Discovery of 5-(2′,4′-difluorophenyl)-salicylanilides as new inhibitors of receptor activator of NF-κ°B ligand (RANKL)-induced osteoclastogenesis

Article abstract of DOI:10.1016/j.ejmech.2015.05.015

To improve the inhibitory potency of lead compound NDMC101 on RANKL-induced osteoclastogenesis, a series of new 5-(2′,4′-difluorophenyl)-salicylanilide derivatives were synthesized and evaluated for osteoclast inhibition by using TRAP-staining assay. Among them, both of compounds 6d and 6i showed three-fold increase in osteoclast-inhibitory activities compared to NDMC101 at half-inhibitory concentration. Further, the mechanistic study showed that 6d and 6i could suppress RANKL-induced osteoclastogenesis-related genes, such as NFATc1, c-fos, TRAP, and cathepsin K. Their inhibitory activities were further confirmed by including specific inhibition of NF-κ°B and NFATc1 expression levels in nucleus. In addition, 6d and 6i also could significantly attenuate bone-resorbing activity of osteoclasts by performing pit formation assay. Thus, a new class of 5-(2′,4′-difluorophenyl)-salicylanilide derivatives may be considered as essential lead structures for the further development of anti-resorptive agents.

Full text of DOI:10.1016/j.ejmech.2015.05.015

Accepted Manuscript
Discovery of 5-(2',4'-difluorophenyl)-salicylanilides as new inhibitors of receptor
activator of NF-κB ligand (RANKL)-induced osteoclastogenesis
Chia-Chung Lee, Fei-Lan Liu, Chun-Liang Chen, Tsung-Chih Chen, Deh-Ming Chang,
Hsu-Shan Huang
PII:
S0223-5234(15)30042-8
10.1016/j.ejmech.2015.05.015
EJMECH 7894
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 March 2015
Revised Date: 30 April 2015
Accepted Date: 10 May 2015
Please cite this article as: C.-C. Lee, F.-L. Liu, C.-L. Chen, T.-C. Chen, D.-M. Chang H.-S. Huang
Discovery of 5-(2',4'-difluorophenyl)-salicylanilides as new inhibitors of receptor activator of NF-κB ligand
(RANKL)-induced osteoclastogenesis, European Journal of Medicinal Chemistry (2015), doi: 10.1016/
j.ejmech.2015.05.015.
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ACCEPTED MANUSCRIPT
Graphical abstract
We synthesized a series of new 5-(2',4'-difluorophenyl)-salicylanilide derivatives. Among
them, 6d and 6i not only suppressed RANKL-induced NF-κB and NFATc1 activation, but
also reduced expression levels of osteoclastogenesis-related genes.

ACCEPTED MANUSCRIPT
Discovery of 5-(2',4'-difluorophenyl)-salicylanilides as new inhibitors of
receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis
a,b,c
d
a
a
d,b*
Chia-Chung Lee , Fei-Lan Liu , Chun-Liang Chen , Tsung-Chih Chen , Deh-Ming Chang ,
a, b, c*
Hsu-Shan Huang
a
Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and
Technology, Taipei Medical University, Taipei 110, Taiwan
b
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan
c
School of Pharmacy, National Defense Medical Center, Taipei 114, Taiwan
d
Rheumatology/Immunology/Allergy, Taipei Veterans General Hospital, Taipei 112, Taiwan
*
Corresponding authors:
Prof. Dr. H.S. Huang (Taipei Medical University); Tel: +886-2-2736-1661 ext.7525, Fax:
886-2-6638-7537, E-mail: huanghs99@tmu.edu.tw; Prof. Dr. D.M. Chang (Taipei Veterans
+
General Hospital); Tel: +886-2-2875-7799, Fax: +886-2-7735-1333, E-mail:
ming0503@ms3.hinet.net
1

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Abstract
To improve the inhibitory potency of lead compound NDMC101 on RANKL-induced
osteoclastogenesis, a series of new 5-(2',4'-difluorophenyl)-salicylanilide derivatives were
synthesized and evaluated for osteoclast inhibition by using TRAP-staining assay. Among
them, both of compounds 6d and 6i showed three-fold increase in osteoclast-inhibitory
activities compared to NDMC101 at half-inhibitory concentration. Further, the mechanistic
study showed that 6d and 6i could suppress RANKL-induced osteoclastogenesis-related
genes, such as NFATc1, c-fos, TRAP, and cathepsin K. Their inhibitory activities were further
confirmed by including specific inhibition of NF-κB and NFATc1 expression levels in
nucleus. In addition, 6d and 6i also could significantly attenuate bone-resorbing activity of
osteoclasts by performing pit formation assay. Thus,
a
new class of
5
-(2',4'-difluorophenyl)-salicylanilide derivatives may be considered as essential lead
structures for the further development of anti-resorptive agents.
Keywords
NDMC101; salicylanilides; RANKL; osteoclastogenesis; TRAP activity assay
2

