Identification of pyrido[1,2-α]pyrimidine-4-ones as new molecules improving the transcriptional functions of estrogen-related receptor α

Article abstract of DOI:10.1021/jm200976s

The nuclear estrogen-related receptor α (ERRα) plays a central role in the regulation of expression of the genes involved in mitochondrial biogenesis and oxidative metabolism. We have successfully identified a series of pyrido[1,2-a]pyrimidin-4-ones as new agonists enhancing the transcriptional functions of ERRα. The compounds potently elevated the mRNA levels and the protein levels of ERRα downstream targets. Consequently, the compounds improved the glucose and fatty acid uptake in C2C12 muscle cells.

Full text of DOI:10.1021/jm200976s

BRIEF ARTICLE
pubs.acs.org/jmc
Identification of Pyrido[1,2-α]pyrimidine-4-ones as New Molecules
Improving the Transcriptional Functions of Estrogen-Related Receptor α
||
Lijie Peng,†,‡,|| Xuefei Gao,†, Lei Duan,† Xiaomei Ren,†,§ Donghai Wu,*,† and Ke Ding*,†
†Key Laboratory of Regenerative Biology and Institute of Chemical Biology, Guangzhou Institutes of Biomedicine and Health,
Chinese Academy of Sciences, No. 190 Kaiyuan Avenue, Guangzhou 510530, China
‡Graduate School of Chinese Academy of Sciences, No. 19 Yuquan Road, Beijing 100049, China
§Zhongshan Medical School, Sun Yat-Sen University, No. 74 2nd Zhongshan Road, Guangzhou 510080 China
S Supporting Information
b
ABSTRACT: The nuclear estrogen-related receptor α (ERRα) plays a central role in the regulation of expression of the genes
involved in mitochondrial biogenesis and oxidative metabolism. We have successfully identified a series of pyrido[1,2-a]pyrimidin-
4-ones as new agonists enhancing the transcriptional functions of ERRα. The compounds potently elevated the mRNA levels and
the protein levels of ERRα downstream targets. Consequently, the compounds improved the glucose and fatty acid uptake in
C2C12 muscle cells.
he estrogen-related receptors (ERRs) belong to an orphan
PGC-1α or RIP-140, etc.15,20 The ligand binding domains of
ERRα is highly occupied by side chains, making it difficult to
accommodate synthetic ligands without disrupting its constitu-
tively active conformation. Although several classes of ERRα inverse
agonists have been identified by high-throughput screening,14À18
small molecular agonists of ERRα are lacking19 likely because of
the tiny binding pocket of the ERRα ligand binding domain
(LBD)20,21 (Figure 1, 1À5). The failure to identify good ERRα
agonists has disappointed many scientists, suggesting that
“ERRα is an intractable molecular target”21 for designing direct
small-molecule ERRα agonists.
Structural feature analysis on the reported ERR modulators
clearly revealed that almost all of the known ERR modulators
possessed a carbonyl group or its isosteric moieties (Figure 1).
An X-ray crystallographic study had also demonstrated that the
carbonyl group contributed greatly to the binding of GSK4716
(Figure 1, 6) with the ERRγ LBD domain by interacting with
Arg-316 in ERRγ.22,23 Therefore, a carbonyl focused library with
diverse scaffolds was constructed for identifying new ERRα
agonists. In this paper, we report the identification of pyrido-
[1,2-a]pyrimidin-4-ones as new molecules improving the tran-
scriptional functions of ERRα by screening our self-constructed
carbonyl focused library.
T
nuclear receptor subfamily.1 Three isoforms of ERRs (ERRα,
-β, and -γ)2 have been identified with distinct expression profiles
and diverse biofunctions.3 ERRα is predominately expressed in
metabolically active tissues such as muscle and adipose and plays
a central role in the regulation of metabolism and energy
homeostasis.3 ERRβ is associated with early development,4 and
its postnatal expression is highly restricted. It could be detected
only at low level in liver, stomach, skeletal muscle, kidney, etc.
