The discovery of novel, potent ERR-alpha inverse agonists for the treatment of triple negative breast cancer

Article abstract of DOI:10.1016/j.ejmech.2017.04.050

The estrogen-related receptor α (ERRα) is an orphan receptor and a novel target for solid tumor therapy, conceivably through effects on the regulation of tumor cell energy metabolism associated with energy stress within solid tumor micro environments. Here we describe the discovery of novel potent inverse agonists of ERRα. In?vitro, compound 11 potently inhibits ERRα’s transcriptional activity by preventing endogenous PGC-1α and ERRα binding and suppresses the proliferation of different human cancer cell lines and the migration of breast cancer cells (MDA-MB-231). In?vivo, compound 11 demonstrates a strong inhibitory effect on the growth of human breast cancer xenografts (MDA-MB-231) and the tumor growth is inhibited by 40.9% after treating with compound 11 (30?mg/kg). The binding mode shows that compound 11 interacts with the binding pocket of ERRα through hydrogen interactions with the residue Gly397 and hydrophobic interactions with the hydrophobic residues. All these results suggest that compound 11 represents a novel potent ERRα inverse agonist and is promising in the discovery of antitumor compounds for the treatment of triple negative breast cancer.

Full text of DOI:10.1016/j.ejmech.2017.04.050

Accepted Manuscript
The discovery of novel, potent ERR-alpha inverse agonists for the treatment of triple
negative breast cancer
Yongli Du, Lianhua Song, Liudi Zhang, Hao Ling, Yanhui Zhang, Haifei Chen, Huijie
Qi, Xiaojin Shi, Qunyi Li
PII:
S0223-5234(17)30316-1
10.1016/j.ejmech.2017.04.050
EJMECH 9401
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 February 2017
Revised Date: 18 April 2017
Accepted Date: 20 April 2017
Please cite this article as: Y. Du, L. Song, L. Zhang, H. Ling, Y. Zhang, H. Chen, H. Qi, X. Shi, Q. Li, The
discovery of novel, potent ERR-alpha inverse agonists for the treatment of triple negative breast cancer,
European Journal of Medicinal Chemistry (2017), doi: 10.1016/j.ejmech.2017.04.050.
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ACCEPTED MANUSCRIPT
Graphic Abstract
4 atom length
linker
O
O
C
N
N
O
O
A
CF3
C
N
O
F3C
S
B
NH
O
H
N
O
A
O
O
B
CF3
XCT790
Design
N
N
H
H
O
A
O
O
Optimization
HN
S
B
O
O
4 atom length linker
N
N
H
H
HN
S
O
O
4 atom length linker
Novel err-alpha Inverse agonist
with high potent activity
in vitro and in vivo
Hit compound

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The Discovery of Novel, Potent ERR-alpha
Inverse Agonists for the Treatment of Triple
Negative Breast Cancer
Yongli Dua, *,†, Lianhua Song a,†, Liudi Zhang b,†, Hao Ling a, Yanhui Zhang a, Haifei
Chenb, Huijie Qib, Xiaojin Shib, Qunyi Li b,*
aSchool of Chemistry and Pharmaceutical Engineering, Qilu University of
Technology, 3501 Daxue Road, Jinan 250353, China
bDepartment of Pharmacy, Huashan Hospital North, Fudan University, 108Luxiang
Road, Shanghai 201907, China
†These three authors contributed equally to this work.
* To whom correspondences should be addressed. Tel.: +86 531 89631208 (YLD) or
86 21 52889303 (QYL); Fax: +86 0531 89631207 (YLD) or 86 21 52889307 (QYL);
E-mail: ylduyjs@163.com orqyli1234@163.com
Abstract:
The estrogen-related receptor α (ERRα) is an orphan receptor and a novel target
for solid tumor therapy, conceivably through effects on the regulation of tumor cell
energy metabolism associated with energy stress within solid tumor micro
environments. Here we describe the discovery of novel potent inverse agonists of
ERRα. In vitro, compound 11 potently inhibits ERRα’s transcriptional activity by
preventing endogenous PGC-1α and ERRα binding and suppresses the proliferation of
different human cancer cell lines and the migration of breast cancer cells
(MDA-MB-231). In vivo, compound 11 demonstrates a strong inhibitory effect on the
growth of human breast cancer xenografts (MDA-MB-231) and the tumor growth is
inhibited by 40.9% after treating with compound 11 (30 mg/kg). The binding mode
shows that compound 11 interacts with the binding pocket of ERRα through hydrogen

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interactions with the residue Gly397 and hydrophobic interactions with the
hydrophobic residues. All these results suggest that compound 11 represents a novel
potent ERRα inverse agonist and is promising in the discovery of antitumor
compounds for the treatment of triple negative breast cancer.
Key words: Estrogen-related receptor α, ERR-alpha, antitumor agent,
Estrogen-related receptor αinverse agonist, triple negative breast cancer
1. Introduction
Estrogen-related receptors (ERRs), comprising three isoforms (ERRα, ERRβ,
and ERRγ), are the member of orphan nuclear receptors whose endogenous ligands
have not been identified[1]. ERRα is a critical regulator involved in the expression of
oxidative metabolism genes and mitochondrial biogenesis by interacting with its
certain
transcriptional
cofactors
PGC-1α
and
RIP140
(peroxisome
proliferator-activated receptor γ coactivators and nuclear receptor-interacting protein
140), etc[2,3]. Substantial evidence shows that ERRα are strongly expressed in various
types of cancerand its high expression has been shown to be involved in the
development, progression and poor prognosis of breast[4], endometrium[5], prostate[6],
ovarian[7], colon[8] and other types of tumors. Therefore, regulating the activity of
ERRα has been considered a promising new therapeutic strategy for the treatment of
human cancers, including those which have acquired a drug resistance to
anti-hormonal therapy accompanied with a pessimistic clinical outcome. Though
hormonal therapy through aromatase and estrogen receptor modulators have obtained
remarkable achievements in the clinical treatment of most breast cancer patients,
about 10%-20% of breast cancer patients are diagnosed as triple-negative (TNBC,
negative for estrogen receptors, progesterone receptors, and HER2) or basal-like
(BLBC), which acquired no therapeutic effect of hormonal therapies[9]. Yet, many
breast cancer patients will progress on these traditional hormone therapies despite the
ER-positive status of their tumors. Therefore, it is urgent to explore a new
comprehensive and effective therapy to meet the clinical needs of breast cancer
patients with different mechanisms of actions. It has been revealed that ERRα is more

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highly expressed in ERα-negative MDAMB-231 cells, a TNBC cell line, compared
with ER-positive MCF-7 cells. Further, the selective inverse agonists of ERRα reduce
the proliferation, migration of MDAMB-231 cells with high expression of ERRα, and
suppress the growth of human breast cancer in an xenograft model[10]. It is promising
that the inverse agonists of ERRα may be a potential strategy in the clinical treatment
of breast cancer including TNBC.
During the past decade, only several classes of inverse agonists of ERRα have
been reported in literatureand their pharmacologicalproperties characterized.
Structural features of the reported ERRα inverse agonists included
thiadiazoleacrylamide and its analogues[11], 1-p-tolyl-1H-indol-3-ylmethyl-amine
analogues[12] and 5-benzylidene-thiazolidine-2,4-dioneanalogues[13] as exemplified by
XCT-790, compounds 1 and 2 shown in Figure 1.
NH
O
N
N
O
O
N
CF3
N
F3C
S
H
N
CF3
compound 1
O
XCT790
O
O
CF3
O
O
O
N
N
H
H
S
HN
S
NH
O
O
N
O
compound 2
compound 3
Figure 1.The structures of the reported ERRα inverse agonists and compound 3.
In our pursuit to discover novel ERRαinverse agonist, compound 3[14] based on
template 1-(2,5-diethoxy-benzyl)-3-phenyl-ureawas identified as ERRα inverse
agonist with moderate activity through screening our in house compounds. Further
optimization of this 1-(2,5-diethoxy-benzyl)-3-phenyl-urea template led to the
discovery of a series of novel ERRα inverse agonists with high potency. In this report,
we will describe in detail the design, syntheses and structure-activity relationship
(SAR) analyses of these novel high potent ERRα inverse agonists.
2. Design
This is the first attempt to develop 1-(2,5-Diethoxy-benzyl)-3-phenyl-urea
containing compounds as novel potential ERRα inverse agonist. Initially, compound 3

