Novel phosphorus-containing 17β-side chain mifepristone analogues as progesterone receptor antagonists

Article abstract of DOI:10.1016/j.steroids.2006.07.003

A novel series of steroidal compounds were designed and synthesized with various phosphorus-containing groups on the 17β-side chain as progesterone receptor antagonists. The structure-activity relationships of these compounds are discussed. Selected compo

Full text of DOI:10.1016/j.steroids.2006.07.003

steroids 7 1 ( 2 0 0 6 ) 949–954
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/steroids
Novel phosphorus-containing 17␤-side chain mifepristone
analogues as progesterone receptor antagonists
Weiqin Jiang∗, George Allan, Xin Chen, James J. Fiordeliso, Olivia Linton,
Pamela Tannenbaum, Jun Xu, Peifang Zhu, Joseph Gunnet,
Keith Demarest, Scott Lundeen, Zhihua Sui
Johnson & Johnson Pharmaceutical Research & Development L.L.C., Drug Discovery, 1000 Route 202, Raritan, NJ 08869, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of steroidal compounds were designed and synthesized with various
phosphorus-containing groups on the 17␤-side chain as progesterone receptor antagonists.
The structure-activity relationships of these compounds are discussed. Selected compounds
were tested in an rat progesterone-sensitive assay. Some of these compounds are more
potent than mifepristone, with a better selectivity profile in differentiating progesterone
receptor from glucocorticoid receptor.
Received 31 May 2006
Received in revised form
19 June 2006
Accepted 4 July 2006
Published on line 28 August 2006
© 2006 Elsevier Inc. All rights reserved.
Keywords:
C-17 phosphorus-containing
steroids
Selective progesterone receptor
antagonist
1.
Introduction
is potentially compromised due to overt glucocorticoid recep-
tor antagonism. The goal of our PR antagonist program is to
The progesterone receptor (PR), a member of the nuclear
receptor superfamily of ligand-dependent transcription fac-
tors, undergoes a conformational change upon ligand binding
to initiate a cascade of regulatory events [1]. Progesterone,
the endogenous ligand for the PR, is involved in the control
of ovulation and preparation of the uterus to support preg-
nancy. Progesterone receptor (PR) antagonists have been tar-
gets of medicinal chemistry programs for decades [2]. Mifepri-
stone (RU-486), a clinically approved PR antagonist, effec-
tively terminates pregnancy and offers therapeutic promise
for endometriosis, uterine fibroids, and breast cancer [3,5].
In addition, a PR antagonist has potential utility as a contra-
ceptive [4]. However, the clinical usefulness of mifepristone
identify a compound with better potency and higher selectiv-
ity than mifepristone in terms of glucocorticoid antagonism
(Fig. 1).
There have been literature reports describing phosphinic
acid as bioisosteres of the carboxylic acid group [6]. Based
on the X-ray structure of hPR-LBD/progesterone [7] the bind-
ing model of mifepristone in the ligand binding domains of
hPR-LBD was built by molecular modeling (cf. Fig. 2). Com-
pared with the X-ray structure of hGR-LBD/mifepristone, [8]
one can tell that at the C17 position of mifepristone there
is available space around the propynyl group. The replace-
ment of the CH3 on the propynyl group with a phosphonyl
group might increase both hydrophobic and hydrophilic inter-
∗
Corresponding author at: Johnson & Johnson Pharmaceutical Research & Development L.L.C., 8 Clarke Dr. Cranbury, NJ 08512, United
States. Tel.: +1 609 409 3496; fax: +1 609 655 6930.
E-mail address: wjiang1@prdus.jnj.com (W. Jiang).
0039-128X/$ – see front matter © 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.steroids.2006.07.003

950
steroids 7 1 ( 2 0 0 6 ) 949–954
[RPMI medium without phenol red (Invitrogen) contain-
ing 5% (v/v) charcoal-treated FBS (Hyclone) and 1% (v/v)
penicillin-streptomycin (Invitrogen)]. Two days later, the
medium was decanted and test compound at various con-
centrations (0.01–1000 nM), 0.1 nM progesterone, or vehi-
cle control (dimethyl sulfoxide) at a final concentration of
0.1% (v/v), was added in fresh assay medium. Twenty-four
hours later, an alkaline phosphatase assay was performed
using a SEAP kit (BD Biosciences Clontech, Palo Alto, CA).
