Synthesis and antiprogestational properties of novel 17-fluorinated steroids

Article abstract of DOI:10.1016/j.steroids.2013.04.003

Progesterone receptor (PR) plays a key role in reproductive functions, and compounds that inhibit progesterone action (antiprogestins) have potential use in the treatment of estrogen- and progesterone-dependent diseases, including uterine leiomyomas and breast cancer. In the present study, we chemically synthesized novel 17-fluorinated steroids and evaluated the cytotoxicity profiles of these compounds in T47D breast cancer cells compared to the activity of known antiprogestins, including ZK230 211, RU-486, CDB2914, CDB4124 and ORG33628. We analyzed in vitro receptor-binding assays and PR-transactivation assays to establish the antiprogestational activity of these molecules. The representative antiprogestin EC304 was found to inhibit in vitro tumorigenicity in a dose-dependent fashion in T47D cells by a colony formation assay at 1 and 10 nM concentrations. The potent in vivo antiprogestational activity of EC304 was also demonstrated in an antinidation assay for the interruption of early pregnancy in rats. The strong antiprogestational activity and absence of antiglucocorticoid activity in EC compounds may demonstrate their utility in the treatment of leiomyoma, endometriosis and breast cancer.

Full text of DOI:10.1016/j.steroids.2013.04.003

Steroids 78 (2013) 909–919
Contents lists available at SciVerse ScienceDirect
Steroids
journal homepage: www.elsevier.com/locate/steroids
Synthesis and antiprogestational properties of novel 17-fluorinated
steroids
c
a
a
b
Klaus Nickisch a, , Hareesh B. Nair , Narkunan Kesavaram , Baishakhi Das , Robert Garfield ,
⇑
Shao-Qing Shi b, Shylesh S. Bhaskaran c, Sandra L. Grimm d, Dean P. Edwards d
a Evestra, Inc., San Antonio, TX, USA
b St. Joseph’s Hospital and Medical Center, Phoenix, AZ, USA
c Texas Biomedical Research Institute, Southwest Primate Research Center, San Antonio, TX, USA
d Departments of Molecular & Cellular Biology and Pathology & Immunology, Baylor College of Medicine, Houston, TX, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Progesterone receptor (PR) plays a key role in reproductive functions, and compounds that inhibit proges-
terone action (antiprogestins) have potential use in the treatment of estrogen- and progesterone-depen-
dent diseases, including uterine leiomyomas and breast cancer. In the present study, we chemically
synthesized novel 17-fluorinated steroids and evaluated the cytotoxicity profiles of these compounds
in T47D breast cancer cells compared to the activity of known antiprogestins, including ZK230 211,
RU-486, CDB2914, CDB4124 and ORG33628. We analyzed in vitro receptor-binding assays and PR-trans-
activation assays to establish the antiprogestational activity of these molecules. The representative anti-
progestin EC304 was found to inhibit in vitro tumorigenicity in a dose-dependent fashion in T47D cells by
a colony formation assay at 1 and 10 nM concentrations. The potent in vivo antiprogestational activity of
EC304 was also demonstrated in an antinidation assay for the interruption of early pregnancy in rats. The
strong antiprogestational activity and absence of antiglucocorticoid activity in EC compounds may dem-
onstrate their utility in the treatment of leiomyoma, endometriosis and breast cancer.
Received 31 October 2012
Received in revised form 14 March 2013
Accepted 6 April 2013
Available online 20 April 2013
Keywords:
Antiprogestin
Fluorine
Breast cancer
Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction
mas in female BALB/c mice [6], and spontaneous mammary tumors
that develop in p53-null mammary gland transplants are highly
Progesterone receptor (PR) is a ligand-dependent transcription
factor that plays a crucial role in the maintenance and develop-
ment of the female reproductive system [1,2]. The biological activ-
ity of progesterone is mediated by a cascade of events induced
upon binding of progesterone with its receptor in the classical
pathway of PR activation. Thus, antiprogestins have considerable
potential as therapeutics for the treatment of several gynecological
disorders and breast cancer [1]. Independent of inhibiting endoge-
nous progesterone levels, antiprogestins have been shown to inhi-
bit estrogen-dependent proliferation in the uterus and in the
mammary gland [3,4]. The mouse mammary gland has served as
an important in vivo surrogate in modeling normal human breast
development and breast cancer. Mouse models have also under-
scored the critical role of PR in mammary tumorigenesis, with PR
knockout mice exhibiting reduced susceptibility to chemically in-
duced mammary tumors [5]. The progestin medroxyprogesterone
acetate (MPA) can induce ER/PR-positive mammary adenocarcino-
dependent on P4 [7].
The majority of breast tumors are of the luminal epithelial phe-
notype, and over 75% of tumors express ER/PR, suggesting that lumi-
nal mammary epithelial cells expressing ER/PR are major targets for
breast tumorigenesis [8,9]. An increased PR-A/PR-B ratio was found
to be associated with high-grade breast tumors, poor clinical out-
come and resistance to endocrine therapy [10]. Transgenic mice that
over-express PR-A exhibit excessive ductal side branching and dis-
ruption of the normal interactions between the epithelial cells and
basement membrane [11]. These data collectively indicate that PR
is potentially a valuable therapeutic target in breast cancer. Preli-
minary clinical trials have been performed with first-generation
PR antagonists (mifepristone and onapristone), with promising re-
sponses obtained with either these molecules alone or in association
with other agents [12–14]. However, the anti-glucocorticoid (anti-
GR) activity of mifepristone and the in vivo hepatic toxicity of
onapristone limit their long-term use [15].
A new finding that a subgroup of breast cancer patients might
especially benefit from a antiprogestin treatment has increased
the interest in antiprogestins for breast cancer again.
Women with mutations in the breast cancer susceptibility gene
BRCA1 are predisposed to breast and ovarian cancers. Treatment of
Abbreviations: PR, progesterone receptor; GR, glucocorticoid receptor; MEM,
minimal essential medium.
⇑
Corresponding author. Tel.: +1 210 673 3300.
E-mail addresses: knickisch@evestra.com, klausnickisch@aol.com (K. Nickisch).
0039-128X/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.steroids.2013.04.003

910
K. Nickisch et al. / Steroids 78 (2013) 909–919
Brca1/p53-deficient mice with the progesterone antagonist mifepri-
stone (RU-486) prevented mammary tumorigenesis. These findings
reveal a tissue-specific function for the BRCA1 protein and raise the
possibility that antiprogesterone treatment may be useful for breast
cancer prevention in individuals with BRCA1 mutations [16].
The field of progesterone receptor antagonists (antiprogestins)
has progressed in two directions since the first compound of this
class, mifepristone (RU-486), was discovered by Teutsch et al [17].
Full receptor antagonists have been investigated for applications
in post-coital contraception, abortion and breast cancer, whereas
mixed agonist/antagonists have been considered for gynecological
indications such as endometriosis and uterine fibroids [18].
Over the years, knowledge about the structure/activity relation-
ship of antiprogestins has grown considerably [19,20]. Mifepri-
stone’s use is limited by its strong antiglucocorticoid activity.
Replacement of the 17-propynyl group in mifepristone with 3-
hydroxypropyl [21], 17-acetoxy [22] or 17,17spiro ether [23] gave
rise to potent antiprogestins with significantly reduced antigluco-
corticoid activity, as shown in Scheme 1.
Recently, 17-perfluoralkyl derivatives have been reported that
exhibit very strong antiprogestational activity and minimal antig-
lucocorticoid activity. The compound ZK230 211 was selected from
this class of compounds based on its extraordinary potency and its
highest receptor selectivity observed to date [24].