Although the exact biological functions of ERRγ still remain
elusive, it is widely expressed in a number of adult human and
mouse tissues, including spinal cord and central nervous system.5
Genetic and functional analysis have demonstrated that ERRα
is a critical regulator of mitochondrial biogenesis and plays a
central role in the regulation of expression of oxidative metabo-
lism genes6 by interacting with transcriptional cofactor PGC-1
(peroxisome proliferator-activated receptor γ coactivators)7 and
RIP-140 (nuclear receptor-interacting protein 140),8 etc. The
downstream target genes of ERRα include MACD (medium-
chain acyl-coenzyme A dehydrogenase),9 PDK4 (pyruvate dehy-
drogenase kinase 4),10,11 ATP5b (ATP synthase 5b),12 and other
key components of oxidative metabolism. Therefore, ERRα has
been considered a novel potential drug target and selective ERRα
agonists might be developed as new therapeutic agents for the
treatment of type II diabetes and other metabolic disorders.13
However, recent results from Patch et al. revealed that a diaryl
ether-based inverse agonist of ERRα potently normalized the
serum triglyceride and insulin levels and improved glucose
tolerance both in diet-induced murine models and in an overt
diabetic rat model.14 These disagreements indicate the complex-
ity of ERRα regulatory pathways. It is highly desired to identify
new ERRα agonists and inverse agonists as chemical probes to
further validate ERRα as a new drug target for metabolic diseases.
X-ray crystallographic studies revealed that ERRα possesses
constitutively active conformations to interact with coactivator
’ CHEMISTRY
The simply substituted pyrido[1,2-a]pyrimidin-4-ones were
synthesized by direct condensation/cyclization of 2-aminopyridines
with substituted β-ketone esters in PPA (polyphosphorus acid) at
100 °C. For the 7-phenyl, 8-phenyl, 7-cyclopropyl, or 8-cyclopropyl
analogues, they were prepared by a palladium catalyzed Suzuki
coupling reaction of 7- or 8-bromopyrido[1,2-a]pyrimidin-4-one
with phenylboronic acid (11) or cyclopropyl acid (10) (Scheme 1).
Received: July 21, 2011
Published: September 29, 2011
r
2011 American Chemical Society
7729
dx.doi.org/10.1021/jm200976s J. Med. Chem. 2011, 54, 7729–7733
|

Journal of Medicinal Chemistry
BRIEF ARTICLE
Figure 2. Pyrido[1,2-a]pyrimidin-4-ones potently agonize the tran-
scriptional function of ERRα. (A) 7aÀg agonize the transcriptional
function of ERRα at 10 μM, while 7j and 7k are inverse agonists of
ERRα. (B) 7b elevates the transcriptional function of ERRα in a dose
dependent manner. Results are the means of at least three independent
experiments (//, p < 0.01).
Figure 1. Chemical structures of reported small molecular modulators
of ERRs: 1À4, ERRα inverse agonists; 5, ERRα agonist; 6, ERRγ
agonist.
replaced by an ethyl, isopropyl or cyclopropyl group, the bioactivity
was obviously improved, which elevated the transcriptional
function of ERRα by 2.7-, 2.5-, and 2.6-fold at 10.0 μM, respec-
tively (Figure 2A, 7bÀd). However, when the 8-position was
substituted by a tert-butyl (7e) or a phenyl (7f), the potency was
decreased. The removal of the 8-methyl group in 7a did not lead
to obvious change of the bioactivity (7g). The results also demon-
stratedthat a substituent at the 3-position might be detrimental to
its agonistic function on ERRα; the 3-methyl (7h) and 3-ethyl
(7i) compounds were obviously less potent than the correspond-
ing 7b. Interestingly, when the hydrophobic group was merged to
the 7-position from the original 8-position, the resulting com-
pounds (7j and 7k) became reverse agonists of ERRα, which
inhibited the transcriptional function of ERRα (Figure 2A).
Further investigation demonstrated that 7b improved the transcrip-
tional function of ERRα in a dose-dependent manner (Figure 2B).
The investigation also demonstrated that 7b effectively elevated
the ERRα-driven luciferase activity in kaempferol pretreated
293FT cells, in which the basal constitutive activity of ERRα was
reduced (Supporting Information). These results further suggested
the agonizing effect of 7b on ERRα.
Scheme 1. Synthesis of Pyrido[1,2-a]pyrimidin-4-one
Derivatives
The selectivity of the new ERRα agonists over other nuclear
receptors (i.e., ERRβ, ERRγ, ERα, and ERβ (estrogen receptor
α and estrogen receptor β)) was also investigated. Not surpris-
ingly, 7b also moderately elevated the transcriptional functions of
ERRγ. However, the compound did not obviously affect the
function of ERRβ in a similar gene reporter assay. Different from
the fact that kaempferol strongly agonized the functions of ERα
and ERβ,18 7b did not show effects on ERα or ERβ at 20.0 μM
(Supporting Information).
