Discovery of potent, nonsteroidal, and highly selective glucocorticoid receptor antagonists

Article abstract of DOI:10.1021/jm0105530

An approach to the computer-assisted, pharmacophore design of nonsteroidal templates for the glucocorticoid receptor (GR) that contained an element of pseudo-C2 symmetry was developed. The enatiomer of the initial design, 1Ra, and not the designed molecul

Full text of DOI:10.1021/jm0105530

J . Med. Chem. 2002, 45, 2417-2424
2417
Discover y of P oten t, Non ster oid a l, a n d High ly Selective Glu cocor ticoid
Recep tor An ta gon ists
Bradley P. Morgan,* Andrew G. Swick, Diane M. Hargrove, J anet A. LaFlamme, Melinda S. Moynihan,
Richard S. Carroll, Kelly A. Martin, Eunsun Lee, Debra Decosta, and J on Bordner
Pfizer Global Research and Development, Pfizer Inc., Eastern Point Road, Groton, Connecticut 06371
Received December 5, 2001
An approach to the computer-assisted, pharmacophore design of nonsteroidal templates for
the glucocorticoid receptor (GR) that contained an element of pseudo-C2 symmetry was
developed. The enatiomer of the initial design, 1Ra , and not the designed molecule, 1S, showed
the desired ligand binding to the GR. The pseudo-C2 symmetry of the template allowed for
rapid improvements in GR activity resulting in potent, selective, nonsteroidal GR antagonists,
CP -394531 and CP -409069.
In tr od u ction
Glucocorticoid receptors (GRs), members of the ster-
oid-thyroid-retinoid superfamily, are soluble, intra-
cellular receptor proteins that act as ligand-regulated
transcription factors controlling specific gene expression
in most mammalian cells.1-7 Glucocorticoids bind to and
activate GRs, and they play a crucial role in the normal
development and maintenance of basal and stress-
related homeostasis.8 The steroid nucleus of typical
glucocorticoids is associated with a rich variety of
hormonal activities and has inspired the development
of an assortment of therapeutic agents.9,10 Steroid
F igu r e 1. RU-486 and RU-43044.
hormones have evolved in nature to be short acting;
therefore, the design of therapeutic agents based on the
the template, and this concept led to rapid improve-
ments in receptor-binding affinity using an analogous
design strategy. This communication reports the design,
synthesis, and evaluation of potent, selective, non-
steroidal GR antagonists, CP -394531 and CP -409069.
steroid skeleton can be plagued with several problems,
including rapid clearance. Nonsteroidal ligands11 have
been developed for the estrogen (ER),12-16 progesterone
(PR),17-20 and androgen receptor (AR).21,22 However,
there are relatively few literature reports of selective,
nonsteroidal GR agonists or antagonists.23 Our efforts
have focused on the rational design of novel, potent, and
selective nonsteroidal GR antagonists for the possible
Mod elin g
A three-dimensional pharmacophore model, based on
steroidal antagonists RU-43044 (selective) and RU-486
(nonselective),44-48 was developed for the design of
selective nonsteroidal GR ligands (Figures 1 and 2). The
initial tricyclic compounds (1S) dramatically simplified
the steroid skeleton, kept the A and B rings and the
angular benzyl pharmocophore of the steroid intact, and
positioned a vector from the phenoxyl moiety of the
C-ring that could occupy aspace similar to that of the
third pharmacophore of interest (Figure 3). For most
steroidal ligands, an A-ring is important for potent and
specific binding to the receptor. A second pharmaco-
phore, the C19-angular benzyl substituent of RU-43044,
is important for GR selectivity and antagonism. From
steroidal SAR,49 the third pharmacophore, the alkyne
on the D-ring of RU-43044, appears to be key for tight-
binding compounds, because large differences in potency
can be observed with relatively subtle changes in
structure. Because the A-ring and the C19-angular
benzyl substituent are important for GR binding, an-
tagonism, and selectivity, the initial design (1S) focused
on positioning these two pharmacophores in the proper
treatment of
a
variety of disorders, including
diabetes,24-27 obesity,28,29 depression,30-33 neurodegenera-
tion,34-37 glaucoma,38,39 and Cushing’s disease.40,41
A potentially general method to simplify the design
of ligands for steroid receptors may be to begin with
templates that contain a C2 or pseudo-C2 symmetric
axis. For the estrogen receptor, pseudo-C2 symmetry
may play a role in the binding orientation of the chosen
ligands.42,43 We recently developed an approach to
pharmacophore design for nonsteroidal ligands for the
GR that contained an element of pseudo-C2 symmetry.
To control the possible involvement of this symmetry
element, we prepared not only the “correct” enantiomer
of our designed template 1S but also its enantiomer 1R.
Interestingly, 1Rb (Ki ) 157 nM) and not 1S (Ki > 10
µM) displayed the desired ligand binding to the GR. This
result was rationalized by the pseudo-C2 symmetry of
* Author to whom correspondence should be addressed. Current
address: Cytokinetics, Inc., 280 E. Grand Ave., South San Francisco,
CA 94080. Tel: (650) 624-3003. Fax: (650) 624-3010. E-mail:
bmorgan@cytokinetics.com.
10.1021/jm0105530 CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/10/2002

2418 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Morgan et al.
1Sb and 1Rb in 87 and 75% yields, respectively.58
Compound 1Rb was treated with lithium metal in liquid
ammonia in dioxane and ether at -78 °C to form the
trans Decalin 4 in 81% yield after purification.59 Addi-
tion of either lithium chloroethyne and lithium propyne
to 4 afforded CP -394531 or CP -409069 in 56 and 69%
yields, respectively. The relative and absolute stereo-
chemistry of CP -394531 was confirmed by X-ray crys-
tallography.60
Biology
The human GR was overexpressed from baculovirus
cells and isolated as a protein extract. Binding affinity
to the human GR was determined by the displacement
of 10 nM radiolabeled dexamethasone, a known GR
agonist, by the test compounds. To assess binding
selectivity, the binding affinity versus human PR, ERR,
ERâ, and AR was determined by the displacement of
the appropriate radiolabeled ligand by the test com-
pounds (Table 1). Compounds 4, CP -395531, and CP -
409069 displayed very potent and selective binding to
the GR over the other steroid receptors that were
assayed.