Briefly, the medium was decanted and the cells were fixed
for 30 min at room temperature with 5% (v/v) formalin
(Sigma). The cells were washed once with room tempera-
ture Hank’s buffered saline solution (Invitrogen). Equal vol-
umes (0.05 mL) of 1X Dilution Buffer, Assay Buffer and 1:20
substrate/enhancer mixture were then added. After 1 h incu-
bation at room temperature in the dark, the lysate was
transferred to a white 96-well plate (Dynex) and lumines-
cence was read using a LuminoSkan Ascent (Thermo Electron,
Woburn, MA).
Fig. 1 – The structure of mifepristone and the designed C17
phosphorus derivatives.
actions between steroid and progesterone receptor. These sub-
tle changes might impact differential binding to progesterone
and glucocorticoid receptors, thus altering the selectivity pro-
file of the target molecules.
2.
Experiment
2.1.
Biological assays
2.1.2. A549 reporter assay
A549 human lung carcinoma cells were washed with OPTI-
MEM I (GIBCO). The medium was removed and lipid-DNA
complex solution (1.5 ␮g/mL of GRE-luciferase reporter DNA in
8.5 mL OPTI-MEM I plus 6 ␮L/mL of DMRIE-C Reagent in 8.5 mL
OPTI-MEM I, combined and mixed, then incubated at room
temperature for 40 min) was overlayed onto the cells in a T160
flask. The cells were incubated for 16 h at 37 ◦C in a CO2 incu-
bator. The DNA-containing medium was removed and 30 mL
of growth medium containing 5% (v/v) charcoal-treated fetal
bovine serum were added. After 5–6 h, the cells were seeded
in 96-well plates and incubated overnight at 37 ◦C. To each
well was then added test compounds followed by dexametha-
sone as a corticoid challenge. The cells were incubated for
24 h. Luciferase assay buffer (Promega) was added to each well
and the cells were incubated for 30 min at room temperature.
Luciferase activity was measured in a DYNEX Microlite plate
on a TopCount (Packard).
2.1.1. T47D alkaline phosphatase assay
T47D human breast cancer cells were plated in 96-well tis-
sue culture plates at 10,000 cells per well in assay medium
2.1.3. Rat complement C3 assay
Ovariectomized 2-month-old Sprague Dawley rats were pur-
chased from Harlan (Indianapolis, IN). Five to 7 days after
surgery, the rats were dosed once with test compound or
control. All test compound-treated animals also received
0.3 mg/kg ethinyl estradiol (EE) and 0.3 mg/kg medroxypro-
gesterone acetate, both given by the oral route. Test com-
pounds were administered orally. About 24 h later, the rats
were euthanized by carbon dioxide asphyxiation. Whole uteri
were removed, trimmed of fat and frozen on dry ice prior to
storage at −80 ◦C. The uteri were homogenized in 1 to 2 mL
each of TRIzol (Invitrogen Life Technologies, Carlsbad, CA);
and the homogenates were processed for RNA preparation
according to the manufacturer’s directions. Quantitative PCR
was performed using rat complement C3 primers and Taq-
Man probe from Applied Biosystems (Foster City, CA) and an
ABI PRISM 7000 Sequence Detection System (Applied Biosys-
tems). The level of 28S ribosomal RNA in each sample was
determined for normalization, and a dilution series of one of
the estrogen-treated samples was used to generate a standard
curve.
Fig. 2 – Illustration of the binding mode of mifepristone in
PR binding site, suggested by molecular modeling. The
ligand and the key residues that act as its hydorgen
bonding partners are represented in the tube model, while
the other residues around binding site in the stick model.
The hydrogen bonds are indicated by the yellow dotted
lines. The molecular surface of binding site is represented
in transparency effect (atom color scheme: hydrogen in
white; oxygen in red; nitrogen in blue; carbon on
mifepristone in purple; and carbon on protein in gray). (For
interpretation of the references to colour in this figure
legend, the reader is referred to the web version of the
article.).

steroids 7 1 ( 2 0 0 6 ) 949–954
951
After 16 h at room temperature, the reaction mixture was par-
titioned between EtOAc/aqueous NH4Cl solution. The organic
layer was separated, dried and concentrated. The crude prod-
uct was purified on silica gel column (3:7 EtOAc/Hex.) to yield
the title compound as a white solid (1.1 g, 90%). 1H NMR (CDCl3)
ı 7.08 (d, 2H, J = 9.1 Hz), 6.68 (d, 2H, J = 9.1 Hz), 4.4 2–3.85 (m, 5H),
2.91 (s, 6H), 2.42–1.56 (m, 19H), 0.52 (s, 3H); MS (m/e): 478 (MH+).
