1-Benzyl-4-phenyl-1H-1,2,3-triazoles improve the transcriptional functions of estrogen-related receptor γ and promote the browning of white adipose

Article abstract of DOI:10.1016/j.bmc.2015.03.082

The estrogen-related receptor γ (ERRγ) is a potential molecular target for the development of small molecules to stimulate the adipose browning process, which may represent a novel attractive strategy to treat obesity related disorders. The receptor possesses a very small ligand binding cavity and therefore identification of small molecule ERRγ modulators is a considerable challenge. We have successfully designed and synthesized a series of 1-benzyl-4-phenyl-1H-1,2,3-triazoles and demonstrated that they improve the transcriptional functions of ERRγ, potently elevating both the mRNA levels and the protein levels of ERRγ downstream targets. One of the most promising compounds, 4-(1-(4-iso-propylbenzyl)-1H-1,2,3-triazol-4-yl)benzene-1,2-diol (2e) was further shown to directly bind with the ERRγ ligand binding domain (ERRγ-LBD) in an isothermal calorimetric (ITC) assay and to thermally stabilize ERRγ-LBD protein by increasing its melting temperature (T_m) as demonstrated by circular dichroism (CD) spectroscopy. Furthermore, 2e potently stimulates the adipocyte browning process and induces mitochondrial biogenesis both in vitro and in vivo, suggesting the considerable therapeutic potential of this compound for the treatment of obesity and related disorders.

Full text of DOI:10.1016/j.bmc.2015.03.082

Accepted Manuscript
1
-Benzyl-4-Phenyl-1H-1,2,3-Triazoles Improve the Transcriptional Functions
of Estrogen-Related Receptor γ and Promote the Browning of White Adipose
Shilin Xu, Liufeng Mao, Ping Ding, Xiaoxi Zhuang, Yang Zhou, Lei Yu,
Yingxue Liu, Tao Nie, Tingting Xu, Yong Xu, Jinsong Liu, Jeff Smaill, Xiaomei
Ren, Donghai Wu, Ke Ding
PII:
S0968-0896(15)00278-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.03.082
BMC 12208
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
22 February 2015
30 March 2015
31 March 2015
Please cite this article as: Xu, S., Mao, L., Ding, P., Zhuang, X., Zhou, Y., Yu, L., Liu, Y., Nie, T., Xu, T., Xu, Y.,
Liu, J., Smaill, J., Ren, X., Wu, D., Ding, K., 1-Benzyl-4-Phenyl-1H-1,2,3-Triazoles Improve the Transcriptional
Functions of Estrogen-Related Receptor γ and Promote the Browning of White Adipose, Bioorganic & Medicinal
Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.03.082
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1
-Benzyl-4-Phenyl-1H-1,2,3-Triazoles Improve the
Transcriptional Functions of Estrogen-Related
Receptor γ and Promote the Browning of White
Adipose
†
Shilin Xu, Liufeng Mao, Ping Ding,
,#
†,#
†,§, #
†
Xiaoxi Zhuang, Yang Zhou, Lei Yu, Yingxue Liu,
†
†
†
†
†
†
†
, ф
,†
Tao Nie, Tingting Xu, Yong Xu, Jinsong Liu, Jeff Smaill, Xiaomei Ren, * Donghai Wu,*
,†
⊥
,†
and Ke Ding*
†
State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health,
Chinese Academy of Sciences, No. 190 Kaiyuan Avenue, Guangzhou 510530, China
§
School of Life Science, Anhui University, Hefei 230601, Anhui, China
⊥Auckland Cancer Society Research Centre, University of Auckland, #92019 Private Bag ,
Auckland 1142, New Zealand
ф
Maurice Wilkins Centre for Molecular Biodiscovery, University of Auckland, #92019 Private
Bag, Auckland 1142, New Zealand
1

KEYWORDS. Estrogen-Related Receptor γ, adipose browning, drug design, triazole
ABSTRACT. The estrogen-related receptor γ (ERRγ) is a potential molecular target for the
development of small molecules to stimulate the adipose browning process, which may represent
a novel attractive strategy to treat obesity related disorders. The receptor possesses a very small
ligand binding cavity and therefore identification of small molecule ERRγ modulators is a
considerable challenge. We have successfully designed and synthesized a series of 1-benzyl-4-
phenyl-1H-1, 2, 3-triazoles and demonstrated that they improve the transcriptional functions of
ERRγ, potently elevating both the mRNA levels and the protein levels of ERRγ downstream
targets. One of the most promising compounds, 4-(1-(4-iso-propylbenzyl)-1H-1,2,3-triazol-4-
yl)benzene-1,2-diol (2e) was further shown to directly bind with the ERRγ ligand binding
domain (ERRγ-LBD) in an isothermal calorimetric (ITC) assay and to thermally stabilize ERRγ-
LBD protein by increasing its melting temperature (Tm) as demonstrated by circular dichroism
(
CD) spectroscopy. Furthermore, 2e potently stimulates the adipocyte browning process and
induces mitochondrial biogenesis both in vitro and in vivo, suggesting the considerable
therapeutic potential of this compound for the treatment of obesity and related disorders.
INTRODUCTION
Two types of adipose tissues have been identified in mammals, i.e. white adipose tissue
1
(
WAT) and brown adipose tissue (BAT). WAT is located predominantly in the subcutaneous
and visceral organs where it stores excess energy in the form of triglycerides. Excessive
accumulation of WAT is the primary cause of obesity and can eventually lead to type 2 diabetes,
2
cardiovascular disease and other metabolic disorders. BAT, on the other hand, stores lipids in a
multilocular droplet morphology and possesses much higher mitochondrial content than WAT.
2

The key functions of BAT are to burn fat for body heat production (adaptive thermogenesis) and
3
to promote weight loss by increasing energy expenditure. Although controversy had been
associated with the occurrence of BAT in adult humans, recent data derived from a positron-
emission-tomography (PET) study convincingly demonstrated the existence of active BAT in
4
adults. Different reports have shown that exposure to cold temperatures or expression of several
5
6
endogenous ligands (e.g. Irisin , FGF21 ) can induce the presence of brown-like adipocytes (also
7
known as beige cells) in WAT tissue and cause the weight loss of animals. The WAT → BAT
transition has been termed biologically as adipose browning. Promoting the browning process in
white adipose tissue and/or activation of the function of BAT by small molecules becomes a
8
highly attractive strategy to treat obesity and its related complications. Several molecules have
been reported to stimulate the adipose browning process with different mechanisms of action
3
b
(
MOA). However, none of these molecules have been advanced into clinical investigation for
the treatment of obesity. It is highly desirable to identify new molecules with different MOA to
promote the browning of white adipose.
Chart 1. Design of 1-benzyl-4-phenyl-1H-1,2,3-triazole derivatives as new ERRγ agonists by
scaffolding hopping of reported compound 1 (GSK4716).
3