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was shown to inhibit constitutive transcriptional activity of ERRα (IC50
=
21.08±8.9µM, Table 1). Thiadiazoleacrylamide based compound XCT790 represents
the most potent and promising inverse agonist for ERRα with inhibiting activities in
the proliferation of estrogen-responsive and estrogen-unresponsive breast cancer cells.
When compared the structure with XCT790, as shown in figure 3, compound 3 has
two aromatic rings A and B similarly to XCT790. The length of the linker
betweenaromatic rings A and B in compound 3 is also four atoms like that in XCT790.
The obvious difference between the structure of compound 3 and XCT790 is that
compound 3 lacks aromatic ring C which in XCT790 interacts with the receptor
ERRα through hydrophobic interactions. To some extent, these explain the weak
activity of compound 3 against receptor ERRα when compared with XCT790. To
further improve the inverse activity of compound 3 against receptor ERRα, -4-OR1
and -3-COR2 groups containing an aromatic ring are introduced into the aromatic ring
B of compound 3, as shown in the template 1 structure in figure 2, to form the
hydrophobic interactions between the new compound andthe receptor ERRα. Further
optimization design give compounds 4-11 with structure characters as shown in
templates 1 and 2 (in figure 2).
linker 4 atom length
O
O
A
O
O
R1
O
O
B
N
N
O
O
A
S
N
N
CF3
C
N
F3C
H
H
B
H
R2
HN
S
O
O
linker
4 atom length
N
CF3
(comd. 4-11)
Template 1
XCT790
O
O
C
O
NH
B
Design
N
N
O
A
O
H
H
O
O
R1
B
HN
O
O
N
H
N
S
linker
4 atom length
A
O
H
HN
Hit compound (comd. 3)
linker
O
O
4 atom length
S
(comd. 6-11)
Template 2
Figure 2.Design of the novel ERRα inverse agonists 4-11.
3. Chemistry
The synthetic steps of the intermediates 6E-11E were shown in scheme 1.
Intermediate B was obtained from intermediate A[15] through hydrolyzation reaction

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with KOH in MeOH at 80°C. Then intermediate B was converted to intermediate C
after treated with SOCl2 and DMF in solvent DCM. The intermediate C effectively
reacted with various amines NH2R2 to give intermediates 6D-11D which was
sequentially subjected to nitro reduction to obtain intermediates 6E-11E.
NH2
NO2
NO2
NO2
NO2
c
b
a
d
O
O
O
O
O
O
HN
O
O
O
HN
O
Cl
O
OH
R2
R2
A
inter. 6D-11D
R2
6D: Bn-
C
B
inter. 6E-11E
R2
6E: Bn-
7E: Ph-
8E: 4-Me-Ph-
9E: 3-MeO-Ph-
10E: i-Pr-
7D: Ph-
8D: 4-Me-Ph-
9D: 3-MeO-Ph-
10D: i-Pr-
11D: 4MeO-Bn-
11E: 4MeO-Bn-
S
cheme 1. Reagents and conditions for the synthesis of intermediates 6I-11I: (a)MeOH,
KOH, 80°C, 3h, then 10%HCl; (b)SOCl2, DMF, DCM, rt, 6h; (c)DCM, Et3N, NH2R2,
3h; (d) NiCl2·6H2O, NaBH4, MeOH, 2h, then 10%HCl.
Intermediates 4H-5H were synthesized as shown in scheme 2. Starting from
material F[15], intermediate 4G-5G was synthesized through alkylation with
bromoethane[16]. Intermediate 4G-5G was sequentially subjected to nitro reduction to
obtain intermediates 4H-5H.
O
O
R1
O
OH
R1
O
g
f
H2N
O
O2N
O2N
O
O
O
inter. 4G-5G
R1
inter. 4H-5H
R1
F
4G: n-Bu--
5G: Bn-
4H: n-Bu--
5H: Bn-
Scheme 2. Reagents and conditions for the synthesis of intermediates 4H-5H: (f)
DMF, K2CO3, R1Br, 80°C, 4h; (g) NiCl2·6H2O, NaBH4, MeOH, 2h, then 10%HCl.
The syntheses of the1-(2,5-Diethoxy-benzyl)-3-phenyl-urea containing
compounds 4–11 were accomplished as depicted in Scheme 3. The intermediate I
effectively reacts with various amines to give the corresponding N-benzyl urea
intermediates 4J-11J in excellent yields [17,18]. Intermediates 4K-11K were prepared
through the reduction of nitro intermediates [18,19]. Methanesulfonyl chloride was

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condensed with intermediates 4K-11K separately to give compounds 4-11.
R
R
1
O
O
1
O
O
O
O
O
O
NH
NH
NH
O
j
NH
O
O
O N
O
2
O
O
H N
2
inter. 4J-5J
inter. 4K-5K
NH
O
i
Et
O
O
O
Et
O
O
inter.
4H-5H
6E-11E
R
2
N
R
N
O
H
2
O
O N
NH
H
2
j
NH
NH
I
NH
O
O
O
O N
H N
O
2
2
inter. 6K-11K
inter. 6J-11J
O
O
O
R
O
O
O
O
O
O
1
N
N
N
H
N
H
H
H
k
O
HN
R
2
HN
S
HN
S
O
O
O
O
O
comp. 4-5
comp. 6-11
inter. /comp.
R
2
inter. /comp.
R
1
6J/6K/ 6:
7J/7K/7:
8J/8K/8:
9J/9K/ 9:
Bn-
Ph-
4-Me-Ph-
3-MeO-Ph-
4J/4K/4:
5J/5K/5:
n-Bu-
Bn-
10J/10K/10: i-Pr-
11J/11K/ 11: 4MeO-Bn-
Scheme 3. Reagents and conditions for the synthesis of compounds 4-11: (i) Et3N,
dioxane, amine intermediate, 80°C, 8h; (j) NiCl2·6H2O, NaBH4, MeOH, rt, 0.5h; (k)
methanesulfonyl chloride, DCM, N2, pyridine, rt, 8h.
4. Results and discussion
4.1. Constitutive transcriptional activity of ERRα and structure–activity relationship
To investigate the inhibiting effects of different groups (R1, R2) on constitutive
transcriptional activity of ERRα, compounds 4–11(Table 1) were designed and
prepared, respectively. Compound 4 (IC50 = 3.43±1.17 µM) with 3-MeOCO- and
4-BuO- groups on aromatic ring B was found to exhibit 6-fold more potent inhibiting
activity against receptor ERRα than compound 3 (IC50 = 21.08±8.9 µM). Similarly,
introducing 3-MeOCO- and 4-BnO- groups to aromatic ring B of compound 3 gave