2.1.4. General information for chemistry
NMR spectra were obtained at 400 and 300 MHz on a Brucker
AVANCE300 and AVANCE400 spectrometer. Chemical shifts
are reported in ppm downfield from TMS as an internal stan-
dard. Thin-layer chromatography was carried out using 2.5-
cm × 7.5-cm silica gel 60 (250 ␮M layer) plates with UV detec-
tion. Magnesium sulfate was employed to dry organic extracts
prior to concentration by rotary evaporation. Flash chromatog-
raphy was done using EM science silical gel 60 (230–400 mesh).
Standard solvents from J.T. Baker were used as received. Anhy-
drous solvents from J.T. Baker or Aldrich and all other com-
mercially available reagents were used without further purifi-
cation. Mass spectra were obtained on a Hewlett-Packard
5989A quadrupole mass spectrometer. Silica gel (E. Merck,
230–400 mesh) was used for all flash chromatography. Thin-
layer chromatography was performed on Analtech silica gel
GF prescored plates (250 ␮m). HPLC analysis was carried on
Agilent 1100 Series LC/MSD equipment.
2.1.8. 3,3-Ethylenedioxy-5a,17b-dihydroxy-11b-[4-(N,N-
dimethylamino)phenyl]-19-nor-17a-pregn-9-ene-21-ethynyl
phosphonic acid diethyl ester (16a)
At room temperature, to a solution of compound 14a (0.10 g,
0.2096 mmol) in THF (10 mL) was added LiHMDS (0.70 mL,
1.0 M in THF, 3.3 equiv.). After 1 h, a solution of phospho-
rochloridic acid diethyl ester (0.0108 g, 3.3 equiv.) in THF (1 mL)
was added. After 16 h, the reaction mixture was partitioned
between EtOAc/water (50/50 mL). The organic layer was sep-
arated, washed with NaHCO3 aqueous solution, brine, and
dried, concentrated. The crude product was purified on sil-
ical gel (60% EtOAc/hexane) to provide the desired product
(110 mg, 89%). 1H NMR (CDCl3) ı 7.04 (d, 2H, J = 9.1 Hz), 6.67 (d,
2H, J = 9.2 Hz), 4.28–3.92 (m, 8H), 2.91 (s, 6H), 2.62–1.25 (m, 29H),
0.52 (s, 3H); MS (m/e): 612 (MH+).
2.1.5. 3,3-Ethylenedioxy-5a,10a-epoxyestr-9,11-en-17-
one (12)
To a solution of ethylene deltanone (4.0 g, 12.74 mmol) in DCM
(100 mL) was added sodium bicarbonate (6.31 g, 75.17 mmol).
The reaction mixture was cooled to less than −40 ◦C and
mCPBA (75%, 3.37 g, 14.65 mmol) in dichloromethane (30 mL)
was added. The reaction mixture was stirred for 30 min in
an ice bath. Water was added and the reaction mixture was
stirred. The layers were separated and the aqueous layer
was extracted with dichloromethane. The organic layers were
washed with saturated sodium bicarbonate solution, brine,
dried, filtered and concentrated. The oil was purified by col-
umn chromatography (30–50% ethyl acetate/hexane) to yield
the title compound as a white solid (1.5 g, 36 %). 1H NMR
(400 MHz, CDCl3) ı 6.06 (br s, 1H), 3.97–3.88 (m, 4H), 2.50–2.45
(m, 2H), 2.19–1.22 (m, 16H), 0.88 (s, 3H); MS (m/e): 331 (MH+).