All these compounds exhibit strong antiprogestational activity
with only marginal agonistic activity. Asoprisnil, however, is the
first representative of a new class of antiprogestins with a dual
antagonistic/agonistic profile [25].
chloroform (CDCl3) using tetramethylsilane (TMS) as an internal
standard (d = 0), unless noted otherwise. ‘Flash column’ chroma-
tography was performed on 32-64 lM silica gel obtained from
EM Science, Gibbstown, New Jersey. Thin-layer chromatography
(TLC) analyses were carried out on prescored silica gel GF (Anal-
tech) glass plates (2.5 cm ꢀ 10 cm with 250
lM silica layer).
Chemicals and solvents were analytical grade and used without
further purification, unless otherwise noted. Most chemicals and
solvents were analytical grade and used without further purifica-
tion. Commercial reagents were purchased from Aldrich Chemical
Company (Milwaukee, WI).
2.2. Chemical synthesis
2.2.1. 3,3-Ethylenedioxy-5a,17b-dihydroxy-17-(3,3,3-trifluoro-1-
propynyl)-110-{40-[10,10-(ethylenedioxy)-ethyl]phenyl}-estr-9-ene (3)
n-Butyllithium (55 mL, 2.5 N, 137.5 mmol) was added to a
solution of diisopropylamine (21.6 mL, 154 mmol) in THF
(40 mL) at ꢁ78 °C under argon over 10 min, and the resulting
mixture was stirred for 30 min. This LDA solution was added
slowly over 20 min to a solution of 2-bromo-3,3,3-trifluoropro-
pene (12 g, 68.5 mmol) in THF (80 mL), precooled to ꢁ78 °C.
The mixture was stirred for 15 min, after which a solution of
3,3-ethylenedioxy-5a
-hydroxy-11b-{40-[10,10-(ethylenedioxy)-ethyl]-
phenyl}-estra-9-ene-17-one (2) (6 g, 12.1 mmol) in THF (80 mL)
was introduced over 15 min, stirred at ꢁ78 °C for an additional
1 h, and slowly warmed to room temperature over 15 h The
reaction mixture was quenched with aqueous ammonium chlo-
ride (50 mL) and extracted with ethyl acetate (3 ꢀ 100 mL). The
organic extracts were combined and washed with water and
brine, dried over sodium sulfate and evaporated in vacuo to af-
ford the crude product, which was purified on a silica gel using
25% ethyl acetate in hexane to afford 3 (5.0 g, 70%).
In an earlier study [26] the authors have reported the synthesis
of steroidal antiprogestins with partial agonistic activity for poten-
tial application in the treatment of fibroids and endometriosis.
In this study the synthesis of pure antiprogestins is described.
The synthesized compounds are evaluated for anti-progestational
activity in vitro and in vivo andcytotoxic activity inhuman breast
cancer T47D cells.
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).
2. Experimental
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
2.1. General
Nuclear magnetic resonance spectra were recorded on
a
Bruker ARX (300 MHz) spectrometer as solutions in deuterated
N
O
N
O
OH
HO
OMe
OMe
OH
N
C C CH3
OH
O
Mifepristone
Onapristone
(ZK-98299)
Asoprisnil
OMe
O
O
O
O
F
F
F
N
O
O
OAc
OH
O
F
F
Proellex
(CDB-4124)
ORG-33628
Lonaprisan
(ZK-230211)
Scheme 1.

K. Nickisch et al. / Steroids 78 (2013) 909–919
911
2.2.2. 11b-(40-Acetyl-phenyl)-17b-hydroxy-17-(3,3,3-trifluoro-1-
propynyl)-estra-4,9-diene-3-one (1a, EC301)
Fifty percent sulfuric acid (2.2 mL) was added to a solution of 3,3-
ethylenedioxy-5a,17 b -dihydroxy-17-(3,3,3-trifluoro-1-propynyl)-
ylenedioxy-5
a
,17b-dihydroxy-17-(3,3,3-trifluoro-1-propynyl)-
11b-{40-[10,10-(ethylenedioxy)-ethyl]phenyl}-estr-9-ene (3) (1.2 g,
2 mmol) in ether (10 mL) and toluene (10 mL) at ꢁ78 °C. The result-
ing reaction mixture was stirred for 3 h at ꢁ78 °C, then warmed to
room temperature over 1 h. The reaction mixture was quenched
with a solution of saturated ammonium chloride (20 mL) and ex-
tracted with ethyl acetate (3 ꢀ 20 mL). The organic layers were com-
bined and washed with water and brine, dried over sodium sulfate
and evaporated under vacuum to afford 7 (1.2 g crude product).
1H NMR (d, 300 MHz) 0.51 (s, 3H), 1.64 (s, 3H), 1.1–2.5 (m, 21H),
3.7–4.1 (m, 8H), 4.30 (d, J = 6 Hz, 1H), 4.45 (s, 1H), 5.80-6.00 (m,
1H), 6.54 (d, J = 15.5 Hz, 1H), 7.19 (d, J = 8.2 Hz, 2H), 7.34 (d,
J = 8.3 Hz, 2H).
11b-{40-[10,10-(ethylenedioxy)-ethyl]phenyl}-estr-9-ene (3) (3.5 g,
6 mmol) in methanol (35 mL) at 0 °C, and the mixture was stirred at
room temperature for 2 h. The reaction mixture was carefully
quenched with a solution of sodium bicarbonate (15 mL) and extracted
with dichloromethane (3 ꢀ 20 mL). The combined organic layer was
washed with water and brine, dried over sodium sulfate and evapo-
rated in vacuo to afford the crude product, which was purified on a sil-
ica gel using 25% ethyl acetate in hexane to afford 1a (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.
2.2.4. 11b-(40-Acetyl-phenyl)-17b-hydroxy-17-(3,3,3-trifluoroprop-
1(E)-enyl)-estra-4,9-diene-3-one (1b, EC315)
Following the procedure outlined for the synthesis of com-
pound 1a, compound 4 (1.2 g) was hydrolyzed in 50% sulfuric acid
to give 700 mg of compound 1b after workup and purification.
1H NMR (d, 300 MHz) 0.59 (s, 3H), 2.57 (s, 3H), 1.3-2.8 (m, 19H),
4.42 (d, J = 7.0 Hz, 1H), 5.80 (s, 1H), 5.80-6.00 (m, 1H), 6.57 (d,
J = 15.5 Hz, 1H), 7.28 (d, J = 8.0 Hz, 2H), 7.87 (d, J = 8.2 Hz, 2H).
2.2.3. 3,3-Ethylenedioxy-5a,17b-dihydroxy-17-(3,3,3-trifluoroprop-
1(E)-enyl)-110-{40-[10,10-(ethylenedioxy)-ethyl]phenyl}-estr-9-ene (4)
A P 65% wt. solution of sodium bis (2-methoxyethoxy) aluminum
hydride in toluene (2.1 mL, 7.1 mmol) was added to a slurry of 3,3-eth-
O
O
O
O
O
O
O
Br
O
OH
OH
CF3
CF3
CF3
LDA, -78o
50% H2SO4
MeOH, H2O
O
O
O
OH
OH
1a (EC301)
2
3
Red-Al
-78o
O
O
O
O
OH
OH
CF3
CF3
50% H2SO4
MeOH, H2O
O
O
OH
4
1b (EC315)
Scheme 2.
O
O
O
O
O
O
F
F
O
O
OH
F
F
Ac2O/Py
DMAP, 70o C
Li
50% H2SO4
O
O
O
O
OH
2
5
1d (EC304)
O
O
OH
F
F
10% Pd/C, H2
ethanol
1e (EC305)
Scheme 3.

912
K. Nickisch et al. / Steroids 78 (2013) 909–919
F
F
F
F
Li
F
O
OH
OH
F
F
H2O2
F
F
BF3
(CF3)2CO 3H2O
O
O
O
O
O
O
O
6
7
8
O
O
O
O
F
F
O
OH
O
OH
MgBr
F
F
50% H2SO4
F
F
CuCl
O
O
OH
9
1c (EC308)
Scheme 4.
