Estrogen receptor β ligands: Design and synthesis of new 2-phenyl-isoindole-1,3-diones

Article abstract of DOI:10.1016/j.bmcl.2006.09.088

The design, synthesis, and biological evaluation of the 2-phenyl-isoindole-1,3-diones will be discussed. Detailed modeling studies with X-ray support were used to understand the ligand binding orientation and observed selectivity.

Full text of DOI:10.1016/j.bmcl.2006.09.088

Bioorganic & Medicinal Chemistry Letters 17 (2007) 118–122
Estrogen receptor b ligands: Design and synthesis of new
2-phenyl-isoindole-1,3-diones
John W. Ullrich,a,* Rayomand J. Unwalla,b, Robert R. Singhaus, Jr.,a
Heather A.Harrisc and Richard E. Mewshawa
aChemical and Screening Sciences Medicinal Chemistry, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
bChemical and Screening Sciences Structural Biology, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
cWomen’s Health and Musculoskeletal Biology, Wyeth Research, 500 Arcola Road, Collegeville, PA 19426, USA
Received 3 August 2006; revised 25 September 2006; accepted 27 September 2006
Available online 1 October 2006
Abstract—The design, synthesis, and biological evaluation of the 2-phenyl-isoindole-1,3-diones will be discussed. Detailed modeling
studies with X-ray support were used to understand the ligand binding orientation and observed selectivity.
Ó 2006 Elsevier Ltd. All rights reserved.
The estrogen receptor (ER) is a ligand-activated tran-
scription factor that belongs to the nuclear hormone
receptor superfamily. Other members of this family
include the progestin, androgen, glucocorticoid, and
mineralocorticoid receptors.1 Until recently estrogen-
mediated events had been thought to be regulated by
only one estrogen receptor which now is identified as
ERa.2,3 With the discovery in 1996 of a second estrogen
receptor subtype (ERb), there was an intense interest in
elucidating ERb function.4,5 The three dimensional
structures of ERa6 and ERb7 have been solved by
co-crystallization with a variety of ligands. The primary
ligand binding domain of these receptors are 58%
homologous. However, the well-recognized ligand-bind-
ing domain (LBD) cavity varies by only two amino
acids7 (i.e., ERb Met336 is replaced by ERa Leu384 and
ERb Ile373 is replaced by ERa Met421). As a conse-
quence of these conservative substitutions, it is not sur-
prising that the many ER ligands known prior to the
discovery of the ERb subtype possessed limited selectiv-
ity for that subtype. Despite the fact that the design of
highly selective subtype ligands appeared to be a
daunting task, there have been many reports of high
affinity and functionally selective ERa and ERb
ligands.8–12 Recently, we have disclosed some of our
efforts toward identifying possible utilities for ERb
selective ligands.13
The phytoestrogen, genistein, has been reported to have
modest ERb selectivity (ꢀ40-fold)14,15 and has been
used by our group as a starting point to design novel
nonsteroidal ERb selective ligands (Fig. 1). Some de-
tailed work focused on substitution around simplified
tricyclic A-BC scaffolds (i.e., the 6-phenylnaphtha-
lenes15 and the 2-phenyl quinolines16). Further varia-
tion on the central ring (ring-B) came about by
modification of the benzofuran 1 (obtained from elabo-
ration of an HTS screening hit),17 with an indenone
core (i.e., 2).18 The authors hypothesized that these
analogs would mimic the binding activity of the phyto-
estrogen genistein as well as allowing a handle to incor-
porate the C-5 hydroxy group of genistein into the
indenone core. Maintaining the positions of the A
and C ring hydroxyls led to two regioisomeric indenone
forms, the 6-hydroxy series 2a and the 5-hydroxy series
2b (not unlike that of genistein). These compounds,
although potent binders to ERb, have moderate <15-
fold ERb-selectivity. The core template, arylindene-1-
one 2 of this series, having the ability to flip 180°
(around the x- and y-axes), allows for multiple binding
modes. To simplify this binding paradigm, we explored
substitution on a central phthalimide core which has
been utilized in the design of a variety of biologically
active molecules.19 The 2-phenyl-isoindole-1,3-dione
core (i.e., 3), bearing two carbonyls, has additional
symmetry along the x-axis.
Keywords: ERa and ERb receptor; Estrogen receptor; Nuclear
hormone receptor.
*
Corresponding author. Tel.: +1 484 865 4266; fax: +1 484 865
9399; e-mail: ullricj@wyeth.com
Corresponding author for molecular modeling. Tel.: +1 484 865
2493; fax: +1 484 865 9398; email: unwallr@wyeth.com.
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.09.088

