Parallel synthesis and SAR study of novel oxa-steroids as potent and selective progesterone receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2007.02.013

Efficient parallel synthesis of novel 7-oxa-steroids 4 has been achieved from the key intermediate 3 via a one-pot four-step sequence. oxa-Steroids 4 with various ortho-, meta-, and para-monosubstituents on the phenyl ring, as well as disubstituted phenyl

Full text of DOI:10.1016/j.bmcl.2007.02.013

Bioorganic & Medicinal Chemistry Letters 17 (2007) 2531–2534
Parallel synthesis and SAR study of novel oxa-steroids as potent
and selective progesterone receptor antagonists
Fu-An Kang,* Jihua Guan, Nareshkumar Jain, George Allan, Olivia Linton,
Pamela Tannenbaum, Xin Chen, Jun Xu, Peifang Zhu, Joseph Gunnet,
Keith Demarest, Scott Lundeen and Zhihua Sui
Johnson and Johnson Pharmaceutical Research and Development, LLC, 665 Stockton Drive, Exton, PA 19341, USA
Received 12 January 2007; revised 5 February 2007; accepted 6 February 2007
Available online 8 February 2007
Abstract—Efficient parallel synthesis of novel 7-oxa-steroids 4 has been achieved from the key intermediate 3 via a one-pot four-step
sequence. oxa-Steroids 4 with various ortho-, meta-, and para-monosubstituents on the phenyl ring, as well as disubstituted phenyl
and heterocycles, were evaluated for progesterone receptor (PR) and glucocorticoid receptor (GR) antagonist activities. SAR study
demonstrated that the para-fluorinated substituents on the phenyl ring not only increased the potency for PR in a T47D cell func-
tional assay, but also improved the selectivity over GR in an A549 cell functional assay. The para-fluorophenyl oxa-steroid 4l and
the para-trifluoromethylphenyl oxa-steroid 4p were found to be remarkably more potent and more selective PR antagonists than
mifepristone, with subnanomolar potency and about 140-fold selectivity over GR. Molecular modeling of the oxa-steroid bound
to PR provided meaningful insight for the SAR study. oxa-Steroids 4a and 4b were found to be more efficacious than mifepristone
in vivo in a rat uterine complement C3 assay via the oral route, although they were less than or equally potent to mifepristone in the
T47D assay.
ꢀ 2007 Elsevier Ltd. All rights reserved.
The progesterone receptor (PR), like other steroid recep-
tors, plays a unique and crucial role in mammalian
development and homeostasis. Progesterone is known
to be required for mammary gland development, ovula-
tion, and the maintenance of pregnancy. It has less
clearly defined functions in bone, the cardiovascular sys-
tem, and the central nervous system. In terms of oppor-
tunities for pharmacological intervention, it has
arguably the greatest unexploited potential of the
steroids. Currently, steroidal progestin agonists and
antagonists are clinically approved for contraception,
hormone replacement therapy, and therapeutic abor-
tion. There is strong preclinical and clinical evidence
for the value of progestin antagonists for treating
endometriosis, uterine fibroids, dysfunctional uterine
bleeding, and breast cancer.1
potent and selective antiprogestins. This has led to the
identification of a variety of steroidal3 and nonsteroidal4
PR modulators in the past years. However, current PR
antagonists, such as mifepristone, are compromised as
clinically useful agents due to overt glucocorticoid
receptor (GR) antagonism.5 Therefore, new compounds
with antiprogestational activity devoid of antiglucocor-
ticoid activity are highly desirable for both clinical appli-
cations and basic endocrine research.6 The recent
discovery that treatment of mifepristone can potentially
prevent BRCA1-mediated breast cancer7 makes it more
urgent to discover novel potent and selective PR antag-
onists to address unmet medical needs.
Me
N
Me
N
The discovery of the first PR antagonist, mifepristone
(RU-486),2 has stimulated an intensive search for more
Me
Me
OH
OH
Me
H
Me
H
Me
R
H
H
O
O
O
Keywords: Progesterone; Receptor; Antagonist; oxa-Steroid; Gluco-
corticoid; Parallel synthesis; SAR study; In vitro; In vivo.
mifepristone
oxa-steroids
*
Corresponding author. Tel.: +1 610 458 4147; e-mail:
fkang@prdus.jnj.com
Figure 1. Structures of mifepristone and oxa-steroids.
0960-894X/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.02.013

2532
F.-A. Kang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2531–2534
We have recently achieved the first synthesis and identi-
fication of novel 7-oxa-steroids as promising potent and
selective PR antagonists (Fig. 1).8 Herein we report the
parallel synthesis and SAR study of a series of new oxa-
steroids along this line for the development of potent
and selective PR antagonists.
chiral centers being excellently controlled by the single
chirality of the starting material, the Hajos–Parrish ke-
tone 1.