2.1.9. 19-Norpregna-4,9-dien-20-yne-21-phosphonic acid
diethyl ester, 11-[4-(N,N-dimethylamino)
phenyl]-17-hydroxy-3-oxo-, (11b,17a)–(9CI) (17a)
Compound 16a (82 mg, 0.13 mmol) was dissolved in acetone
(10 mL) and then mixed with p-TSA·H2O (5 mg). This was
stirred at 50 ◦C for 30 min. The reaction mixture was parti-
tioned between EtOAc/water (50/50 mL). The organic layer was
separated, washed with saturated NaHCO3 aqueous solution,
brine, dried and concentrated. The crude product was purified
on preparative TLC (50% EtOAc/hexane) to provide the prod-
uct as a white solid (13 mg, 18%). 1H NMR (CDCl3) ı 7.02 (d, 2H,
J = 9.4 Hz), 6.62 (d, 2H, J = 9.4 Hz), 5.78 (s, 1H), 4.38 (m, 1H), 4.10
(m, 4H), 2.94–1.26 (m, 29H), 0.61 (s, 3H); MS (m/e): 552 (MH+);
HRMS: calc’d. MH+ for C32H42NO5P 551.2800; found 551.2806.
2.1.6. 3,3-Ethylenedioxy-5a-hydroxy-11b-[4-(N,N-
dimethylamino)phenyl]-estr-9-en-17-one (13a)
To
a round-bottomed flask with copper chloride (1.24 g,
2.1.10. Estra-5(10),9(11)-dien-3-one, 11-[4-(N,N-
dimethylamino)phenyl]-17-hydroxy-17-(1-(dimethyl-
phosphinoyl)-ethynyl)-, (17b)–(9CI) (17b)
The title compound was prepared according to the proce-
dure to prepare 17a, starting from the compound 14a (108 mg,
0.2264 mmol). The title compound was obtained as a white
solid (70 mg, 63% for 2 steps). 1H NMR (CDCl3) ı 7.02 (d, 2H,
J = 9.4 Hz), 6.68 (d, 2H, J = 9.4 Hz), 5.74 (s, 1H), 4.38 (m, 1H),
2.94–1.26 (m, 29H), 0.61 (s, 3H); MS (m/e): 492 (MH+), 514 (MNa+);
HRMS: calc’d. M+ for C30H38NO3P 491.2589; found 491.2606.
12.51 mmol, 1.3 equiv.) under nitrogen was added 4-(N,N-
dimethyl)anilinemagnesium bromide (50 mL, 0.5 M in THF,
25 mmol, 2.6 equiv.). The reaction mixture was stirred vig-
orously for 5 min until all of the copper chloride dissolved.
To the reaction mixture was then added a solution of the
steroid epoxide compound 12 (3.2 g, 9.62 mmol) in THF (50 mL).
The reaction mixture became white and cloudy. After 1 h at
room temperature and saturated ammonium chloride solu-
tion was added. The aqueous solution was extracted twice
with ethyl acetate, dried, filtered and concentrated. The
residue was purified by column chromatography (10–100%
ethyl acetate/hexanes) to yield an off-white solid (1.95 g, 45%).
1H NMR (CDCl3) ı 7.02 (d, 2H, J = 9.2 Hz), 6.62 (d, 2H, J = 9.2 Hz),
4.38–3.85 (m, 5H), 2.92 (s, 6H), 2.48–1.56 (m, 19 H), 0.51 (s, 3H);
MS (m/e): 478 (MH+).
2.1.11. Estra-5(10),9(11)-dien-3-one, 11-[4-(N,N-
dimethylamino)phenyl]-17-hydroxy-17-(1-(diphenyl-
phosphinoyl)-ethynyl)-, (17b)–(9CI) (17c)
The title compound was prepared (31 mg, 22% for 2 steps)
according to the procedure to prepare compound 17a starting
from the compound 14a (0.10 g, 0.21 mmol). 1H NMR (CDCl3) ı
7.78 (m, 5H), 7.48 (m, 5H), 7.02 (d, 2H, J = 9.4 Hz), 6.62 (d, 2H,
J = 9.4 Hz), 5.74 (s, 1H), 4.38 (m, 1H), 2.94–1.26 (m, 23H), 0.72
(s, 3H); MS (m/e): 616 (MH+); HRMS: calc’d. M+ for C40H42NO3P
615.2902; found 615.2899.
2.1.7. 3,3-Ethylenedioxy-5a,17b-dihydroxy-11b-[4-(N,N-
dimethylamino)phenyl]-19-nor-17a-pregn-9-ene-21-ethyne
(14a)
To a solution of compound 13a (1.1 g, 2.44 mmol) in THF (20 mL)
was added ethynyl MgBr (0.5 M in THF, 11.76 mL, 5.88 mmol).

