Identification and structure-activity relationship of phenolic acyl hydrazones as selective agonists for the estrogen-related orphan nuclear receptors ERRβ and ERRγ

Article abstract of DOI:10.1021/jm050161j

The first small molecule agonists of the estrogen-related receptors have been identified. GSK4716 (3) and GSK9089 (4) show binding to ERRγ with remarkable selectivity over the classical estrogen receptors. Notably, in cell-based reporter assays, 3 mimics

Full text of DOI:10.1021/jm050161j

J. Med. Chem. 2005, 48, 3107-3109
3107
Chart 1. Structures of ERRγ Ligands:
4-Hydroxytamoxifen (1), Diethylstilbestrol (2), and 3
Identification and Structure-Activity
Relationship of Phenolic Acyl
Hydrazones as Selective Agonists for the
Estrogen-Related Orphan Nuclear
Receptors ERRâ and ERRγ
William J. Zuercher,*,† Ste´phanie Gaillard,‡
Lisa A. Orband-Miller,† Esther Y. H. Chao,†
Barry G. Shearer,† David G. Jones,† Aaron B. Miller,†
Jon L. Collins,† Donald P. McDonnell,‡ and
Timothy M. Willson†
Discovery Research, GlaxoSmithKline, Five Moore Drive,
Research Triangle Park, North Carolina 27707, and
Department of Pharmacology and Cancer Biology, Duke
University, Levine Science Research Center, Research Drive,
Durham, North Carolina 27710
stitutive ERR activity) have been reported. The potent
ER ligands, 4-hydroxytamoxifen (1, 4-OHT) and dieth-
ylstilbestrol (2, DES) (Chart 1) are inverse agonists of
ERRγ, with 2 being relatively weak in potency.9 Second,
the X-ray crystal structures of unliganded ERRR and
ERRγ reveal the presence of small ligand binding
pockets.10,11 We recently disclosed the identification of
the phenolic acyl hydrazones GSK4716 (3) and GSK9089
(4) as the first reported ERRâ/γ agonists and as the first
ERR ligands with selectivity over the classical estrogen
receptors.12 Herein we report the discovery of these
ligands, their structure-activity relationship, and char-
acterization of potency and selectivity as ERR agonists.
Examination of in house crystal structures for the
orphans ERRγ and ERRR and the classical ERR and
ERâ reveals that the ligand binding pockets contains
some common elements although they differ in size and
shape. Notably, the glutamate and arginine residues in
ERR and ERâ that form the essential hydrogen bond
interaction with the phenolic A-ring of estradiol are
conserved in all three of the ERRs. Using this observa-
tion combined with positive results from diversity
screening, we proposed the synthesis of targeted arrays
of small, phenolic compounds as ERR ligands. Phenolic
acyl hydrazones were selected based on their simple
preparation through condensation of a hydrazide and
aldehyde and their potential to undergo oxidative cy-
clization to heterocyclic oxadiazole derivatives.
Initially, a solid-phase approach was developed using
the phenol building block as an anchor point (Figure
1A). Wang lanterns were reacted with CBr4 and dis-
placed with 4-hydroxybenzaldehyde. Subsequent chlo-
rite oxidation, followed by pentafluorophenyl trifluoro-
acetate activation of the acid and reaction with hydrazine,
led to the immobilized hydrazide. Condensation with an
aldehyde was followed by cleavage with CF3CO2H to
produce the targeted acyl hydrazones in excellent purity
and reasonable yield (slightly under 50% compared with
reported theoretical loading). An array of approximately
100 compounds was prepared by this methodology.
To expand the number of accessible analogues, a
solution-phase route for the preparation of acyl hydra-
zones was developed (Figure 1B). Equimolar amounts
of hydrazide and aldehyde were condensed in an alco-
holic solvent at elevated temperature for 2 h. Minor
Received February 19, 2005
Abstract: The first small molecule agonists of the estrogen-
related receptors have been identified. GSK4716 (3) and
GSK9089 (4) show binding to ERRγ with remarkable selectiv-
ity over the classical estrogen receptors. Notably, in cell-based
reporter assays, 3 mimics the protein ligand PGC-1R in
activation of human ERRâ and ERRγ.
We have pioneered the use of chemical genomics for
the functional analysis of orphan nuclear receptors
through the identification of small molecule chemical
tools using high throughput chemistry and structure-
guided design.1 Examples include the identification of
chemical tools for PPARδ,2 FXR,3 and LXR.4 In each
case the chemical tool enabled the use of reverse
endocrinology5 to identify therapeutic utility of receptor
ligands and has led to the development of new clinical
candidates.
The orphan estrogen-related receptor exists as three
subtypes (ERRR, ERRâ, and ERRγ) in the human
genome.6 ERRR is expressed in metabolically active
tissues such as muscle and adipose. ERRγ is expressed
in the spinal cord and CNS. ERRâ appears to play a
role in development, and its postnatal expression is
highly restricted, being detected only at low levels in
the liver, stomach, skeletal muscle, heart, and kidney.
These orphan nuclear receptors are most closely related
in sequence to the classical estrogen receptors ERR and
ERâ but do not bind to estradiol or other related
steroidal estrogens. In contrast, the ERRs show varying
levels of constitutive transcriptional activity, and no
endogenous ligand has been identified to date.7 Notably,
the fasting-induced cofactor PGC-1R has been reported
as an endogenous protein ligand for the ERRs,8 raising
the possibility that the receptors may not be regulated
by steroid hormones. Despite these observations, two
lines of evidence demonstrate that the ERRs still have
the potential to be pharmacologically regulated by small
molecules. First, inverse agonists (antagonists of con-
* Address correspondence to William J. Zuercher, GlaxoSmithKline,
High Throughput Chemistry, NTH-M2127, Five Moore Dr., Research
Triangle Park, NC 27709; tel:
william.j.zuercher@gsk.com.
† GlaxoSmithKline.
(919) 483-5275; e-mail:
‡ Duke University.
10.1021/jm050161j CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/12/2005