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compound 5 which improved 5.2-fold (IC50 = 4.06±0.12 µM) constitutive
transcriptional inhibition activity of ERRα compared with compound 3. Compound 6
with a smaller 4-EtO- group and a more steric 3-Bn-NHCO- group was also shown to
produce more potent (IC50 = 3.81±0.17 µM) inhibition activity of ERRα when
compared with compound 5. Compounds 4-6 exhibited excellent inhibitory activity of
ERRα perhaps because the introduced groups improved the hydrophobic interactions
with receptor ERRα LBP. In consideration of having the more stable group
3-Bn-NHCO- and high potent inhibitory activity of ERRα, compound 6 was chosen
for further optimization to improve the inhibitory activity of ERRα. Several
substituted groups, such as Ph-, 4-Me-Ph, 3-MeO-Ph-, i-Pr- and 4-MeO-Bn- groups,
were separately introduced into the ring of aromatic ring B to instead the Bn- in the
3-Bn-NHCO- group in compound 6 which produced analogues 7-11 (table 1) and the
structureactivity relationships (SAR) were also studied. All the compounds (7-11)
showed high ERRα inhibitory activity (IC50< 4.23 µM). The trend observed for
analogues 7-9 was that the substituted 4-Me-Ph- group (in compound 8, IC50
=
3.04±0.16 µM) was preferred to the Ph- group (in compound 7, IC50 = 3.72±2.55 µM)
and the 3-MeO-Ph- (in compound 9, IC50 = 4.23±0.95 µM) group. Compound 8 with
4-Me-Ph- group also demonstrated more potent ERRα inhibitory activity than
compound 10 possessing a i-Pr- group (IC50 = 3.48±1.55µM). These probably
indicated that the para-position of phenyl with hydrophobic group, such as
para-methyl in compound 8, was more suitable for ERRα LBP and was beneficial to
ERRα inhibition. Correspondingly, compound 11 (IC50 = 1.90±0.39µM) with
hydrophobic substitute group at para position of benzyl (4-MeO-Bn-) exhibited
greatly improved (2-fold) ERRα inhibition activity when compared with its
analogouscompound 6(IC50 = 3.81±0.17µM).
Table1. The effects of compounds 3-11 on constitutive transcriptional activity of
ERRα.a

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O
R2
O
O
A
O
O
R1
B
N
N
H
H
HN
S
O
O
Compounds 4-11
IC50（µM）
Compound
R1
R2
mean ± SEM
XCT790
/
/
/
0.27±0.045
21.08±8.9
/
3
n-Bu-
Bn-
Et-
MeO-
4
3.43±1.17
4.06±0.12
3.81±0.17
3.72±2.55
3.04±0.16
4.23±0.95
MeO-
5
BnNH-
PhNH-
6
Et-
7
Et-
4-MePhNH-
3-MeOPhNH-
i-PrNH-
4-MeO-BnNH-
8
Et-
9
Et-
10
11
3.48±1.55
1.90±0.39
Et-
aValues represent triplicate determinations.
4.2.The inhibition activities of compound 11 against human cancer cell proliferation
According to the constitutive transcriptional activity of ERRα, the most potent
ERRα inverse agonist (compound 11) were selected to test the effects on the
proliferation of a panel of selective human tumor cell lines. As shown in Table 2,
compound 11 exhibited strong and widely antiproliferative activities toward eight
different cancer cell lines, including ER-positive and ER-negative breast cancer cell
lines after treatment with compound 11 for 48 h. IC50 values for all eight cancer cell

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lines in response to compound 11 were calculated (Table 2). These data showed that
compound 11 exhibited lowest IC50 values (1.46 ± 0.12µM) in ER-negative
MDA-MB-231 cells compared with ER-negative MDA-MB-435 and ER-positive
T47D with IC50 values of 2.53 ± 0.21 and 8.31 ± 1.16µM, respectively, which
demonstrated that ER-negative MDA-MB-231 cells were the most sensitive breast
cancer cells responded to compound 11 treatment. These data suggested that
compound 11 is a more potent and sensitive inhibitor of breast cancer cell
proliferation. All these results suggested that compound 11 is a more potent and
sensitive inhibitor of breast cancer cell proliferationin a manner that was independent
of the presence of ER.
Table 2. IC50 values of tumor cell lines treated with compound 11.a
Compound 11
Cell line
IC50 (µM)
MDA-MB-231[ER(-)]
MDA-MB-435[ER(-)]
T47D[ER(+)]
MCF-7[ER(+)]
HELA
1.46 ± 0.12
2.53 ± 0.21
8.31 ± 1.16
14.13 ± 1.97
6.25 ± 0.89
5.01 ± 0.66
11.37 ± 2.32
4.84 ± 0.29
PC3
LNCap
A549
aValues represent triplicate determinations.
4.3. The inhibition activity of compound 11 against the migration of MDA-MB-231
cells
To further evaluate effects onthe migration of compound 11, in vitro transwell
cell migration was designed to assess the capacity and rate of MDA-MB-231cell
migration after the incubation of different concentrations of compound 11. As shown
in figure 3, incubation of MDA-MB-231 cells with increasing concentrations of

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compound 11 markedly reduced the number of migrated cells on the lower part of the
chamber, indicating that treatment with compound 11 strongly inhibits the migration
of MDA-MB-231cells. The migration rate of 5, 10 and 20 µM compound 11 treated
MDA-MB-231cells was 53.16%, 39.29% and 17.81%, respectively. The inhibition in
migration among 5, 10 and 20µM treated MDA-MB-231cells were statistically
significant. The inhibition of compound 11 treatment on MDA-MB-231 cells
migration are dose-dependent with the highest concentration of compound 11 (20µM)
producing the fewest number of cells that migrated toward the lower chamber.
Figure 3. Effects of compound 11 on the the migratory capacity of MDA-MB-231
cells using in vitro transwell cell migration assay. MDA-MB-231 were treated with
compound 11 for 48 h. Compound 11 does-dependently decreased the cell numbers of
MDA-MB-231 cells that migrated toward the upper chamber. Data (mean ± S.E.M.)
are representative of three independent experiments. *P<0.05, **P<0.01 and ***P <
0.001 vs. control (10% FBS).
4.4. Compound 11 induced ROS generation and apoptosis in MDA-MB-231 cells.
Intracellular reactive oxygen species (ROS) analysis was conducted to study the
effects of compound 11 in MDA-MB-231 cells. As shown in figure 4A, treatment
with compound 11 for 24 h resulted in the significantly increased production of ROS
in MDA-MB-231 cells (P < 0.05) in a dose-dependent manner. Furthermore,
compound 11 induced significant apoptotic activity at the concentration of 10 µM
using Annexin V-FITC apoptosis assay (Figure. 4B).