It was recently reported that transfection of estrogen-related receptor γ (ERRγ) could
remarkably enhance the expression of uncoupling protein 1 (UCP1), a brown-fat specific gene,
and improve fatty acid oxidation in differentiating white pre-adipocytes and/or BAT, suggesting
ERRγ could be a new promising target for development of small molecules to induce adipose
9
10
browning. ERRγ belongs to an orphan nuclear receptor subfamily and is expressed mainly in
1
1
energy-dependent tissues including the brain, heart, kidney, skeletal muscle and BAT. It has
3
been demonstrated that ERRγ possesses a small ligand binding pocket less than 280 Å in
1
2
volume, highlighting the challenge to identify small molecules regulating this receptor. Only
recently, GSK4716 (1, Chart 1) and closely related derivatives were reported to selectively
1
3
upregulate the transcriptional functions of ERRγ. However, to the best of our knowledge, there
has been no report of using a small molecule ERRγ modulator to induce adipose browning.
Herein, we describe the design of new molecules improving the transcriptional functions of
ERRγ. Furthermore, one of the representative molecules, 2e, potently stimulates the browning
process of white adipose.
CHEMISTRY
The rationally designed compounds were readily synthesized using the procedures shown in
scheme 1. Briefly, the hydroxyl substituted bromobenzene starting materials 3 were protected
with 3,4-dihydro-2H-pyran (DHP) to give the compounds 4. Pd-catalyzed Sonogashira coupling
of these with trimethylsilylacetylene (TMSA) followed by treatment of K CO in methanol gave
2
3
the acetylene derivatives 5. These underwent a Cu-catalyzed “click” cycloaddition with the self-
prepared benzyl azides to obtain the triazole intermediates 6, which were further deprotected to
provide the products 2a-2k.
a
Scheme 1. Synthesis of compounds 2a-2k
4

a
Reagents and conditions: (a) 3,4-Dihydro-2H-pyran, pyridinium p-toluenesulfonate, CH Cl , rt;
2
2
(
b) Trimethylsilylacetylene, PdCl (PPh ) , CuI, triethylamine, 80 ℃; (c) K CO , MeOH, rt; (d)
2
3
2
2
3
Sodium ascorbate, CuSO , t-BuOH/H O (1:1), rt; (e) Pyridinium p-toluenesulfonate, EtOH, 50
4
2
℃
.
RESULTS AND DISCUSSION
Structural feature analysis of the ERRγ-compound 1 complex (PDB: 2GPP) clearly revealed a
conformational rearrangement of ERRγ induced by compound 1, resulting in an accessible rear
pocket originally shielded by a salt-bridge between Glu275 and Arg316. The phenolic hydroxyl
and acyl hydrazone moieties in 1 participate in critical hydrogen bond networks with various
residues of the protein, while the isopropyl group is held in a small hydrophobic cavity by Van
12
der Waals interactions. Based on the structural information of the compound 1-ERRγ complex,
we successfully designed compound 2a as a new potential ERRγ modulator in which the 1,2,3-
triazole moiety could be considered as a non-classical bioisostere of the acyl hydrazone moiety
1
4
of compound 1. Modeling studies suggested that, similarly to 1, compound 2a could bind the
conformationally rearranged ligand binding domain of ERRγ (ERRγ-LBD, Figure 1a). However,
5

the hydroxyl substituent of 2a is predicted to form a strong hydrogen bond with Glu247 of the
protein, while the corresponding residue for compound 1 is Asp328.
A
B
Asp328
Asp328
Glu247
Glu247
Figure 1. Predicted binding modes of compounds 2a and 2e with ERRγ-LBD. a) Predicted
binding mode of 2a (magenta carbons) in the LBD of ERRγ (PDB: 2GPP), and superposition of
2
a with compound 1 (dark gray carbons). b) Predicted binding mode of 2e (magenta carbons)
with ERRγ-LBD. The ligands are showed in stick model, while the ERRγ protein is shown in
cartoon model. The key residues are shown in a line model, and the water molecules are depicted
as red spheres.
Preliminary determination of the modulating effect of 2a on ERRγ was accomplished by a
well-established mammalian two-hybrid reporter gene assay at 2.0 µM using compound 1 as a
15
positive control for parallel comparison (Figure 2). Under the screening conditions, compound
1
potently improved the transcriptional function of ERRγ by a factor of 1.85-fold which is
13a
comparable to the reported results. Compound 2a displayed an almost identical effect on ERRγ
transcriptional activity. Further structure-activity relationship (SAR) investigation revealed
that the 4-hydroxyl group in ring A of 2a might be critical for its effect on ERRγ. When the 4-
hydroxyl group is removed (2b) or is moved to the 2- position as in 2c, the resulting compounds
6

showed weaker regulatory effects on ERRγ. Interestingly, the 3-hydroxyl derivative (2d)
displayed slightly improved potency, potentially resulting from this substituent forming a
favorable hydrogen bond with Asp328 of the protein. The 3,4-dihydroxyl compound 2e
significantly elevated the transcriptional function of ERRγ by a factor of 5-fold at 2.0 µM, and is
obviously more potent than 2a or 2d. A preliminary computational investigation suggests 2e may
form two hydrogen bonds with Asp328 and Glu247, respectively, and thus achieve improved
bioactivity (Figure 1b). It is noteworthy that the catechol scaffold in 2e has been identified in a
number of FDA approved drugs including Dopamine and Northera, despite the general belief
that it is a pharmaceutically unfavorable group. Further investigation demonstrated that the
compound’s effect on ERRγ is dose-dependent (Supporting Information, Figure S3). The impact
of the substituent in ring B was also investigated and it was clearly revealed that a para-
isopropyl group is optimal; when the isopropyl group in 2e is moved to the m- (2f) or the o-
position (2g), the potency decreases considerably. Other compounds with smaller or larger
substituents, such as H (2h), methyl (2i), tert-butyl (2j) and phenyl (2k), are also less potent.
Thus, 2e represents one of the most potent compounds for further biological investigation.
7