Taking 7b as an example, the agonizing effect of pyrido[1,2-a]-
pyrimidin-4-ones on ERRα was further validated by investigating
the expression changes of ERRα targeted genes. MCAD, PDK4,
and ATP5b are key components of oxidative metabolism, and the
transcription of these genes are regulated by transcriptional
factor ERRα. Highly consistent with our primary reporter
screening results (Figure 2), 7b obviously increased the mRNA
levels of MCAD, PDK4, and ATP5b in a dose dependent manner
monitored in a quantitative real-time PCR assay (parts A, B, and
C of Figure 3). Not surprisingly, the protein levels of PDK4
and ATP5b were also elevated as determined by a Western blot
analysis (Figure 3D) after a 24 h treatment.
’ RESULTS AND DISCUSSION
The compounds were primarily screened by using a well
established cell-based reporter gene assay to monitor their regula-
tory functions on the transcriptional activity of ERRα.14À19,22
The heterologous reporter assay was performed in 293FT cells
by transient transfection with a vector expressing LBD domain of
human ERRα fused to Gal4 DNA binding domain and a reporter
plasmid containing firefly luciferase gene. A well characterized
ERRα inverse agonist kaempferol18 was used as a reference
compound to validate the screening conditions.
Among all of the compounds tested, 8-methyl-2-phenyl-4H-
pyrido[1,2-a]pyrimidin-4-one (7a, Figure 2) effectively enhanced
the ERRα-driven luciferase activity at 10.0 μM after a 24 h
incubation, indicating that 7a was an agonist that up-regulated
the transcriptional functions of ERRα (Figure 2A). By use of 7a
as a lead compound, new pyrido[1,2-a]pyrimidin-4-one deriva-
tives were designed, synthesized, and evaluated (Scheme 1). The
results suggested that the 8-methyl group could be replaced by a
slightly larger hydrophobic group to achieve a better agonizing
effect on ERRα. For instance, when the 8-methyl group was
ERRα plays a central role in mitochondrial biogenesis and
oxidative metabolism. Theoretically, a cell permeable small molecule
7730
dx.doi.org/10.1021/jm200976s |J. Med. Chem. 2011, 54, 7729–7733

Journal of Medicinal Chemistry
BRIEF ARTICLE
Figure 4. Compound 7b improves the insulin-stimulated glucose uptake
and fatty acid uptake in well differentiated C2C12 mouse muscle cells.
(A) 7b dose-dependently improves the insulin-stimulated glucose
uptake. (B) 7b improves the fatty acid uptake in dose dependent man-
ner. Results are the mean of at least three independent experiments
(/, p < 0.05; //, p < 0.01).
phenylboronic acid (0.55 mmol), Cs2CO3 (1.0 mmol), and PdCl2-
(dppf) (0.05 mmol) in dioxane (2.0 mL) was stirred at 80 °C under an
argon atmosphere. The mixture was cooled to room temperature after
complete consumption of starting material as monitored by TLC. Water
(10 mL) and EtOAc (10 mL) were added to the reaction mixture. The
organic phase was separated, and the aqueous phase was further extracted
with EtOAc (10 mL). The combined organic layers were dried over
anhydrous Na2SO4 and concentrated. The residue was purified by flash
chromatography to provide the desired product.
Figure 3. Compound 7b potently up-regulates the transcription of
ERRα genes. (AÀC) 7b dose-dependently increased the mRNA levels
of MCAD, PDK4, and ATP5b in a quantitative real-time PCR assay. (D)
7b elevates the protein levels of PDK4 and ATP5b in dose dependent
manner determined by a Western blot analysis. Results are representa-
tive and the mean of at least three independent experiments (/, p < 0.05;
//, p < 0.01).
2,8-Diphenyl-4H-pyrido[1,2-a]pyrimidin-4-one (7f). 1H NMR
(400 MHz, DMSO-d6) δ 9.00 (d, J = 7.6 Hz, 1 H), 8.26À8.21 (m, 2 H),
8.07 (d, J = 2.0 Hz, 1 H), 8.02À8.00 (m, 2 H), 7.77 (dd, J = 2.0, 7.6 Hz, 1
H), 7.62À7.53 (m, 6 H), 6.98 (s, 1 H); 13C NMR (125 MHz, DMSO-
d6) δ 160.7, 157.5, 151.0, 147.7, 136.7, 135.2, 130.7, 130.3, 129.4, 128.7,
127.4, 127.2, 127.2, 121.7, 114.7, 98.4. HRMS (ESI): exact mass calcd
for C20H14N2O [M + H]+, 299.1179, found 299.1182. Anal. (C20H14-
N2O) C, H, N.