952
steroids 7 1 ( 2 0 0 6 ) 949–954
4.16–4.04 (m, 4H), 2.80–1.25 (m, 26H), 0.58 (s, 3H); MS (m/z): 555
2.1.12. Estra-5(10),9(11)-dien-3-one, 11-[4-(N,N-
(MH+), 577 (MNa+); HRMS: calc’d. MH+ for C33H41O5P 555.2334;
found 555.2329.
dimethylamino)phenyl]-17-hydroxy-17-(1-(bis(2-oxo-3-
oxazolidinyl)-phosphinoyl)-ethynyl)-, (17b)–(9CI) (17d)
The title compound was prepared (15 mg, 66% for two steps)
according to the procedure to prepare compound 17a, starting
from the compound 14a (50 mg, 0.10 mmol). 1H NMR (CDCl3)
ı 7.02 (d, 2H, J = 9.4 Hz), 6.62 (d, 2H, J = 9.4 Hz), 5.78 (s, 1H), 4.41
(m, 4H), 4.10 (m, 4H), 2.94–1.42 (m, 24H), 0.61 (s, 3H); MS (m/e):
634 (MH+), 656 (MNa+).
2.1.18. 3,3-Ethylenedioxy-5a-hydroxy-11b-[4-
(methoxy)phenyl]-estr-9-en-17-one (13c)
The title compound was prepared according to the procedure
for compound 13a, starting from the compound 12 (2.85 g,
8.66 mmol). The title compound was obtained as white solid
(1.28 g, 34%). 1H NMR (CDCl3) ı 7.14 (d, 2H, J = 9.1 Hz), 6.81 (d,
2H, J = 9.1 Hz), 4.3 (m, 1H), 4) (m, 4H), 3.78 (s, 3H), 2.42–1.56 (m,
18H), 0.5 (s, 3H); MS: 421 [(M-18)H]+, 461 (MNa+).
2.1.13. Estra-5(10),9(11)-dien-3-one, 11-[4-(N,N-
dimethylamino)phenyl]-17-hydroxy-17-(1-(dimethoxy-
thiophosphinoyl)-ethynyl)-, (17b)–(9CI) (17e)
The title compound was prepared (27 mg, 20% for two steps)
according to the procedure to prepare compound 17a, starting
from the compound 14a (0.050 g, 0.10 mmol). 1H NMR (CDCl3)
ı 7.02 (d, 2H, J = 9.4 Hz), 6.68 (d, 2H, J = 9.4 Hz), 5.74 (s, 1H), 4.38
(m, 1H), 3.75 (m, 6H, 2-OMe), 2.94–1.26 (m, 23H), 0.61 (s, 3H); MS
(m/e): 540 (MH+), 562 (MNa+).
2.1.19. 3,3-Ethylenedioxy-5a,17b-dihydroxy-11b-[4-
(methoxy)phenyl]-19-nor-17a-pregn-9-ene-21-ethyne (14c)
The title compound was prepared according to the procedure
for compound 14a, starting from the compound 13c (1.26 g,
2.88 mmol). The title compound was obtained as white solid
(1.05 g, 79%).
2.1.14. Estra-5(10),9(11)-dien-3-one, 11-[4-(N,N-
dimethylamino)phenyl]-17-hydroxy-17-(1-bis(N,N-
dimethyl)-phosphinoyl)-ethynyl)-, (17b)–(9CI) (17f)
2.1.20. 19-Norpregna-4,9-dien-20-yne-21-phosphonic acid
diethyl ester, 11-(4-methoxy-phenyl)-17-hydroxy-3-oxo-,
(11b,17a)–(9CI) (17h)
The title compound was prepared (14 mg, 24% for two steps)
according to the procedure to prepare compound 17a, starting
from the compound 14a (0.050 g, 0.10 mmol). 1H NMR (CDCl3)
ı 7.08 (m, 2H), 6.62 (m, 2H), 5.72 (s, 1H), 4.38–3.62 and 2.98–1.29
(m, 36H), 0.55 (s, 3H); MS (m/e): 550 (MH+); HRMS: calc’d. M+ for
The title compound was prepared according to the proce-
dures to prepare compound 17a, starting from the compound
14c (85 mg, 0.183 mmol). The title compound was obtained as
white solid (29 mg, 30% for 2 steps). 1H NMR (CDCl3) ı 7.09 (d,
2H, J = 9.4 Hz), 6.81 (d, 2H, J = 9.4 Hz), 5.78 (s, 1H), 4.38 (m, 1H),
4.28–4.08 (m, 7H), 2.82–1.26 (m, 23H), 0.61 (s, 3H); MS (m/e):
539 (MH+); HRMS: calc’d. MH+ for C31H39O6P 539.2563; found
539.2565.