F igu r e 2. Overlap of RU-486 (yellow) and RU-43044 (red);
the differences in the angular benzyl substituent of RU-43044
and the angular phenyl substituent of RU-486 are believed
to impart GR selectivity to RU-43044.44-48
To assess agonism versus antagonism and the func-
tional selectivity of GRs versus other closely related
steroid receptors (e.g., PR and MR), human cell lines
containing endogenous steroid receptors (SW1352-GR,
T47D-GR, MR, and PR) were identified and character-
ized. Glucocorticoid receptor antagonist/agonist activity
was determined in SW1353/MMTV-8 cells in the pres-
ence and absence of a half-maximal concentration of
dexamethasone (40 nM). Selectivity for the GR versus
the MR and PR was performed in transiently trans-
fected T47D cells. In all cases, the compounds were
tested alone for agonist activity and in the presence of
the appropriate ligand for antagonist activity. None of
the new, reported compounds displayed functional ago-
nism above basal levels at concentrations up to 1 µM
at the GR, MR, or PR under the assay conditions. CP -
409069 and CP -394531 completely blocked (100%) the
half-maximal agonistic effects of dexamethasone (40
nM) at concentrations g1 µM. Antagonism data for the
GR, MR, and PR are reported in Table 2. CP -395531
and CP -409069 displayed very potent, complete, and
selective antagonism to the GR over the other steroid
receptors that were assayed.
F igu r e 3. Overlap of RU-43044 (red) and 1Sa (green).
binding orientation. The third pharmacophore, the
substituent attached to the D-ring, could then be used
to increase the GR receptor affinity. This third phar-
macophore could easily be varied by alkylation of the
C-ring phenoxide. Finally, both enantiomers of 1 (R and
S) were synthesized so that 1R could serve as a control
for the testing of 1S which possesses the natural steroid
configuration in the A- and B-rings.
Ch em istr y
Discu ssion
Alkylation of the eneamine of 6-methoxy-2-tetralone50
with benzyl bromide followed by hydrolysis and purifi-
cation resulted in an 85% yield of 1-benzyl-6-methoxy-
2-tetralone (2, Scheme 1).51 Treatment of 2 with either
(R)-(+)-R-methylbenylamine or (S)-(-)-R-methylbenzyl-
amine with the azeotropic removal of water followed by
treatment with methyl vinyl ketone and hydrolysis
resulted in the bridged bicyclic ketols 3S and 3R,
respectively. Treatment of 3S and 3R with sodium
methoxide in methanol resulted in the initial targets of
1Sa and 1Ra with overall yields of 48 and 33%,
respectively.52-56 Compounds 1Sa and 1Ra were ob-
tained in >98% ee after a single recrystallization.57
Cleavage of the methyl ethers was accomplished by the
treatment of 1Sa and 1Ra with tetrabutylammonium
iodide and boron trichloride in dichloromethane to form
The enantiomeric phenol 1Rb (Ki ) 157 nM), included
as a control, and not 1Sa (Ki > 10 µM) displayed the
desired ligand binding to the GR (Table 1). Stereoiso-
mers 1S and 1R contain a pseudo-C2 symmetric axis.
That is, 1Rb may bind to the receptor with the phenol
acting as the A-ring. From this orientation, the angular
benzyl substituent can still occupy a space similar to
that envisioned for it in the 3-D pharmacophore model
(Figure 4). Addition of the third pharmacophore, in-
tended to improve the GR potency, was now planned
from a different site. By molecular modeling, an axial
propyne moiety on the C-ring occupies a space similar
to that of the propyne of RU-43044 or RU-486. More-
over, an equatorial hydroxyl could participate in hydro-
gen-bonding interactions comparable to that of the C17
hydroxyl of RU-43044 or RU-486 (Figure 5). Reduction

Glucocorticoid Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2419
Sch em e 1a
a
Reagents: (a) (i) pyrrolidine, toluene, Dean-Stark trap, reflux; (ii) benzyl bromide, dioxane, reflux; (iii) H2O. (b) (i) (R)-(+)-R-
Methylbenzylamine, methyl vinyl ketone, toluene, 40 °C; (ii) acetic acid. (c) (i) (S)-(-)-R-Methylbenzylamine, methyl vinyl ketone, toluene,
40 °C; (ii) acetic acid. (d) NaOMe, MeOH, 70 °C; (e) BCl3, tetrabutylammonium iodide, CH2Cl2, -78 °C to rt. (f) Li, NH3, dioxane/ether,
-78 °C. (g) Propyne, LDA, THF. (h) cis-1,2-Dichloroethene, LDA, THF.
Ta ble 1. Binding Affinities versus Human GR, PR, AR, ERR, and ERâa
compound
GR
PR
AR
ERR
ERâ
RU-486
RU-43044
1Sa
1Sb
1Ra
1Rb
4
CP -394531
CP -409069
0.24 ( 0.02
0.10 ( 0.01
15 ( 2.6
30% at 10 µM
>10000
>10000
>10000
>10000
>10000
>10000
>10000
4.6 ( 0.41
90 ( 7.8
54 ( 1.1
44% at 10 µM
>10000b
92% at 10 µM
>10000
>10000
>10000
>10000
>10000
>10000
>10000
36% at 10 µM
27% at 10 µM
70% at 10 µM
45% at 10 µM
62% at 10 µM
50% at 10 µM
24% at 10 µM
>10000
39% at 10 µM
54% at 10 µM
75% at 10 µM
57% at 10 µM
690 ( 42
180 ( 42
157 ( 13
5.96 ( 0.49
0.10 ( 0.02
0.17 ( 0.02
1300 ( 220
130 ( 26
650 ( 140
a
(
b
Reported as Ki’s SEM (n ) 3) in nM or % inhibition at dose. >10000 ) e20% inhibition at 10 µM.