Figure 2. 1-Benzyl-4-phenyl-1H-1,2,3-triazole derivatives upregulate the transcriptional
function of ERRγ at 2.0 µM in a mammalian two-hybrid reporter gene assay. Results are the
mean (± SEM) of at least three independent experiments.
The effect of 2e on the improvement of the transcriptional function of ERRγ was further
validated by investigating its impact on the expression of ERRγ targeted genes, such as SHP
1
6
17
11c, 18
(
small heterodimer partner) , ERRα , ATP5b (ATP synthase 5b)
and MCAD (medium-chain
1
8
acyl-coenzyme A dehydrogenase). It was shown that 2e dose-dependently elevated the mRNA
levels of these genes in a quantitative real-time PCR assay, while the less potent compound 2c
failed to show much effect at 1.0 µM. It was also noteworthy that the mRNA level of ERRγ is
19
also increased after exposure to compound 2e. Further investigation revealed that 2e also
significantly increased the levels of ATP5b and MCAD proteins in a western blot assay (Figure
3
).
8

Figure 3. Compound 2e upregulates the transcriptional functions of ERRγ. a-e) 2e elevated the
mRNA levels of SHP, ERRα, ATP5b, MCAD and ERRγ in a quantitative real-time PCR assay.
f) 2e increased the protein levels of ATP5b and MCAD determined by a western blot assay.
9

ATP5b and MCAD proteins were detected at a size of 52 and 43 kDa, respectively. Results are
the mean (± SEM) or representative of three independent experiments (**P < 0.01).
The direct binding of compound 2e with ERRγ-LBD was also examined in an isothermal
calorimetric (ITC) assay. It was confirmed that 2e indeed could bind to the protein with an
experimental K
d
value of 46.0 µM at 20 °C (Figure 4), about 10-fold weaker than compound 1
(
K
d
= 5.0 µM, data not shown) under the same assay conditions. Further investigation by circular
dichroism (CD) assay showed a small increase in the melting temperature (Tm) by 0.7 °C when
compound 2e was added (Supporting Information, Table S1). A comparable increase in the Tm
12
for compound 1 was reported. The discrepancy between the weak protein binding affinity and
its relatively strong effect on improving transcriptional function suggests that compound 2e may
achieve ERRγ modulating activity though a different mechanism than that of compound 1.
10

Figure 4. Isothermal calorimetric analysis of the binding interaction between ERRγ-LBD and
compound 2e.
The ability of 2e to drive adipose browning was also examined by studying its effect on the
expression of UCP1 in white adipocytes derived from mouse embryonic fibroblasts (MEF). It
was shown that 2e dose-dependently induced the elevation of UCP1 both at the mRNA and
protein level (Figure 5a and 5b). Further study revealed that 2e also markedly increased the
number of UCP1-positive adipocytes in an immunofluorescence staining assay (Figure 5c). It is
20
noteworthy that another brown-fat-selective gene, Prdm16 , was also significantly elevated in
MEF-derived adipocytes after 2e treatment (Supporting Information, Figure S4). Moreover, a
similar browning effect of 2e could be observed in the white adipocytes differentiated from
11

primary pre-adipocytes of the subcutaneous adipose tissue of mice (Supporting Information,
Figure S5).
Figure 5. Compound 2e induces browning of white adipose in vitro. a) 2e enhances the
expression level of UCP1 in a dose-dependent manner in a real-time PCR assay. FGF21 (1
µg/mL) was used as a positive control. b) 2e increases the level of UCP1 protein as determined
by a western blot assay. The UCP1 protein was found to be 33 kDa in size. c) Representative
images from immunofluorescence against UCP1. Results are the mean (± SEM) or representative
of three independent experiments (**P < 0.01).
12

One of the most significant features in brown fat development is mitochondrial biogenesis and
7
remodelling. Consequently, we further evaluated the effect of 2e on mitochondrial biogenesis by
assessing the expression of several mitochondria-related genes including PGC-1α (Peroxisome
proliferator-activated receptor-γ coactivator 1α), CS (Citrate Synthase), CPT1 (Carnitine
11c, 19, 21
Palmytoyltransferase-1) and COXII (Cytochrome c Oxidase subunit II),
and found that 2e
significantly improves the mRNA levels of these genes. More significantly, the protein level of
1
9
SDH (Succinate Dehydrogenase) , an important mitochondrial function protein, is also increased
Figure 6). The role of compound 2e in the induction of mitochondrial biogenesis was further
supported by electron microscopic analysis (SI, Figure S6).
(
13

Figure 6. Compound 2e promotes expression of mitochondrial genes. a-e) 2e induces the mRNA
levels of PGC-1α, CS, CPT1, COXII and SDH in a real-time PCR assay. (f) 2e elevates the
protein level of SDH as determined by western blot analysis. SDH protein was found to be 70
14

kDa in size. Results are the mean (± SEM) or representative of three independent experiments
(
**P < 0.01).
The in vivo adipose browning effect of 2e was also investigated in a mouse model. Similar to
the in vitro investigation, repeated oral administration of 2e at 20 mg/kg/day induced UCP1
elevation in mouse subcutaneous white adipose tissue (Supporting Information, Figure S7). The
in vivo browning effect of 2e was further confirmed in an immunohistochemistry assay where a
robust increase in the UCP1-positive stained area of subcutaneous white adipose tissue was
observed (Figure 7). It was also noteworthy that compound 2e caused a similar increase in UCP1
in mouse interscapular brown adipose tissues, suggesting its potential to improve the biological
functions of BAT (Supporting Information, Figure S8).
Figure 7. Compound 2e induces browning of the subcutaneous white adipose tissue in vivo in
mice. a) Representative images of immunohistochemistry against UCP1. b) The quantitative
ratio of the UCP1-positive stained area. Results are the mean (± SEM) or representative of two
independent experiments (*p < 0.05).
15

