Identification of an agonist ligand for estrogen-related receptors ERRβ/γ

Article abstract of DOI:10.1016/j.bmcl.2005.01.025

In order to develop agonist ligands that are specific for the estrogen-related receptors ERRβ/γ, a hydrazone with a 4-hydroxy group at one phenyl ring and a 4-diethylamino moiety at the other phenyl ring was synthesized. We demonstrate that compound 3 (DY131; N′-{(1E)-[4- (diethylamino)phenyl]methylene}-4-hydroxybenzohydrazide) effectively and selectively activates ERRβ/γ. DY131 had no effect on the structurally related receptors ERRα or the estrogen receptors α and β (ERα/β). This work defines a convenient synthesis for a novel and selective pharmacologic tool that can be used to elucidate the biological activities of ERRβ/γ.

Full text of DOI:10.1016/j.bmcl.2005.01.025

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1311–1313
Identification of an agonist ligand for estrogen-related
receptors ERRb/c
Donna D. Yu and Barry Marc Forman*
Department of Gene regulation and Drug Discovery, The Beckman Research Institute at The City of Hope National Medical Center,
The Gonda Diabetes and Genetic Research Center, Duarte, CA 91010, USA
Received 23 December 2004; revised 10 January 2005; accepted 12 January 2005
Abstract—In order to develop agonist ligands that are specific for the estrogen-related receptors ERRb/c, a hydrazone with a 4-
hydroxy group at one phenyl ring and a 4-diethylamino moiety at the other phenyl ring was synthesized. We demonstrate that com-
pound 3 (DY131; N0-{(1E)-[4-(diethylamino)phenyl]methylene}-4-hydroxybenzohydrazide) effectively and selectively activates
ERRb/c. DY131 had no effect on the structurally related receptors ERRa or the estrogen receptors a and b (ERa/b). This work
defines a convenient synthesis for a novel and selective pharmacologic tool that can be used to elucidate the biological activities
of ERRb/c.
Ó 2005 Elsevier Ltd. All rights reserved.
Metabolic disease, including obesity, diabetes and ath-
erosclerosis is the leading cause of mortality in industri-
alized nations. It is estimated that over one-third of the
United States population is obese and these individuals
are at risk for developing diabetes and atherosclerosis.
These disorders are responsible for over 500,000 deaths
in the United States each year. The growing incidence of
metabolic disease has led to an intense interest in identi-
fying new molecular targets and new pharmacologic
agents to treat and/or prevent these disorders.
and both proteins form a complex that stimulates the
expression of genes involved in mitochondrial fatty acid
oxidation (medium-chain acyl coenzyme A dehydroge-
nase,4 peroxisome proliferator activated receptor a5)
and oxidative phosphorylation.6,7 ERRa has also been
shown to regulate genes involved in intestinal lipid
absorption, satiety signals (apolipoprotein A-IV8) and
vascular relaxation (endothelial nitric oxide synthase9).
An important advance in the elucidation of orphan
receptor biology is the identification of synthetic ligands
that can be used to modulate specific receptors in cells
and in vivo. For example, a synthetic ERRa antagonist
known as XCT790 (Fig. 1) has recently been de-
scribed.10,11 This chemical reagent facilitated the discov-
ery of ERRa as a regulator of oxidative phosphorylation6
and of monoamine oxidase B expression.10 XCT790 has
no activity on ERRc; its activity on the related receptors
ERRb, ERa and ERb has not been reported.
Orphan nuclear receptors provide an example of an
important class of molecular targets for the treatment
of metabolic disease. Nuclear receptors are ligand-
dependent transcription factors that regulate gene
expression in response to small-molecule ligands. The
orphan receptor ERRa (estrogen-related receptor a) is
known to be a key regulator of lipid homeostasis. For
example, mice lacking ERRa are lean and are resistant
to developing obesity when challenged with a high-fat
diet.1 ERRa is expressed in tissues with a high capacity
for b-oxidation of fatty acids including the heart, kid-
neys, brown adipose tissue and skeletal muscle.2 ERRa
expression is stimulated by the coactivator PGC-1a3
Although there has been significant progress in under-
standing the physiologic activity of ERRa, little is
known about the biological activity of ERRb and
ERRc. A major factor that is limiting progress in this
field is the lack of selective ERRb/c ligands that can
be utilized in cell culture and in vivo studies. For exam-
ple, while (Z)-4-hydroxytamoxifen has been reported to
be an antagonist of both ERRb and ERRc,12,13 this
compound is not useful experimentally because it is
several orders of magnitude more potent as an ERa
Keywords: Estrogen-related receptor; Nuclear hormone receptor;
Agonist.
*
Corresponding author. Tel.: +1 626 256 4673; fax: +1 626 256
8704; e-mail: bmforman@earthlink.net
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.025

