Identification of a selective inverse agonist for the orphan nuclear receptor estrogen-related receptor α

Article abstract of DOI:10.1021/jm049334f

The estrogen-related receptor α (ERRα) is an orphan receptor belonging to the nuclear receptor superfamily. The physiological role of ERRα has yet to be established primarily because of lack of a natural ligand. Herein, we describe the discovery of the fi

Full text of DOI:10.1021/jm049334f

© Copyright 2004 by the American Chemical Society
Volume 47, Number 23
November 4, 2004
Letters
Identification of a Selective Inverse
Agonist for the Orphan Nuclear Receptor
Estrogen-Related Receptor r
Brett B. Busch,§ William C. Stevens, Jr.,§
Richard Martin,§ Peter Ordentlich,‡ Sihong Zhou,‡
Douglas W. Sapp,‡ Robert A. Horlick,‡ and
Raju Mohan*,§
Figure 1. Reported structure of the HTS lead compound.
muscle mitochondria and whole-body oxygen consump-
tion. In contrast, exercise improves mitochondrial pro-
liferation. In recent publications, changes in the expres-
sion of genes involved in oxidative phosphorylation have
been found in individuals with NIDDM and it has been
suggested that a nuclear receptor coactivator, peroxi-
some proliferator-activated receptor γ coactivator-1
(PGC-1), is responsible for orchestrating these genetic
modifications.5 This implicates PGC-1 as an important
regulator of energy metabolism through metabolic regu-
lation in skeletal muscle and the liver. PGC-1 is a potent
regulator of ERRR,6-8 and it is postulated that ligands
modulating ERRR activity should regulate oxidative
phosphorylation and increase mitochondrial respiration,
which is severely compromised in type II diabetes.9
ERRR is a constitutively active receptor because it can
function as a transcriptional activator in the absence
of any added ligand.10 Despite its high homology with
ERs, ERRR does not respond to estradiol and no
endogenous ligands have been reported to date. While
ERRR was the first orphan nuclear receptor identified
over a decade ago,11 the lack of a chemical tool has
severely hampered the ability to associate its function
with endocrine physiology, especially in the regulation
of energy metabolism and type II diabetes. Our goal was
to identify a potent and selective ligand for ERRR and
establish the biology and physiological relevance of this
receptor via reverse endocrinology.12
Departments of Medicinal Chemistry and Lead Discovery,
X-Ceptor Therapeutics, Inc., 4757 Nexus Center Drive,
San Diego, California 92121
Received August 11, 2004
Abstract: The estrogen-related receptor R (ERRR) is an
orphan receptor belonging to the nuclear receptor superfamily.
The physiological role of ERRR has yet to be established
primarily because of lack of a natural ligand. Herein, we
describe the discovery of the first potent and selective inverse
agonist of ERRR. Through in vitro and in vivo studies, these
ligands will elucidate the endocrine signaling pathways medi-
ated by ERRR including association with human disease
states.
The estrogen-related receptors (ERRs R,â,γ) structur-
ally and functionally belong to the subfamily of estrogen
receptors (ERs), and recent discoveries suggest that the
ERR and ER families may be more closely related than
previously thought. Such evidence includes findings that
ERRs have a role in controlling proliferation and dif-
ferentiation of cells including osteoclasts/osteoblasts and
cells implicated in mammary carcinoma.1 ERRR is
principally expressed in tissues involved in fatty acid
metabolism, and ERR target genes include genes in-
volved in energy metabolism.2,3 Recent experiments
have also demonstrated that deletion of ERRR results
in mice that are lean.4
Our work was initiated by the high-throughput
screening (HTS) of our compound library and the
identification of thiadiazolopyrimidinone 1a as an ERRR
inverse agonist.13 Compound 1a was found using a
fluorescence polarization (FP) assay, which measures
the ligand-dependent displacement of a labeled steroid
receptor coactivator-1 (SRC-1) fragment peptide from
ERRR. The selection of the SRC-1 peptide followed the
Symptoms of type II diabetes (non-insulin-dependent
diabetes mellitus (NIDDM)) include a reduction in
energy metabolism that is in part due to a decrease in
* To whom correspondence should be addressed. Phone: 858-458-
4537. Fax: 858-458-4501. E-mail: rmohan@x-ceptor.com.
§ Department of Medicinal Chemistry.
‡ Department of Lead Discovery.
10.1021/jm049334f CCC: $27.50 © 2004 American Chemical Society
Published on Web 10/01/2004