Table 1
EC compounds inhibit cell proliferation of T47D cells: Cytotoxicity screening of EC
compounds in 47D cells demonstrates their potent antiproliferative activity com-
pared with known antiprogestins in the MTT assay.
Compounds
IC50 (EC300)
RU-486
CDB 2914
CDB 4124
ORG33628
ZK230211
EC301
>100 nM
>100 nM
>100 nM
100 pM
5.1 nM
10 nM
EC304
EC305
0.5 nM
<1 pM
EC308
EC315
5.9 nM
1 pM
Table 2
MMTV-Luc regulation.
Compound
EC50 [nM]
IC50 [nM]
Progesterone
RU-486
EC300
EC304
EC315
0.43
–––
ND
ND
ND
–––
0.124
0.034
0.047
0.259
ND = none detected.
Table 3
PR and GR-antagonist screen.
CMPD
Invitrogen receptor profiling
Progesterone
Glucocorticoid
Antagonist (Rel to RU-486)
Antagonist (Rel to RU-486)
Fig. 1. (A) Estrogen stimulated 3-dimensional growth of T47D cells: The 3-
dimensional structures of T47D cells in Matrigel were completely destroyed within
48 h of EC304 treatment. The treated cells were determined to be cytostatic and did
not grow further. (B) EC compounds induce cell-cycle arrest at G1 phase: One of the
leading candidates, EC304, induced apoptosis and G1-phase cell-cycle arrest when
co-incubated with E2 for 24 h. The percentage of cells gated for G1 phase in a
control reaction compared to those treated with 1 or 10 nM EC304 were 40.45,
50.21 and 54.22, respectively. (C) EC304 inhibits cell proliferation at protein level:
Antiprogestin EC304 inhibits cell proliferation by inhibiting cyclin D1 and stabi-
lizing the cyclin-dependent kinase inhibitor p21.
EC301
EC304
EC305
EC308
EC315
ZK230 211
CDB 4124
<6%
ND
21%
10%
5%
22%
3
119%
191%
21%
33%
81%
186%
ND
ND = none detected.

K. Nickisch et al. / Steroids 78 (2013) 909–919
913
A
B
cntl.001 Gate 1
800
600
400
200
0
M5
M1
M2
M3
M4
102
M1
102
102
103
103
FL3-Height
1nM 304.001 Gate 1
500
375
250
125
0
M5
M2
M3
M4
102
102
102
103
103
FL3-Height
10nM 304.001 Gate 1
800
600
400
200
0
M5
M1
M2
M3
M4
102
102
102
103
103
FL3-Height
Fig. 2. MMTV-Luciferase reporter gene assay in breast cancer cells. (A) Agonist mode. T47D cells infected with adenovirus MMTV-Luc (MOI = 3) were treated with increasing
amounts of progesterone (0.1, 1, 5, 10, 100 or 500 nM) or EC compounds (5, 20, 100 or 500 nM) for 24 h. Luciferase activity was measured in triplicate as described in the
Experimental section, with data normalized to protein concentration and values indicating the average Luc units per mg protein SEM. The data were graphed as Luc units/
mg protein vs. log of the concentration (nM) of compounds and were fit by non-linear regression using GraphPad Prism 5.0. (B) Antagonist mode. T47D cells infected with
adenovirus MMTV-Luc (MOI = 3) were treated for 24 h with 5 nM P4 alone or 5 nM P4 supplemented with increasing amounts of EC304 (0.01, 0.03, 0.06, 0.1 or 0.3 nM) or RU-
486 (0.03, 0.1, 0.3, 1 or 3 nM). Luciferase activity was measured and the data were quantified and graphed as in panel A.
2.2.5. 3,3-Ethylenedioxy-11b-{40-[10,10-(ethylenedioxy)-ethyl]phenyl}-
estra-4,9-diene-17-one (5)
phenyl}-estra-4,9-diene-17-one (5) (400 mg, 0.84 mmol) and 3-
bromo-3,3-difluoropropene (530 mg, 3.4 mmol) in (4:1:1)
a
DMAP (150 mg) and acetic anhydride (3 mL) were added to a
THF:ether:pentane mixture (6 mL) at ꢁ100 °C. The reaction mixture
was stirred for 90 min at ꢁ95 °C and warmed to room temperature
over 3 h, then quenched with ammonium chloride solution
(20 mL) and extracted with ethyl acetate (3 ꢀ 15 mL). The combined
organic layers were concentrated to dryness, dissolved in methanol
(5 mL) and treated with 50% sulfuric acid (0.25 mL) at 0 °C. The reac-
tion was stirred at room temperature for 2 h and carefully quenched
with a solution of sodium bicarbonate (15 mL). The solution was ex-
tracted with dichloromethane (3 ꢀ 10 mL) and the combined
dichloromethane layers were dried over sodium sulfate, concen-
trated under vacuum, and purified on a silica gel column using
25% ethyl acetate in hexane to afford 1d (160 mg, 40%).
solution of 3,3-ethylenedioxy-5a
-hydroxy-11b-{40-[10,10-(ethyl-
enedioxy)-ethyl]phenyl}-estra-9-ene-17-one (2) (3 g, 6 mmol) in
pyridine (30 mL), and the resulting reaction mixture was heated
at 70 °C for 30 h. The reaction mixture was concentrated under
vacuum and directly purified on a silica gel using 10% ethyl acetate
in hexane containing 1% TEA to afford 5 (2.3 g, 80%).
1H NMR (d, 300 MHz) 0.48 (s, 3H), 1.2–2.8 (m, 17H), 1.63 (s, 3H),
3.7–4.1 (m, 8H), 4.31 (d, J = 7.1 Hz, 1H), 5.4 (s, 1H), 7.18 (d,
J = 8.2 Hz, 2H), 7.34 (d, J = 8.3 Hz, 2H).
13C NMR (75 MHz) 14.5, 22.0, 24.3, 27.4, 27.5, 30.5, 33.2, 35.5,
37.5, 37.7, 39.6, 47.8, 51.0, 64.5, 64.6, 64.7, 106.2, 108.9, 121.9,
125.4, 127.0, 130.4, 137.5, 139.3, 140.7, 144.6, 219.6.
1H NMR (d, 300 MHz) 0.52 (s, 3H), 1.2-2.8 (m, 17H), 2.52 (s, 3H),
4.42 (bs, 1H), 5.50 (d, J = 11.1 Hz, 1H), 5.68 (d, J = 17.3 Hz, 1H), 5.74
(s, 1H), 6.0-6.3 (m, 1H), 7.26 (d, J = 8.1 Hz, 2H), 7.83 (d, J = 8.3 Hz,
2H).
2.2.6. 11b-(40-Acetyl-phenyl)-17b-hydroxy-17-(1,1-difluoroprop-2-
enyl)-estra-4,9-diene-3-one (1d, EC304)
13C NMR (75 MHz) 16.9, 24.5, 25.8, 26.5, 27.7, 31.1, 33.6, 36.7,
38.8, 39.5, 41.0, 48.2, 51.0, 85.1 (t, J = 26 Hz), 120.7 (t, J = 9.5 Hz),
n-BuLi (1.3 mL, 2.5 M, 3.2 mmol) was added drop wise to a solu-
tion of 3,3-ethylenedioxy-11b-{40-[10,10-(ethylenedioxy)-ethyl]-

914
K. Nickisch et al. / Steroids 78 (2013) 909–919
Fig. 3. Regulation of endogenous PR-target genes: T47D cells were treated for 24 h with 10 nM P4, or 20 nM of either an EC compound or RU-486 alone, or, 5 nM P4 plus
antagonist (1 nM of EC300 or EC304 or 3 nM of EC315, EC317 or RU-486). Total RNA was isolated and used in a PanomicsQuantiGenePlex 2.0 assay to quantify expression of
RNA from twenty-seven different endogenous target genes. The RNA expression of four representative genes, normalized to GAPDH expression, are shown as panels A–D: (A)
FKBP5, (B) HSD11ß2, (C) SGK, and (D) mRas. The factor by which P4 is induced is indicated for each gene.