Ta ble 2. Functional Antagonism versus Human GR, MR, and
PRa
compound
GR
2.0 ( 0.7
MR
PR
RU-486
<10
11.8 ( 3.5
(100% at 1 µM)
(100% at 1 µM)
(69.7 ( 3.6%
at 10 nM)
RU-43044 12 ( 0.8
(100% at 1 µM)
>1000b
>1000
1Sa
1Sb
1Ra
1Rb
4
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
>1000
(35% at 1 µM)
CP -394531 4.1 ( 0.8
(100% at 1 µM)
CP -409069 10 ( 0.7
(100% at 1 µM)
>1000
>1000
a
Reported as Kif ( SEM (n ) 3) in nM (% inhibition at dose) of
a half-maximal dexamethasone response.69 >1000 ) no inhibi-
tion at 1 µM.
b
F igu r e 4. Computer-assisted designs.
of 1Rb to 4 followed by a nucleophilic attack on the
C-ring carbonyl of 4 with lithium propyne or lithium
chloroethyne from the face opposite the angular benzyl
moiety afforded CP -409069 and CP -394531. This posi-
tions the third phamacophore in the appropriate relative
orientation. As anticipated, the addition of this phar-
macophore provided a dramatic increase in GR binding
and antagonistic potency with exquisite selectivity. The
GR binding potency of CP -409069 and CP -394531
increased approximately 1000-fold over the original lead
(1Rb). CP -409069 and CP -394531 have GR binding
and antagonism potencies similar to those of RU-486
and selectivities similar to that of RU-43044 (Tables 1
and 2).
The recognition of the pseudo-C2 symmetric axis
helped to refine the 3-D pharmacophore model. The fact

2420 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Morgan et al.
rected. Nuclear magnetic resonance spectra (1H-NMR at 300
or 400 MHz and 13C-NMR at 75 MHz) were run using the
residual solvent as an internal standard. Mass spectra deter-
minations were performed by the Exploratory Medicinal
Sciences Department of Pfizer Global Research and Develop-
ment at Groton, CT. Elemental analyses were performed by
Schwarzkopf Microanalytical Laboratory, Inc., Woodside, NY.
Optical rotations were measured at 20 °C in MeOH. Analytical
thin-layer chromatography was done on Kieselgel 60 F-254
plates precoated with 0.25 mm thick silica gel distributed by
E. Merck. Column chromatography was performed on silica
gel (Kieselgel 60, 230-400 mesh) from E. Merck. Reagents
were purchased and used without further purification unless
otherwise stated. Both (S)-(-)-R-methylbenzylamine and (R)-
(+)-R-methylbenzylamine were freshly distilled from CaH at
atmospheric pressure under nitrogen directly prior to use.
Methyl vinyl ketone was placed over solid K2CO3 for 30 min,
decanted, and distilled at 200 mmHg pressure. The colorless
heart of the distillate was collected (40 °C) and used directly
in the reaction. Human PR, ERR, and ERâ binding affinities
were performed by Cerep (Rueil-Malmaison, France;
www.cerep.com).
F igu r e 5. Overlap of RU-43044 (red) and CP -409069 (blue).
that 1S did not bind to the GR at concentrations less
than 10 µM and that 1Rb had modest binding affinity
to the GR (Ki ) 157 nM) suggested that the nature of
the C-ring of the tricyclic skeleton has a much greater
effect on binding potency than we had initially antici-
pated. Saturation of the C-ring double bond of 1Rb to
the trans Decalin 4 increased GR binding potency by
greater than an order of magnitude. Furthermore, 4
began to demonstrate antagonism in the GR whole-cell
luciferase assay (35% block of a half-maximal dexa-
methasone activity, Table 2). This activity seemed to
be selective for the GR versus the closely related MR
and PR, and 4, like CP -394531 and CP -409069, did not
display any GR, MR, or PR agonist activity. These
results suggest that there are significant hydrophobic
interactions between the saturated C-ring of our tricyclic
template and the GR. Retrospectively, this result may
be explained by at least two potential interactions with
the GR. First, the aforementioned hydrophobic inter-
action of the saturated C-ring of 4, CP -394531, and CP -
409069 may help to mimic the hydrophobic interaction
of the C-D ring and its angular methyl group of the
steroids RU-486 and RU-43044. Second, the equatorial
hydroxyl in the C-ring of CP -394531 and CP -409069
may fit into a similar hydrogen-bonding network with
the GR as the hydroxyl moiety in the D-ring of RU-486
and RU-43044 (Figures 4 and 5).