C32H44N3O3P 549.3120; found 549.3110.
2.1.15. 3,3-Ethylenedioxy-5a-hydroxy-11b-[4-
(thiomethoxy)phenyl]-estr-9-en-17-one (13b)
The title compound was prepared according to the proce-
dure for compound 13a, starting from the compound 12 (6.4 g,
19.37 mmol) and 4-thioanisolemagnesium bromide (0.5 M,
100 mL, 50 mmol). The title compound was obtained as a white
solid (3.3 g, 38%). 1H NMR (400 MHz, CDCl3) ı 7.14 (s, 4H), 4.38
(s, 1H), 4.28 (d, J = 6.9 Hz, 1H), 4.04–3.91 (m, 4H), 2.46–2.25 (m,
10H), 2.11–1.21 (m, 11H), 0.50 (s, 3H); MS (m/e): [(M-water)H]+
437, (MNa+) 477.
3.
Results and discussion
Commercially available ethylene deltanone 11, was converted
into epoxide 12 under mCPBA/NaHCO3 conditions in about
50% yield, with an ˛:ˇ ratio of 3:1, which is in consistency with
the literature reported ratio in the similar reactions [9]. We
found that the rest of the side product is the epoxide on C9–C11
double bond, which could easily be separated from the desired
product. The cuprate reaction is amenable to install the C11
side chain of 4-N,N-dimethyl group to furnish compound 13.
Addition of ethynyl magnesium bromide to the C17 carbonyl
group led to intermediate 14. The literature reported quite a
few methods of alkylation of alkyne. We tried different bases,
such as NaH, MeMgBr, and nBuLi, where little or no product
was obtained. Installation of the phosphorus group was finally
accomplished by using LiHMDS and phosphonyl chloride 15 to
provide target 17 after acid assisted deprotection of C3 ketal
and hydrolysis of C5 hydroxy group [10].
2.1.16. 3,3-Ethylenedioxy-5a,17b-dihydroxy-11b-[4-
(thiomethoxy)-phenyl]-19-nor-17a-pregn-9-ene-21-ethyne
(14b)
The title compound was prepared according to the procedure
for compound 14a, starting from the compound 13b (1.77 g,
3.89 mmol). The title compound was obtained as white solid
(1.24 g, 66%). 1H NMR (400 MHz, CDCl3) ı 7.14 (s, 4H), 4.39 (s, 1H),
4.29 (d, J = 7.4 Hz, 1H), 4.04–3.88 (m, 4H), 2.60 (s, 1H), 2.47–1.49
(m, 22H), 0.47 (s, 3H); MS (m/e): 503 (MNa+), 463 [(M-water)H]+.
2.1.17. 19-Norpregna-4,9-dien-20-yne-21-phosphonic acid
diethyl ester, 11-[4-(thiomethoxy) phenyl]-17-hydroxy-
3-oxo-, (11b,17a)–(9CI) (17g)
The title compound was prepared according to the proce-
dures to prepare compound 17a, starting from the compound
14c (50 mg, 0.104 mmol). The title compound was obtained as
white solid (7 mg, 12% for 2 steps). 1H NMR (400 MHz, CDCl3)
ı 7.49 (d, J = 8.3 Hz, 1H), 7.32 (d, J = 8.4 Hz, 1H), 7.18 (d, J = 8.7 Hz,
1H), 7.10 (d, J = 8.3 Hz, 1H), 5.78 (s, 1H), 4.40–4.36 (m, 1H),
The compounds were evaluated for PR antagonist activity
based on their ability to block progesterone-induced alkaline
phosphatase activity in the human breast cancer cell line
T47D (Table 1). The compounds were also evaluated in the
human lung carcinoma cell line A549 for their ability to inhibit
corticoid-induced transcription from a GRE-linked luciferase
reporter gene as a measure of their glucocorticoid antagonist
activities. All compounds are potent progesterone receptor
antagonists, with nanomolar (17b, 17d–17h) or subnanomo-

steroids 7 1 ( 2 0 0 6 ) 949–954
953
Table 1 – The activities of compounds 17a–17h in T47D and A549 cells
Comd. #
Rꢀ
X
R
T47D IC50 (nM)
A549 IC50 (nM)
Ratio
Mifepristone
–
–
–
0.2
0.28
7.3
2.6
>100
70.42
41.61
13
>357
10
17a
17b
17c
Me2N
Me2N
Me2N
O
O
O
OEt
Me
Ph
0.33
126
17d
Me2N
O
7.25
64.05
9
17e
17f
17g
17h
Me2N
Me2N
MeS
S
OMe
NMe2
OEt
1.06
5.5
5.4
3.8
55.87
120.94
453.78
326.96
53
22
84
86
O
O
O
MeO
OEt