The mifepristone-like oxa-steroid 4a has an IC50 of
7.5 nM for PR and over 10-fold selectivity over GR.
Although it is less potent than mifepristone, it is slightly
more selective than the equipotent mifepristone (Table
1). A preliminary SAR study at the C17-ethynyl position
with various substituents has led to the discovery of the
remarkably more potent and more selective PR antago-
nist 4b with the phenyl group at the C17-ethynyl
position. Compound 4b has an IC50 of 1.4 nM for PR
and over 200-fold selectivity over GR.8 Apparently,
The oxa-steroids 4 were synthesized from (8S,13S,14R)-
7-oxa-estra-4,9-diene-3,17-dione 2 (Scheme 1),8 which
was prepared from the Hajos–Parrish ketone 1.9 It is
interesting to note that the synthesis of oxa-steroids 4
with five chiral centers in the congested heteropolycyclic
structure was efficiently achieved in a highly stereoselec-
tive fashion, with the stereochemistry of the four new
Me
N
Me
N
O
Me
Me
O
Me
O
Me
H
OH
Me
Me
H
c
a
b
H
O
R
H
O
H
O
H
O
O
O
O
O
OH
1
2
3
4
Scheme 1. (a) Ref. 9; (b) Ref. 8; (c) parallel synthesis of oxa-steroids 4 via the one-pot four-step sequence. Reagents and conditions: (1) RCCH,
LiHMDS, THF, rt, 15 min; (2) compound 3, THF, rt, 4 h; (3) 3 N HCl aq, acetone, rt, 4 h, 60–80%.
Table 1. SAR study of oxa-steroids 4
Me
N
Me
OH
Me
R
H
H
O
O
Compound
R
T47D (PR), IC50 (nM)
A549 (GR), IC50 (nM)
Selectivity (GR/PR)
Mifepristone
—
Methyl
Phenyl
1.4
7.5
1.6
86.5
304.0
65.1
64.8
62.7
113.2
51.0
56.6
48.9
36.3
45.4
37.6
43.5
31.3
22.1
111.0
80.3
41.3
65.8
34.4
175.3
341.6
246.5
56.0
1
4a
4b
4c
4d
4e
4f
12
217
47
59
7
1.4
1.4
2-F–phenyl
2-Cl–phenyl
2-Br–phenyl
2-Me–phenyl
2-F3C–phenyl
3-F–phenyl
3-Cl–phenyl
3-Me–phenyl
3-F3C–phenyl
4-F–phenyl
4-Cl–phenyl
4-Br–phenyl
4-Me–phenyl
4-F3C–phenyl
4-MeO–phenyl
4-NC–phenyl
4-Me3C–phenyl
3,5-di-F–phenyl
2-Pyridyl
1.1
8.6
5.8
3.2
20
16
57
4
4g
4h
4i
1.0
12.5
2.9
4j
13
17
139
71
33
14
148
25
11
3
4k
4l
2.7
0.27
0.61
0.94
1.6
4m
4n
4o
4p
4q
4r
4s
4t
0.75
3.2
3.7
19.5
0.91
34.0
21.0
39.0
1.6
38
5
4u
4v
4w
4x
3-Pyridyl
4-Pyridyl
3-Thienyl
16
6
35

F.-A. Kang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2531–2534
2533
compound 4b is a new PR antagonist that is as potent
as, but much more selective than, mifepristone. There-
fore, it became the new lead for further SAR study.
For a focused SAR study, we were interested in fine-tun-
ing the substitution pattern at the C17-ethynyl position
of these oxa-steroids by installing various substituted
phenyl groups and heterocycles, while keeping the rest
of the molecule the same as that of mifepristone.
with subnanomolar potency (0.27–0.94 nM). The F
and CF3 substituents also lead to about 140-fold selec-
tivity for PR over GR. Therefore, compounds 4l and
4p are promising potent and selective PR antagonists
identified in the oxa-steroid series after compound 4b.
In addition, the bulky tert-butyl group causes more than
10-fold reduction in PR potency suggesting a size limita-
tion at this position.