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Figure 4. Compound 11 induced ROS generation and apoptosis in MDA-MB-231
cells. A) effect of compound 11 on ROS generation in MDA-MB-231 cells. The cells
were pretreated with compound 11 (1-30 µmol/l) for 24 h at 37 °C. Then, the cells
were stained with DCFH-DA (10 µmol/l) and analyzed by a fluorescence plate reader.
Relative fluorescence intensities were calculated using untreated control cells as a
standard. Results are presented as mean ± SEM. n=4. B) Apoptotic effect of
compound 11 on MDA-MB-231 cells. The cells were treated by compound 11 for 48
h and stained using an Annexin V-FITC apoptosis detection kit. *P<0.05 and **P <
0.01 vs. control (Ctl). Data (mean ± S.E.M.) are representative of three independent
experiments.
4.5. The inhibition activity of compound 11 against the expression of ERRα-regulated
target genes
To evaluate the transcriptional level of ERRα-regulated target genes, we further
adapt quantitative real-time PCR to investigate the expression of peroxisome
proliferator-activated receptor coactivator 1α (PGC-1α), pyruvate dehydrogenase
kinase 4 (PDK4), osteopontin (SPP1) and pS2 (TFF1) in MDA-MB-231 cells
incubated with compound 11 for 48 h, showing that PGC-1α, PDK4, osteopontin and
pS2mRNA expression in 5 and 10µM compound 11 treated MDA-MB-231 cells were
all distinctly reduced (Figure 5). Taken together, these results suggested that
compound 11 decreases expression of ERRα-regulated target genes. However, as

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determined by quantitative real-time PCR, the steady-state level of ERRα mRNA was
not reduced upon compound 11 treatment for 48 h (Figure 5).
Figure 5. Compound 11 down-regulates the expression of ERRα-regulated target
genes. A&B&C, compound 11 decreases the mRNA levels of ERRα target genes
including PDK4, pS2 and osteopontin in a quantitative real-time PCR assay. Cells
were under the treatment of vehicle (DMSO) or various concentration of compound
11 for 48 h followed by RT-PCR analysis of mRNA expression of ERRα target genes
D, Effect of compound 11 on the expression of PGC-1α at the mRNA level in human
breast cancer cells. *P<0.05, **P<0.01 and ***P < 0.001 vs. control. Data (mean ±
S.E.M.) are representative of three independent experiments.
4.6. The inhibition activity of compound 11 against the binding of ERRα with PGC-1
The transcriptional function of ERRα relies on the interaction with
coactivatorPGC-1α. We developed a TR-FRET (time-resolved fluorescence energy
transfer) assay to monitor the interaction between the ERRα LBD with a terbium
chelate and a fluorescein-labeled fragment of the PGC-1α co-activator peptide. Ability
to disrupt the interaction of ERRα with PGC-1α of compound 11 was tested using a
TR-FRET assay in which compound 11 was measured for its receptor affinity with
ERRα. As shown in figure 6, the binding affinity in terms of FRET signal between
ERRα-LBD and PGC-1a was decreased by synthesized compound 11 in a
dose-dependent manner. A potent ERRα specific inverse agonist XCT790 was used
as a positive control in the ERRα TR-FRET cell-free assay. Compound 11 inhibited
co-activator binding with an IC50 value of 0.61 ± 0.11 µM. Therefore, it is likely that
compound 11 acts as an ERRα specific inverse agonist through directly binding to
ERRα and suppressing the interaction with its co-activators.

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Figure 6. Effects of compound 11 on the interaction between ERRα and PGC-1α.
Compound 11 inhibited the interaction between ERRα-LBD with PGC-1α in a
dose-dependent manner which was in according with the positive control of ERRα
(using a TR-FRET assay).
4.7. The inhibition activity of compound 11 against the growth of breast tumors in
mice xenograft models
Given its promising in vitro biological activity, the efficacy of compound 11 in
vivo for treating breast cancer was studied. We adapted human breast cancer
xenograft model in nude mice to determine the antitumor effect of compound 11.
MDA-MB-231 (1 × 107) cells/sites were injected subcutaneously into the right flank
of nude mice. After 2 weeks implantation, tumors reached an average size
of >100ꢀmm3, mice were divided into three treatment groups: control vehicle, 30
mg/kg of compound 11, and 30 mg/kg of cyclophosphamide.
After 2 weeks of treatment, a decrease in growth was observed in both treatment
groups of compound 11 and cyclophosphamide. And untreated tumor-bearing mice or
mice treated with vehicle, increased in size at a steady rate over time within 15 days
reaching 1400 mm3 experienced rapid growth of MDA-MB-231 flank xenografts
within 15 days. At the final measurement, vehicle group tumor volume exceed 1500
mm3 (P = 0.004) whereas the compound 11 tumor volume was 720 mm3. At the end of
the study, the tumor volumes from the mice treated with 30 mg/kg compound 11 and
30 mg/kg cyclophosphamide were 40.9% and 50.1% of the size of the tumors in
vehicle group, respectively (Figure 7). Taken together, these results indicate that
compound 11 has promising antitumor effect for treating human breast cancer.

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Figure 7. Compound 11 inhibits tumor growth in a subcutaneous xenograft
model of human breast cancer. MDA-MB-231 cells were implanted subcutaneously
into the right flank of nude mice and allowed to grow to 100ꢀmm3 before treatments
with compound 11, cyclophosphamide or with vehicle alone. Treatments were
continued for 30 days, and the differences in tumor growth rates between the control
group and each treatment group were evaluated statistically. Tumor volumes were
significantly reduced when compared with control, *P<0.05 and **P < 0.01.
5. Molecular docking model
The ERRα ligand binding pocket (LBP, Fig. 1B) is delimited by the 22 amino
acids Leu324, Cys325, Leu327, Phe328, Glu331 (all on H3), Trp361, Met362,
Leu365, Val366, Val369, Arg372 (all on H5), Phe382 (on S1), Leu398, Leu401,
Gly402, Leu405 (all on H7), Val491, Leu492, His494, Phe495 (all on H11), Phe510,
and Leu514 (all on H12)[20]. The crystal structure of estrogen-related receptor α in
complex with a synthetic inverse agonist compound 1(PDB:2PJL) reveals its novel
molecular mechanism[12]. The structure reveals the dramatic multiple conformational
changes in the LBP, which create the necessary space for the ligand. As a
consequence, the new side chain conformation of Phe328 (on helix H3), Phe510(H12)
hasto move away, and thus the activation helix H12 is displaced from its agonist
position and subsequently filling the coactivator groove of the AF-2 domain. The
amino-nitrogen of compound 1 makes a direct salt bridge and a water-mediated
hydrogen bond interaction with Glu331. All other interactions are hydrophobic,

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mainly with the side chains of Phe328, Phe382, Leu398, Phe495, Leu500, Val321 and
Leu324.
The 3D docking model of compound 1, XCT-790 and compound 11 to ERRα
was constructed by Surflex-dock geomx docking module within the software
Sybyl-X2.0 using human ERRα crystal structure (PDB entry 2PJL) as the model. The
key features of the binding mode of XCT-790 are summarized as follows (shown in
figure 8). First, hydrogen bonds existed between the NH of amide group attached to
A-ring of XCT-790and the side chain Gly397 of the receptor. Second, B-ring and
C-ring of XCT-790 interacted with the receptor through hydrophobic interactions,
strongly resembled the hydrophobic interactions of compound 1 with ERRα LBP.
Figure 8. Superimposition of docking model of compound 1 (cyan) and XCT-790
(reddish brown) docked into ERRα crystal structure (2PJL). Hydrogen bonding
interactions are shown with dotted yellow lines. These images were generated using
the sybyl-x2.0 program.
According the docking prediction, the docking model of compound 11 mostly
resembled the binding model of XCT790 on the ERRα ligand-binding domain of
2PJL (Figure.9).The B and C aromatic rings in compound 11 occupied the positions
nearly where B and C aromatic rings in XCT790 were located separately and
interacted with the receptor ERRα through hydrophobic interactions. Similar to
hydrogen binding between the NH of amide group attached to A-ring of XCT-790 and
the side chain Gly397 of the receptor, the two NH in the ureido group of compound
11 formed strong hydrogen binding interactions with the residue Gly397 of ERRα
receptor. The A-ring in compound 11 stretched upwards and covered the opening of

