re-
sponse curve.
Agonist (Rel to
R5020)
Antagonist (Rel to
RU486)
Antagonist (Rel to
RU486)
EC301 <2.85%
<6.45%
20.87%
243.4%
20.67%
383.7%
165.4%
243.9%
79.1%
ND
EC306 <2.85%
EC307 <2.85%
EC308 <2.85%
EC310 <0.28%
EC311 ND
EC312 <0.28%
EC313 <0.28%
EC316 ND
11.66%
<5.83%
<5.83%
14.1%
ND
26.6%
6.4%
ND
16.13%
3.2.2.2. Histology of the ovaries, corpora lutea. Of eight vehicle-trea-
ted control animals, seven had fresh corpora lutea (CL) in their ova-
ries on day 18 of the treatment cycle, which confirms reliable
control of the cycle in the laboratory and normal ovulation in these
animals.
activity in the antiglucocorticoid assay, indicating a 10-fold better
dissociation between antiprogestational and antiglucocorticoid
activity. Fluorination in other positions of the steroid led to signif-
icantly lower activity in the antiprogestational assay compared to
Table 2
Molecular properties of new PRM and orientating study concerning the ratio of PR-agonistic and antagonistic properties at a single high dose (10.0 mg/day s.c.) in cycling guinea
pigs.
Code of
substance ovulation
Rate of
ut weight mean (g) ER-PR-balance: proliferation,
cornification versus mucification
of vagina epithelium
Ovary/corpora lutea Classification
EC301
EC306
EC307
EC308
EC310
EC311
EC312
EC313
EC316
CDB2914
CDB4124
RU486
Onapr.
controls
0/3
1/3
0/8
0/3
1/3
0/3
0/8
0/8
0/3
0/3
1/3
0/3
0/3
7/8
1.3
prol ++
corn +
muc (+)
prol+
corn absent
muc +
prol (+)
corn absent
muc+
prol ++
corn (+)
muc+
prol +
corn (+)
muc+
prol ++
corn+
muc (+)
prol absent
corn absent
muc+
prol (+)
corn absent
muc+
prol ++
corn++
muc absent
prol ++
corn+
muc (+)
prol ++
corn ++
muc absent
prol ++
corn ++
muc (+)
prol ++
corn ++
muc absent
prol +
(lf CL)
Almost pure antiprogestin
2.16⁄
1.26
1.8⁄
lf CL
State of vagina, uterus and CL inconsistent-mesoprogestin
deg CL
Mesoprogestin
(lf CL)
Mesoprogestin
1.37
0.94
1.03
1.13
2.35⁄
1.17
1.41
1.83*
0.98
1.05
deg CL
Mesoprogestin
deg CL
Blunted antiprogestin
deg CL
Mesoprogestin, complete suppression of proliferation
Mesoprogestin, almost complete suppression of proliferation
Almost pure antiprogestin
Blunted antiprogestin
deg CL
(lf CL)
(lf and deg CL)
(lf and deg CL)
(lf CL)
Almost pure antiprogestin
Blunted antiprogestin
lf CL
Pure antiprogestin
fresh CL
–
corn absent (metestrus)
muc absent
Abbreviations: CL, corpora lutea; lf, large and functional; deg, degenerating.
Vag, vaginal epithelium; prol, proliferation of basal layers; corn, cornification; muc, mucification of upper layer; ut, uterus; n.t., not tested; (+)-++ indicate presence and
dominance degree of feature in the vaginal histology.
*
statistical significant difference.

K. Nickisch et al. / Steroids 78 (2013) 255–267
265
With the exception of single animals, all tested compounds pre-
vented ovulation. Old CL showed a substance-specific histological
appearance. Some compounds led to the maintenance of large
and apparently functional CL (Onapristone, EC306). Under other
substances, a degeneration of CL or its complete disappearance
took place, which indicates the completion of luteolysis as in the
normal ovulatory cycle (see Table 2).
nists: Strong proliferation of the basal squamous cell layer and a
thick cornified upper without any signs of mucification were seen
after treatment with EC316, CDB4124, and Onapristone. RU486 in-
duces strong proliferation and cornification of the vaginal epithe-
lium; however, the mucification of the upper layers of the
vaginal epithelium indicates a disturbance of the cornification pro-
cess by PR-agonistic activity. RU486 may thus not be classified as
‘‘pure’’ antagonist in this animal model.
3.2.2.3. Uterine weight. Some compounds elevated the uterine
weight dramatically. This increase versus controls was statistically
significant (p < 0.05) for EC306, EC308, EC316, and RU486. Signifi-
cant reductions of the uterine weight were not recorded. In ex-
tended studies (data not shown) including lower doses, uterine
weight tended to be lower at higher doses of the following sub-
stances: EC307, EC312, and EC313. However, these differences
were not statistically significant.
3.2.3.1. Diminished ER-dominance. Compounds EC301, EC308,
EC310, EC311, and CDB2914 led to a reduced and/or atypical corni-
fication of the vaginal epithelium. The response varies among the
animals; e.g., one animal of the EC308 group lacks vaginal cornifi-
cation (see Fig. 11), contrary to the others in this group. After treat-
ment with EC306, EC307, EC312, EC313, no induction of an
estrogen dominated vagina, specifically no induction of cornifica-
tion of the upper layers of the epithelium, which is typical of anti-
progestins, is seen.
3.2.3. Histology of the vaginal mucosa (see Table 2 and Figs. 8–10)
Very different states of the vaginal mucosa and compound-wise
transitions from ER- to PR-dominance were seen. Pure PR-antago-
Marginal estrogenic effects prevail under most of these com-
pounds, as indicated by basal layers of proliferating squamous cells
Fig. 8. Histology of vagina, day 18 of treatment cycle, dose cycle day 10–17, reference substances; (a) RU486 and (b) CDB2914, 10.0 mg/animal/day s.c.: proliferation of basal
and cornification of upper epithelial layers indicate largely unopposed indirect estrogenic effects. Mucified cells on the surface of epithelium probably reflect an interference
of PR-agonistic properties of both compounds.
Fig. 9. Histology of the vagina in cycling guinea pigs on day 18 of the treatment cycle, dose cycle day 10–17: 10.0 mg/day s.c.: (a) EC316, full unopposed indirect estrogenic
effect with proliferation and cornification of the upper cell layers. Gradually blunted estrogenic effects in case of (b) EC306, (c) EC301, and (d) EC310. Note: Compound-related
reduction of cornified cell layer compared to (a) and the appearance of mucified epithelial cells as upper layer (c and d).