5594 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Letters
Scheme 1
Scheme 2
FP assay of 15 peptides comprising individual LxxLL
motifs from nuclear receptor coactivators including
SRC-1, SRC-2, SRC-3, TIF1, and CBP. The greatest
signal-to-noise was achieved with the ILRKLLQE pep-
tide motif from SRC-1. By use of the FP assay, 1a was
found to exhibit an IC50 of 1.4 µM. The activity of the
compound was confirmed in a cell-based cotransfection
assay using the Gal4-ERR format and displayed an IC50
of 2.3 µM.
Table 1. Analogues of the ERRR Inverse Agonist Lead
We synthesized several batches of pyrimidinone 1a
using the reported methodology shown in Scheme 114
and several closely related analogues where the R1
group of the thiadiazole ring was varied. We were able
to obtain crystals of the tert-butyl analogue 1b for X-ray
diffraction. To our surprise, the structure indicated that
the product obtained from our synthetic sequence was
the monocyclic thiadiazoloacrylamide 2b and not the
thiadiazolopyrimidinone 1b (Scheme 1). Subsequent IR
analyses of other thiadiazoleacrylamides showed the
characteristic nitrile stretch in the region 2260-2220
ERRR Gal4
IC50 (µM)a
% inhibition
compd
R
X
Z
(10 µM)
2a
2b
2c
2d
2e
2f
Et
N
S
S
S
S
S
S
S
O
2.0
1.7
2.6
1.5
2.0
1.4
NAb
3.3
96
104
92
112
74
103
tBu
MeS
CF3
N
N
N
p-Me2NPh
N
H
N
2g
2h
H
CH
N
Et
88
a Unless otherwise specified, IC50 values were generated by
nonlinear regression from titration curves of compounds from 10
doses and reported as an average of at least two experiments.
Standard error of the mean was typically less than 30% for each
experiment. b NA: not active.
cm-1
.
We believe that the reported methodology15 to prepare
the thiadiazolopyrimidinone precursor 3 from the 2-alkyl-
5-amino[1,3,4]thiadiazole derivatives 5 provides only the
transamidation product 4. The spectral data for the
resynthesized HTS hit 1a were identical to the com-
mercially obtained compound, suggesting that the struc-
ture of the commercial compound was also assigned
incorrectly. Indeed, our thorough perusal of literature
data on the synthesis and characterization of these
compounds led us to the conclusion that our assign-
ments are consistent with the open acrylamide struc-
tures 2a,b. With the correct lead structure elucidated,
we initiated lead optimization to develop potent, selec-
tive, and orally bioavailable ERRR inverse agonists.
Thiadiazoloacrylamide analogues reported in this
work were prepared by the synthetic route shown in
Scheme 2. The 2-alkyl-5-amino[1,3,4]thiadiazole deriva-
tives 5 were commercially available or readily prepared
from acid chlorides and thiosemicarbazide, via cyclode-
hydration of the acylthiosemicarbazide intermediate
under acidic conditions.16 The thiadiazoloacetamides 4
were prepared from the amino thiadiazoles 5 and ethyl
cyanoacetate in a refluxing MeONa/MeOH mixture.
Standard alkylation conditions were used to prepare the
chloroethyl ether derivative 6, which was reacted with
vanillin 7 to provide the benzaldehyde 8. Thiadiazolo-
acrylamides 2a-f were synthesized by Knoevenagel
condensation of the benzaldehyde analogue 8 with the
cyanoacetamide 4 under basic or acidic conditions using
triethylamine or acetic acid, respectively. The thiazole
analogue 2g and the oxadiazole analogue 2h were
prepared from a similar sequence using commercially
available 2-ethyl-5-aminothiazole and 2-ethyl-5-ami-
nooxadiazole, respectively. On the basis of the X-ray
structure of 2b, only the E regioisomer is observed under
our condensation and purification conditions. The screen-
ing results for synthetic compounds are shown in Table
1.
Variation of the R group had little effect on ERRR
t
potency. For example, 2b, R ) Bu (IC50 ) 1.7 µΜ),
exhibited potency similar to that of 2f, R ) H (IC50
)
1.4 µM). Compound 2g was found to be inactive,
potentially showing the importance of the endocyclic
3-postion nitrogen to ERRR activity. The oxadiazole 2h
was comparable in activity to the corresponding thia-
diazole 2a.
In earlier efforts toward lead validation, we had
established that the vanillin core is beneficial for ERRR