The oxa-steroids 4 were originally synthesized in two
steps from compound 3, including stereoselective Grig-
nard addition and subsequent deprotection and dehy-
dration.8 For a more efficient parallel synthesis of oxa-
steroids 4, we developed a one-pot four-step sequence
from compound 3, which includes in situ generation of
arylethynyl lithium, stereoselective addition to the
C17-carbonyl group, deprotection of the C3-ketal
group, and dehydration of the C5-hydroxy group. Thus,
new oxa-steroids 4c–4x were efficiently synthesized
through this one-pot reaction in 60–80% yields after pre-
parative TLC purification.10
The PR and GR activities of oxa-steroids 4t–4x with
disubstituted phenyl and heterocycles at the C17-ethynyl
position are listed in Table 1. It appears that 2-, 3-, and 4-
pyridyl groups result in up to 20-fold loss of PR potency,
while 3,5-difluorophenyl and 3-thienyl groups exhibit
similar potency but less selectivity for PR versus GR.
On the basis of the SAR of the minor modifications at
the C17-ethynyl position, the trend is that small substit-
uents such as F, Cl, Me, and CF3 at the ortho-, meta-,
and para-positions of the phenyl group retain or in-
crease PR potency, compared to the unsubstituted phen-
yl group in compound 4b. Interestingly, the small and
electron-withdrawing substituents, such as F and CF3,
on the para-substituted phenyl groups not only enhance
PR potency, but retain good selectivity over GR.
Compounds 4c–4x were evaluated for PR antagonist
activity based on their ability to block progesterone
induction of alkaline phosphatase activity in the human
breast cancer cell line T47D. They were also tested for
GR antagonist activity based on their ability to inhibit
corticoid-induced transcription from a glucocorticoid re-
sponse element (GRE)-linked luciferase reporter gene in
the human lung carcinoma cell line A549. The IC50 values
of the compounds from the T47D and A549 assays are
listed in Table 1. The ratio of their IC50 values was calcu-
lated as a measure of the separation of PR and GR antag-
onist activities. Mifepristone was tested as a control.
The possible binding modes of compound 4a in the li-
gand-binding domain of PR suggested by molecular mod-
eling are shown in Figure 2. The model was built based on
the X-ray crystal structures of hPR-norenthindrone and
hGR-mifepristone complexes.8 The molecular modeling
suggests that there is an open space available around the
C17-ethynylmethyl group, which is consistent with our
SAR study. Therefore, bigger groups, such as phenyls
and heterocycles in compounds 4b–4x, can be accommo-
dated at this binding site. Compared to the methyl groups
at the C17-ethynyl position of mifepristone and com-
pound 4a with up to about 10-fold selectivity toward
The PR and GR activities of oxa-steroids 4c–4g with
ortho-substituted phenyl groups at the C17-ethynyl posi-
tion are listed in Table 1. It seems that PR activity toler-
ates the ortho-substitution with F, Cl, Br, Me, and CF3
well, which results in potent PR antagonists with IC50s
ranging from 1 to 10 nM. However, the ortho-substitution
with these substituents also gives rise to relatively potent
GR antagonists, with only a small separation between
PR and GR. Compared to the phenyl group of compound
4b, the small substituents on the phenyl group such as F
and Cl retain PR potency (1–2 nM) and moderate selec-
tivity over GR (about 50-fold selectivity).
The PR and GR activities of oxa-steroids 4h–4k with
meta-substituted phenyl groups at the C17-ethynyl posi-
tion are listed in Table 1. Similar to the ortho-substitution,
the meta-substitution with F, Cl, Me, and CF3 leads to po-
tent but less selective PR antagonists. Interestingly, the F
substituent makes the oxa-steroid slightly more potent
and selective than the Cl substituent, with an IC50 of
1.0 nM for PR and over 50-fold selectivity over GR.
The PR and GR activities of oxa-steroids 4l–4s with
para-substituted phenyl groups at the C17-ethynyl posi-
tion are listed in Table 1. One half of these para-substi-
tuted phenyl groups make the oxa-steroids even more
potent than mifepristone and compound 4b. The F,
Cl, Br, and CF3 substituents result in PR antagonists
Figure 2. Molecular modeling of compound 4a bound to PR.

2534
F.-A. Kang et al. / Bioorg. Med. Chem. Lett. 17 (2007) 2531–2534
PR over GR, the phenyl groups in compounds 4b, 4l, and
4p with up to about 200-fold selectivity for PR over GR
should play an important role in increasing PR potency
and simultaneously decreasing GR potency. In contrast
to the equipotent mifepristone, this potency and selectiv-
ity profile might result from the unique combination of
ligand–protein interactions between the O7-oxygen atom
and the nearby MET756, VAL760, LEU887, and
MET801, as well as those between the C17-ethynylphenyl
group and the phenyl groups of the nearby TYR890 and
PHE794.