In summary, we have designed and synthesized a series of 1-benzyl-4-phenyl-1H-1,2,3-
triazole derivatives as novel small molecules that improve the transcriptional functions of ERRγ.
One of the most promising compounds 2e directly binds with ERRγ-LBD and potently
stimulates the adipose browning process both in vitro and in vivo. Compound 2e also
significantly improves mitochondrial biogenesis and the biological functions in MEF-
differentiated white adipocytes although a detailed mechanism is yet to be confirmed. Our
studies suggest that compound 2e has excellent potential to serve as a lead compound for the
development of new therapeutic agents for the treatment of obesity and related disorders.
EXPERIMENTAL SECTION
General Methods for Chemistry. All reagents and solvents used as were purchased from
commercial sources. Flash chromatography was performed using silica gel (300 mesh). All
reactions were monitored by TLC, using silica gel plates with fluorescence F254 and UV light
1
13
visualization. H NMR and C NMR spectra were recorded on a Brucker AV-400 spectrometer
at 400MHz and Brucker AV-500 spectrometer at 125 MHz. Coupling constants (J) are expressed
in hertz (Hz). Chemical shifts (δ) of NMR are reported in parts per million (ppm) units relative to
an internal control (TMS). High resolution ESI-MS were recorded on an Applied Biosystems Q-
STAR Elite ESI-LC-MS/MS mass spectrometer. The purity of compounds was determined by
reverse-phase high performance liquid chromatography (HPLC) analysis to be >95%. HPLC
instrument: Dionex Summit HPLC (Column: Diamonsil C18, 5.0 µm, 4.6 × 250mm (Dikma
Technologies); detector: PDA-100 photodiode array; injector: ASI-100 autoinjector; pump: p-
6
80A). A flow rate of 1.0 mL/min was used with mobile phase of MeOH in H O with 0.1%
2
modifier (ammonia or trifluoroacetic acid e, v/v).
16

2
-(4-Bromophenoxy)tetrahydro-2H-pyran (4a). General procedure for the synthesis of 4a-k.
,4-Dihydro-2H-pyran (4.0 mL, 43.4 mmol) was added to a solution of 4-bromophenol (5 g, 28.9
mmol) in anhydrous CH Cl (100 mL) under argon, followed by pyridinium p-toluenesulfonate
75 mg, 0.3 mmol). The reaction mixture was stirred at room temperature overnight. The crude
reaction mixture was washed three times with saturated aqueous NaHCO and brine, dried with
Na SO , filtered, and concentrated in vacuo. The resultant crude material was purified by column
chromatography (SiO
400 MHz, CDCl ) δ 7.37 (d, J = 8.8 Hz, 2H), 6.94 (d, J = 8.8 Hz, 2H), 5.37 (t, J = 3.0 Hz, 1H),
.90-3.84 (m, 1H), 3.62-3.57 (m, 1H), 2.05-1.94 (m, 1H), 1.87-1.83 (m, 2H), 1.74-1.59 (m, 3H).
-(4-Ethynylphenoxy)tetrahydro-2H-pyran (5a). General procedure for the synthesis of 5a-
k.To a solution of 4a (1g, 3.9 mmol), PdCl (PPh (55 mg, 0.078 mmol) and CuI (7.4 mg, 0.040
mmol) were added trimethylsilylacetylene (572 mg, 5.84 mmol) with syringe under argon. After
the reaction mixture was stirred at 80℃ overnight, it was diluted with CH Cl (50 mL) and
3
2
2
(
3
2
4
1
2
, PE/EtOAc, 10:1) to give the title compound (7.3g, 98% yield). H NMR
(
3
3
2
2
)
3 2
2
2
filtered through a pad of Celite. The filtrate was diluted with H
with CH Cl . The combined organic extracts were washed with brine, dried over anhydrous
Na SO , filtered, and concentrated in vacuo. The residue was dissolved in methanol (150 mL),
followed by adding K CO (978 mg, 7.09 mmol). The reaction mixture was stirred at room
temperature for 4 h, and concentrated in vacuo. The residue was diluted with H O, and extracted
several times with CH Cl . The combined organic phases were washed with brine and dried over
anhydrous Na SO . Filtration and removal of the solvent gave a crude oil that was subjected to
2
O, and extracted several times
2
2
2
4
2
3
2
2
2
2
4
column chromatography (SiO
2
, PE/EtOAc, stepwise elution, PE to 10:1) to give the title product.
) δ 7.42 (d, J = 8.8 Hz, 2H), 6.99 (d, J = 8.8
1
(
759 mg, 97% yield). H NMR (400 MHz, CDCl
3
17

Hz, 2H), 5.43 (t, J = 3.2 Hz, 1H), 3.90-3.84 (m, 1H), 3.64-3.58 (m, 1H), 2.99 (s, 1H), 2.04-1.95
m, 1H), 1.88-1.84 (m, 2H), 1.74-1.59 (m, 3H).
-(4-Isopropylbenzyl)-4-(4-(tetrahydro-2H-pyran-2-yloxy)phenyl)-1H-1,2,3-triazole (6a).
General procedure for the synthesis of 6a-k. Sodium ascorbate (60 mg, 0.3 mmol) and CuSO
(
1
4
(
16 mg, 0.1 mmol) were added to a solution of 5a (202 mg, 1 mmol) and 1-(azidomethyl)-4-
isopropylbenzene (193 mg, 1.1 mmol) in 1:1 t-BuOH/H O (20 mL). The reaction mixture was
2
stirred at room temperature under argon overnight and then was concentrated in vacuo, diluted
with H O, and extracted with CH Cl . The combined organic phases were dried over anhydrous
Na SO
2
2
2
2
4
, filtered, and concentrated in vacuo. The residue was subjected to column
1
chromatography (SiO
NMR (400 MHz, CDCl
Hz, 2H), 5.52 (s, 2H), 5.44 (t, J = 3.2 Hz, 1H), 3.93-3.87 (m, 1H), 3.63-3.58 (m, 1H), 2.96-2.86
m, 1H), 2.07-1.96 (m, 1H), 1.88-1.85 (m, 2H), 1.71-1.59 (m, 3H), 1.24 (d, J = 7.2 Hz, 6H).
-(1-(4-Isopropylbenzyl)-1H-1,2,3-triazol-4-yl)phenol (2a). General procedure for the
synthesis of 2a-k. Pyridinium p-toluenesulfonate (5 mg, 0.02 mmol) was added to a solution of
a (147 mg, 0.39 mmol) in EtOH (20 mL). The reaction mixture was then warmed to 50 ℃ and
2
, PE/EtOAc, 5:1) to give the title compound (147 mg, 40% yield). H
3
) δ 7.71 (d, J = 8.4 Hz, 2H), 7.57 (s, 1H), 7.24 (s, 4H), 7.08 (d, J = 8.8
(
4
6
stirred for 2 h. After the complete consumption of the starting material, the solution was
concentrated in vacuo and subjected to flash column chromatography (SiO
2
, CH
2
Cl /EtOAc,
2
1
1
d
2
0:1) to give the title compound (106 mg, 90% yield, white solid). H NMR (400 MHz, DMSO-
6
) δ 9.55 (s, 1H), 8.42 (s, 1H), 7.63 (d, J = 8.8 Hz, 2H), 7.28-7.23 (m, 4H), 6.81 (d, J = 8.8 Hz,
13
H), 5.55 (d, 2H), 2.90-2.83 (m, 1H), 1.17 (d, J = 7.2 Hz, 6H). C NMR (125 MHz, DMSO-d )
6
δ 157.21, 148.34, 146.93, 133.53, 127.90, 126.63, 126.53, 121.69, 120.00, 115.55, 52.70, 33.10,
18