Procedure for Synthesis of 7d and 7j. To a solution of 7-bromo
or 8-bromo 2-phenyl-4H-pyrido[1,2-a]pyrimidin-4-one (0.5 mmol),
cyclopropylboronic acid (0.6 mmol), potassium phosphate (1.75 mmol),
and tricyclohexylphosphine (0.05 mmol) in toluene (2.0 mL) and water
(100 uL) under a nitrogen atmosphere was added palladium acetate
(0.025 mmol). The mixture was heated to 100 °C for 3 h and then
cooled to room temperature. Water (5 mL) was added and the aqueous
portion extracted with EtOAc (2 Â 10 mL). The combined organics
were washed with brine (10 mL), dried over MgSO4, and concentrated
in vacuo. The residue was purified by column chromatography to afford
the desired compounds.
ERRα agonist would enhance the lipid metabolism and improve
the glucose and fatty acid uptake in muscle cells.24À26 Therefore,
the influence of 7b on glucose and fatty acid uptake was also
investigated in well differentiated C2C12 mouse muscle cells.
Indeed, 7b moderately improved the glucose and fatty acid uptake in
C2C12 muscle cells in a dose-dependent manner (Figure 4A,B).
In summary, pyrido[1,2-a]pyrimidin-4-ones were successfully
identified as new small molecular agonists of ERRα receptor.
Although the precise mechanism remains elusive, our study
demonstrated that pyrido[1,2-a]pyrimidin-4-ones could en-
hance the transcription of ERRα downstream target genes and
improve the glucose and fatty acid uptake in C2C12 muscle cells.
Our results may provide an interesting basis for further validation
of ERRα as a new drug target for metabolic diseases.
’ EXPERIMENTAL SECTION
8-Cyclopropyl-2-phenyl-4H-pyrido[1,2-a]pyrimidin-4-one
(7d). H NMR (400 MHz, DMSO-d6) δ 8.83 (d, J = 7.2 Hz, 1 H),
1
General Procedure for Synthesis of Substituted Pyrido-
[1,2-a]pyrimidin-4-ones 7aÀc,e,gÀi. A mixture of 2-aminopyr-
idine 8 (1.00 mmol) and the substituted β-keto ester 9 (1.50 mmol) in
polyphosphorus acid (2.00 g) was heated at 100 °C with vigorously
stirring. After 1.0 h, the mixture was cooled in an ice bath and neutralized
with 5% aqueous sodium hydroxide. The solid precipitate was collected
by filtration, washed with water. The crude products were purified by
recrystallization from ethanol.
8.20À8.15 (m, 2 H), 7.53À7.50 (m, 4 H), 7.04 (dd, J = 2.0, 7.6 Hz, 1 H),
6.86 (s, 1 H), 2.21À2.15 (m, 1 H), 1.22À1.17 (m, 2 H), 1.03À0.99 (m, 2
H); 13C NMR (125 MHz, DMSO-d6) δ 160.7, 157.6, 155.9, 150.5,
136.8, 130.5, 128.6, 127.1, 126.6, 120.3, 114.2, 97.3, 15.1, 11.1. HRMS
(ESI): exact mass calcd for C17H14N2O [M + H]+, 263.1179, found
263.1176. Anal. (C17H14N2O) C, H, N.
Cell Lines and Cell Culture. See Supporting Information.
8-Methyl-2-phenyl-4H-pyrido[1,2-a]pyrimidin-4-one (7a).
1H NMR (400 MHz, CDCl3) δ 8.90 (d, J = 7.2 Hz, 1 H), 8.06À8.03 (m,
2 H), 7.49À7.43 (m, 4 H), 6.90 (dd, J = 1.6, 7.2 Hz, 1 H), 6.80 (s, 1 H),
2.43 (s, 3 H); 13C NMR (100 MHz, CDCl3) δ 162.1, 158.5, 151.0, 148.3,
137.4, 130.5, 128.7, 127.4, 126.5, 124.7, 117.8, 99.1, 21.3. HRMS (ESI):
exact mass calcd for C15H12N2O [M + H]+, 237.1022, found 237.1024.
Anal. (C15H12N2O) C, H, N.