References and notes
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V. J.; Yen, S. S. J. Clin. Endocrinol. Metab. 1993, 76, 513;
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S. Fertil. Steril. 1995, 64, 187; (c) Kettel, L. M.; Murphy,
A. A.; Morales, A. J.; Yen, S. S. Am. J. Obstet. Gynecol.
1998, 178, 1151; (d) Kettel, L. M.; Murphy, A. A.;
Morales, A. J.; Yen, S. S. Hum. Reprod. 1994, 9 (Suppl. 1),
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2. Brogden, R. N.; Goa, K. L.; Faulds, D. Drugs 1993, 45,
384.
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2005, 26, 423; (c) Chabbert-Buffet, N.; Meduri, G.;
Bouchaard, P.; Spitz, I. M. Hum. Reprod. Update
2005, 11, 293.
4. (a) Allan, G. F.; Sui, Z. Mini-Rev. Med. Chem. 2005, 5,
701; (b) Zhang, P.; Fensome, A.; Wrobel, J.; Winneker,
R.; Zhang, Z. Expert Opin. 2003, 13, 1839.
5. Lazar, G.; Lazar, G., Jr.; Husztik, E.; Duda, E.; Agarwal,
K. K. Ann. NY Acad. Sci. 1995, 277, 248.
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Lee, E. Y.-H. P. Science 2006, 314, 1467; (b) Marx, J.
Science 2006, 314, 1370.
8. Kang, F.-A.; Allan, G.; Guan, J.; Jain, N.; Linton, O.;
Tannenbaum, P.; Xu, J.; Zhu, P.; Gunnet, J.; Chen, X.;
Demarest, K.; Lundeen, S.; Sui, Z. Bioorg. Med. Chem.
Lett. 2007, 17, 907.
To investigate the in vivo profile of this novel series of
oxa-steroids, compounds 4a and 4b were tested orally in
ovariectomized Sprague–Dawley rats in a rat uterine
complement C3 assay.11 In this assay, ethynyl estradiol
(EE) is used to stimulate C3 expression. Progestins inhibit
EE-induced expression. In turn, antiprogestins counter-
act progestin-dependent inhibition. When compounds
4a and 4b were administered via the oral route along with
EE and progesterone, they were found to be more
efficacious than mifepristone, although they were less
than or equally potent to mifepristone in vitro. This out-
come demonstrated that these oxa-steroids, particularly
the more potent ones with the relatively large phenyl
groups at the C17-ethynylmethyl position, still have good
oral exposure from the pharmacokinetic perspective.
In summary, efficient parallel synthesis via a one-pot four-
step sequence led to a series of novel oxa-steroids 4. SAR
study on the C17-ethynyl position with various phenyl
groups and heterocycles resulted in new PR antagonists
4l and 4p that are more potent and more selective than
mifepristone. Molecular modeling of the oxa-steroid in
PR provided meaningful insight for the SAR study.
oxa-Steroids 4a and 4b were found to be more efficacious
than mifepristone in vivo in a rat uterine complement C3
assay via the oral route, although they were less than or
equally potent to mifepristone in vitro. Our study of 7-
oxa-steroids as potent and selective PR antagonists dem-
onstrated that the unnatural 7-oxa-steroids not only
excellently mimic the natural steroids in terms of shape
and activity, but also exhibit significant differentiation
from similar steroid receptors and excellent in vivo
efficacy. Further profiling of selected compounds in this
series will be reported in due course.
9. Kang, F.-A.; Jain, N.; Sui, Z. Tetrahedron Lett. 2007, 48,
193.
10. General procedure for the one-pot parallel synthesis of
oxa-steroids 4: 1 M LiHMDS in THF (48.6 lL,
0.048 mmol) was added to the stirred solution of the aryl
alkyns (0.06 mmol) in THF (0.5 mL) at room temperature.
After 15 min, compound 3 (5.5 mg, 0.012 mmol) in THF
(0.5 mL) was added to the reaction mixture. After 4 h,
acetone (1.0 mL) and 3 M HCl (0.15 mL) were added to
the reaction mixture. After 4 h, sat. NaHCO3 aq solution
(5 mL) and EtOAc (5 mL) were added to the reaction
mixture. The aqueous layer was extracted with EtOAc (2·
5 mL) and the combined organic layer was dried with
brine and MgSO4. Purification by preparative TLC eluted
with 50–70% EtOAc in hexane gave compounds 4 in 60–
80% yields.
11. Lundeen,S.G.;Zhang,Z.;Zhu,Y.;Carver,J.M.;Winneker,
R. C. J. Steroid Biochem. Mol. Biol. 2001, 78, 137.