123.0 (t, J = 247 Hz), 123.2, 127.1, 128.7, 129.9, 131.2 (t,
J = 25.2 Hz), 134.9, 144.3, 150.7, 156.3, 197.7, 199.3.
(ethylenedioxy)-5(10),9(11)-estradiene] (6) (1 g, 3.04 mmol) in
ether (7 mL) was added to the mixture, followed by the drop wise
addition of boron trifluorideetherate (0.38 mL, 3.04 mmol). The
reaction mixture was stirred at ꢁ78 °C for 1 h and warmed to room
temperature over 1 h, then quenched with a solution of sodium
2.2.7. 11b-(40-Acetyl-phenyl)-17b-hydroxy-17-(1,1-difluoropropyl)-
estra-4,9-diene-3-one (1e, EC305)
solution of 11b-(40-acetyl-phenyl)-17b-hydroxy-17-(1,1-
bicarbonate (20 mL) and extracted with ethyl acetate (3
X
A
15 mL). The organic layers were combined and washed with water
and brine, dried over sodium sulfate and evaporated in vacuo to af-
ford the crude product, which was purified on silica using 20%
ethyl acetate in hexane to afford 7 (430 mg, 35%).
difluoroprop-2-enyl)-estra-4,9-diene-3-one
(1d)
(160 mg,
0.34 mmol) in ethanol (5 mL) containing 10% Pd/C (20 mg) was
stirred under positive hydrogen pressure for 2 h. The catalyst
was removed by filtration through a cotton plug, the solvent was
removed under vacuum, and the crude product was purified on sil-
ica gel to yield 1e (120 mg, 75%).
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).
1H NMR (d, 300 MHz) 0.53 (s, 3H), 1.03 (t, J = 7.4 Hz, 3H), 1.1–
2.8 (m, 19H), 2.55 (s, 3H), 4.44 (bs, 1H), 5.76 (s, 1H), 7.28 (d,
J = 8.4 Hz), 7.86 (d, J = 8.4 Hz).
13C NMR (75 MHz) 1.0, 5.5 (t, J = 6.1 Hz), 17.1, 24.7, 25.9, 26.1,
26.6, 27.7, 31.1, 34.1, 36.8, 39.1, 39.4, 41.2, 48.6, 51.4, 85.7 (t,
J = 26 Hz), 123.3, 127.2, 127.8 (t, J = 249 Hz), 128.8, 130.0, 135.0,
144.3, 150.9, 156.3, 197.7, 199.3.
2.2.9. 3,3-Ethylenedioxy-5a,10a-epoxy-17b-hydroxy-17-(2,3,3-
trifluoroprop-2-enyl)-estr-9(11)-ene (8)
Hydrogen peroxide (0.18 mL, 30%, 1.6 mmol) was added to an
ice-cold solution of hexafluoroacetonetrihydrate (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 (7)
(410 mg, 1 mmol) in dichloromethane (3 mL) was added, and the
2.2.8. 3,3-Ethylenedioxy-17b-hydroxy-17-(2,3,3-trifluoroprop-2-
enyl)-5(10),9(11)-estradiene (7)
n-BuLi (2.5 M, 2.4 mL, 6.1 mmol) was added to a solution of
1,1,1,2-tetrafluoroethane (820 mg, 8 mmol) in ether (10 mL) at
ꢁ78 °C over 10 min, and the mixture was stirred for 1 h at
ꢁ78 °C. A solution of spiro-20-(10-oxacyc1opropane)-17(S)-[3,3-

K. Nickisch et al. / Steroids 78 (2013) 909–919
915
Table 4
Endogenous gene regulation in T47D breast cancer cells.
Gene
Accession
Agonist mode: fold induction
Antagonist mode: % inhibition
RU-486
P4
EC304
EC304
S100P
SGK1
ZBTB16
HSD11B2
NDRG1
F3
FKBP5
CDKN1C
KLF15
MT2A
MRAS
CALD1
FGF18
ARHGAP26
FOXO1
EGFR
SOX4
GREB1
NFIC
RGS2
IGF1
MAP3K6
TRAF5
CCND1
E2F1
MYC
NDRG2
NM_005980.2
NM_005627.3
NM_006006.4
NM_000196.3
NM_006096.3
NM_001993.4
NM_004117.3
NM_000076.2
NM_014079.3
NM_005953.3
NM_012219.4
NM_033138.3
NM_003862.2
NM_015071.4
NM_002015.3
NM_005228.3
NM_003107.2
NM_014668.3
NM_205843.2
NM_002923.3
NM_000618.3
NM_004672.3
NM_004619.3
NM_053056.2
NM_005225.2
NM_002467.4
NM_201535.1
125.2
117.9
660.0
41.4
30.0
16.2
12.0
22.9
12.2
11.4
7.9
3.9
12.3
4.6
4.2
3.0
4.0
2.0
2.0
1.6
3.7
2.2
1.1
1.3
0.9
2.0
6.6
1.1
1.0
0.4
0.7
1.3
1.6
1.1
1.3
0.7
1.5
1.3
0.9
1.1
1.9
0.8
1.0
0.6
2.9
1.4
1.0
0.9
1.1
0.5
0.3
98.8
98.1
97.8
97.2
96.8
95.9
93.0
90.9
89.9
86.6
83.8
83.6
80.7
77.3
72.4
69.4
68.0
61.5
58.6
51.1
50.9
44.3
NA
98.2
98.6
98.3
96.2
95.7
96.1
91.1
94.3
84.9
86.9
83.3
76.1
84.3
76.4
73.5
60.2
68.8
47.1
57.3
56.6
25.9
32.3
NA
NA
NA
NA
NA
NA
NA
NA
NA
1.4
1.1
0.5
NA – not applicable (not induced by P4).
2.2.10. 3,3-Ethylenedioxy-5a,17b-dihydroxy-17-(2,3,3-trifluoroprop-
2-enyl)-11b-{40-[10,10-(ethylenedioxy)-ethyl]phenyl}-estr-9-ene (9)
A slurry of magnesium (220 mg, 9 mmol) in THF (10 mL) con-
taining a crystal of iodine was heated to reflux for 10 min until col-
orless. A solution of 2-(4-bromophenyl)-2-methyl-1,3-dioxolane
(2.1 g, 8.5 mmol) in THF (5 mL) was added to the slurry over
5 min and the resulting mixture refluxed for 1 h. The mixture
was cooled on ice prior to the addition of solid CuCl (150 mg,
1.5 mmol). The reaction was stirred at 0 °C for 30 min. Finally, a
solution of 3,3-ethylenedioxy-5a,10a-epoxy-17b-hydroxy-17-
(2,3,3-trifluoroprop-2-enyl)-estr-9(11)-ene (8) (730 mg, 1.7 mmol)
in THF (5 mL) was added to the cuprate solution and stirred for 2 h
at 0 °C, after which the reaction was quenched with aqueous
ammonium chloride solution (30 mL) and extracted with ethyl
acetate (3ꢀ 25 mL). The organic layers were combined and washed
with water and brine, dried over sodium sulfate and evaporated in
vacuo to afford the crude product, which was purified on silica gel
using 25% ethyl acetate in hexane to afford 9 (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).
Fig. 4. Down-regulation and phosphorylation of PR: T47D cells grown in medium
supplemented with 2% dextran-coated charcoal-treated FBS were treated for 24 h
with 100 nM of P4, R5020, RU-486, or an EC compound. Cell lysates were analyzed
by immunoblot assay for total PR protein (PR-A and PR-B) content with the
monoclonal antibody (MAb) 1294 (top panel) or with a MAb that recognizes
phosphorylation of serine 294 of PR-B (middle panel). An immunoblot to b-actin is
shown as a loading control (bottom panel).