1312
D. D. Yu, B. M. Forman / Bioorg. Med. Chem. Lett. 15 (2005) 1311–1313
OCH
3
3
N
H
3
3
N
XCT790
DY131
(Z)-4-hydroxytamoxifen
Figure 1. Chemical structures of ERR ligands. XCT790 is an ERRa antagonist. (Z)-4-Hydroxytamoxifen is a high affinity antagonist for ERa/b and
a low affinity antagonist for ERRb/c. DY131 is an ERRb/c agonist.
and ERb antagonist. Moreover, no agonist or activating
ligands have been reported for any of the ERRs; only
antagonists have been identified. We therefore sought
to develop a simple and efficient synthesis for com-
pounds that could selectively activate ERRb/c with little
activity on ERRa and ERa/b. In a recent symposia,
GlaxoSmithKline suggested acyl hydrazone derivatives
as potential ligands for ERRc.14 As part of our ongoing
programme, we developed two alternative routes for the
synthesis of the acyl phenylhydrazone derivative DY131
(Fig. 1) and evaluated its potential to act as a selective
ERRb/c agonist.
by the simultaneous demethylation and deprotection of
the N-benzyl group of 2 by ether cleavage with boron
tribromide. This produced compound 3 (DY131, 4-hy-
droxy-benzoic acid (4-diethylamino-benzylidene)-hydr-
azide), with an overall yield of 27%.
We next sought a more efficient procedure for the syn-
thesis of DY131 (3), for example, one that replaced
the three-step procedure of Scheme 1 with a single step
synthesis. To do so, we synthesized DY131 by direct
condensation of 4-hydroxybenzoic acid hydrazide with
4-diethylaminobenzaldehyde (Scheme 2). The phenolic
hydroxyl and the hydrogen of the secondary amine re-
mained unchanged during the reaction and DY131 (3)
was successfully synthesized in this one-step reaction
with a yield of 65%.
The simplest synthesis of DY131 would be a direct con-
densation of 4-hydroxybenzoic acid hydrazide with 4-
diethylaminobenzaldehyde. However, because of the
uncertainty in using an unprotected hydroxyl, we ini-
tially used 4-methoxybenzoic acid hydrazide. 4-Dieth-
ylaminobenzaldehyde was condensed with 4-
methoxybenzoic acid hydrazide by refluxing in absolute
ethanol with a catalytic amount of acetic acid according
to the method of Bahadur et al.15 This resulted in a 76%
yield of compound 1. We found that direct conversion
of 1 to 3 with boron tribromide gave low yields. Thus,
a two-step procedure was explored whereby the amine
of 1 was protected prior to conversion to 3. Specifically,
the amino group of 1 was protected by reacting with a-
bromo-p-xylene to give compound 2. This was followed
DY131 (3) was evaluated for selectivity and efficacy in
modulating the transcriptional activity of ERRa/b/c
and ERa/b. CV-1 cells were transfected with appropri-
ate reporter constructs and expression vectors and the
fold activation of the reporter construct was determined
at several concentrations of DY131 (Table 1). DY131
failed to activate the reporter construct or ERRa at
any of the concentrations tested. In contrast, DY131 re-
sulted in three- to fourfold activation of ERRb at con-
centrations of 10–30 lM. Activity on ERRc was even
more pronounced with fivefold activation at 3 lM and
N
N
N
O
O
O
Br
K2CO3
OHC
N
N
NHNH2
N
N
H
EtOH
H3CO
H3CO
H3CO
1
2
BBr3
N
O
N
N
H
HO
3 (DY131)
Scheme 1. Three-step synthesis of 3 (DY131) with an overall yield of 27%.