1-Be n zyl-6-m e t h oxy-3,4-d ih yd r o-1H -n a p h t h a le n -2-
on e [247936-61-4] (2).51 A solution of 51 g (0.289 mol) of
6-methoxy-2-tetralone and 24.2 mL (0.289 mol) of pyrrolidine
in 1.5 L of toluene was heated to reflux under a Dean-Stark
trap overnight. After removal of the azeotroped water, the
reaction mixture was cooled to rt, concentrated to an oil, and
dissolved in 725 mL of dioxane. To this solution was added 52
mL (0.434 mol) of benzyl bromide, and the resulting solution
was heated to reflux overnight. Water (100 mL) was added to
the solution, and heating to reflux of the resultant mixture
was continued for an additional 2 h. The mixture was cooled
to rt, poured into a solution of 1 N HCl, and extracted 3 times
with EtOAc. The organic layers were washed with H2O and
saturated NaHCO3 and then dried over Na2SO4, filtered, and
evaporated to dryness. The crude product was purified by flash
chromatography over SiO2 using 10-15% EtOAc in hexanes
as the gradient eluant to give 65.2 g of 2 as a yellow oil (85%):
1
IR (neat) 2937, 1712, 1500 cm-1; H-NMR (400 MHz, CDCl3,
δ) 2.41-2.59 (m, 3H), 2.76 (dt, 1H, J ) 5.4, 15.5 Hz), 3.15-
3.70 (m, 2H), 3.67 (t, 1H, J ) 6.3 Hz), 3.77 (s, 3H), 6.67-6.70
(m, 2H), 6.81 (d, 1H, J ) 8.1 Hz), 6.87-6.89 (m, 2H), 7.13-
7.17 (m, 3H); 13C-NMR (100 MHz, CDCl3, δ) 27.44, 38.19,
39.19, 54.13, 55.14, 112.11, 112.96, 126.30, 128.07, 128.26,
129.35, 129.53, 138.05, 138.20, 158.30, 212.41; MS m/z 267 (M
+ H)+.
1(S)-Ben zyl-6-m eth oxy-1(R)-(3-oxo-bu tyl)-3,4-d ih yd r o-
1H-n a p h th elen -2-on e (3R). A solution of 62 g (0.23 mol) of
1-benzyl-6-methoxy-3,4-dihydro-1H-naphthalen-2-one (2) and
28 mL (0.23 mol) of freshly distilled (S)-(-)-R-methylbenzyl-
amine in 100 mL of toluene was heated to reflux under a Dean-
Stark trap overnight. After removal of the azeotroped water,
the imine solution was cooled to 0 °C, and 21 mL (0.26 mol) of
freshly distilled methyl vinyl ketone was added dropwise to
the solution. The solution was stirred at 0 °C for 30 min and
then heated to 40 °C overnight. The reaction solution was
cooled to 0 °C. Acetic acid (17 mL) and H2O (14 mL) were
added, and the resultant solution was allowed to warm to rt
for 2 h. The solution was poured into H2O and extracted 3
times with EtOAc. The combined organic layers were washed
with 1 N HCl, H2O, and saturated NaHCO3 and then dried
over Na2SO4, filtered, and evaporated to dryness. The crude
product was purified by chromatography over SiO2 using 15-
35% EtOAc in hexanes as the gradient eluant to give 48 g of
3R as a yellow solid: 1H-NMR (400 MHz, CDCl3, δ) 1.38 (s,
3H), 1.40-1.51 (m, 2H), 1.64 (ddd, 1H, J ) 2.1, 4.5, 13 Hz),
1.97 (broad s, 1H), 2.20 (dt, 1H, J ) 4.5, 13 Hz), 2.59 (d, 1H,
J ) 6.6 Hz), 3.08 (d, 1H, J ) 18 Hz), 3.16 (d, 1H, J ) 16 Hz),
3.33 (dd, 1H, J ) 6.6, 18 Hz), 3.62 (d, 1H, J ) 16 Hz), 3.72 (s,
3H), 6.57 (d, 1H, J ) 2.5 Hz), 6.67 (dd, 1H, J ) 2.5, 8.8 Hz),
7.00-7.23 (m, 6H); 13C-NMR (100 MHz, CDCl3, δ) 27.90, 32.79,
34.40, 38.43, 41.49, 53.51, 55.12, 58.47, 79.06, 112.05, 113.09,
Con clu sion
In summary, a 3-D pharmacophore model for the
design of selective GR antagonists that do not contain
the steroid nucleus has been developed. The resultant
templates contained a pseudo C2-symmetric axis that
incorporated two of the three pharmacophores impor-
tant for GR selectivity and functional handles to append
the third pharmacophore to increase GR potency. This
model assisted in the rapid design of CP -394531 and
CP -409069, potent, selective, nonsteroidal GR antago-
nists. Additional in vitro and in vivo studies with CP -
394531, CP -409069, and related molecules are ongoing
and will be presented in due course.61
Exp er im en ta l P r oced u r es
Gen er a l Meth od s. Molecular modeling was performed on
a Silicon Graphics workstation using SYBYL version 6.2
(Tripos, Inc., St. Louis, MO) with the TRIPOS force field.
Molecular overlaps were executed manually. Melting points
were determined with a capillary apparatus and are uncor-

Glucocorticoid Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2421
125.37, 127.63, 127.69, 130.27, 132.21, 135.45, 138.65, 157.88,
was used to form compound 1Sb. Starting with 1.8 g of 4a-
(R)-benzyl-7-methoxy-4,4a,9,10-tetrahydro-3H-phenanthren-
2-one (1Sa ) produced 1.3 g of 1Sb as a white solid (75%). All
physical constants are the same as reported for 1Sb except
optical rotation, [R]20D +213° (c 0.0114, MeOH). Anal. (C21H20O2)
C, H, N.
213.49; MS m/z 337 (M + H)+, 319 (M - OH)+.
1(R)-Ben zyl-6-m eth oxy-1(R)-(3-oxo-bu tyl)-3,4-d ih yd r o-
1H-n a p h th elen -2-on e (3S). The compound 3S was formed
by the same procedure as 1(S)-benzyl-6-methoxy-1(R)-(3-oxo-
butyl)-3,4-dihydro-1H-naphthelen-2-one (3R) using (R)-(+)-R-
methylbenzylamine in the initial imine formation. Starting
with 4.64 g 1-benzyl-6-methoxy-3,4-dihydro-1H-naphthalen-
2-one (2) produced 3.58 g of 3S as a yellow solid. All physical
constants are the same as reported for 3R.