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ERRα LBP like a lid. These interations between the receptor ERRα and compound 11
probably explained the activities of compound 11 against ERRα.
Figure 9. Superimposition of docking model of compound 11(white) and XCT-790
(reddish brown) docked into ERRα crystal structure (2PJL). Hydrogen binding
interactions are shown with dotted yellow lines. These imageswere generated using
the sybyl-x2.0 program.
6. Conclusion
A
novel
structural
class
of
ERRα
inverse
agonists,
1-(2,5-diethoxy-benzyl)-3-phenyl-urea analogues (compounds 3-11), are discovered
through structural optimization and ERRα in vitro and in vivo assays. Among them,
compound 11 is found to be the most promising ERRα inverse agonist. First,
compound 11 demonstrates strong inhibitory effects on the transcriptional activity of
ERRα in human MDA-MB-231 cells in a dose dependent manner. Second, compound
11 reveals broadly anti-proliferative effect toward multiple tumor cell lines in which
ER-negative MDA-MB-231 cells are the most sensitive to compound 11 treatment.
Third, compound 11 does-dependently inhibits the migration of breast cancer cells.
Forth, compound 11 decreases the expression of ERRα-regulated target genes. Fifth,
compound 11 acts as an inverse agonist to selectively disrupt the interaction between
ERRα LBD and its nuclear co-activator PGC-1α in a dose dependent manner. Sixth,
compound 11 reveals significant inhibitory effect on the growth of ER-negative
MDA-MB-231 human breast cancer xenografts in vivo. The binding characteristics
between ERRα domain and ligand show that compound 11 interacts with the binding

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pocket through hydrogen interactions with the residue Gly397 and hydrophobic
interactions with the hydrophobic residues.These novel compounds reported in this
study will provide new insights for the design and development of potent ERRα
inverse agonists and for the treatment of triple negative breast cancer.
7. Experimental section
7.1. Chemistry
7.1.1. General synthetic methods
General synthetic methods: Unless otherwise noted, all materials were obtained
from commercial suppliers and used without further purification.All reactions were
monitored using thin-layer chromatography (TLC) on silica gel plates. NMR spectra
were determined using a Bruker AVANCE II 400spectrometers in CDCl3or DMSO
solution. The final compound purity was determined by high performance liquid
chromatography (HPLC: thermo fisher scientific ODS column, 4.6×250, 8min, flow
rate=2.0ml/min, gradient=90-10% CH3OH/10-90% water.)
7.1.2.2-Butoxy-5-[3-(2,5-diethoxy-4-methanesulfonylamino-benzyl)-ureido]-benzoic
acid methyl ester(4)
The mixture of 2-Hydroxy-5-nitro-benzoic acid methyl ester (F) (1.0 g,
5.07mmol) and K2CO3 (1.39 g, 10.1 mmol) in N,N-dimethylformamide (DMF) was
heated at 80℃for 10 min, then the bromobutane (0.77g, 5.98mmol) was added
dropwise to the solution followed by stirring at 80°C for 4h. DMF was removed by
distillation and the residue was dissolved in dichloromethane and extracted with water
(3×200 ml). The combined organic phases were dried with Na2SO4and concentrated
under reduced pressure. The remaining solid was recrystallized from methanol to give
the pale yellow solid (4G, 1.1g, yield 85.9%).
To a solution of 2-Butoxy-5-nitro-benzoic acid methyl ester (4G, 1.0 g, 3.95
mmol) in MeOH (15 ml) was added NiCl2·6H2O (1.61 g, 6.8mmol), NaBH4 (0.51 g,
13.6mmol), and the mixture was stirred at room temperature for 2h. The mixture was
made acidic with a 10% HCl solution and extracted with ethyl acetate (3×150 ml), the

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organic layer was washed with a saturated NaCl solution (3×100 ml), and the
combined organic phases were dried with Na2SO4, filtered, concentrated, and purified
by flash column chromatography to give 5-Amino-2-butoxy-benzoic acid methyl ester
(4H, 0.78 g, yield 88.6% ) as a white solid.
To a solution of (2,5-diethoxy-4-nitro-benzyl)-carbamic acid phenyl ester 29 (I,
0.4 g, 1.11mmol ) in dioxane was added 4H(0.245 g, 1.1mmol) and triethylamine
(1.12 g, 11.1mmol), and the mixture was heated at 80oC for 8h. After the solvent was
removed, the residue was dissolved in dichloromethane and extracted with water
(3×100 ml). The combined organic phases were dried with Na2SO4, filtered,
concentrated, and recrystallized from methanol to give pure product
2-butoxy-5-[3-(2,5-diethoxy-4-nitro-benzyl)-ureido]-benzoic acid methyl ester (4J,
0.462g, yield 86.1%) as yellow solid.
To a stirring solution 1-(2,5-Diethoxy-4-nitro-benzyl)-3-phenyl-urea (4J, 0.391g,
0.80 mmol) in MeOH (15ml),NiCl2·6H2O (0.353 g, 1.48mmol) and NaBH4 (0.112
g,2.96mmol ) were added and stirred at room temperature for 0.5h.The mixture was
made acidic with a 10% HCl solution and extracted with ethyl acetate (3×150 ml), the
organic layer was washed with a saturated NaCl solution (3×100 ml), and the
combined organic phases were dried with Na2SO4, filtered, concentrated, and purified
by
flash
column
chromatography
to
give
5-[3-(4-Amino-2,5-diethoxy-benzyl)-ureido]-2-butoxy-benzoic acid methyl ester (4K,
0.312g, yield 85.0% ) as white solid.
To a stirring solution of 4K (0.321 g, 0.70mmol) in dichloromethane (15ml) and
pyridine (0.0608g, 0.77mmol), methanesulfonyl chloride (0.080g, 0.70mmol) was
added dropwise followed by stirring at room temperature for 8h.The solid residue was
dissolved in dichloromethane (100 ml) and washed with a saturated NaCl solution
(3×50 ml). The combined organic phases were dried with Na2SO4, filtered, and
purified by flash column chromatography to give pure product (compound 4,0.201g,
yield53.7% ) as white solid.1H NMR (400 MHz, DMSO) δ 8.84 (s, 1H), 8.57 (s, 1H),
7.72 (d, J = 2.6 Hz, 1H), 7.44 (dd, J = 8.9, 2.6 Hz, 1H), 7.01 (d, J = 9.0 Hz, 1H), 6.93
(s, 1H), 6.87 (s, 1H), 6.32 (t, J = 5.8 Hz, 1H), 4.21 (d, J = 5.7 Hz, 2H), 3.97 (ddd, J =