Letters
Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23 5595
Table 3. Data for Library Resynthesis Compounds
Scheme 3
ERRR Gal4
IC50 (µM)a
% inhibition
(10 µM)
compd
R
10a
10b
10c
10g
10h
2,4-CF3
0.61
1.7
104
86
5-F, 2-CF3
4-F, 2-CF3
2,5-CF3
1.5
90
NAb
NAb
NAb
NAb
2-F, 6-CF3
a Unless otherwise specified, IC50 values were generated by
nonlinear regression from titration curves of compounds from 10
doses and reported as an average of at least two experiments.
Standard error of the mean was typically less than 30% for each
experiment. b NA: not active under assay conditions.
Table 2. Data for Selected Library Analogues
ERRR Gal4
Table 4. Functionalization of the Thiadiazole Core
% inhibition
(10 µM)
compd
R
IC50 (µM)a
10a
10b
10c
10d
10e
10f
2,4-CF3
0.4
106
112
109
101
90
5-F, 2-CF3
4-F, 2-CF3
2-OCF3
0.80
0.92
1.8
1-naphthyl
2-Br
1.8
2.2
57
ERRR Gal4
IC50 (µM)a
% inhibition
a Unless otherwise specified, IC50 values were generated by
nonlinear regression from titration curves of compounds from 10
doses and reported as an average of at least two experiments.
Standard error of the mean was typically less than 30% for each
experiment.
compd
R
(10 µM)
10a
11
12
13
14
15
Et
Ph
0.54
0.50
99
105
109
99
105
43
CF3
H
0.37
0.75
MeS
0.44
tBu
>10
activity. We therefore decided to explore the SAR
around the ether linkage via a focused library derived
from an alkylation-condensation sequence as shown in
Scheme 3.
a Unless otherwise specified, IC50 values were generated by
nonlinear regression from titration curves of compounds from 10
doses and reported as an average of at least two experiments.
Standard error of the mean was typically less than 30% for each
experiment.
The target compounds 10 were synthesized via a two-
step procedure starting from vanillin 7. Vanillin was
alkylated with commercially available alkyl and benzyl
halides (185 reagents including R-halo ketones, esters,
and acetamides) to provide aldehydes 9, which were
then condensed with the thiadiazoloacetamide 4 to
provide the target compounds 10. Excess alkylating
agents and excess aldehydes 9 were scavenged using
silica gel based thiol and amino resins, respectively.
The samples were purified using reverse-phase HPLC
and were quantified using evaporative light scattering
detection to afford 2-5 mg of 100 discrete vanillin ethers
with a purity criteria of >85% pure by HPLC-MS. The
samples were formatted to a 10 µM concentration in
DMSO and assayed at a single 3 µM concentration in
the Gal4-ERRR assay. Dose response was carried out
on any compound exhibiting >50% inhibition at 3 µM.
Table 2 shows data for selected library compounds.
The three most potent compounds identified from the
library were the 2-(trifluoromethy)benzyl derivatives
10a-c, and these were resynthesized on a 10 mg scale
for full analytical and biological characterization. In
addition, two related trifluoromethyl analogues 10g and
10h (not originally synthesized in the library) were also
prepared. ERRR inverse agonist activity was measured
in the Gal4-ERRR assay, and the data are shown in
Table 3.
Figure 2. Inverse agonist activity of 12 in GAL4-ERRR cell-
based transfection assay.
we then decided to optimize the R groups on the
thiadiazole ring. To this end we selected functional
groups identified in the initial set of compounds 2a-h
as shown in Table 1 and incorporated them into
template 10 using procedures developed for the library
synthesis.
As shown in Table 4, a wide range of functionality is
tolerated on the thiadiazole ring. The trifluoromethyl
analogue 12 displays the highest potency and inverse
agonist activity in the cell-based GAL4-ERRa transfec-
tion assay (Figure 2). Additionally, 12 was inactive
against ERRγ and the estrogen receptors ERR and ERâ.
When profiled for cross-reactivity against a broader
panel of nuclear receptors, 12 was also highly selective
except for weak agonist activity against PPARγ (EC50
) 1.3 µM; 15% efficacy vs agonist control).
The bis-trifluoromethyl analogue 10a was recon-
firmed as the most potent analogue from the ether
library with a 4-fold improvement in potency over the
initial lead 2a. Analogues 10b and 10c were weaker as
resynthesized compounds, while 10g and 10h were
inactive.
In summary, thiadiazoleacrylamide 12 (XCT790)
represents the first potent inverse agonist for ERRR.
Identification of a ligand for an orphan nuclear receptor
Having optimized the ether linkage on the lead
compound with the bis-trifluoromethyl analogue 10a,

5596 Journal of Medicinal Chemistry, 2004, Vol. 47, No. 23
Letters
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is a significant milestone that should allow for reverse
endocrinology studies directed toward understanding
the biology of ERRR. Using XCT790, we have demon-
strated that ERRR inverse agonists alter the ERRR/
PGC-1 signaling pathways and are currently validating
ERRR as a target for the regulation of energy metabo-
lism and type II diabetes (results published else-
where).6,17 Discovery of ligands specific to ERRR will
help to elucidate the role of ERRR in the endocrine and
metabolic signaling pathways. Through the identifica-
tion of novel ligands such as 12 (XCT790), it is likely
that new classes of selective ERRR modulators will be
identified. As in the case of other orphan nuclear
receptors, these ligands may eventually lead to the
discovery of new drugs targeting ERRR and as potential
new therapeutics for diabetes and cancer.
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Acknowledgment. The authors are grateful to Mr.
Tao Wang for the pharmacokinetic data and mass
spectrometry assistance and to Ms. Robin Felts-Sum-
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Supporting Information Available: Experimental pro-
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HRMS) for cyanoacetamide 4a, benzaldehydes 8 and 9a, and
thiadiazoloacrylamides 2a-h and 11-16, X-ray structure data
for 2b, general procedures for the library synthesis and
resynthesis of identified active compounds from the thiadiazole
library 10a-f, and protocols for the ERRR FP and cotrans-
fection assays. This material is available free of charge via
the Internet at http://pubs.acs.org.
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JM049334F