J. W. Ullrich et al. / Bioorg. Med. Chem. Lett. 17 (2007) 118–122
119
OH
O
O
OH
OH
C
B
C
X
5
O
A
A
A
H
HO
HO
HO
17β-Estradiol (E2)
Genistein
X = -CH-, 6-phenylnaphthalenes
X = -N-, 2-phenylquinolines
X
5
OH
HO
OH
6
HO
O
O
1
2a and 2b X = halogen, alkyl, aryl
C2
O
OH
OH
HO
C2
C
HO
A
N B
X
O
3
X = O or C(O)
Figure 1. Compounds of interest.
Table 1. Binding affinity for 2-phenyl-isoindole-1,3-dione core
If compound 3 rotates along the x-axis the ‘B-ring’ of
this A-BC ring system will not be altered and has the
advantage of allowing the template to serve as a vehicle
to rapidly evaluate and design ERb selectivity into the
new structural motif.
O
R
OH
4'
N
B
A
C
2'
3'
7
O
The synthesis of the phthalimides (i.e., 3) was straight-
forward and is outlined in Scheme 1. It was accom-
plished by reacting a mixture of 4-hydroxyphthalic
acid and N-methyl-2-pyrrolidine (NMP) in xylenes at
reflux temperature with an appropriately substituted
amine. The free hydroxyls were obtained by deprotec-
tion of the methyl ethers with pyridine–HCl (190 °C)
to provide the desired hydroxy phenyl-phthalimides
(i.e., 3). Alternatively, a copper-mediated coupling of
an appropriately substituted N–H phthalimide and a
suitably substituted iodoanisole may construct the core
phthalimide.
a
a
Compound
R
A ring
ERb IC50
(nM)
ERa IC50
(nM)
E2
Genistein
—
—
—
—
3.6 1.6
9.7 4.3
84 31 (7)
3.2 1.0
395 181
1980 719 (7)
3
4
5
6
7
8
9
OH 40-OH
OH 30-OH
2900 346 (3) >5000 (3)
H
OH
OH 40-OMe
OH 20,40-OH 2470
OH 30,50-OH >5000
40-OH
>10000
2700
3300
>10000
>5000
>5000
>5000
>5000
H
a Values are means of independent determinations (number in
parentheses) SD.
A competitive radioligand binding assay was used to
assess the relative binding affinity (IC50) of compounds
for the human ligand binding domains (LBD) of ERb
and ERa.20 Table 1 shows the binding affinities for
analogs with variations of the hydroxyl group on the
N-phenyl ring. The C-40 placement of the hydroxyl
is optimal for binding to the ER.
saturated six-membered ring (i.e., 10) and observed a
significant decrease in affinity. In addition, a fused aryl
system (i.e., 11 and 12) also decreased affinity. Intro-
duction of a C-20 electron-withdrawing substituent
enhanced binding affinity (i.e., 14 and 16) which has
been previously observed in the literature for ERb selec-
tive ligands.8,9,12,18 Although an increase in affinity was
Table 2 shows structural variations on the appended
N-phenyl ring where we replaced the aryl ring with a
H2N
O
a, b
HO
OH
+
OH
R
OR
O
O
or HCl Salt
R = H, CH3
OH
HO
N
R
O
O
OMe
c,b
RO
N
H
+
3
R = H and 4'-OH
I
R
O
R = H
Scheme 1. Reagents and conditions: (a) NMP, xylene, 150 °C; (b) Py–HCl, 190 °C; (c) Cu2O, collidine.