ered that changing the amide functionality to a thio-amide
resulted in compounds displaying potent PR agonist activ-
ity [11b]. It was also found that the functional activity of
6-aryl benzoxazinone-based PR antagonism [11c] changed to
PR agonism when the 2-carbonyl group was replaced by a 2-
thiocarbonyl moiety [11d]. From X-ray crystal structures of the
binding pockets of (a) PR complexed with Tanaproget and (b)
PR complexed with progesterone [12], it was observed, that
the amide carbonyl group resides at the same position of
the C17 side chain carbonyl on progesterone. While it is true
for the non-steroidal series that antagonism switches to ago-
nism when the carbonyl group is switched to the thio-carbonyl
group, for the steroidal compounds both appeared to be potent
lar (17a, 17c) potencies in T47D assays. Except for compound
17a, which is as potent as mifepristone, the rest of compounds,
17b–17h, are slightly less potent than mifepristone. In A549
assay, all compounds were less potent than mifepristone, with
IC50 values greater than 41 nM. Mifepristone 1 is a potent GR
antagonist (Table 1). The PR and GR antagonist activities of
compound 17 were calculated as the ratio of the IC50 in the
A549 assay divided by that in the T47D assay. Compounds 17a,
17c, 17e, 17g, and 17h showed better selectivity than mifepri-
stone. Compounds 17a and 17c were as potent as and more
selective than mifepristone (Scheme 1).
There are several reports of non-steroidal progesterone
antagonists with an improved selectivity profile relative to
mifepristone. In studies of 3,3-disubstituted-5-aryloxindoles
progesterone receptor (PR) antagonists, [11a] it was discov-
PR antagonists with a P O group at C17 (17a) or with a P
(17e).
S
Scheme 1 – The synthetic route leading to compounds 17a–17h. (i) mCPBA, NaHCO3, 35%; (ii) CuCl, 4-Rꢀ-phenyl magnesium
bromide, 50%; (iii) ethynyl magnesiumbromide, quantitative; (iv) LiHMDS, then, CI(O)PR2 15; 15–50%; (v) p-TSA, acetone, 85%.

954
steroids 7 1 ( 2 0 0 6 ) 949–954
[4] Ulmann A, Peyron R, Silverstre L. Clinical uses of
Table 2 – The results of in vivo C3 assay for compounds
17a, 17c and 17d
Mifepristone (MFP). Ann N Y Acad Sci 1995;761:248–60.
[5] (a) Murphy AA, Kettel LM, Morales AJ, Roberts VJ, Yen SS.
Regression of Uterine Leiomyomata in Response to the
Antiprogesterone RU-486. J Clin Endo Metab 1993;76:
513–7;
ID50 (mg/kg)
Mifepristone
3.0
3.0
∼10
∼10
17a
17c
17e
(b) Kettel LM, Murphy AA, Mortola JF, Liu JH, Ulmann A, Yen
SS. Endocrine response to long-term administration of the
antiprogesterone RU-486 in patients with pelvic
endometriosis. Fertil Steril 1991;56:402–7.
[6] Kehler J, Ebert B, Dahl O, Krogsgarrd-Larsen P. Syntheses and
GABA receptor binding properties of 4-amino-1-,2-, and
3-hydroxybutylphosphinic acids. Tetrahedron
1999;55:771–80.
The three most potent and selective compounds (17a, 17c,
17e) were tested in vivo in ovariectomized Sprague Dawley
rats. Lundeen et al. [13] reported the development and char-
acterization of the rat uterine complement component C3
assay for the evaluation of the activity of progestins in the
uterus. In this assay, EE is used to stimulate C3 expression and
progestins inhibit expression; in turn, antiprogestins coun-
teract progestin-dependent inhibition. When the compound
was administered via the oral route along with EE and pro-
gesterone, 17a was as potent and efficacious as mifepristone,
with an ID50 of 3 mg/kg. Consistent with the in vitro T47D data,
compound 17a was more potent in vivo than compound 17c
or 17e (Table 2).