4a (S)-Ben zyl-7-h yd r oxy-3,4,4a ,9,10,10a (R)-h exa h yd r o-
1H-p h en a n th r en -2-on e (4). Ammonia (1.5 L) was condensed
into a round-bottom flask equipped with a dry-ice reflux
condenser at -78 °C and a mechanical stirrer. To this flask
was added 0.7 g (99 mmol) of lithium wire, and the solution
turned dark blue. A solution of 10 g (32.8 mmol) of 4a(S)-
benzyl-7-hydroxy-4,4a,9,10-tetrahydro-3H-phenanthren-2-
one (1Rb) in 400 mL of 1:1 dioxane/ether was added to the
mixture slowly in order to keep the mixture a dark blue. As
the blue color dissipated, a small amount of lithium wire was
added to the mixture to regenerate the blue color. The total
amount of lithium added to the reaction mixture did not exceed
3.5 g (495 mmol). After the complete addition of 1Rb, the
reaction mixture was stirred for an additional 30 min, then
14 g of solid ammonium chloride was added, and an immediate
dissipation of the blue color was observed. H2O was added to
the mixture, and it was extracted with EtOAc, dried over Na2-
SO4, filtered, and concentrated to dryness. The crude product
was purified by flash chromatography over SiO2 using 15-
20% EtOAc in hexanes as the gradient eluant to afford 8.16 g
of 4 as a white solid (81%): 1H-NMR (400 MHz, CD3OD, δ)
1.52 (dt, 1H, J ) 4.5, 13 Hz), 1.64-1.71 (m, 1H), 1.90-2.15
(m, 2H), 2.27 (ddd, 1H, J ) 2.5, 3.7, 15 Hz), 2.39 (dm, 1H, J )
15 Hz), 2.48 (ddd, 1H, J ) 2.0, 6.5, 13 Hz), 2.72 (t, 1H, J ) 14
Hz), 2.84 (d, 1H, J ) 13 Hz), 2.89-3.01 (m, 3H), 3.22 (d, 1H,
J ) 13 Hz), 6.17 (d, 1H, J ) 8.5 Hz), 6.24 (dd, 1H, J ) 2.5, 8.5
Hz), 6.53 (d, 1H, J ) 2.5 Hz), 6.65-6.68 (m, 1H), 7.04-7.13,
(m, 3H); 13C-NMR (100 MHz, CD3OD, δ) 27.9, 33.7, 34.8, 36.0,
37.6, 39.4, 43.6, 44.0, 111.3, 114.6, 125.7, 127.0, 127.9, 130.5,
4a (S)-Ben zyl-7-m eth oxy-4,4a ,9,10-tetr a h yd r o-3H-p h en -
a n th r en -2-on e (1Ra ). A solution of 48 g (143 mmol) of 1(S)-
benzyl-6-methoxy-1(R)-(3-oxo-butyl)-3,4-dihydro-1H-naphthelen-
2-one (3R) and 71 mL of 1 M sodium methoxide in 100 mL of
methanol was stirred at rt for 15 min and then heated to 75
°C for 3 h. The solution was cooled to 0 °C, treated dropwise
with 8.2 mL of acetic acid, and concentrated to an oil. The oil
was dissolved in EtOAc, washed with saturated NaHCO3 and
brine, dried over Na2SO4, filtered, and evaporated to dryness.
The crude product was purified by chromatography over SiO2
using 15-35% EtOAc in hexanes as the gradient eluant to give
44 g of 1Ra as an off-white powder. Recrystallization from
EtOAc/hexane afforded 35 g of 1Ra as a white crystalline solid
(48% from 1-benzyl-6-methoxy-3,4-dihydro-1H-naphthalen-2-
one, 2): mp 101-102 °C; IR (neat) 1667, 1500 cm-1; 1H-NMR
(300 MHz, CDCl3, δ) 1.83-1.90 (m, 1H), 2.02 (dt, 1H, J ) 5.5,
14 Hz), 2.27 (dt, 1H, J ) 4.3, 14 Hz), 2.44-2.51 (m, 2H), 2.64-
2.79 (m, 3H), 3.14 (d, 1H, J ) 13 Hz), 3.21 (d, 1H, J ) 13 Hz),
3.78 (s, 3H), 5.96 (s, 1H), 6.54 (d, 1H, J ) 2.6 Hz), 6.71 (d, 2H,
J ) 7.1 Hz), 6.77 (dd, 1H, J ) 2.6, 8.7 Hz), 7.06-7.23 (m, 4H);
13C-NMR (100 MHz, CDCl3, δ) 30.71, 32.10, 34.62, 36.09, 43.62,
46.36, 55.20, 112.78, 112.84, 125.53, 126.68, 127.96, 128.12,
130.08, 133.01, 137.24, 137.28, 157.75, 169.16, 198.81; MS m/z
319 (M + H)+; [R]20D -220° (c 0.0102, MeOH). Anal. (C22H22O2)
C, H, N.
133.4, 136.8, 138.0, 155.1, 212.7; MS m/z 307 (M + H)+; [R]20
-120° (c 0.0104, MeOH). Anal. (C21H22O2) C, H, N.