2.2.11. 11a
-(40-Acetyl-phenyl)-17b-hydroxy-17-(2,3,3-trifluoroprop-
2-enyl)-estra-4,9-diene-3-one (1c, EC308)
Following the procedure outlined for the synthesis of com-
pound 1a, compound 9 (1.5 g) was hydrolyzed in 50% sulfuric acid
to give 1.1 g of compound 1c after workup and purification.
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).
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), washed
with a 10% sodium sulfite solution (15 mL) and water, dried over
sodium sulfate and concentrated under vacuum to obtain a mix-
ture of crude epoxides. Separation of the epoxide isomers was car-
ried out on a silica gel column using 20% ethyl acetate in hexane to
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.
afford the pure a-isomer 8 (240 mg, 56%).
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).

916
K. Nickisch et al. / Steroids 78 (2013) 909–919
LUMIstar Omega luminometer. Luciferase light units were normal-
ized to protein concentrations in cell lysates as determined by
Bradford assay [2,28].
100
90
80
70
60
50
40
30
20
10
0
2.6. Quantitative RNA assays
The total RNA from T47D cells was isolated using RNeasy col-
umns (Qiagen) according to the manufacturer’s instructions.
QuantigenePlex 2.0 assays [3] were performed using 100 ng of
RNA from each treatment group according to the manufacturers
recommendation. RNA concentration and quality were deter-
mined by NanoDrop assay. Assays were performed with the help
of the Genomic and RNA Expression Profiling Core at Baylor Col-
lege of Medicine [29]. Relative RNA levels were normalized to
GAPDH, and values obtained in triplicate were reported as the
average SEM.
E
E
E
V
O
P
n
C
C
C
e
r
o
n
h
a
3
3
3
e
ic
p
0
0
1
l
l
r
1
8
5
l
e
e
i
s
x
to
e
Fig. 5. EC compounds exhibit potent anti-nidation activity. The antinidation
activities EC compounds were screened in Sprague-Dawley rats. Three control rats
(vehicle treated, s.c.), three rats treated (s.c.) with a known progestin antagonist and
three rats treated (s.c.) with the test compound (3 mg/day) were used. Female rats
were placed with male rats for three to four days, and their vaginas were examined
for sperm plugs every morning. The presence of a sperm plug indicated day one of
pregnancy. Pregnant rats were treated daily with 3 mg of an EC compound
beginning on day five of pregnancy. On day 9, the rats were euthanized, and their
nidation sites were counted. Since the sample size of this experiment is limited to
calculate statistical significance, the observations from this experiment may be
considered as a pilot data.
2.7. Western blot analysis
Cell monolayers were washed with PBS and lysed directly with
200 lL of a buffer containing 50 mM Tris pH 7.4, 50 mM KCl, 4 mM
EDTA, 2 mM EGTA, 1% NP-40 and a protease inhibitor tablet
(Roche). The lysates were centrifuged at 14,000 rpm for 20 min at
4 °C, and protein concentrations were measured by Bradford assay.
2.3. Biological assays
Equal amounts of protein (30 lg) were subjected to electrophore-
Antiprogestational and antiglucocorticoid activity of EC304 was
determined as previously described using select screen assay sys-
tem (Invitrogen-Life Technologies) [26,27]. Briefly, PR-UAS-bla
HEK 293T and GR-UAS-bla HEK 293T cells were used for the PR
antogonistic screen and GR antagonistic screen respectively. Cells
were activated by R5020 (PR agonist) and dexamethasone (GR ago-
sis on 7.5% SDS-polyacrylamide gels and immunoblotted with a
mouse monoclonal antibody to total PR-A and PR-B (clone 1294)
or to phosphoserine 294 of PR (clone B608) and detected by chemi-
luminescence, as previously described [2,30,31]. PR-A and B spe-
cific antibodies were indigenouslyprepared in Dr. Edwards’s lab,
p21 actin and cyclin D1 were purchased from Santacruz laborato-
ries, Santacruz, CA.
nist) for anti-PR and anti-GR screening. 32 lL of cell suspension
was added to the wells and pre-incubated at 37 °C/5% CO2 in a
humidified incubator with compounds and control antagonist
2.8. Cytotoxicity screening
titration for 30 min. 4
l
L of 10ꢀ control agonist R5020 at the
pre-determined EC80 concentration was added to wells containing
The in vitro cytotoxicity of the newly synthesized antiprogestins
was analyzed by the MTT (ATCC, Manassas, VA) assay. Briefly, 5000
human breast cancer T47D cells were seeded per well in a 96-well
plate (Corning, Lowell, MA) containing phenol red-free RPMI-1640
medium (Invitrogen, Austin, TX) supplemented with 0.1 nM estra-
diol (Sigma-Aldrich, St. Louis, MO), 10% charcoal-stripped serum
(Invitrogen) and various concentrations of the test compounds.
T47D cells were selected for use in this experiment based on their
logarithmic growth phase at 70% confluence, and cultivated for
48 h in phenol red-free charcoal-stripped RPMI medium supple-
mented with 1% penicillin-streptomycin (Invitrogen). Once seeded,
the cells were grown for 3 days before supplementing the medium
with test compounds and E2. The inhibition of cell proliferation
the control antagonist or compounds. The plate was incubated for
16–24 h at 37 °C/5% CO2 in a humidified incubator. 8 lL of 1 lM
Substrate Loading Solution was added to each well and the plate
was incubated for 2 h at room temperature. The plate was read
on a fluorescence plate reader.
2.4. Cell culture
T47D breast cancer cells were maintained in
a-MEM media
containing 5% fetal bovine serum (FBS) as previously described
[2,28]. For MMTV-Luc assays, cells were plated in 12-well dishes
at a density of 150,000 cells/well and, after 24 h, an adenovirus
vector expressing MMTV-Luciferase (Luc) was added at an MOI
of 3 particles/cell, as previously described [2]. Twenty-four hours
after transduction, cells were switched to MEM supplemented
with 2% dextran-charcoal-treated FBS and were subjected to
the new compounds for 24 h at the concentrations indicated in
the figure legends. Identical experiments were conducted in
three wells. For RNA expression and protein immunoblot assays,
cells were plated at 350,000–400,000 cells/well in 6-well dishes
and treated with the new compounds in triplicate wells, as de-
scribed above.
was measured on the 7th day by the addition of 20 lL of MTT solu-
tion to each well. After 4 h, the formation of the tetrazolium salt
was measured with a Thermo Multiskan Spectrum spectrophotom-
eter (Thermo Labsystems, Waltham, MA). Six replicates were mea-
sured for each concentration of each compound tested, from which
the IC50 growth inhibition values for all compounds were
determined.
2.8.1. 3-Dimensional spheroid culture
T47D cell spheroids were prepared using the liquid overlay
method (Offner et al., 1993). Briefly, a T47D cell suspension of
2.5. Luciferase assay
2.5 ꢀ 105 cells/ml in 100
lL of RPMI 1640 medium supplemented
with 10% FBS was plated on 1% agarose-coated 96-well culture
plates. After 5 days of incubation at 37 °C in a humidified atmo-
sphere with 5% CO2, the cells formed spheroids that were then
used to analyze the antiproliferative activity of the test compounds
compared with known antiprogestins.