Transient Transfection and Dual Luciferase Reporter As-
say. 293FT cells were seeded in 96-well plate at a density of 10 000
cells/well in DMEM containing 10% FBS. At 18À24 h after plating,
transit transfections were performed with Lipofectamine 2000 (Invitrogen)
according to the manufacturer’s instruction. Total DNA for transfec-
tions included Gal4-DBD-ERRα-LBD vector (0.5 ng), pcDNA-PGC-
1α vector (0.5 ng), pFRlaczeo vector (25 ng), and internal control
vector pRLTK renilla (3 ng). After cells were transfected for 6 h, com-
pounds were then added for an additional 24 h. Luciferase activity was
Procedure for Synthesis of 7f and 7k. A mixture of 7-bromo
or 8-bromo 2-phenyl-4H-pyrido[1,2-a]pyrimidin-4-one (0.5 mmol),
7731
dx.doi.org/10.1021/jm200976s |J. Med. Chem. 2011, 54, 7729–7733

Journal of Medicinal Chemistry
BRIEF ARTICLE
measured by dual luciferase reporter assay system (Promega) according
to the manufacturer’s instruction on a Veritas microplate luminometer.
Relative luciferase units were the ratio of the absolute activity of firefly
luciferase to that of renilla luciferase. The experiment was done in triplicate,
and results are representatives of at least three independent experiments.
Western Blot Analysis. For myotube formation, C2C12 myo-
blasts at ∼100% confluence was changed to DMEM supplemented with
2% donor equine serum and the cells were maintained for an additional
5À7 days. During myotube formation, the medium was changed every
day. Then cells were treated with 7b of indicated concentration or vehicle
control. After an indicated period of time, Western blot was carried out
according to the protocol provided by Cell Signaling Technology Ltd.
Real-Time PCR Analysis. RNA isolation and real-time analysis
were performed according to a previous report.27 Briefly, total RNA was
isolated from C2C12 myocytes pretreated with 7b for 20 h using Trizol
reagent (Invitrogen), and isolated total RNA was reverse-transcribed
with Superscript III reverse transcriptase (Invitrogen). For real-time
PCR analysis, cDNA samples were used in quantitative PCR reaction in
the presence of fluorescent dye Cybergreen (Bio-Rad, Benicia, CA, U.S.).
The following PCR conditions were applied: 5 min, 95 °C; 40 Â (10 s,
95 °C; 20 s, 60 °C; 1 s, 70 °C). After each elongation step, the reaction
was quantified in a reading step and the product quality tested by melting
curve analysis. Relative abundance of mRNA was calculated after
normalization to Gapdh mRNA. Sequences for the primers used in this
study are shown in the table in Supporting Information.
Analysis of Cellular Glucose and Fatty Acid Uptake. After a
24 h treatment of 7b, C2C12 myocytes were glucose-starved for 10 min
in Krebs Ringer Hepes (KRH) buffer (pH 7.5) containing 120 mM
NaCl, 4.7 mM KCl, 2.2 mM CaCl2, 10 mM Hepes, 1.2 mM KH2PO4,
1.2 mM MgSO4, 0.1% BSA. Fifteen minutes after incubation with insulin
(100 nM), tracer [1,2-3H]-2-deoxy-D-glucose (0.93 GBq/mmol, Amer-
sham Pharmacia, Buckinghamshire, U.K.) was then added for 15 min
and glucose uptake was assayed in triplicate for each condition. Fatty
acid uptake assays were initiated by incubating cells for 20 min in KRH
buffer containing 5.4 mM glucose and [1-14C]oleic acid (2.04 GBq/
mmol, Amersham Pharmacia, Buckinghamshire, U.K.) bound to fatty
acid-free BSA. The cells were washed and cellular incorporated [3H] and
[14C] radioactivity was determined by liquid scintillation counting. The
abundance of radioactivity was normalized to protein content.
’ ABBREVIATIONS USED
PCR, polymerase chain reaction; DMEM, Dulbecco’s modified
Eagle medium; FBS, fetal bovine serum; DNA, deoxyribobucleic
acid; cDNA, complementary DNA; RNA, ribobucleic acid; mRNA,
messenger RNA; BSA, bovine serum albumin
’ REFERENCES
(1) Mangelsdorf, D. J.; Thummel, C.; Beato, M.; Herrlich, P.; Schutz,
G.; Umesono, K.; Blumberg, B.; Kastner, P.; Mark, M.; Chambon, P.;
Evans, R. M. The nuclear receptor superfamily: the second decade.
Cell 1995, 83, 835–839.