D
4a(R)-Ben zyl-7-m eth oxy-4,4a,9,10-tetr ah ydr o-3H-ph en -
a n th r en -2-on e (1Sa ). The compound 1Sa was formed by the
same procedure used for 4a(S)-benzyl-7-methoxy-4,4a,9,10-
tetrahydro-3H-phenanthren-2-one (1Ra ). Starting with 3.53
g of 1(R)-benzyl-6-methoxy-1(R)-(3-oxo-butyl)-3,4-dihydro-1H-
naphthelen-2-one (3S) produced 2.78 g of 1Sa as an off-white
powder. Recrystallization from EtOAc/hexane afforded 2.15 g
of 1Sa as a white crystalline solid (33% from 1-benzyl-6-
methoxy-3,4-dihydro-1H-naphthalen-2-one, 2). All physical
constants are the same as reported for 4a(S)-benzyl-7-methoxy-
4,4a,9,10-tetrahydro-3H-phenanthren-2-one (1Ra ) except opti-
4a (S)-Ben zyl-2(R)-ch lor oeth yn yl-1,2,3,4,4a ,9,10,10a (R)-
octah ydr o-ph en an th r en e-2,7-diol (CP -394531). To a stirred
solution of 95 mg of cis-dichloroethylene (0.98 mM) in 5 mL of
THF at 0 °C was added 2.5 mL of 0.5 M lithium diisopropyl-
amide in THF, and the resultant mixture was allowed to warm
to rt for 30 min under a nitrogen atmosphere. A solution of 30
mg (0.098 mmol) of 4a(S)-benzyl-7-hydroxy-3,4,4a,9,10,10a(R)-
hexahydro-1H-phenanthren-2-one (4) in 0.65 mL of THF was
added dropwise, and the reaction mixture was stirred for an
additional 2 h. Saturated aqueous ammonium chloride was
added, and the mixture was extracted with EtOAc (3 times).
The combined organic layers were dried over Na2SO4, filtered,
and concentrated to dryness. Initial purification by flash
chromatography over SiO2 using 20% EtOAc in hexanes as
the eluant afforded 30 mg of a light brown solid. Further
purification by flash chromatography over SiO2 using 2-4%
acetone in dichloromethane as a gradient eluant afforded 20
cal rotation, [R]20 +225° (c 0.0114, MeOH). Anal. (C22H22O2)
D
C, H, N.
4a (S)-Ben zyl-7-h yd r oxy-4,4a ,9,10-tetr a h yd r o-3H-p h en -
a n th r en -2-on e (1Rb). To a stirred solution of 40 g (0.126 mol)
of 4a(S)-benzyl-7-methoxy-4,4a,9,10-tetrahydro-3H-phenan-
thren-2-one (1Ra ) and 46.5 g (0.126 mol) of tetrabutylammo-
nium iodide in 630 mL of dichloromethane was added 300 mL
of 1 M boron trichloride in methylene chloride at -78 °C under
an N2 atmosphere. The resultant solution was allowed to warm
to rt for 1.5 h and then poured into excess ice and stirred
vigorously overnight. The mixture was extracted with dichlo-
romethane (3 times), dried over Na2SO4, filtered, and concen-
trated to dryness. Purification by flash chromatography over
SiO2 using 20-60% EtOAc in hexanes as the gradient eluant
afforded 33.3 g of an off-white powder (87%): 1H-NMR (400
MHz, CD3OD, δ) 1.81-2.00 (m, 2H), 2.26 (dt, 1H, J ) 4.2, 13
Hz), 2.40 (dd, 1H, J ) 4.5, 18 Hz), 2.53 (ddd, 1H, J ) 1.7, 5.6,
14 Hz), 2.58-2.80 (m, 3H), 3.20 (d, 1H, J ) 13 Hz), 3.26 (d,
1H, J ) 13 Hz), 5.92 (s, 1H), 6.45 (d, 1H, J ) 2.5 Hz), 6.67
(dd, 1H, J ) 2.5, 8.5 Hz), 6.76 (d, 2H, J ) 6.6 Hz), 7.05-7.14
(m, 4H); 13C-NMR (100 MHz, CD3OD, δ) 30.22, 32.03, 34.08,
36.04, 43.73, 45.97, 113.76, 113.91, 124.50, 126.25, 127.49,
127.94, 129.84, 131.86, 137.0, 137.71, 155.34, 171.73, 200.33;
1
mg (56%) of CP -394351: mp 230-232 °C dec; H-NMR (300
MHz, CD3OD, δ) 1.40 (mt, 1H, J ) 14 Hz), 1.64-1.70 (m, 1H),
1.80-2.13 (m, 7H), 2.59 (d, 1H, J ) 13 Hz), 2.93-2.97 (m, 3H),
6.13 (d, 1H, J ) 8.5 Hz), 6.25 (dd, 1H, J ) 2.6, 8.5 Hz), 6.54-
6.57 (m, 3H), 7.00-7.07 (m, 3H); 13C-NMR (75 MHz, CD3OD,
δ) 25.5, 28.7, 31.8, 37.2, 40.52, 41.71, 43.43, 63.1, 70.7, 74.1,
112.4, 116.1, 126.8, 128.3, 138.9, 132.1, 136.2, 138.3, 139.5,
156.3; MS m/z 366 (M + H)+, 349 (M - OH)+. [R]20 -98° (c
D
0.0103, MeOH). Anal. (C23H23ClO2) C, H, N.
4a (S)-Ben zyl-2(R)-p r op -1-yn yl-1,2,3,4,4a ,9,10,10a (R)-oc-
ta h yd r o-p h en a n th r en e-2,7-d iol (CP -409069). To a stirring
solution of 30 mL of THF saturated with propyne gas at 0 °C
was added 52 mL of 0.5 M lithium diisopropylamide in THF,
and the resultant mixture was stirred under a nitrogen
atomosphere for 20 min. A solution of 1.34 g (4.3 mmol) of 4a-
(S)-benzyl-7-hydroxy-3,4,4a,9,10,10a(R)-hexahydro-1H-phenan-
thren-2-one (4) in 18 mL of THF was added dropwise, and the
reaction mixture was warmed to rt and stirred for 1 h.
Saturated aqueous ammonium chloride was added, and the
mixture was extracted with EtOAc (3 times). The combined
MS m/z 305 (M + H)+; [R]20 -210° (c 0.0100, MeOH). Anal.
D
(C22H22O2‚1/3CH3CO2C2H5) C, H, N.