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19.3, 9.7, 5.9 Hz, 6H), 3.75 (s, 3H), 2.92 (s, 3H), 1.73 – 1.54 (m, 2H), 1.48 – 1.37 (m,
2H), 1.32 (dd, J = 12.8, 6.7 Hz, 6H), 0.91 (t, J = 7.4 Hz, 3H).13C NMR (101 MHz,
DMSO) δ 166.2, 155.2, 152.3, 149.6, 145.4, 133.3, 126.2, 125.0, 123.0, 120.3, 120.1,
114.6, 113.5, 110.6, 68.5, 64.5, 64.0, 51.7, 40.1, 38.1, 30.8, 18.6, 14.9, 14.6, 13.6.
7.1.3. 2-Benzyloxy-5-[3-(2,5-diethoxy-4-methanesulfonylamino-benzyl)-ureido]-benzo
-icacid methyl ester (5)
Compound 5as a white solid was prepared by a procedure similar to that
described for the synthesis of compound
4
using the corresponding
bromomethyl-benzene instead of bromobutane.1H NMR (400 MHz, CDCl3) δ 7.66 (d,
J = 2.5 Hz, 1H), 7.49 – 7.41 (m, 3H), 7.38 (t, J = 7.4 Hz, 2H), 7.07 (s, 1H), 6.92 (t, J
= 9.5 Hz, 1H), 6.88 (s, 1H), 6.77 (s, 1H), 5.13 (s, 2H), 4.34 (s, 2H), 4.05 – 3.90 (m,
4H), 3.87 (s, 3H), 2.93 (d, J = 10.6 Hz, 3H), 1.40 – 1.32 (m, 3H), 1.28 (t, J = 7.0 Hz,
3H).13C NMR (101 MHz, DMSO) δ 166.0, 155.2, 151.9, 149.6, 149.6, 145.4, 137.2,
133.7, 128.3(2C), 127.5, 127.0(2C), 126.2, 125.1, 122.9, 120.2, 115.2, 113.6, 110.6,
70.3, 64.6, 64.1, 51.8, 40.2, 38.1, 14.7, 14.6.
7.1.4.
N-Benzyl-5-[3-(2,5-diethoxy-4-methanesulfonylamino-benzyl)-ureido]-2-ethoxy-benza
mide(6)
To a solution of 2-ethoxy-5-nitro-benzoic acid methyl ester[15](A, 6g, 26.7 mmol)
in methanol (80 ml) was added KOH (3g, 53.6 mmol) and the solution was stirred at
80°C for 3h. Then the mixture was made acidic with a 10% HCl solution and the
white solid was precipitated. The solid was collected by filtration and washed with
water, then dried in vacuum over 18 h to give the 2-ethoxy-5-nitro-benzoic acid (B,
5.5 g, yield 97.7%). To a stirring solution of compound B (5.5 g, 26.1 mmol) in
dichloromethane was added N,N-dimethylformamide (0.1 ml) followed by thionyl
chloride (6.2 ml, 52.1 mmol), and the mixture was stirred at room temperature for
6h.Dichloromethane (3×100 mL) was used asazeotrope for the residue and the solvent
was removed to give 2-ethoxy-5-nitro-benzoyl chloride (C) as a yellow oil

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(0.55g,yield 97%), which was used without further purification. To a stirring solution
of compound C(0.50 g, 2.18 mmol) in dichloromethane was added benzylamine
(0.233g, 2.18 mmol) followed by triethylamine(0.242 g, 2.40 mmol), and the mixture
was stirred at room temperature for 3h.Then water (50ml ) was added and the mixture
was extracted with dichloromethane(3 x 50 mL). The combined extracts were dried
over Na2SO4, filtered, concentrated and purified by flash column chromatography to
give pure product N-benzyl-2-ethoxy-5-nitro-benzamide (6D, 0.550 g, yield 84.1%)
as white solid. To a stirring solution of 6D(0.520g, 1.73mmol) in MeOH (8 ml) was
added NiCl2·6H2O (0.707g, 2.97mmol) followed by NaBH4 (0.226g, 5.94mmol), and
the mixture was stirred at room temperature for 2h. Then the mixture was made acidic
with a 10% HCl solution and extracted with ethyl acetate(2×150ml), the organic layer
was washed with a saturated NaCl solution (3×80ml), the combined organic phases
were dried with Na2SO4, filtered, concentrated and purified by flash column
chromatography to give the pure product 5-amino-N-benzyl-2-ethoxy-benzamide (6E,
0.420g, yield 89.9%) as white solid. Then compound 6 (white solid, 0.170g ) was
prepared by a procedure similar to that described for the synthesis of compound 4
using the corresponding 5-amino-N-benzyl-2-ethoxy-benzamide (6E) instead of
5-amino-2-butoxy-benzoic acid methyl ester (4H).1H NMR (400 MHz, DMSO) δ
8.82 (s, 1H), 8.57 (d, J = 7.6 Hz, 2H), 7.69 (d, J = 2.7 Hz, 1H), 7.54 (dd, J = 8.9, 2.6
Hz, 1H), 7.33 (dd, J = 8.7, 5.2 Hz, 4H), 7.28 - 7.19 (m, 1H), 7.01 (d, J = 9.0 Hz, 1H),
6.94 (s, 1H), 6.87 (s, 1H), 6.28 (t, J = 5.8 Hz, 1H), 4.49 (d, J = 5.8 Hz, 2H), 4.21 (d, J
= 5.7 Hz, 2H), 4.07 (q, J = 6.9 Hz, 2H), 3.98 (qd, J = 6.8, 2.5 Hz, 4H), 2.92 (s, 3H),
1.36 -1.24 (m, 9H).13C NMR (101 MHz, DMSO) δ 164.9, 155.2, 150.8, 149.6, 145.4,
139.3, 133.7, 128.2(3C), 127.1(3C), 126.8, 126.2, 125.0, 121.7, 120.0, 113.6, 113.5,
110.6, 64.5, 64.0, 42.6, 40.1, 38.1, 14.7, 14.6, 14.5. HPLC purity = 96.0%.
7.1.5.5-[3-(2,5-Diethoxy-4-methanesulfonylamino-benzyl)-ureido]-2-ethoxy-N-phenyl
-benzamide(7)
Compound 7 (white solid, 0.170 g)was prepared by a procedure similar to that
described for the synthesis of compound 6.1H NMR (400 MHz, DMSO) δ 10.16 (s,