(2) (a) Giguꢀere, V.; Yang, N.; Segui, P.; Evans, R. M. Identification
of a new class of steroid hormone receptors. Nature 1988, 331, 91–94.
(b) Eudy, J. D.; Yao, S.; Weston, M. D.; Ma-Edmonds, M.; Talmadge,
C. B.; Cheng, J. J.; Kimberling, W. J.; Sumegi, J. Isolation of a gene
encoding a novel member of the nuclear receptor superfamily from the
critical region of Usher syndrome type IIa at 1q41. Genomics 1998,
50, 382–384.
(3) (a) Bookout, A. L.; Jeong, Y.; Downes, M.; Yu, R. T.; Evans,
R. M.; Mangelsdorf, D. J. Anatomical profiling of nuclear receptor
expression reveals a hierarchical transcriptional network. Cell 2006,
126, 789–799. (b) Yang, X.; Downes, M.; Yu, R. T.; Bookout, A. L.; He,
W.; Straume, M.; Mangelsdorf, D. J.; Evans, R. M. Nuclear receptor
expression links the circadian clock to metabolism. Cell 2006, 126, 801–810.
(4) Luo, J.; Sladek, R.; Bader, J. A.; Matthyssen, A.; Rossant, J.;
Giguꢀere, V. Placental abnormalities in mouse embryos lacking the
orphan nuclear receptor ERR-β. Nature 1997, 388, 778–782.
(5) Heard, D. J.; Norby, P. L.; Holloway, J.; Vissing, H. Human
ERRgamma, a third member of the estrogen receptor-related receptor
(ERR) subfamily of orphan nuclear receptors: tissue-specific isoforms
are expressed during development and in the adult. Mol. Endocrinol.
2000, 14, 382–392.
(6) Recent reviews on ERRa: (a) Giguꢀere, V. Transcriptional control
of energy homeostasis by the estrogen-related receptors. Endocr. Rev.
2008, 29, 677–696. (b) Villena, J. A.; Kralli, A. ERRα: a metabolic
function for the oldest orphan. Trends Endocrinol. Metab. 2008, 19,
269–276.
(7) (a) Huss, J. M.; Kopp, R. P.; Kelly, D. P. Peroxisome proliferator-
activated receptor coactivator-1 α (PGC-1α) coactivates the cardiacen-
riched nuclear receptors estrogen-related receptor-α and -γ. Identifica-
tion of novel leucine-rich interaction motif within PGC-1α. J. Biol. Chem.
2002, 277, 40265–40274. (b) Schreiber, S. N.; Knutti, D.; Brogli, K.;
Uhlmann, T.; Kralli, A. The transcriptional coactivator PGC-1 regulates
the expression and activity of the orphan nuclear receptor ERRα. J. Biol.
Chem. 2003, 278, 9013–9018. (c) Huss, J. M.; Torra, I. P.; Staels, B.;
Giguꢀere, V.; Kelly, D. P. Estrogen-related receptor alpha directs peroxi-
some proliferator-activated receptor alpha signaling in the transcrip-
tional control of energy metabolism in cardiac and skeletal muscle. Mol.
Cell. Biol. 2004, 24, 9079–9091. (d) Schreiber, S. N.; Emter, R.; Hock,
M. B.; Knutti, D.; Cardenas, J.; Podvinec, M.; Oakeley, E. J.; Kralli, A.
The estrogen-related receptor alpha (ERRalpha) functions in PPAR-
gamma coactivator 1alpha (PGC-1alpha)-induced mitochondrial bio-
genesis. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 6472–6477.
’ ASSOCIATED CONTENT
S
Supporting Information. Chemical data for 7b,c,e,gÀk,
b
general information for chemistry and biology, and procedure for
Western blot analysis. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*For D.W. and K.D.: phone, +96-20-3201-5276; fax, +96-20-
3201-5299; e-mail, ding_ke@gibh.ac.cn.
Author Contributions
L.P. and X.G. contributed equally to this work.
(8) (a) Seth, A.; Steel, J. H.; Nichol, D.; Pocock, V.; Kumaran, M. K.;
Fritah, A.; Mobberley, M.; Ryder, T. A.; Rowlerson, A.; Scott, J.;
Poutanen, M.; White, R.; Parker, M. The transcriptional corepressor
RIP140 regulates oxidative metabolism in skeletal muscle. Cell Metab.