+
2
3.76. HRMS (ESI) for C18
H
19
N O [M + H] , calcd: 294.1601. Found: 294.1598. HPLC
3
analysis: MeOH-H
2
O (78:22) with 0.1% ammonia, 6.39 min, 97.17% purity.
1
1
-(4-Isopropylbenzyl)-4-phenyl-1H-1,2,3-triazole (2b). H NMR (400 MHz, DMSO-d
6
) δ
8
7
.62 (s, 1H), 7.84 (d, J = 7.6 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.33 (d, J = 7.6 Hz, 1H), 7.30-
1
3
.24 (m, 4H), 5.59 (s, 2H), 2.92-2.82 (m, 1H), 1.17 (d, J = 7.2 Hz, 6H). C NMR (125 MHz,
DMSO-d
6
) δ 148.39, 146.59, 133.39, 130.65, 128.82, 127.93, 127.81, 126.65, 125.10, 121.40,
+
5
2.79, 33.09, 23.74. HRMS (ESI) for C18
H
19
N
3
[M + H] , calcd: 278.1652. Found: 278.1650.
HPLC analysis: MeOH-H
2
O (78:22) with 0.1% ammonia, 10.68 min, 95.12% purity.
1
2
-(1-(4-Isopropylbenzyl)-1H-1,2,3-triazol-4-yl)phenol (2c). H NMR (400 MHz, DMSO-d
6
)
δ 10.17 (s, 1H), 8.45 (s, 1H), 8.00 (dd, J = 1.6, 7.6 Hz, 1H), 7.28 (d, J = 8.4 Hz, 2H), 7.24 (d, J =
8
2
1
2
.0 Hz, 2H), 7.18-7.13 (m, 1H), 6.95 (d, J = 8.0 Hz, 1H), 6.89 (t, J = 7.4 Hz, 1H), 5.62 (s, 2H),
1
3
.92-2.81 (m, 1H), 1.17 (d, J = 6.8 Hz, 6H). C NMR (125 MHz, DMSO-d ) δ 153.80, 148.32,
6
43.12, 133.60, 128.61, 127.94, 126.61, 126.43, 123.19, 119.24, 116.94, 115.92, 52.53, 33.09,
+
3.75. HRMS (ESI) for C18
H
19
N
3
O [M + H] , calcd: 294.1601. Found: 294.1599. HPLC
analysis: MeOH-H
2
O (78:22) with 0.1% ammonia, 9.90 min, 96.42% purity.
1
3
-(1-(4-Isopropylbenzyl)-1H-1,2,3-triazol-4-yl)phenol (2d). H NMR (400 MHz, DMSO-d
6
)
δ 9.52 (s, 1H), 8.56 (s, 1H), 7.29-7.19 (m, 7H), 6.73-6.70 (m, 1H), 5.57 (s, 2H), 2.92-2.81 (m,
1
3
1
1
H), 1.17 (d, J = 6.8 Hz, 6H). C NMR (125 MHz, DMSO-d ) δ 157.69, 148.37, 146.67, 133.43,
6
31.84, 129.86, 127.93, 126.64, 121.35, 116.01, 114.86, 111.82, 52.75, 33.10, 23.75. HRMS
+
(
(
ESI) for C18
H
19
N
3
O [M + H] , calcd: 294.1601. Found: 294.1598. HPLC analysis: MeOH-H
2
O
78:22) with 0.1% ammonia, 6.90 min, 98.01% purity.
-(1-(4-Isopropylbenzyl)-1H-1,2,3-triazol-4-yl)benzene-1,2-diol (2e). H NMR (400 MHz,
DMSO-d ) δ 9.02 (s, 2H), 8.38 (s, 1H), 7.28-7.23 (m, 5H), 7.06 (dd, J = 1.6, 8.0 Hz, 1H), 6.76
1
4
6
19