4a (R)-Ben zyl-7-h yd r oxy-4,4a ,9,10-tetr a h yd r o-3H-p h en -
a n th r en -2-on e (1Sb). The same procedure for 4a(S)-benzyl-
7-hydroxy-4,4a,9,10-tetrahydro-3H-phenanthren-2-one (1Rb)

2422 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12
Morgan et al.
organic layers were dried over Na2SO4, filtered, and concen-
trated to dryness. Purification by flash chromatography over
SiO2 using 2-4% acetone in dichloromethane as the eluant
afforded 1.05 g (69%) of CP -409069: mp 227-229 °C dec); 1H-
NMR (400 MHz, CD3OD, δ) 1.42 (mt, 1H, J ) 14 Hz), 1.61
(ddd, 1H, J ) 3.4, 4.1, 8.8 Hz), 1.72 (s, 3H), 1.73-1.82 (m, 2H),
1.84-2.10 (m, 5H), 2.55 (d, 1H, J ) 13 Hz), 2.83-2.93 (m) and
2.94 (d, 3H, J ) 13 Hz), 6.10 (d, 1H, J ) 8.3 Hz), 6.23 (dd, 1H,
J ) 2.5, 8.4 Hz), 6.52-6.55 (m, 3H), 7.00-7.05 (m, 3H); 13C-
NMR (62 MHz, CD3OD, δ) 2.5, 24.1, 27.3, 30.5, 35.8, 36.1, 39.1,
40.2, 42.4, 68.9, 79.5, 82.3, 110.9, 114.7, 125.4, 126.8, 127.5,
130.7, 135.1, 136.9, 138.3, 154.8; MS m/z 346 (M + H)+, 329
EDTA, 10 mM Na2MoO4, 1 mM Pefabloc, 10% glycerol) at a
concentration of 6.7 × 107 cells/mL. Following sonication, cell
debris was pelleted in a microfuge, and cell extract was frozen
in aliquots in liquid N2 and stored at -80 °C. Binding assays
were carried out in 96-well filter plates (Millipore MHVPN45)
containing 45 µL of hydroxylapatite (Calbiochem 391947) in
a final volume of 200 µL of binding buffer (previously described
without Pefabloc) containing 5 µL of hAR extract, 1 nM [3H]-
dihydrotestosterone (Perkin-Elmer NET453), and the test
compounds at concentrations ranging from 0.01 nM to 30 µM.
Each concentration was run in triplicate. Nonspecific binding
in triplicate wells was determined on each plate by the addition
of excess unlabeled dihydrotestosterone (final concentration,
10 µM). The reaction mixture was incubated for 1 h at 4 °C.
Unbound radioactivity was removed by filtration and three
washes with buffer at pH 7.2 (50 mM HEPES, 10 mM NaPO4,
0.1% Triton X-100, 100 mM KCl). Fifty microliters of scintil-
lation fluid (Packard Microscint 20) was added to each well,
and radioactive counts per minute were determined using a
TopCount micro scintillation counter (Packard). The dose-
response data for specifically bound counts were fitted using
a sigmoidal equation to calculate IC50, and Ki was calculated
using the equation of Cheng and Prusoff.63 The Kd for the
radioligand is 0.4 nM.65-68
(M - OH)+; [R]20 -120° (c 0.0104, MeOH). Anal. (C24H26O2)
D
C, H, N.
Hu m a n Glu cocor ticoid Recep tor (h GR) Bin d in g Assa y
P r otocol. Binding displacement assays were run using cyto-
solic preparations from Sf9 cells infected with the human
glucocorticoid receptor (hGR).62 Briefly, Sf9 cells were inocu-
lated at a density of 3 × 105/mL and grown to ∼2 × 106/mL in
an SF900 II medium. Cells were infected at a multiplicity of
infection (MOI) of 1, harvested 72 h postinfection, and batch
centrifuged at 3500 rpm for 10 min. The cell pellets were stored
at -75 °C. Cytosol was prepared by suspending the Sf9 cell
pellets in 80 mL of lysing buffer (10 mM K2HPO4, pH 7.6, 100
µM EDTA, 5 mM dithiothreitol, 20 mM sodium molybdate, 200
µM AEBSF (lysing reagent from Calbiochem-Novabiochem
Corporation, LaJ olla, CA), and 80 µg/mL leupeptin), centrifug-
ing at 100 000g for 30 min at 4 °C, and collecting the
supernatant. Protein concentration was determined using an
automated analyzer (Abbott CCX). Binding assays were car-
ried out in 96-well v-bottom polypropylene plates with a final
volume of 130 µL containing 200-600 µg of cytosolic protein,
assay buffer (113 µL, containing 20 mM Tris, 1 µM EDTA, 5
mM DTT, 10 mM sodium molybdate), [3H]dexamethasone
(final concentration, 10 nM), and test compounds at concentra-
tions ranging from 0.3 nM to 30 µM or test compound vehicle
(to give total bound counts). Each concentration was run in
duplicate. Nonspecific binding in duplicate wells was deter-
mined on each plate by the addition of excess unlabeled
dexamethasone (final concentration, 11.5 µM). Cytosols were
incubated for 18 h at 4 °C. Unbound radioactivity was removed
by the addition of dextran-coated charcoal and centrifugation.
One hundred microliters of the supernatant from each well
was transferred to a 96-well PET plate with 200 µL of
scintillation fluid and counted (1450 micro â counter from
Perkin-Elmer Wallac, Gaithersburg, MD). The dose-response
data for specifically bound counts were fitted using a sigmoidal
equation to calculate IC50, and Ki was calculated using the
equation of Cheng and Prusoff.63 The Kd for dexamethasone,
determined by saturation binding, was 2.5 nM.
Wh ole Cell F u n ct ion a l Assa ys, GR E . SW1353 human
chondrosarcoma cells expressing the endogenous glucocorticoid
receptor were transfected with a plasmid containing the
murine mammary tumor virus (MMTV) promoter linked to
luciferase and conferring neomycin resistance (Clontech).
Neomycin resistant (500 µg/mL) cell clones were isolated and
characterized by generating dexamethasone response curves.