120
J. W. Ullrich et al. / Bioorg. Med. Chem. Lett. 17 (2007) 118–122
Table 2. Structural variations on 2-phenyl-isoindole-1,3-dione
O
R
R1
N
B
C
7
O
R1
ERb IC50 (nM)
ERa IC50 (nM)
ERa/ERb ratio
a
a
Compound
R
3
10
11
12
13
14
15
16
17
18
OH
OH
40-OH-phenyl
84 31 (7)
>5000
802
1980 719 (7)
>5000
2810
24
n/a
4
40-OH-C6H11 (trans)
50-OH-1-naphthyl
40-OH-1-naphthyl
20-NO2-40-OMe-phenyl
20-NO2-40-OH-phenyl
20-Me 40-OH-phenyl
20-F 40-OH-phenyl
40-OH-phenyl
OH
OH
2640
560
>5000
0.2
n/a
4
OH
OH
>5000
21
802
92
2810
OH
OH
4
53 13 (2)
>5000
36 2.1 (2)
339 92 (2)
>5000
1625 7.1 (2)
6
OMe, 7-Br
OH, 7-Br
n/a
45
40-OH-phenyl
a Values are means of independent determinations (number in parentheses) SD.
observed with substitution at the C-20 position, the com-
pounds did not exhibit increased ERb-selectivity when
compared to the core template, 3 .
Looking closely at the simplified core, there appeared to
be an opportunity to increase selectivity by substituting
at the 7-position of the phthalimide core. Previous stud-
ies in our laboratories23,24 suggested that substitution at
the 7-position may satisfy the binding requirements as
well as probe the Met/Ile selectivity pocket. Figure 3
shows the 7-bromo phthalimide 1825 docked into the
ERb/genistein pocket.
Docking calculations21 were used to predict the orienta-
tion of our novel core phthalimide 3 in the ERb binding
site.7 Figure 2 shows that the 40-OH group makes a
critical interaction with Arg346 and Glu305, while the
5-OH group makes an interaction with the His475
residue. This binding mode is consistent with that of
other A-B/C ring systems, such as benzofuran and inde-
none for which ERb X-ray structures are known.17,18
As discussed earlier, only two residues are different
within the ligand binding pockets of ERa and ERb.
The B ring of this template lies in close proximity to the
ERa Leu384/ERb Met336 residue mutation and it
appears that the core ERb selectivity (ꢀ24-fold) of this
scaffold is due to a favorable interaction of this aromatic
ring with ERb Met336, similar to what has been
described for genistein22 and other compounds.23
O
OH
HO
N
7
O
Br
Figure 2. Docked structure of compound 3 in the ERb/genistein
binding site. Only key residues are shown for simplicity. Residue
differences within the binding pocket between ERa and ERb are
highlighted. Hydrogen bonds are shown by a yellow dotted line. Arrow
indicates opportunity for increasing selectivity by targeting region of
residue difference.
Figure 3. Compound 18 docked into the ERb/genistein pocket. Only
key residues are shown for simplicity. Residue differences within the
binding pocket between ERa and ERb are highlighted. Hydrogen
bonds are shown by a yellow dotted line.

J. W. Ullrich et al. / Bioorg. Med. Chem. Lett. 17 (2007) 118–122
121
Acknowledgments
We thank the Wyeth Discovery Analytical Chemistry
department for physical analysis. Special thanks to Dr.
Chris P. Miller for serving as team leader and providing
insight into the SERM arena. Thanks to Drs. Jay
Wrobel and Gene Trybulski for advice and discussions.
Special thanks to Dr. John Yardley.
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˚
imity (2.1 A) to the sulfur of the ERa Met421 residue
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˚
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not expected to have significant electronic or steric
repulsion. The slight increase in ERb potency for this
compound (ꢀ2.3-fold) could be attributed to the in-
creased nature of the lipophilic group (i.e., bromine
occupying a hydrophobic pocket). This differential
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selectivity (ꢀ45-fold) observed for the bromo-substi-
tuted phthalimide 18.
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substituted phthalimides was prepared. This dihy-
droxy-substituted core offers a symmetric B-ring that
mimics genistein and binds to ERb with modest ERb-se-
lectivity (ꢀ24-fold) compared to genistein. The binding
mode for this series of compounds was consistent with
that of other previously reported A-B/C ring systems,
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18 was prepared and found to be slightly more ERb
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122
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