Cell monolayers were washed with PBS before scraping the cells
into 200
tion, cell lysates (30
reagent (Promega), and light output was measured for 2 s using a
l
L of 1ꢀ reporter lysis buffer (Promega). After centrifuga-
l
L were injected with 50 L of luciferase assay
l

K. Nickisch et al. / Steroids 78 (2013) 909–919
917
2.8.2. Gene expression studies
3. Results and discussion
For gene expression analyses, RNA was isolated from T47D cells
treated with progesterone and antiprogestins using the RNeasy kit
(no. 74104; QIAGEN, Valencia, CA) and was reverse transcribed using
SuperScript III (no. 18080-093; Invitrogen, Carlsbad, CA). Real-time
PCR and subsequent analyses were carried out with 0.25% Sybr Green
using an ABI Prism 7700 Sequence Detector System (PE Applied Biosys-
tems, Foster City, CA) according to the manufacturer’s instructions. The
expression of PR-regulated genes and housekeeping genes was de-
tected as described earlier. Melting curve analysis was performed after
each real-time PCR to ascertain PCR product specificity. Relative
expression was determined using the formula 2_DDCt. Real-time
PCR assays were performed in duplicate per experiment, and each
experiment was repeated at least three times.
3.1. Chemistry
The field of progesterone receptor antagonists (antiprogestins)
has progressed in two directions since the first compound of this
class, mifepristone (RU-486), was discovered by Teutsch et al [17].
Full receptor antagonists have been investigated for applications
in post-coital contraception, abortion and breast cancer, whereas
mixed agonist/antagonists have been considered for gynecological
indications such as endometriosis and uterine fibroids [18].
Over the years, knowledge about the structure/activity relation-
ship of antiprogestins has grown considerably [19,20]. Mifepri-
stone’s use is limited by its strong antiglucocorticoid activity.
Replacement of the 17-propynyl group in mifepristone with 3-
hydroxypropyl [21], 17-acetoxy [22] or 17,17 spiro ether [23] gave
rise to potent antiprogestins with significantly reduced antigluco-
corticoid activity, as shown in Scheme 1.
2.9. In vivo antiprogestational activity
In vivo antiprogestational activity was measured by an anti-nida-
tion assay in which the antiprogestin test compounds were screened
in Sprague–Dawley rats. Briefly, three control rats (vehicle treated,
s.c.), three rats treated (s.c.) with a known progestin antagonist
and three rats treated (s.c.) with the test compound (3 mg/day) were
studied. The female rats were placed with male rats for three to four
days, and their vaginas were examined for sperm plugs every morn-
ing. The presence of a sperm plug indicated day one of pregnancy.
Pregnant rats were treated daily with 3 mg of the antiprogestin com-
pounds beginning on day five of pregnancy. On day nine, the rats
were euthanized, and their nidation sites were counted.
Recently, 17-perfluoralkyl derivatives have been reported that
exhibit very strong antiprogestational activity and minimal antig-
lucocorticoid activity. The compound ZK230 211 was selected from
this class of compounds based on its extraordinary potency and its
highest receptor selectivity observed to date [24].
All these compounds exhibit strong antiprogestational activity
with only marginal agonistic activity. Asoprisnil, however, is the
first representative of a new class of antiprogestins with a dual
antagonistic/agonistic profile [25].
We report here the synthesis and biological characterization of a
new class of antiprogestins derived from ZK230 211. The high po-
tency and selectivity of the latter compound is predominantly
attributed to its pentafluoro side chain [24]. To investigate the influ-
ence of partially fluorinated 17 sidechains on antiprogestin potency
and selectivity, the following new antiprogestins were synthesized:
2.10. Cell cycle analysis
T47D cells were synchronized to G0–G1 phase by serum depri-
vation for three days in 0.5% dextran-coated charcoal-treated ser-
um-containing medium, then released into the cell cycle by the
addition of 10ꢁ8 mol/L E2 in 10% fetal bovine serum–containing
medium (Balasenthil et al). Flow cytometry was performed to ana-
lyze the cell cycle progression.
O
OH
R1
2.11. Protein analysis
The expression of proteins was determined using Western blot
analysis. Total protein was isolated from the cervical tissues by
homogenization in lysis buffer. Equal amounts of protein (60 lg)
O
from representative samples were separated on a denaturing poly-
acrylamide gel and transferred to a nylon membrane. Nonspecific
binding of antibodies was blocked by incubation (overnight, 4 °C)
in TBS containing 0.1% Triton X-100 (TBST) and 5% nonfat dry milk.
The membranes were then incubated with their respective primary
antibodies in TBST and 5% milk overnight at 4 °C. The specific bind-
ing of antibodies to target proteins was visualized using species-
specific IgG followed by chemiluminescent detection and exposure
to enhanced chemiluminescencehyperfilm (Amersham, Piscata-
way, NJ). The antibody for cyclin D1 was purchased from NeoMar-
kers (Fremont, CA), whereas p21 and beta-actin antibodies were
procured from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).
CF3
1a (EC301) R1 =
CF3
1b (EC315) R1 =
F
F
F
1c (EC308) R1 =
1d EC(304) R1 =
1e (EC305) R1 =
F
F
F
F
2.12. Statistical analysis
All experiments were carried out in triplicate, with the results
are expressed as the means SEM where applicable. A Stu-
dent’st-test was used to determine the validity of the differences
between control and treatment data sets. A P-value of less than
0.05 was considered significant.

918
K. Nickisch et al. / Steroids 78 (2013) 909–919
The syntheses of EC301 and EC315 are presented in Scheme 2.
The intermediate 2 may be synthesized according to a previ-
ously reported protocol [32]. Treatment of 2-bromo-3,3,3-triflu-
taining progesterone response elements (PREs) in the promoter
and LTR of the MMTV gene [1,2]. When cells were treated with
EC compounds over a wide concentration range (5–500 nM), no
induction of MMTV-Luc was detectable with EC304. This finding
contrasts with P4, which dramatically induced expression of
MMTV-Luc with an EC50 of 0.43 nM (Fig. 2A). Cells were also trea-
ted in the antagonists with a single dose of P4 (5 nM) in the ab-
sence orpresence of increasing concentrations of EC compounds.
As exemplified with EC304, all EC compounds inhibited P4 induc-
oropropene with
2
eqs of LDA at ꢁ78 °C generated 3,3,3-
trifluoropropynyllithium, which was added to 2 to give the desired
product, 3. Red-Al reduction of 3 at ꢁ78 °C selectively yielded the
trans-double bond. Upon hydrolysis, compounds 3 and 4 yielded
1a (EC301) and 1b (EC315), respectively.
The syntheses of EC304 and EC305 are presented in Scheme 3.
Dehydration of the 17-keto derivative (2) was achieved by
treatment with excess of acetic anhydride in pyridine at 70 °C for
30 h to afford 5. Addition of difluoroallyllithium to 5 at ꢁ100 °C
generated the addition product, which upon acid hydrolysis
yielded 1d (EC304). The side chain double bond at C-17 can be
selectively hydrogenated using 10% Pd/C under a hydrogen atmo-
sphere to provide 1e (EC305).
tion of MMTV-Luc expression in
a dose-dependent manner
(Fig. 2B), with calculated IC50s between 0.034 and 0.259 nM (Ta-
ble 2). A direct comparison with the PR antagonist RU-486 revealed
that ZK 230211 (EC300) and EC304 had lower IC50s than RU-486
(0.124 nM), whereas EC315 had a higher IC50 (Table 2). Thus, under
these conditions, the EC compounds are all PR antagonists either
comparable to or more potent than RU-486.
The synthesis of EC308 is presented in Scheme 4.
The intermediate 6 may be synthesized according to a previ-
ously reported protocol [33]. Ring-opening of the epoxide 6 with
trifluorovinyllithium generated from 1,1,1,2-tetrafluoroethane
and n-BuLi at ꢁ78 °C in the presence of boron trifluorideetherate
afforded 7. Subsequent epoxidation, conjugate Grignard addition
and hydrolysis yielded 1c.