Clone SW1353/MMTV-8, having a median response (data not
shown), was used to determine the transcription activity of
test compounds. Briefly, cells were transferred to 96-well
microtiter plates in complete media (Dulbecco’s Modified Eagle
Media, 10% fetal bovine serum, gentamicin reagent, and 500
µg/mL neomycin), and after an overnight incubation, the
medium was changed to a serum-free formulation (Dulbecco’s
Modified Eagle Media, 1 mg/L insulin, 0.5 mg/L ascorbate, 2
g/L lactalbumin hydrosylate) 4 h prior to treatment with
various concentrations (10-10 to 10-6 M) of test compounds in
the absence or presence of 40 nM dexamethasone (to determine
agonist or antagonist activity, respectively). After 16 h, cell
lysates were prepared, and luciferase activity was quantified
using a LucLite reagent (Packard) and a luminometer. Agonist
activity was expressed as the relative luciferase activity from
cells treated with test compounds to those treated with
dexamethasone. Antagonism was determined by the relative
inhibition of luciferase activity from cells cotreated with the
test compounds and a constant amount of dexamethasone (40
nM, that which produced a half-maximal response). The EC50
(concentration that produced 50% of the maximal response)
for dexamethasone was calculated for the dose-response
curves. The Kif was calculated using the modified equation of
Cheng and Prusoff Kif ) IC50/1 + ([Dex]/EC50Dex).63
Wh ole Cell F u n ction a l Assa ys, MRE a n d P RE. T47D
cells (from ATCC) containing endogenous human progesterone
and mineralocorticoid receptors were transiently transfected
with 3 × GRE-luciferase plasmid generated using Lipofectin
plus (GIBCO-DRL, Gaithersburg, MD). Twentyfour hours post-
transfection, cells were maintained in 10% charcoal-stripped
serum and transferred to 96-well microtiter plates. The next
day, cells were treated with various concentrations (10-12 to
10-5 M) of test compounds in the absence or presence of 10
nM progesterone and 10 nM aldosterone in triplicate for up
to 24 h. Cell lysates were prepared, and luciferase activity was
determined using a luminometer. Agonist activity was as-
sessed by comparing the luciferease activity from cells treated
with test compounds alone to cells treated with either proges-
terone or aldosterone. Antagonist activity was addressed by
comparing the luciferease activity of an EC50 concentration of
progesterone or aldosterone in the absence and presence of
the test compound. The EC50 for dexamethasone was calcu-
lated for the dose-response curves. The Kif was calculated
H u m a n An d r ogen R ecep t or (h AR ) Bin d in g Assa y
P r otocol. Binding displacement assays were run using whole
cell preparations from Sf9 cells infected with the human
androgen receptor (hAR). The human androgen receptor
cDNA64 was obtained from Dr. Chawnshang Chang (University
of Chicago, Chicago, IL) and subcloned into the baculovirus
transfer vector pBlueBac4.5 (Invitrogen, Carlsbad, CA). The
transfer vector was cotransfected into Sf9 cells with Bac-N-
Blue DNA (Invitrogen), and plaques containing recombinant
virus were selected based on the expression of â-galactosidase.
Appropriate recombination was confirmed by PCR, and the
recombinant virus was subjected to an additional round of
plaque purification. This virus (AR22) was expanded and
titered. A MOI and time-course experiment was conducted by
infecting Sf9 cells growing in spinner culture in Grace’s Insect
medium supplemented with 10% FCS and 0.1% F68 (Invitro-
gen) at 2 × 106 cells/mL with MOIs ranging from 0.1 to 10.
Optimal expression of the AR was found by Western blot to
be 48 h after infection with an MOI ) 2. AR for binding assays
was then produced by infecting 1 L Sf9 cultures with AR22
virus under these conditions of pelleting the cells by centrifu-
gation 48 h after infection and resuspending and sonicating
the cell pellet in binding buffer at pH 7.2 (5 mM dithiothreitol,
10 mM NaF, 100 µg/mL bacitracin, 50 mM HEPES, 1.5 mM

Glucocorticoid Receptor Antagonists
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 12 2423
(16) Rosati, R. L.; DaSilva-J ardine, P.; Cameron, K. O.; Thompson,
D. D.; Zhu Ke, H.; Toler, S. M.; Brown, T. A.; Pan, L. C.;
Ebbinghaus, C. F.; Reinhold, A. R.; Elliott, N. C.; Newhouse, B.
N.; Tjoa, C. M.; Sweetnam, P. M.; Cole, M. J .; Arriola, M. W.;
Gauthier, J . W.; Crawford, D. T.; Nickerson, D. F.; Pirie, C. M.;
Qi, H.; Simmons, H. A.; Tkalcevic, G. T. Discovery and Preclini-
cal Pharmacology of a Novel, Potent, Nonsteroidal Estrogen
using the modified equation of Cheng and Prusoff [Kif ) IC50
(1 + [ligand]/EC50ligand)].63
/
Ack n ow led gm en t. The authors thank Mr. Eben D.
Salter and Dr. J ames P. O’Malley for providing andro-
gen binding data, Ms. Michele L. Millham and Dr.
Leonard Buckbinder for providing GR functional data,
and Ms. Celeste J ohnson for coordinating progesterone
and estrogen binding data from Cerep on the above
compounds. Additionally, the authors thank Ms. Mar-
garet A. Rushing, Mr. Graeme F. Woodworth, Ms.
Melissa H. Rockwell, Mr. Richard Trilles, and Mr. Ivan
Samardjiev for their technical assistance, Dr. Alan M.
Mathiowetz for graphics support, Dr. J ohn A. Lowe and
Prof. Andrew G. Myers for their helpful suggestions in
drafting the manuscript, and Drs. Robert L. Dow, Kevin
K. Liu, Peter A. McCarthy, and Paul DaSilva-J ardine
for their insightful discussions.
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