3108 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Letters
Figure 1. A. Solid-phase synthesis of acyl hydrazones. B. Solution phase synthesis of acyl hydrazones.
Table 1. ERRγ FRET Activity of Acyl Hydrazones
compd
R1
R2
R3
ERRγ FRETa EC50 (µM)
ERRγ FRET efficacy (%)b
1
-
-
-
4-OH
4-OH
4-OH
4-OH
4-H
4-NH2
4-F
4-OH
4-OH
4-OH
-
0.015
0.63
1.3
0.13
i.a.
i.a.
1.6
0.63
i.a.
-100
-100
+40
+55
-
2
-
-
3
4-iPr
H
H
H
H
H
H
H
Me
-
4
4-NEt2
2-OH, 4-NEt2
4-OtBu
4-NEt2
4-NEt2
4-NEt2
4-NEt2
4-iPr
5
6
-
7
+55
+50
-
+40
-
-
8
9
10
11
12
0.50
i.a.
i.a.
4-NEt2
-
a Recruitment of the RIP140 NR-box peptide to the ERRγ ligand binding domain; i.a. ) inactive at 3 µM; all data n ) 2 in triplicate,
variance <15%. b Change in peptide recruitment relative to basal interaction ) 0 and 1 ) -100.
amounts of unreacted starting materials were observed,
and resin bound scavenger reagents were employed for
their removal. The optimized reaction conditions rou-
tinely produce compounds in high yield and purity
following simple filtration without further purification.
The solution-phase route offers the advantage of com-
patibility with a wider range of functionality at the
aromatic acyl substituent (R2 in Table 1). Moreover, this
route allows for the synthesis of sulfonyl hydrazones and
use of ketones in the condensation reaction. Approxi-
mately 200 compounds were prepared using this solu-
tion-phase methodology.
The compounds were screened using an established
biochemical assay that monitored the recruitment of a
coactivator peptide to the purified ligand binding do-
main of ERRγ by fluorescence resonance energy transfer
(FRET).13 Analogous assays have been described in the
characterization of ERR inverse agonists.9 A subset of
the acyl hydrazones from the solid and solution phase
arrays showed activity in the ERRγ FRET assay with
an 40-55% increase in coactivator peptide recruitment
(Table 1). The ERRγ activity is highly sensitive to
substitution at R1. The optimal substituents were 4-iPr
(3) and 4-NEt2 (4) (Table 1 and data not shown).
Introduction of polarity (5) or large nonpolar groups (6)
at R1 decreased ERRγ activity. At the R1 aryl substitu-
ent, derived from the hydrazide, the nonphenolic com-
pound 7 showed reduced ERRγ activity while the
4-amino analogue 8 retained activity. While a 4-fluoro
group has been shown as a suitable replacement for the
hydroxyl group in some estrogens,14 compound 9 was
inactive on ERRγ. Small alkyl substitution on the
hydrazone (R3) was tolerated, as demonstrated by 10.
The analogous diacyl hydrazine (12) and sulfonyl hy-
drazone (13) were inactive in the ERRγ FRET assay.
As an independent measure of the activity of acyl
hydrazones, analogues 3 and 4 were assayed in a
scintillation proximity assay for competition with [3H]-
4-OHT for binding to ERRγ. The binding affinities of
were consistent with the values from the ERRγ FRET
assay, with IC50 values of 2.0 and 0.66 µM for 3 and 4,
respectively. Remarkably neither compound bound to
the classical ERR or ERâ at concentrations up to 50 µM
despite the presence of the phenol chemotype. Thus, acyl

Letters
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3109
Katso for collection of ER binding data, and Dudley Rose
for assistance in compound management. S.G. and
D.P.M. acknowledge the support of the NIH (NURSA
U19 DK062434).
Supporting Information Available: Analytical data and
experimental protocols described in this letter. This material
is available free of charge at http://pubs.acs.org.
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Acknowledgment. We thank Randy Bledsoe and
Thomas Consler for generation of assay reagents, Roy
JM050161J