In summary, a series of novel phosphorus-containing C-17
side chain mifepristone analogues were prepared by a five-
step synthetic route. Some of these compounds were as potent
as mifepristone, with better selectivities in terms of gluco-
corticoid antagonist activity. While maintaining similar PR
potencies to mifepristone, compound 17a and 17c showed 30-
and 10-fold more selectivity than mifepristone, respectively.
Compound 17a was as potent as mifepristone in rats when
administered orally. These discoveries show promise in terms
of clinical applications and basic endocrine research for more
potent and more selective steroidal antiprogestins.
[7] Williams SP, Sigler PB. Atomic structure of progesterone
complexed with its receptor. Nature 1998;393:392–6.
[8] Kauppi B, Jacob C, Farnegardh M, Yang J, Ahola H, Alarcon M,
et al. The three dimensional structures of antagonistic and
agonistic forms of the glucocorticoid receptor
ligand-binding domain. J Bio Chem 2003;278:22748–54.
[9] Rao PN, wang Z, Cessac JW, Rosenberg RS, Jenkins DJA,
Diamandis EP. New 11␤-aryl-substituted steroids exhibit
both progestational and antiprogestational activity. Steroids
1998;63:523–30.
[10] Blanchard C, Vaultier M, Mortier J. Lithium
Bis-(diisopropylamino)boracetylide [LiC C· · ·B(Ni· · ·Pr2)2]. A
New Reagent Preparation Term Alkynes. Tet Lett
1997;38:8863–6.
[11] (a) Fensome A, Bender R, Cohen J, Collins MA, Mackner VA,
Miller LL, et al. New progesterone receptor antagonists:
3,3-disubstituted-5-aryloxindoles. Bio Med Chem Lett
2002;12:3487–90;
(b) Fensome A, Koko M, Wrobel J, Zhang P, Zhang Z, Cohen J,
et al. Novel 5-aryl-1,3-dihydro-indole-2-thiones: potent,
orally active progesterone receptor agonists. Bio Med Chem
Lett 2003;13:1317–20;
(c) Zhang P, Terefenko EA, Fensome A, Wrobel J, Winneker R,
Lundeen S, et al.
6-aryl-1,4-dihydro-benzo[d][1,3]oxazin-2-ones: a novel class
of potent, selective, and orally active nonsteroidal
progesterone receptor antagonists. J Med Chem
2002;45:4379–82;
r e f e r e n c e s
[1] Spitz IM, Chwalisz K. Progesterone receptor modulators and
progesterone antagonists in women’s health. Steroids
2000;65:807–15.
[2] (a) Teutsch G, Philibert D. History and perspectives of
antiprogestins from the chemist’s point of view. Human
Repro 1994;9(Suppl. 1):11–30;
(d) Zhang P, Terefenki EA, Fensome A, Wrobel J, Winneker R,
Zhang Z. Novel
6-aryl-1,4-dihydrobenzo[d][1,3]oxazine-2thiones as potent,
selective, and orally active nonsteroidal progesterone
receptor agonists. Bio Med Chem Lett 2003;13:1313–6.
[12] Fensome A, Bender R, Chopra R, Cohen J, et al. Synthesis
and SAR of novel
(b) DaManno, Debroah, et al. Asoprisnil (J867): a selective
progesterone receptor modulator for gynecological therapy.
Steroids 2003;68:1019–32;
(c) Zhang ZM, et al. Molecular and pharmacological
properties of a potent and selective novel non-steroidal
progesterone receptor agonist Tanaproget. J Bio Chem
2005;280:28468–75.
6-aryl-1,4-dihydrobenzo[d][1,3]oxazine-2-thiones as
progesterone receptor modulators leading to the potent and
selective nonsteroidal PR agonist Tanaproget. J Med Chem
2005;48:5092–5.
[13] Lundeen SG, Zhang Z, Zhu Y, Carver JM, Winneker RC. Rat
uterine complement C3 expression as a model for
progesterone receptor modulators: characterization of the
new progestin trimegestone. J Steroid Biochem Mol Biol
2001;78:137–43.
[3] Hess-Stumpp H, Hoffmann J, Fuhrmann U. Progesterone
receptor antagonist (antiprogestins). Drugs Future
2002;27:1113–23.