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1H), 8.84 (s, 1H), 8.61 (s, 1H), 7.71 (dd, J = 12.2, 5.2 Hz, 3H), 7.55 (dd, J = 8.9, 2.5
Hz, 1H), 7.34 (t, J = 7.8 Hz, 2H), 7.08 (t, J = 8.3 Hz, 2H), 6.95 (s, 1H), 6.87 (s, 1H),
6.32 (t, J = 5.7 Hz, 1H), 4.22 (d, J = 5.6 Hz, 2H), 4.13 (q, J = 6.8 Hz, 2H), 3.99 (qd,
J= 6.7, 2.4 Hz, 4H), 2.93 (s, 3H), 1.45 - 1.25 (m, 9H).13C NMR (101 MHz, DMSO) δ
163.7, 155.2, 150.6, 149.6, 145.4, 138.9, 133.9, 128.8 (2C), 126.2, 125.1, 123.8, 123.5,
121.9, 119.7, 119.5 (2C), 113.8, 113.6, 110.6, 64.8, 64.6, 64.1, 40.1, 38.1, 14.7, 14.6,
14.6. HPLC purity = 97.1%.
7.1.6.5-[3-(2,5-Diethoxy-4-methanesulfonylamino-benzyl)-ureido]-2-ethoxy-N-p-tolyl
-benzamide(8)
Compound 8 (white solid, 0.160 g ) was prepared by a procedure similar to that
described for the synthesis of compound 6.1H NMR (400 MHz, DMSO) δ 10.08 (s,
1H), 8.82 (s, 1H), 8.61 (s, 1H), 7.72 (d, J = 2.6 Hz, 1H), 7.56 (dd, J = 14.5, 5.5 Hz,
3H), 7.14 (d, J = 8.2 Hz, 2H), 7.06 (d, J = 9.0 Hz, 1H), 6.95 (s, 1H), 6.87 (s, 1H), 6.33
(t, J = 5.7 Hz, 1H), 4.22 (d, J = 5.7 Hz, 2H), 4.12 (q, J = 6.8 Hz, 2H), 3.99 (qd, J = 6.8,
3.4 Hz, 4H), 2.92 (s, 3H), 2.26 (s, 3H), 1.41 - 1.31 (m, 9H).13C NMR (101 MHz,
DMSO) δ 163.4, 155.3, 150.6, 149.6, 145.4, 136.4, 133.9, 132.4, 131.5, , 129.2, 128.6,
126.2, 125.0, 123.8, 121.9, 119.4, 113.8, 113.5, 110.6, 67.4, 65.0, 64.8, 64.6, 64.1,
40.1, 38.1, 20.4, 14.7, 14.6, 14.6. HPLC purity = 96.7%.
7.1.6.5-[3-(2,5-Diethoxy-4-methanesulfonylamino-benzyl)-ureido]-2-ethoxy-N-(3-met
hoxy-phenyl)-benzamide(9)
Compound 9 (white solid, 0.110 g ) was prepared by a procedure similar to that
described for the synthesis of compound 6.1H NMR (400 MHz, DMSO) δ 10.14 (s,
1H), 8.82 (s, 1H), 8.59 (s, 1H), 7.72 (d, J = 2.4 Hz, 1H), 7.55 (dd, J = 8.9, 2.5 Hz, 1H),
7.41 (s, 1H), 7.27 - 7.17 (m, 2H), 7.06 (d, J = 9.0 Hz, 1H), 6.95 (s, 1H), 6.88 (s, 1H),
6.67 (d, J = 7.3 Hz, 1H), 6.32 (t, J = 5.7 Hz, 1H), 5.75 (s, 1H), 4.22 (d, J = 5.6 Hz,
2H), 4.12 (q, J = 6.8 Hz, 2H), 3.99 (qd, J = 6.8, 2.9 Hz, 4H), 3.74 (s, 3H), 2.93 (s, 3H),
1.42 -1.26 (m, 9H).13C NMR (101 MHz, DMSO) δ163.7, 159.6, 155.2, 150.6, 149.6,
145.4, 140.0, 133.9, 129.6, 126.2, 125.1, 123.8, 121.9, 119.7, 113.8, 113.6, 111.8,

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110.6, 108.8, 105.4, 64.7, 64.6, 64.1, 55.0, 40.1, 38.1, 14.7, 14.6, 14.6. HPLC purity =
98.4%.
7.1.7.5-[3-(2,5-Diethoxy-4-methanesulfonylamino-benzyl)-ureido]-2-ethoxy-N-isopro
pyl-benzamide (10)
Compound 10 (white solid, 0.180 g )was prepared by a procedure similar to that
described for the synthesis of compound 6.1H NMR (400 MHz, DMSO) δ 8.81 (s,
1H), 8.54 (s, 1H), 8.02 (d, J = 7.4 Hz, 1H), 7.70 (d, J = 2.7 Hz, 1H), 7.54 (dd, J = 8.9,
2.7 Hz, 1H), 6.99 (d, J = 8.9 Hz, 1H), 6.94 (s, 1H), 6.87 (s, 1H), 6.27 (t, J = 5.8 Hz,
1H), 4.21 (d, J = 5.7 Hz, 2H), 4.09 -4.06 (m, 2H), 3.98 (tt, J = 6.8, 3.3 Hz, 4H), 2.93
(s, 3H), 1.40 -1.28 (m, 9H), 1.15 (d, J = 6.5 Hz, 6H).13C NMR (101 MHz, DMSO) δ
163.6, 155.2, 150.9, 149.6, 145.4, 133.8, 126.2, 125.1, 122.9, 121.6, 120.0, 113.8,
113.6, 110.6, 64.8, 64.6, 64.1, 40.8, 40.1, 38.1, 22.4 (2C), 14.7, 14.6, 14.5.
7.1.8.5-[3-(2,5-Diethoxy-4-methanesulfonylamino-benzyl)-ureido]-2-ethoxy-N-(4-met
hoxy-benzyl)-benzamide(11)
Compound 11 (white solid, 0.110 g )as a white solid was prepared by a
procedure similar to that described for the synthesis of compound 6.1H NMR (400
MHz, DMSO) δ 8.80 (s, 1H), 8.59 -8.48 (m, 2H), 7.70 (d, J = 2.7 Hz, 1H), 7.54 (dd, J
= 8.9, 2.7 Hz, 1H), 7.24 (t, J = 7.9 Hz, 1H), 7.01 (d, J = 9.0 Hz, 1H), 6.91 (dd, J =
17.8, 10.4 Hz, 4H), 6.81 (d, J = 9.1 Hz, 1H), 6.27 (t, J = 5.7 Hz, 1H), 4.46 (d, J = 5.8
Hz, 2H), 4.21 (d, J = 5.7 Hz, 2H), 4.07 (q, J = 6.9 Hz, 2H), 3.99 (qd, J = 6.8, 2.4 Hz,
4H), 3.73 (s, 3H), 2.93 (s, 3H), 1.38 – 1.23 (m, 9H).13C NMR (101 MHz, DMSO) δ
164.6, 158.2, 155.2, 150.8, 149.6, 145.4, 133.8, 131.2, 128.5(3C), 126.2, 125.0, 123.0,
121.7, 120.0, 113.7(2C), 113.5, 110.6, 64.6, 64.5, 64.0, 55.0, 42.1, 40.1, 38.1, 14.7,
14.6, 14.5. HPLC purity = 96.9%.
7.2.Biologicalassays
7.2.1. Cell culture and reagents
All chemicals were purchased from Sigma Chemical Company (St. Louis, MO,
USA) unless otherwise noted. MDA-MB-231 cells were grown in Leibovitz’s L-15

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Medium (Gaitherburg, MD, USA) enriched with 10% fetal bovine serum (FBS), 1%
L-glutamine, 1% sodium pyruvate and 100 U/ml penicillin/streptomycin and
maintained at 37°C in a 5% CO2 humidified incubator. BCA Protein Assay Kit, RIPA
buffers, cell counting kit (CCK-8) were purchased from Beyotime Biotechnology
(Shanghai, China). Antibodies against ERRα, and β-actin were purchased from
Abcam (Cambridge, MA, UK).
7.2.2.Cell proliferation assay
The effects of compounds on the proliferation of tumor cells were detected by
CCK-8 assay. Briefly, cells were seeded into 96-well plates and cultured overnight
using the conditions described above for the cell culture. Then, cells were treated with
different concentrations of compounds for 48 h. CCK-8 (10 µl) was subsequently
added to each well and the cells were further incubated at 37°C for 2 h prior to
measuring the optical density (OD) in each well at a wavelength of 450 nm using
Spectramax M2 microplate Reader (Molecular Devices).
7.2.3. TR-FRET assay
The interaction of the coactivator proteins with ERRs is regulated by
ligand-induced conformation of the ERRs ligand-binding domain (LBD) where
coactivator proteins are bound to ERRs. A time-resolved fluorescence resonance
energy transfer (TR-FRET) co-activator assay was used to analyze the interaction of
the LBD of ERRs with PGC-1α peptide according to the manufacturer’s
instructions(Invitrogen)10. To determine the one-half maximal effective concentrations
(IC50), nonlinear regression, sigmoidal dose-response (variable slope) curves were
performed, using GraphPad Prism version 6.0 (GraphPad Software, San Diego, CA).
7.2.4.Transient transfection luciferase reporter assay
A well-established mammalian two-hybrid reporter gene assay was used to
determine biological activities of compounds [10,19]. HEK 293 cells were plated in
96-well plate at a density of 1×105 cells/well maintained in DMEM medium
supplemented with 10% FBS (v/v). After plating at 70-80 percent confluency, cells
were transiently co-thransfected using Lipofectamine 2000 (Invitrogen). Cells were