2007, 6, 236–245. (b) Caste, A.; Herledan, A.; Bonnet, S.; Jalaguier, S.;
Vanacker, J. M.; Cavaillꢀes, V. Receptor-interacting protein 140 differen-
tially regulates estrogen receptor-related receptor transactivation de-
pending on target genes. Mol. Endocrinol. 2006, 20, 1035–1047.
(9) Sladek, R.; Bader, J. A.; Giguꢀere, V. The orphan nuclear receptor
estrogen-related receptor α is a transcriptional regulator of the human
medium-chain acyl coenzyme A dehydrogenase gene. Mol. Cell. Biol.
1997, 17, 5400–5409.
’ ACKNOWLEDGMENT
We thank National Natural Science Foundation (Grant
90813033), National Basic Research Program of China (Grants
2010CB529706, 2009CB940904), and the 100-Talent Program
of Chinese Academy of Sciences (CAS) for their financial
support. We also thank Micky Tortorella for the help on the
preparation of the manuscript.
7732
dx.doi.org/10.1021/jm200976s |J. Med. Chem. 2011, 54, 7729–7733

Journal of Medicinal Chemistry
BRIEF ARTICLE
(10) Zhang, Y.; Ma, K.; Sadana, P.; Chowdhury, F.; Gaillard, S.;
Wang, F.; McDonnell, D. P.; Unterman, T. G.; Elam, M. B.; Park, E. A.
Estrogen-related receptors stimulate pyruvate dehydrogenase kinase
isoform 4 gene expression. J. Biol. Chem. 2006, 281, 39897–39906.
(11) Wende, A. R.; Huss, J. M.; Schaeffer, P. J.; Giguꢀere, V.; Kelly,
D. P. PGC-1alpha coactivates PDK4 gene expression via the orphan
nuclear receptor ERRalpha: a mechanism for transcriptional control of
muscle glucose metabolism. Mol. Cell. Biol. 2005, 25, 10684–10694.
(12) Dufour, C. R.; Wilson, B. J.; Huss, J. M.; Kelly, D. P.; Alaynick,
W. A.; Downes, M.; Evans, R. M.; Blanchette, M.; Giguꢀere, V. Genome-
wide orchestration of cardiac functions by the orphan nuclear receptors
ERRalpha and γ. Cell Metab. 2007, 5, 345–356.
(13) (a) Mootha, V. K.; Handschin, C.; Arlow, D.; Xie, X.; Pierre,
J. S.; 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. ERRα and GABPAα/β specify PGC-1α-dependent oxidative
phosphorylation gene expression that is altered in diabetic muscle. Proc.
Natl. Acad. Sci. U.S.A. 2004, 101, 6570–6575. (b) Handschin, C.;
Mootha, V. K. Estrogen-related receptor a (ERRa): a novel target in
type 2 diabetes. Drug Discovery Today: Ther. Strategies 2005, 2, 151–156.
(c) Soriano, F. X.; Liesa, M.; Bach, D.; Chan, D. C.; Palacín, M.; Zorzano,
A. Evidence for a mitochondrial regulatory pathway defined by peroxi-
some proliferator-activated receptor-gamma coactivator-1 alpha, estro-
gen-related receptor-alpha, and mitofusin 2. Diabetes 2006, 55, 1783–1791.
(d) Ariazi, E. A.; Jordan, V. C. Estrogen-related receptors as emerging
targets in cancer and metabolic disorders. Curr. Top. Med. Chem. 2006,
6, 181–193.
binding domain in three functional states reveal the molecular basis of
small molecule regulation. J. Biol. Chem. 2006, 281, 37773–37781.
(24) Michael, L. F.; Wu, Z.; Cheatham, R. B.; Puigserver, P.;
Adelmant, G.; Lehman, J. J.; Kelly, D. P.; Spiegelman, B. M. Restoration
of insulin-sensitive glucose transporter (GLUT4) gene expression in
muscle cells by the transcriptional coactivator PGC-1. Proc. Nat. Acad.
Sci. U.S.A. 2001, 98, 3820–3825.
(25) Carrier, J. C.; Deblois, G.; Champigny, C.; Levy, E; Giguꢀere, V.
Estrogen-related receptor α (ERRα) is a transcriptional regulator of
apolipoprotein A-IV and controls lipid handling in the intestine. J. Biol.
Chem. 2004, 279, 52052–52058.
(26) Hagberg, C. E.; Falkevall, A.; Wang, X; Larsson, E.; Huusko, J.;
Nilsson, I.; Meeteren, L. A.; Samen, E.; Lu, L.; Vanwildemeersch, M.;
Klar, J.; Genove, G.; Pietras, K.; Stone-Elander, S.; Claesson-Welsh, L.;
Yla-Herttuala, S.; Lindahl, P.; Eriksson, U. Vascular endothelial growth
factor B controls endothelial fatty acid uptake. Nature 2010, 464, 917–919.