266
K. Nickisch et al. / Steroids 78 (2013) 255–267
Fig. 10. Histology of the vagina in cycling guinea pigs on day 18 of the treatment cycle, dose cycle day 10–17: (a) EC308, 10.0 mg/day s.c. and (b) EC313, 10.0 mg/day s.c.:
mucification, no cornification, almost complete suppression of basal proliferation; (c) EC312 10.0 mg/day s.c.: no cornification, complete suppression of basal cell
proliferation, formation of a mucified epithelium; (d) control at metestrous, epithelium after desquamation of cornified layers.
Table 3
under a superficial layer of mucified epithelial cells (see Figs. 9 and
10).
Under EC308 and EC313 this basal proliferation was largely
suppressed, under EC312 a complete suppression of basal prolifer-
ation prevails (EC312 see Fig. 10).
Uterus weights and standard deviations (Comment: EC313 (EC303?) and EC 315
missing; check Meister’s data).
4. Discussion
Antiprogestins abolish inhibitory functions of the PR by acting
on the proliferation inducing effects of estrogens in the genital
tract. This inhibition is sufficient to induce massive estrogenic ef-
fects in the vagina leading to proliferation and the ceratinization
of the vaginal epithelium reaching or exceeding the degree of stim-
ulation which is normally seen at proestrus. Within the uterus a
proliferation of uterine tissues and uterine growth may occur
(see Tables 2 and 3). In the presence of the ovaries, despite the
inhibition of follicular maturation and ovulation, a basal ovarian
estrogen secretion may be sufficient for these growth events (see
Table 4).
Table 4
With regard to the therapeutic intention to block ovulation for
all kinds of indications, fibroid disease, endometriosis, contracep-
tion, the issue of endometrial safety in humans has to be consid-
ered seriously. Considering the powerful estrogenic effects of
pure PR-antagonists in some animal species and those of RU486
in some clinical studies [27] there appears to be an urgent need
to shape PRMs in such a way, that the proliferation inducing poten-
tial of this class of compounds, via the ER, is controlled by the
introduction of a PR-agonistic component. PRMs with substantial
PR-agonistic properties for the arrest of endometrial proliferation,
like EC312 and EC313, fulfill all criteria of such a mesoprogestin. By
definition a mesoprogestin should be characterized by a reduced or
absent abortifacient potential when compared to RU486. These
studies remain to be done for the selected clinical candidates.
PRM for labor induction: It was shown in prior studies that the
elevated responsiveness of the myometrium in pregnancy to pros-
Statistical evaluation of uterus weights: multiple comparisons of the substances to
the vehicle control. Raw and adjusted p-values are displayed, with sig = 1 indicating
significant differences. Non-adjusted confidence intervals are provided for the fold-
change of weight in comparison to the control group.
Substance
p Value
p-Adjust
Sign
l.CL
Fold
Up. CL
CDB.2914
CDB.4124
EC.301
EC.306
EC.307
EC.308
EC.310
EC.311
EC.312
EC.316
RU486
0.42468
0.06528
0.16090
0.00096
0.72662
0.00280
0.11283
0.40999
0.88308
0.00005
0.00160
0.74701
1.00000
0.84859
1.00000
0.01535
1.00000
0.03926
1.00000
1.00000
1.00000
0.00089
0.02403
1.00000
0
0
0
1
0
1
0
0
0
1
1
0
0.80
1.00
0.90
1.30
0.70
1.20
0.90
0.70
0.80
1.50
1.30
0.70
1.20
1.40
1.30
1.90
1.10
1.70
1.30
0.90
1.00
2.20
1.80
1.00
1.60
2.00
1.80
2.60
1.50
2.50
1.90
1.20
1.30
3.10
2.60
1.30
ZK98.299

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Acknowledgements
We thank Katarina Jewgenow for valuable advice and help per-
forming the morphological evaluation, Mario Moreno for perform-
ing the animal studies and Monika Blankenburg for skillful help
during the preparation of the manuscript.
the novel selective progesterone receptor modulator (SPRM) J867.
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