3.4. Effects on endogenous PR target gene expression
To examine the relative agonist and antagonist activities of EC-
compounds with endogenous PR target genes, the RNA expression
of a panel of 27 known PR targets in T47D cells was assayed by the
Quantigene branched DNA multiplex bead technology [3]. The
genes were selected from previous microarray studies in the liter-
ature as well-characterized, robust targets of PR in breast cancer
cells [2,4–11]. Cells were treated for 24 h with P4 or EC compounds
alone or with P4 in the presence of a single concentration of EC
compound. The single dose concentration of each EC compound
that exhibit PR antagonism was chosen from the MMTV-Luc assay
as the lowest concentration that gave maximal inhibition of induc-
tion by P4 (Fig. 2). Using a 2-fold increase in RNA expression as the
criteria for an induced target gene, P4 induced expression of 22 of
the 27 genes analyzed (Fig. 3, Table 4). Genes (RGS2, E2F1, CCND1,
MYC) that were not significantly increased by P4 were early re-
sponse genes that are not likely to be maintained at induced levels
after 24 h of treatment [2,9,10]. The effects of the EC compounds
compared with RU-486 in the agonist and antagonist mode for four
representative genes (FKBP5, SGK, HSD11b2 and mRas) are shown
in Fig. 3. None of the EC compounds alone significantly induced
expression of these endogenous target genes, whereas two com-
pounds, EC300 and EC304, completely inhibited induction by P4
(Fig. 3). Similar to the results obtained with MMVT-Luc assays,
EC315 was a slightly less potent antagonist and did not completely
inhibit P4 induction of target genes such as FKBP5, HSD11b2 and
SGK. Table 4 summarizes the results with all 27 targets, and high-
lights the effects of EC304 and P4 as single compound, and of
EC304 vs RU-486 as P4 antagonists. EC304 exhibited very little
agonist activity with this set of target genes, with the exception
of a 2.9 fold induction of IGF-I and a 6.6 fold induction of ZBTB16.
However, ZBTB16 expression was induced 660-fold by P4, indicat-
ing that EC304 has less than 1% the activity of P4 on this gene. In
the antagonist mode, EC304 inhibited P4 induction in a manner
that was indistinguishable from that of RU-486 for each target
gene (Table 4).
3.2. Biological characterization
3.2.1. EC compounds showed potent antiprogestational activity and
inhibited growth of T47D cells
We sought to determine the agonist and antagonistic activity of
the EC compounds before testing their cytotoxicity in breast cancer
cells, as the most promising currently known antiprogestins exhi-
bit progestational and antiglucocorticoid activity. GeneBLAzerÒ
beta-lactamase reporter technology was used to detect receptor
agonism or antagonism induced by the compounds. Our results
show that three of the initial five newly synthesized antiprogestins
have significant antiprogestational activity, without significant
progesterone agonist activity or glucocorticoid antagonist activity.
The strong antiprogestational effect was confirmed by antinidation
assays in rats (Fig. 5). Moreover, several of our test compounds
were superior to the known antiprogestins, ZK230211 (internal
code EC300) or RU-486 (Tables 1 and 3). Based on the cytotoxicity
profiles and progestational/antiglucocorticoid activity results, we
have selected EC304 as one of the lead candidate antiprogestins
for more extensive study.
T47D cell 3D spheroids treated with EC304 exhibited a dose-
dependent inhibition of spheroid formation after 6 days in culture.
Treatment with EC304 dissociated the 3D cell spheroids, which
were rendered cytostatic. Cell cycle analysis revealed that the cyto-
static cells were arrested in G1 phase upon 24 h treatment with
EC304. The percentage of gated cells in the untreated control
T47D cells was 40.45%, whereas 50.21 and 54.22%, respectively,
of cells treated with 1 nM and 10 nM EC304 were arrested in G1
phase (Fig. 1B). Mechanistically, the protein levels of the cyclin-
dependent kinase inhibitor p21 were increased with EC304 treat-
ment, further confirming that G1 phase arrest gives rise to the
cytostatic effect (Fig. 1C).
3.5. Down-regulation and phosphorylation of PR
3.3. Functional activity for progesterone receptor (PR) in breast cancer
cells by MMTV-Luc reporter gene assay
A hallmark of progesterone agonists is to down-regulate PR pro-
tein upon prolonged treatment, whereas the antagonist RU-486
stabilizes PR protein in the cell [11,15,34]. To test how EC com-
pounds affect PR down-regulation, T47D cells were treated for 24
h with P4, the synthetic progestin R5020, or each of the EC com-
pounds, and steady-state PR protein levels were monitored by
immunoblot assay of whole cell lysates with an antibody to both
the PR-A and PR-B isoforms. As expected, substantial down-regula-
tion of PR was observed with P4 and R5020 compared with vehicle
To determine the relative agonist and antagonist activities of
the EC compounds with respect to PR-mediated transcriptional
activation, the MMTV-Luciferase (Luc) reporter gene assay was
utilized. T47D human breast cancer cells that express PR (both A
and B isoforms) and are responsive to progesterone (P4) were
transduced with an adenoviral vector expressing MMTV-Luc con-

K. Nickisch et al. / Steroids 78 (2013) 909–919
919
[10] Kobayashi S, Stice JP, Kazmin D, Wittmann BM, Kimbrel EA, Edwards DP, et al.
Mechanisms of progesterone recpetor inhibition of inflammatory responses in
cellular models of breast cancer. Mol Endocrinol 2010;24:2292–302.
[11] Berrodin TJ, Jelinsky SA, Graciani N, Butera JA, Zhang Z, Nagpal S, et al. Novel
progesterone receptor modulators with gene selective and context-dependent
partial agonism. Biochem Pharmacol 2009;77:204–51.
controls (EtOH, DMSO). Similar to results with RU-486, PR protein
was stabilized and was not down-regulated by treatment with the
EC compounds (Fig. 4, top panel). Progesterone binding in cells
leads to a rapid increase of PR phosphorylation on several serine
residues including serine 294, a residue that is predominantly
phosphorylated on the PR-B isoform [35]. Although the role of PR
phosphorylation is not well defined, data indicates that phosphor-
ylation modulates nuclear translocation, PR protein turnover and
PR-mediated transcriptional regulation in a target gene-specific
manner [30,36]. The antagonist RU-486 has been shown previously
to stimulate phosphorylation of PR on the same sites as are stimu-
lated by the hormone agonist, whereas Onapristone (ZK98299)
fails to increase PR phosphorylation [29,31,37]. Thus, RU-486 does
not exert its antagonist action by interfering with or altering PR
phosphorylation. By immunoblotting with an antibody that recog-
nizes phospho-PR serine 294 [34], it was determined that the EC
compounds permitted PR phosphorylation of serine 294 and thus
affected phosphorylation in a manner similar to that of RU-486
(Fig. 4).
[12] Robertso JFR, Willscher PC, Witterbotton L, Blamly RW. Onapristone,
a
progesterone receptor antagonist, as first-line therapy in primary breast
cancer. Eur J Cancer 1999;35:214–8.
[13] Klijn JGM, Setyono-Han B, Foekens JA. Progesterone antagonists and
progesterone receptor modulators in the treatment of breast cancer. Steroids
2000;65:825–30.
[14] Klij G, De Jomg F, Bakker G. Antiprogestins, a new form of endocrine therapy
for human breast cancer. Cancer Res 1989;49:2851–6.
[15] Wardell SE, Edwards DP. Mechanisms controlling agonist and antagonist
potential of selective progesterone receptor modulators (SPRMs). Semin
Reprod Med 2005;23:9–21.
[16] Poole AJ, Li Y, Kim Y, Lin S-CJ, Lee W-H, Lee P. Prevention of Brca1-mediated
mammary tumorigenesis in mice by
2006;314:1467.
a progesterone antagonist. Science
[17] Belanger A, Philibert D, Teutsch G. Regio and steriospecific synthesis of 11b-
substituted 19-norsteroids. Steroids 1981;37:361–82.