13
(
d, J = 8.0 Hz, 1H), 5.53 (s, 2H), 2.91-2.81 (m, 1H), 1.17 (d, J = 6.8 Hz, 6H). C NMR (125
MHz, DMSO-d
6
) δ 148.32, 147.07, 145.46, 145.30, 133.55, 127.90, 126.61, 122.13, 120.01,
+
1
3
1
16.59, 115.87, 112.67, 52.66, 33.10, 23.76. HRMS (ESI) for C18
H
19
N
3
O
2
[M + H] , calcd:
10.1550. Found: 310.1549. HPLC analysis: MeOH-H
2
O (65:35) with 0.1% trifluoroacetic acid,
2.68 min, 96.90% purity.
1
4
-(1-(3-Isopropylbenzyl)-1H-1,2,3-triazol-4-yl)benzene-1,2-diol (2f). H NMR (400 MHz,
DMSO-d
6
) δ 9.03 (s, 2H), 8.39 (s, 1H), 7.31-7.20 (m, 4H), 7.12-7.06 (m, 2H), 6.76 (d, J = 8.4
1
3
Hz, 1H), 5.56 (s, 2H), 2.92-2.82 (m, 1H), 1.18 (d, J = 7.2 Hz, 6H). C NMR (125 MHz, DMSO-
d
1
3
1
6
) δ 148.91, 147.04, 145.47, 145.32, 136.04, 128.72, 125.97, 125.91, 125.26, 122.11, 120.10,
+
16.60, 115.87, 112.68, 52.97, 33.29, 23.77. HRMS (ESI) for C18
H
19
N
3
O
2
[M + H] , calcd:
10.1550. Found: 310.1549. HPLC analysis: MeOH-H
2
O (65:35) with 0.1% trifluoroacetic acid,
1.83 min, 98.27% purity.
1
4
-(1-(2-Isopropylbenzyl)-1H-1,2,3-triazol-4-yl)benzene-1,2-diol (2g). H NMR (400 MHz,
DMSO-d
.75 (d, J = 8.4 Hz, 1H), 5.65 (s, 2H), 3.36-3.30 (m, 1H), 1.12 (d, J = 6.8 Hz, 6H). C NMR
125 MHz, DMSO-d ) δ 146.91, 146.87, 145.47, 145.31, 132.51, 129.25, 128.72, 125.99, 125.65,
22.10, 120.05, 116.60, 115.86, 112.68, 50.59, 28.03, 23.55. HRMS (ESI) for C18 [M +
O (85:15) with 0.1%
6
) δ 9.02 (s, 2H), 8.27 (s, 1H), 7.38-7.31 (m, 2H), 7.25-7.15 (m, 3H), 7.08-7.05 (m, 1H),
13
6
(
6
1
H
19
N
3
O
2
+
H] , calcd: 310.1550. Found: 310.1551. HPLC analysis: MeOH-H
2
trifluoroacetic acid, 3.88 min, 96.48% purity.
1
4
-(1-Benzyl-1H-1,2,3-triazol-4-yl)benzene-1,2-diol (2h). H NMR (400 MHz, DMSO-d
6
) δ
9
1
1
.03 (s, 2H), 8.38 (s, 1H), 7.40-7.32 (m, 5H), 7.25 (d, J = 1.6 Hz, 1H), 7.07 (dd, J = 2.0, 8.0 Hz,
1
3
H), 6.76 (d, J = 8.4 Hz, 1H), 5.59 (s, 2H). C NMR (125 MHz, DMSO-d
6
) δ 147.07, 145.46,
45.32, 136.12, 128.71, 128.04, 127.80, 122.09, 120.12, 116.59, 115.87, 112.67, 52.84. HRMS
20

+
(
ESI) for C15
65:35) with 0.1% trifluoroacetic acid, 5.00 min, 95.06% purity.
-(1-(4-Methylbenzyl)-1H-1,2,3-triazol-4-yl)benzene-1,2-diol (2i). H NMR (400 MHz,
DMSO-d ) δ 9.02 (s, 2H), 8.34 (s, 1H), 7.25-7.17 (m, 5H), 7.06 (dd, J = 0.8, 8.0 Hz, 1H), 6.76
H
13
N
3
O
2
[M + H] , calcd: 268.1081. Found: 268.1079. HPLC analysis: MeOH-H
2
O
(
1
4
6
1
3
(
d, J = 8.0 Hz, 1H), 5.53 (s, 2H), 2.28 (s, 3H). C NMR (125 MHz, DMSO-d ) δ 147.04, 145.46,
6
1
45.30, 137.38, 133.12, 129.24, 127.86, 122.13, 119.97, 116.58, 115.87, 112.67, 52.66, 20.65.
+
HRMS (ESI) for C16
MeOH-H
-(1-(4-Tert-butylbenzyl)-1H-1,2,3-triazol-4-yl)benzene-1,2-diol (2j). H NMR (400 MHz,
DMSO-d ) δ 9.03 (s, 2H), 8.37 (s, 1H), 7.39 (d, J = 8.4 Hz, 2H), 7.28-7.25 (m, 3H), 7.08-7.05
H
15
N
3
O
2
[M + H] , calcd: 282.1237. Found: 282.1236. HPLC analysis:
2
O (65:35) with 0.1% ammonia, 6.71 min, 95.57% purity.
1
4
6
1
3
(
m, 1H), 6.76 (d, J = 8.4 Hz, 1H), 5.54 (s, 2H), 1.25 (s, 9H). C NMR (125 MHz, DMSO-d ) δ
6
1
5
50.54, 147.07, 145.46, 145.30, 133.19, 127.62, 125.47, 122.12, 120.02, 116.59, 115.86, 112.66,
+
2.57, 34.23, 31.01. HRMS (ESI) for C19
H
21
N
3
O
2
[M + H] , calcd: 324.1707. Found: 324.1706.
HPLC analysis: MeOH-H
2
O (85:15) with 0.1% trifluoroacetic acid, 4.40 min, 96.70% purity.
1
4
-(1-(Biphenyl-4-ylmethyl)-1H-1,2,3-triazol-4-yl)benzene-1,2-diol (2k). H NMR (400
MHz, DMSO-d6) δ 9.04 (s, 2H), 8.43 (s, 1H), 7.69-7.64 (m, 4H), 7.48-7.41 (m, 4H), 7.38-7.34
(
(
m, 1H), 7.26 (d, J = 2.0 Hz, 1H), 7.08 (dd, J = 2.0, 8.4 Hz, 1H), 6.77 (d, J = 8.0 Hz, 1H), 5.64
1
3
s, 2H). C NMR (125 MHz, DMSO-d6) δ 147.12, 145.48, 145.33, 139.95, 139.56, 135.27,
1
28.90, 128.44, 127.55, 127.03, 126.65, 122.10, 120.16, 116.61, 115.88, 112.69, 52.54. HRMS
+
(
(
ESI) for C21
H
17
N
3
O
2
[M + H] , calcd: 344.1394. Found: 344.1390. HPLC analysis: MeOH-H
2
O
65:35) with 0.1% ammonia, 14.09 min, 95.61% purity.
Computational Protein-Ligand Docking. The published crystal structure of ERRγ with
compound 1 (PDB: 2GPP) was used for the modelling of possible binding modes. All
21