(27) Gao, X. F.; Chen, W.; Kong, X. P.; Xu, A. M.; Wang, Z. G.;
Sweeney, G.; Wu, D. Enhanced susceptibility of Cpt1c knockout mice
to glucose intolerance induced by a high-fat diet involves elevated
hepatic gluconeogenesis and decreased skeletal muscle glucose uptake.
Diabetologia 2009, 52, 912–920.
(14) Patch, R. J.; Searle, L. L.; Kim, A. J.; De, D.; Zhu, X.; Askari,
H. B.; O’Neill, J. C.; Abad, M. C.; Rentzeperis, D.; Liu, J.; Kemmerer, M.;
Lin, J.; Kasturi, J.; Geisler, J. G.; Lenhard, J. M.; Player, M. R.; Gaul, M. D.
Identification of diaryl ether-based ligands for estrogen-related receptor
α as potential antidiabetic agents. J. Med. Chem. 2011, 54, 788–808.
(15) Greschik, H.; Wurtz, J. M.; Sanglier, S.; Bourguet, W.; van
Dorsselaer, A.; Moras, D.; Renaud, J.-P. Structural and functional
evidence for ligand-independent transcriptional activation by the estro-
gen-related receptor 3. Mol. Cell 2002, 9, 303–313.
(16) Busch, B. B.; Stevens, W. C., Jr.; Martin, R.; Ordentlich, P.;
Zhou, S.; Sapp, D. W.; Horlick, R. A.; Mohan, R. Identification of a
selective inverse agonist for the orphan nuclear receptor estrogenrelated
receptor α. J. Med. Chem. 2004, 47, 5593–5596.
(17) Kallen, J.; Lattmann, R.; Beerli, R.; Blechschmidt, A.; Blommers,
M. J.; Geiser, M.; Ottl, J.; Schlaeppi, J. M.; Strauss, A.; Fournier, B. Crystal
structure of human estrogen-related receptor α in complex with a syn-
thetic inverse agonist reveals its novel molecular mechanism. J. Biol. Chem.
2007, 282, 23231–23239.
(18) Wang, J.; Fang, F.; Huang, Z.; Wang, Y.; Wong, C. Kaempferol
is an estrogen-related receptor α and γ inverse agonist. FEBS Lett. 2009,
583, 643–647.
(19) Suetsugi, M.; Su, L.; karlsberg, K.; Yuan, Y.-C.; Chen, S. Flavone
and isoflavone phytoestrogens are agonists of estrogen-related recep-
tors. Mol. Cancer Res. 2003, 1, 981–991.
(20) Kallen, J.; Schlaeppi, J. M.; Bitsch, F.; Filipuzzi, I.; Schilb, A.;
Riou, V.; Graham, A.; Strauss, A.; Geiser, M.; Fournier, B. Evidence for
ligand-independent transcriptional activation of the human estrogen-
related receptor α (ERRα). J. Biol. Chem. 2004, 279, 49330–49337.
(21) Hyatt, S. M.; Lockamy, E. L.; Stein, R. A.; McDonnell, D. P.;
Miller, A. B.; Orband-Miller, L. A.; Willson, T. M.; Zuercher, W. J. On
the intractability of estrogen-related receptor α as a target for activation
by small molecules. J. Med. Chem. 2007, 50, 6722–6724.
(22) Zuercher, W. J.; Gaillard, S.; Orband-Miller, L. A.; Chao,
E. Y. H.; Shearer, B. G.; Jones, D. G.; Miller, A. B.; Collins, J. L.;
McDonnell, D. P.; Willson, T. M. Identification and structureÀactivity
relationship of phenolic acyl hydrazones as selective agonists for the
estrogen-related orphan nuclear receptors ERRbeta and ERRgamma.
J. Med. Chem. 2005, 48, 3107–3109.
(23) Wang, L.; Zuercher, W. J.; Consler, T. G.; Lambert, M. H.;
Miller, A. B.; Orband-Miller, L. A.; McKee, D. D.; Willson, T. M.; Nolte,
R. T. X-ray crystal structures of the estrogen-related receptor-γ ligand
7733
dx.doi.org/10.1021/jm200976s |J. Med. Chem. 2011, 54, 7729–7733