[18] Garcia T, Benhamou B, Gofflo D, Vergezac A, Philibert D, Chambon P, et al.
Switching agonistic, antagonistic, and mixed transcriptional responses to 11
beta-substituted progestins by mutation of the progesterone receptor. Mol
Endocrinol 1992;6:2071–8.
[19] Teutsch G, Philibert D. History and perspectives of antiprogestins from the
chemist’s point of view. Hum Reprod 1994;9(Suppl 1):12–31.
4. Conclusion
[20] Schoonen WGEH, Vermeulen GJ, Deckers GH, Verbost PM, Klooserboer HJ.
Antiprogestins: their mechanism of action and the consequences for
compound selection by in vitro and in vivo studies. Cur Top Steroid Res
1999;2:15–54.
Substitution of the 17 pentafluor ethyl group of the steroid mol-
ecule ZK230 211 with partially fluorinated unsaturated side chains
gave rise to compounds with significantly higher cytotoxicity in
the T47D cell line model. EC304 was selected for further studies
based on its satisfactory differentiation between progesterone
and glucocorticoid binding. EC304 was shown to degrade PR recep-
tors through BRCA1-mediated ubiquitination and subsequent pro-
teasomaldegradation. In experiments with endogenous PR target
genes, EC304 showed similar effects as RU-486.
[21] Neef G, Beier S, Elger W, Henderson D, Wiechert R. New steroids with
antiprogestational and antiglucocorticoid activities. Steroids 1984;44:349–72.
[22] Attardi BJ, Burgenson J, Hild SA, Reel J. In vitro antiprogestational/
antiglucocorticoid activity and progestin and glucocorticoid receptor binding
of the putative metabolites and synthetic derivatives of CDB-2914, CDB-4124,
and mifepristone. J. Steroid Biochem. Mol. Biol. 2004;88:277–88.
[23] Kloosterboer HJ, Deckers GH, Schoonen WGEJ, Hanssen RGJM, Rose UM,
Verbost PM, et al. Preclinical experience with two selective progesterone
receptor modulators on breast and endometrium. Steroids 2000;65:733–40.
[24] Cleve A, Klar U, Schwede W. Beneficial effects of fluorine in the anti-progestin
ZK 230211. J Fluorine Chem 2005;126:217–20.
Ongoing studies are focused on developing EC304 as a chemo-
therapeutic agent for PR-positive breast cancer patients, as well
as a chemopreventive for BRCA1- and p53-mutated patients.
[25] Elger W, Bartley J, Schneider B, Kaufmann G, Schubert G, Chwalisz K. Endocrine
pharmacological characterization of progesterone antagonists and
progesterone receptor modulators with respect to PR-agonistic and
antagonistic activity. Steroids 2000;65:713–23.
Acknowledgements
[26] Nickisch K, Elger W, Cessaca J, Kesavarama N, Dasa B, Garfield R, et al.
Synthesis and biological evaluation of partially fluorinated antiprogestins and
mesoprogestins. Steroids 2013;78(2):255–67.
[27] Guo G, Smith A. A genome-wide screen in EpiSCs identifies Nr5a nuclear
receptors as potent inducers of ground state pluripotency. Development
2010;137(19):3185–92.
This work was supported in part by NIH Grant ROI DK40903
(DPE).
[28] Wardell SE, Narayanan R, Weigel NL, Edwards DP. Partial agonist activity of the
progesterone receptor antagonist RU486 mediated by an amino-terminal
domain co-activator and phosphorylation of serine-400. Mol Endocrinol
2010;24:335–45.
[29] Beck CA, Zhang Y, Weigel NL, Edwards DP. Two types of anti-progestins have
distinct effects on site-specific phosphorylation of human progesterone
receptor. J Biol Chem 1996;271:1209–17.
References
[1] Lieberman BA, Bona BJ, Edwards DP, Nordeen SK. The constitution of
progesterone response element. Mol Endocrinol 1993;7:515–27.
[2] Boonyaratanakornkit V, McGowan E, Sherman L, Mancini MA, Cheskis BJ,
Edwards DP. The role of extranuclear signaling actions of progesterone
receptor in mediating progestrone regulation of gene expression and the cell
cycle. Mol Endocrinol 2007;21:359–75.
a
[30] Ward RD, Weigel NL. Steroid receptor phosphorylation: assigning function ot
site-specific phosphorylation. BioFactors 2009;35:528–36.
[31] Beck CA, Zhang Y, Altmann M, Weigel NL, Edwards DP. Stoichiometry and site-
specific phosphorylation of human progesterone receptor in native target cells
and in the baculovirus expression system. J Biol Chem 1996;271:19546–55.
[32] Rao PN, Wang Z, Cessac JW, Rosenberg RS, Jenkins DJA, Diamandis EP. New
[3] Canales RD, Luo Y, Willey JC, et al. Evaluation of DNA microarray results with
quantitative gene expression platforms. Nat Biotechnol 2006;24:1115–22.
[4] Jacobsen BM, Richer JK, Sartorius CA, Horwitz KB. Expression profiing of
human breast cancers and gene regulation by progesterone receptors.
Mammary Gland Biol Neoplasia 2003;8:257–68.
J
11b-aryl-substituted
steroids
exhibit
both
progestational
and
antiprogestational activity. Steroids 1998;63:523–30.
[5] Graham JD, Mote PA, Salagame U, van Dijk JH, Balleine RL, Huschtscha LI, et al.
DNA replication licensing and progenitor numbers are increased by
progesterone in normal human breast. Endocrinology 2009;150:3318–26.
[6] Yudt MR, Russo LA, Berrodin TJ, et al. Discovery of a novel mechanism of
steroid receptor antagonism: WAY-255349 modulates progesterone receptor
cellular localization and promoter interactions. Biochem Pharmacol
2011;82:1709–19.
[33] Liu A, Carlson E, Katzenellengogen JA. Synthesis of high affinity fluorine-
substituted ligands for the androgen receptor. Potential agents for imaging
prostatic cancer by positron emission tomography.
1992;35:2113–29.
J
Med Chem
[34] Leonhardt SA, Edwards DP. Mechanism of action of progesterone antagonists.
Exp Biol Med 2002;227:969–80.
[35] Press M, Spaulding B, Groshen S, Kaminsky D, Hagerty M, Sherman L, et al.
Comparison of different antibodies for detection of progesterone receptor in
breast cancer. Steroids 2002;67:799–813.
[36] Hagan CR, Daniel AR, Dressing GE, Lange CA. Role of phosphorylation in
progesterone receptor signaling and specificity. Mol Cell Endocrinol
2012;357:43–9.
[37] Gass EK, Leonhardt SA, Nordeen SK, Edwards DP. The antagonists RU486 and
ZK98299 stimulate progesterone receptor binding to deoxyribonucleic acid
in vitro and in vivo, but have distinct effects on receptor conformation.
Endocrinology 1998;139:1905–19.
[7] Subtil-Rodriguez A, Millan-Arino L, Quiles I, Ballare C, Beato M, Jordan A.
Progesterone induction of the 11beta-hydroxysteroid dehydrogenase type 2
promoter in breast cancer cells involves coordinated recruitment of STAT5A
and progesterone receptor to a distal enhancer and polymerase tracking. Mol
Cell Biol 2008;28:3830–49.
[8] Magklara A, Smith CL. A composite intronic element directs dynamic binding
of the progesterone receptor and GATA-2. Mol Endocrinol 2009;23:61–73.
[9] Moore MR, Zhou JL, Blankenship KA, Strobl JS, Edwards DP, Gentry RN. A
sequence in the 50 flanking region confers progestin responsiveness on the
human c-myc gene. J Steroid Biochem Mol Biol 1997;62:243–52.