crystallographic water molecules were deleted except two water molecules which mediate the
hydrogen bonds between 1 and ERRγ. The crystal structure was prepared for docking using the
protein preparation wizard in Maestro (Version 9.6, Schrödinger), which assigns bond
orders,adds hydrogens and missing atoms. The active site was defined by manually selecting the
original ligand and using the default of box length with a Receptor Grid Generation model. Glide
was used for the protein-ligand docking in an XP protocol. Multiple stereoisomers, ionization
states, and 3D structures of each ligand as the input to the docking calculation, were initially
generated by LigPrep using the OPLS_2005 force field. Docked binding modes were ranked
using the Docking Score and manually inspected for a reasonable interaction. All docking figures
were generated using PyMOL (Schrödinger, USA).
Cell Lines and Cell Culture. The human 293FT cell line was purchased from Invitrogen.
Cells were grown in the medium recommended by Invitrogen, and cultured in a complete
medium containing 10% FBS in a 5% CO2 incubator at 37℃.
Primary mouse embryonic fibroblast (MEF). MEF were harvested, according to the
2
2
standard protocol. Briefly, uteri were obtained at E13.5 days of pregnancy and embryos were
isolated. Heads and viscera were removed and the remaining bodies were washed in PBS,
transferred to 10 cm petridishes and minced with a pair of scissors before being digested with
0
.1% trypsin (GIBCO) for 40min at 37℃. Following digestion, the suspension was centrifuged
at 1000 g for 5 min, then 10 mL DMEM (Hyclone), supplemented with 10% FBS (Hyclone) and
% P/S (Hyclone), was added and the tissue pipetted up and down to get a single cell suspension.
1
MEF were cultured in 10 cm cell culture dishes until 80–85% confluent and then were passaged
at a ratio of 1: 2.
22

Isolation of preadipocytes from adipose tissues. Isolation of stromal cells from fat tissue was
6
, 23
performed as previously described.
Subcutaneous fat was dissected out, rinsed in PBS,
minced, and digested for 40 min at 37 ℃ in 0.1% (w/v) collagenase solution (Collagenase type I,
dissolved in D-Hanks buffer). The digested tissue was filtered through a 100 µm nylon mesh to
remove undigested tissues before being centrifuged at 1000 g for 5 min. The pellet
(
preadipocytes) was collected and washed again in PBS, then 5 mL DMEM, supplemented with
5% FBS and 1% P/S, was added and the tissue pipetted up and down to get a single cell
1
suspension. The cells were cultured in 4cm cell culture dishes and the cell medium was changed
every day.
For white adipocyte differentiation assays, MEF and preadipocytes from subcutaneous adipose
tissue were plated in 6 well plates and cultured in DMEM with 10 % FBS. Two days after
reaching confluence (day 0), differentiation was induced by incubation of the cells in
differentiation medium containing 5 µg/mL insulin (Sigma), 1 µM dexamethasone (Sigma), and
0
.5 mM isobutylmethylxanthine (Sigma). After 2 days, the media was replaced with DMEM
supplemented with 10% FBS, 5 µg/ml insulin and 1µM rosiglitazone, and the cells were re-fed
every 2 days until day 8. Compounds 2e and 2c were dissolved in demethylsulfoxide (DMSO)
and FGF21 was dissolved in PBS for cell culture studies. At day 8, cells were exposed to
compounds at the concentrations indicated with DMEM supplemented with 10% FBS for
additional two days.
Transient Transfection Mammalian Two-hybrid Reporter Gene Assay. 293FT cells were
seeded in 96-well plate at a density of 10,000 cells/well in DMEM containing 10% FBS. After
plating for 18-24 hrs, transient transfections were performed with Lipofectamine 2000
(
Invitrogen) according to the manufacturer’s instructions. Total DNA for transfections included
23

GAL4-DBD-ERRγ-LBD vector (0.5 ng), pcDNA-PGC-1α vector (0.5 ng), pFR vector (25 ng),
and internal control vector pRLTK renilla (3 ng). After cells were transfected for 6 h, 2 µM of
the test compounds were then added for an additional 24 hrs. (When the concentration of test
compounds was up to 10 µM or higher, the luciferase light units of both the firefly luciferase and
renilla luciferase groups were significantly reduced, which may be caused by the toxicity of
these compounds against the 293FT cells at such high concentration. Therefore, the
concentration of these compounds was set to a relative low value of 2 µM.) Luciferase activity
was measured by dual luciferase reporter assay system (Promega) according to the
manufacturer’s instruction on a Veritas Microplate Luminometer. Relative luciferase light units
(
RLU) 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.
Real-Time PCR Assay. Total RNA was isolated from cells and tissues using Trizol Reagent
(
Invitrogen). First-strand cDNA synthesis was performed with Superscript™ III Reverse
Transcriptase (Invitrogen). Quantification of mRNA levels was performed using SYBR® Premix
Ex Taq™ (TaKaRa) under optimized conditions following the manufacturer’s instruction. The
reference gene chosen was GAPDH. The primers were synthesized by Invitrogen in Shanghai,
China.
Western Blot Assay. Cells were washed twice with ice-cold PBS and lysed with RIPA buffer
(
Beyotime) for 30 min on ice. Cell lysates were centrifuged at 12,000 g for 15 min at 4℃ and
supernatants were collected. 40 µg of cellular proteins were resolved by 12% SDS-PAGE gel and
transferred to PVDF membrane (Millipore). The membranes were probed overnight with specific
antibodies at 4℃, washed three times with TBST followed by incubation with rabbit radish
24