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separately transfected with 20 ng per well expression plasmids of either
GAL4-DBD-ERRα-LBD, -ERRβ-LBD, -ERRγ-LBD and -ERα-LBD together with 20
ng per well luciferase reporter UAS-Luc and 5 ng per well internal control vector
pRL-TK. Following incubation with transfection reagents for 6 h at 37°C, cells were
treated with vehicle (0.05% DMSO) or test compounds for another 48 h. Subsequently,
the cells were lysed and luciferase activity was measured by dual luciferase reporter
assay system according to the manufacturer’s instruction (Promega, Madison, WI,
USA).
Full-length human ERRα vector (pCMV Tag2-hERRα) and pERRE-Luc reporter
plasmid were co-transfected into HEK-293 with Lipofectamine 2000 (Invitrogen) and
the procedure was carried out as directed by the manufacturer. The
pSV-β-galactosidase plasmid (pCH110) was co-transfected as an internal control.
Luciferase activity was assayed according to the manufacturer’s protocol using the
luciferase assay system (Promega, Madison, WI, USA). Relative luciferase units
(RLU) normalized to transfection efficiency was calculated as the ratio of luciferase
activity to β-galactosidase.
7.2.5.Quantitative real-time PCR
MDA-MB-231 cells were grown in 6-well plate (in 2 ml media) at a density of
5×106 cells/well and pretreated in the presence or absence of compounds for 24 h.
Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA) as
instructed by the manufacturer. 2 µg of total mRNA was reverse-transcribed with the
Superscript™ reverse transcription system (Takara, Dalian, Japan). Quantitative
real-time reverse transcription polymerase chain reaction (RT-PCR) reaction was
carried out on ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City,
CA, USA) using SYBR Green PCR master mix reagents (Takara, Dalian,
Japan).Relative quantification of target gene mRNA level was calculated after
normalization to GAPDH mRNA using the 2-△△Ct method. The primer sequences were
as follows:
GAPDH

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5’-AGAAGGCTGGGGCTCATTTG-3’ (forward)
5’-AGGGGCCATCCACAGTCTC-3’ (reverse);
ERRα
5’-ACTGCAGGATGAGCTGG -3’ (forward)
5’-TGCACAGAGTCTGAATTG -3’ (reverse);
PGC-1α
5’-TGCCACCACCATCAAAGAAGC -3’ (forward)
5’-TCACCAAACAGCCGCAGACT -3’ (reverse)
PDK4
5’- CGCTTAGTGAACACTCCTTCG -3’ (forward)
5’-CTTCTGGGCTCTTCTCATGG -3’ (reverse)
pS2
5’-ATGGCCACCATGGAGAACAAGG -3’ (forward)
5’-CTAAAATTCACACTCCTCTTCTGG -3’ (reverse)
Osteopontin
5’-CATCTCAGAAGCAGAATCTCCTA -3’ (forward)
5’-GGAAAGTTCCTGACTATCAATCA -3’ (reverse)
7.2.6. Western blot
Briefly, whole cell lysate (MDA-MB-231) was prepared in RIPA buffer and then
centrifuged for 15 min at 14000 rpm[19]. The protein concentration in each sample was
quantified using the BCA Protein Assay kit. Equal amounts of proteins were loaded
per lane in a 12% sodium dodecyl sulfate-polyacrylamide electrophoresis
(SDS-PAGE), and then transferred onto PVDF membranes. The membranes were
blocked with 0.5% skimmed milk in TBST at room temperature for 2 h. Then,
membranes were probed with primary antibodies (ERRα) overnight at 4°C followed
by incubation with corresponding horseradish peroxidase-conjugated secondary
antibodies for 1 h at room temperature on the next day. β-actin was served as the
internal control.
7.2.7. Transwell migration assay

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The in vitro cell migration assay was conducted using a transwell chamber (8-µm
pore size) as instructed by the manufacturer[10]. MDA-MB-231 cells were seeded in
the upper part of the transwell plate in the absence or presence of different compounds
concentrations for 24 h in the medium containing 1% FBS. Cells were allowed
migrating for 24 h at 37°C/5% CO2. After incubation, the cells in the upper surface of
the transwell plate were carefully removed with a wet cotton swab, migrated cells
which passed through the filters on the lower part of the membrane was fixed with
70% ethanol, stained with 0.02% crystal violet and photographed at magnification
×200.
7.2.8. Assessment of intracellular reactive oxygen species (ROS) production
Reactive
oxygen
species
production
was
quantified
by
the
2’,7’-dichlorofluorescin diacetate (DCFH-DA) method[21], based on the reactive
oxygen species-dependent oxidation of DCFH-DA to 2’,7’-dichlorofluorescein (DCF).
MDA-MB-231 cells grown in 96-well plates were then incubated with DCFH-DA (10
µmol/l) for 30 min. Fluorescence of the samples was monitored at an excitation
wavelength of 488 nm and an emission wavelength of 535 nm.
7.2.9. Apoptosis assay
To evaluate the proapoptotic effect of compound 11, MDA-MB-231 cells were
stained using an Annexin V-FITC apoptosis detection kit (Sigma) [21]. Briefly, 1×106
cells were washed twice, resuspended in PBS, and stained with 5 µL of Annexin
V-FITC according to the manufacturer’s instruction. At least 300 cells were observed
by fluorescent microscopy in each slide, and the percentage of Annexin V-FITC
positive cells was counted in each sample. The number of apoptotic cells among 300
cells was counted and expressed as percentage in compound 11- or vehicle-treated
cells, respectively.
7.2.10. Breast cancer xenografts
Human breast cancer xenografts were generated by subcutaneously injecting
2ꢀ×ꢀ106 MDA-MB-231 cells suspended with PBS in the right flank region of
6-week-old mice BALB/c nude mice[10]. Mice were monitored for tumor growth at

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indicated times after cell implantation. When subcutaneous tumors reached an average
size of>100ꢀmm3, mice were randomized into appropriate groups of 10 mice each and
treatments with HSP1604, cyclophosphamide or with vehicle alone were then
initiated. Treatment regimens consisted of a total of 2 doses of HSP1604, with doses
at 30mg/kg or 100 mg/kg administered every other day during 28-day period. During
the study, Mice were weighed 3 times per week during and after the drug-treatments,
with no toxicities. Tumor volumes were calculated using the formula V =
lengthꢀ×ꢀwidth2ꢀ×ꢀ0.5, where length and width represent the longer and shorter tumor
diameters, respectively. All procedures were performed in accordance to approved
protocols by the Huashan Hospital Institutional Animal Care and Use Committee.
7.3.Molecular docking
The crystal structure of the ERRα ligand binding domain in complex with
compound 1 was retrieved from the RCSB Protein Data Bank (PDB entry2PJL). All
the docking simulations of the studied compounds within the binding pocket of ERRα
have been performed using surflex-dock geomx program (Sybyl X2.0) with the
default settings.
Acknowledgment
This work was supported by Grants from the National Natural Science
Foundation of China (81202389 and 81302853) and Shanghai Municipal Commission
of Health and Family Planning (201540261).
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