D. D. Yu, B. M. Forman / Bioorg. Med. Chem. Lett. 15 (2005) 1311–1313
1313
N
O
N
O
OHC
EtOH
NHNH2
N
N
H
HO
HO
3 (DY131)
Scheme 2. One-step synthesis of 3 (DY131) with an overall yield of 65%.
Table 1. Cell-based reporter gene assays examining the fold activation
of ERR and ER in response to DY131 and b-estradiol
Supplementary data
Receptora
DY131 (3)
b-Estradiol
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2005.01.025. The supplementary data provide an experi-
mental description of the preparation of the compounds
as well spectral, physical and elemental analyses.
3 lM
10 lM
30 lM
100 nM
—
0.8
1.5
1.9
5.0
0.7
1.7
3.1
5.9
0.9
1.6
3.8
6.6
1.1
1.1
1.0
0.8
ERRa
ERRb
ERRc
References and notes
—
1.0
1.1
1.1
1.1
1.0
1.0
1.0
0.9
1.2
1.0
5.2
6.2
ERa
ERb
1. Luo, J.; Sladek, R.; Carrier, J.; Bader, J. A.; Richard, D.;
Giguere, V. Mol. Cell. Biol. 2003, 23, 7947.
2. Giguere, V. Trends Endocrinol. Metab. 2002, 13,
220.
3. Schreiber, S. N.; Knutti, D.; Brogli, K.; Uhlmann, T.;
Kralli, A. J. Biol. Chem. 2003, 278, 9013.
4. Huss, J. M.; Kopp, R. P.; Kelly, D. P. J. Biol. Chem. 2002,
277, 40265.
5. Huss, J. M.; Torra, I. P.; Staels, B.; Giguere, V.; Kelly, D.
P. Mol. Cell. Biol. 2004, 24, 9079.
a Cell based transactivation assays were performed in CV-1 cells as
described.16 ERR activity was assayed with a GAL4 reporter con-
struct and fusion proteins containing the ligand binding domains of
human ERRa, human ERRb and mouse ERRc linked to the DNA
binding domain of yeast GAL4. Human ERa and ERb were exam-
ined as full-length proteins using an estrogen receptor responsive
reporter construct. Fold activation is reported.
6. Mootha, V. K.; Handschin, C.; Arlow, D.; Xie, X.; St
Pierre, J.; Sihag, S.; Yang, W.; Altshuler, D.; Puigserver,
P.; Patterson, N.; Willy, P. J.; Schulman, I. G.; Heyman,
R. A.; Lander, E. S.; Spiegelman, B. M. Proc. Natl. Acad.
Sci. U.S.A. 2004, 101, 6570.
7. Schreiber, S. N.; Emter, R.; Hock, M. B.; Knutti, D.;
Cardenas, J.; Podvinec, M.; Oakeley, E. J.; Kralli, A. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 6472.
8. Carrier, J. C.; Deblois, G.; Champigny, C.; Levy, E.;
Giguere, V. J. Biol. Chem. 2004.
9. Sumi, D.; Ignarro, L. J. Proc. Natl. Acad. Sci. U.S.A.
2003, 100, 14451.
10. Willy, P. J.; Murray, I. R.; Qian, J.; Busch, B. B.; Stevens,
W. C., Jr.; Martin, R.; Mohan, R.; Zhou, S.; Ordentlich,
P.; Wei, P.; Sapp, D. W.; Horlick, R. A.; Heyman, R. A.;
Schulman, I. G. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
8912.
11. Busch, B. B.; Stevens, W. C., Jr.; Martin, R.; Ordentlich,
P.; Zhou, S.; Sapp, D. W.; Horlick, R. A.; Mohan, R. J.
Med. Chem. 2004, 47, 5593.
12. Coward, P.; Lee, D.; Hull, M. V.; Lehmann, J. M. Proc.
Natl. Acad. Sci. U.S.A. 2001, 98, 8880.
13. Tremblay, G. B.; Bergeron, D.; Giguere, V. Endocrinology
2001, 142, 4572.
maximal 6.6-fold activity observed at 30 lM. Thus,
DY131 is an ERRb/c-specific ligand that displays pref-
erential selectivity for ERRc at lower concentrations.
Due to the overlap in recognition of (Z)-4-hydroxytam-
oxifen by both ERR and ER, we also examined the
activity of DY131 on ERa and ERb. DY131 failed to
activate or inhibit either of these receptors whereas the
control ligand, b-estradiol, resulted in the expected five-
to sixfold activation. These data demonstrate that
DY131 is a selective agonist of ERRb/c with no activity
on the related receptors ERRa, ERa and ERb. This
selective activity establishes DY131 as a novel pharma-
cologic tool to study the biological activities of ERRb/c.
In summary, we designed a simple, one-step procedure
for the synthesis of DY131, a novel ERRb/c selective
agonist. This compound provides a unique chemical tool
for studying ERRb/c action in human cells to elucidate
the physiological activities of ERRb/c.
14. Willson, T. M.; Williams, S. P.; Shewchuk, L.; Xu, R.;
Nolte, R. T. Are all Orphan Nuclear Receptors Good Drug
Targets? Nuclear Receptors: Orphan Brothers, Keystone,
CO, Feb 28–Mar 4, 2004; Keystone Symposia: Silver-
thorne, CO, 2004; p 042.
Acknowledgements
15. Bahadur, S.; Varma, R. S.; Saxena, M. J. Indian Chem.
Soc. 1980, LVII, 918.
16. Wang, H.; Chen, J.; Hollister, K.; Sowers, L. C.; Forman,
B. M. Mol. Cell 1999, 3, 543.
The authors thank Irina Ianculescu for technical assis-
tance and the Gonda Family for support and
encouragement.