peroxidase conjugated secondary antibody for 4 h at 4℃. The membranes were developed by
applying ECL Plus developing system (GE Healthcare). Membranes were stripped with stripping
buffer (Comwin biotech) and reprobed with other antibodies if necessary. The primary
antibodies used in this experiment are UCP1 (1:1000; abcam; ab10983); SDH (1:4000;
epitomics; p31040); MCAD (1:5000; epitomics; P11310); ATP5b (1:1000; abcam; ab150291);
β-actin (1:2000; abcam; ab8227).
Immunofluorescence Staining Assay. For immunofluorescence staining, coverslips plated
with white adipocyte derived MEF cells were fixed in 4% formaldehyde and were blocked with
5
% bovine serum albumin (BSA) in PBS at room temperature for 1 h and then incubated with
rabbit anti-UCP1 antibody (1:1000; Abcam; ab10983) at 4℃ overnight. Then the sections were
incubated with Alexa Fluor 647-conjugated anti-rabbit secondary antibody (1:200; Abcam;
ab150079) at room temperature for 1 h before the nuclei were stained by DAPI. Coverslips were
mounted onto glass slides. Labeled samples were imaged using Leica microscope.
Electron Microscopy Detection. At day 10 the white adipose cells differentiated from MEF
were washed 3 times in PBS, fixed for 1 hour with 2.5% glutaraldehyde, and postfixed for 1 hour
with 1% osmic acid. After dehydration in a graded series of cold ethanol, cells were
subsequently embedded in epoxy resin. Ultrafine sections were performed, using an
ultramicrotome, before staining with uranyl acetate and counterstaining with lead citrate.
Sections were then examined with a transmission electron microscope (Hitachi-600; Hitachi Ltd,
Tokyo, Japan).
Animal Study. Male mice (8 weeks old) in C57BL6 background were housed in SPF mice
room and maintained on a 12 h light-dark cycle at 23°C and fed a high-fat diet (21.9 kJ/g, 60%
25

of energy as fat, 20% of energy as protein, 20% of energy as carbohydrate; D12492; Research
Diet, New Brunswick, NJ, USA) for 4 weeks. At the same time mice were given 2e (20 mg/kg
body weight) or vehicle (5% alcohol in PBS) every day, by oral gavage. The animals were killed
4
weeks later, and tissues were isolated and kept in -80°C for RNA extraction or fixed in 4%
formaldehyde for morphology.
Immunohistochemistry Assay. White adipose tissues and brown adipose tissues were fixed in
4
% formaldehyde overnight at room temperature immediately after the mice were sacrificed.
Tissues were paraffinized and sectioned by microtome. Paraffin embedded sections of adipose
tissues were stained with anti-rabbit UCP1 (1:1000; Abcam; ab10983) to assess the expression of
U Experimental section CP1. Sections were examined by light microscopy.
Cloning, protein expression and purification. The gene encoding ERRγ 229-458 amino
acids was cloned in pET15b. The target protein was expressed with an N-terminal hexahistidine
tag and a thrombin cleavage site. E. coli BL21 (DE3) cells transformed with expression plasmids
were grown in Luria-Bertani (LB) medium to an OD600nm of 0.6-0.8 at 310 K. Expression of
the recombinant proteins was induced with 0.3 mM isopropyl β-D-1-thiogalactopyranoside
(
IPTG) and the cells were further grown for 20 h at 289 K. The target protein was first purified
from a nickel-nitrilotriacetic acid (Ni-NTA) affinity column (Qiagen) in an eluting buffer
containing 25 mM Tris–HCl, pH 8.0, 150 mM NaCl, 200 mM imidazole. Further purification
was then achieved by anion exchange chromatography (Source Q, GE healthcare) and gel
filtration (Hiload 16/60 Superdex 75 size exclusion column, GE Healthcare) equilibrated against
a phosphate buffer at pH8.0.
Isothermal Titration Calorimetry (ITC). Isothermal titration calorimetry experiments were
performed using a MicroCal iTC200 (GE Healthcare) at 20 °C. The protein eluted from gel
26

filtration was concentrated by ultrafiltration (Millipore Amicon) and supplemented with
dimerthyl sulfoxide (DMSO) to a final concentration of 0.24%. Small molecules were diluted in
the same buffer from 50 mM stock in DMSO. All solutions were degassed before titrations were
performed. The sample cell was filled with 200 µL small molecule in 122 µM and the syringe
was filled with 60 µL protein in 1.36 mM. The titration consisted of an initial injection of 0.5 µL
followed by 19 injections of 2 µL. To determine the baseline, the protein was titrated into the
same buffer without the small molecules under the same conditions. Data was analyzed with the
software Origin 7.0 (OriginLab) using the one set of sites model.
Circular dichroism (CD) Spectroscopy. CD experiments were carried out on a circular
dichroism spectrometer (Chirascan, Applied Photophysics Ltd.). Purified protein in phosphate
buffer was diluted to 0.2 mg/mL. The ligands were dissolved in DMSO to 200 mM before being
added to the protein solution and incubated overnight. Measurements were made using a quartz
cuvette with 1 mm path length. For thermal sensitivity experiments, ellipticity from 200 nm to
2
60 nm was measured at one-degree increments of the temperature from 30 °C to 60 °C.
ASSOCIATED CONTENT
Supporting Information. Experimental procedures, characterization data, biological data, and
1
13
copies of H NMR and C NMR spectra for compounds 2a-k. This material is available free of
charge via the Internet.
AUTHOR INFORMATION
Corresponding Author
*
Tel: +86-20-32015276. Fax: +86-20-32015299. E-mail: ding_ke@gibh.ac.cn,
wu_donghai@gibh.ac.cn, ren_xiaomei@gibh.ac.cn
27

Author Contributions
#
These authors contributed equally.
Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
We thank the Strategic Collaboration grant between China and New Zealand from Ministry of
Science and Technology of China (#2014DFG32100), National Basic Research Program of
China (2011CB504004 and 2010CB94550), the National Natural Science Foundation
(
#81425021, #21272235), 100-Distinguished Scientist Award of Guangdong province (Nanyue-
Baijie award) and the Key Project on Innovative Drug of Guangdong Province
#2012A080201014).
(
ABBREVIATIONS
ERR: estrogen-related receptor, WAT: white adipose tissue, BAT: brown adipose tissue, PET:
positron-emission-tomography, FGF21: fibroblast growth factor 21, MOA: mechanisms of
action, UCP1: uncoupling protein 1, SHP: small heterodimer partner, ATP5b: ATP synthase 5b,
MCAD: medium-chain acyl-coenzyme A dehydrogenase, MEF: mouse embryonic fibroblasts,
Prdm16: PR domain containing 16, PGC-1α: peroxisome proliferator-activated receptor-γ
coactivator 1, CS: citrate synthase, CPT1: carnitine palmytoyltransferase-1, COXII: cytochrome
C oxidase subunit II, SDH: succinate dehydrogenase, ITC: Isothermal Titration Calorimetry, CD:
Circular dichroism Spectroscopy, LBD: ligand binbding domain, Tm: melting temperature.
REFERENCES
28

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