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1
. Introduction
Bone remodeling process is mediated by the balance between bone formation and
resorption, which are regulated by osteoblasts and osteoclasts respectively [1-3]. When the
balanced bone remodeling is disturbed in favor of bone resorption, it would lead to
pathological diseases, such as Paget’s disease [4], osteoporosis [5, 6], and rheumatoid arthritis
(RA) [7-9]. Osteoclasts are multinucleated cells (MNCs) of hematopoietic origin that are
formed from monocyte/macrophage lineage precursor cells [10-13]. The receptor activator of
NF-κB ligand (RANKL), a tumor necrosis factor (TNF) family molecule, is an essential
factor for the osteoclast differentiation and activation [14-16]. When RANKL binds to its
receptor RANK, TNF receptor-associated factor 6 (TRAF 6) is recruited into the intracellular
domain of RANK and then activates various intracellular signaling pathways involved in
osteoclastogenesis [17, 18]. Among these transcriptional factors, the major one is activating
NF-κB pathway [19-23]. Further, it has been reported that the RANKL signaling is associated
with the expression of osteoclast-associated genes, including nuclear factor of activated T
cells c1 (NFATc1) [24-26], c-fos [27, 28], tartrate-resistant acid phosphatase (TRAP) [29, 30],
cathepsin K [31, 32], matrix metalloprotease-9 (MMP-9) [33, 34], and dendritic cell-specific
transmembrane protein (DC-STAMP) [35, 36]. Either gene expression of NFATc1 or c-fos
appears to be an important to induce osteoclast differentiation and formation [25-28]. For
example, the NFATc1-deficient embryonic stem cells are unable to differentiate into
osteoclasts and the mice lacking c-fos develop into severe osteopetrosis due to an early block
of osteoclastogenesis in the osteoclast lineage [24, 25]. During the RANKL-induced
osteoclast differentiation, NFATc1 not only can be activated by NF-κB but also
auto-amplified by its own gene [24-26]. Based on these mechanistic findings, osteoclast
differentiation in RANKL-RANK signaling pathway provides different molecular targets for
the development of therapeutics to bone loss related diseases. Hence, numerous natural
3

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products and small molecules such as paeonol [37], magnolol [38], (-)-DHMEQ [39],
salicylanilide derivative NDMC101 [40, 41], and biphenyl-carboxylate derivative ABD345
[
42], have been developed to potential osteoclastogenesis inhibitors (Fig. 1) In addition, a
diverse range of pharmacophores including the salicylanilide core [40, 41], the
biphenyl-containing scaffold [42], and the phosphonate moiety [43, 44] explore their
osteoclast-inhibitory activities in drug discovery that encourages medicinal chemists to further
optimize lead compounds.
In our previous study, NDMC101 was observed exhibiting moderate in vitro inhibitory
activity (IC50 ≈ 15 µM) on RANKL-induced osteoclast formation in RAW264.7 cells
(osteoclast precursor cells) [40]. NDMC101 not only effectively ameliorated
inflammation-induced bone destruction but also significantly reduced the number of
TRAP-staining osteoclasts at sites of articular bone erosions in collagen-induced arthritis
(CIA) mice in vivo [40]. On the basis of these findings, the salicylanilide core scaffold
derivative (NDMC101) could be a lead structure that warrants further optimization. More
recently, van’t Hof developed a series of biphenyl-based derivatives as inhibitors of
RANKL-induced osteoclast formation, which potentially exhibited anti-resorptive effects in
vitro and in vivo [42]. It is notable that 2’,4’-dihalophenyls among these derivatives,
especially 2’,4’-difluoro substituted, showed an increase in inhibitory effects on
RANKL-induced osteoclast formation. The aforementioned encouraging results stimulated
our efforts in pursuing new inhibitors of RANKL-induced osteoclastogenesis. In the present
work, we designed and synthesized a series of salicylanilide analogues 6a-6l together with a
2
’,4’-difluorophenyl scaffold by modifying the core structure of salicylanilide (Fig. 2). The
inhibitory activities of all synthesized compounds 6a-6l on RANKL-induced osteoclast
differentiation from RAW264.7 cells were evaluated by using TRAP-staining assay. On the
basis of this preliminary screening, we constructed structure-activity relationships (SARs) of
4

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all compounds according to the inhibition of RANKL-induced osteoclastogenesis. Among
them, compounds 6d and 6i were the most potent derivatives that could effectively inhibit
RANKL-induced osteoclastogenesis. Thus, we further investigated the inhibitory effects of
compounds 6d and 6i on RANKL-induced osteoclast formation and anti-resorptive effects by
suppressing osteoclastogenesis-related genes, blocking NF-κB nuclear translocation, and
decreasing NFATc1 expression levels. The detailed description of synthetic procedure,
biological evaluation, and SARs studies of these new compounds is presented as follows.
2
. Chemistry
A series of 5-(2',4'-difluorophenyl)-salicylanilides were synthesized and the synthetic
procedures are depicted in Scheme 1. Firstly, methyl 5-iodo-2-methoxybenzoate (2) was
prepared from protected 5-iodosalicylic acid (1) by the etherification with methyl iodide (MeI)
and
potassium
carbonate
(K CO ).
In
order
to
obtain
methyl
2
3
5
-(2',4'-difluorophenyl)-2-methoxybenzoate (3), 2 was refluxed and coupled with
2
’,4’-difluorophenyl boronic acid in the presence of K CO , palladium (II) acetate, and
2
3
toluene through Suzuki-coupling reaction. To obtain 5-(2',4'-difluorophenyl)-salicylic acid (4),
the protected 3 was subsequently deprotected by using lithium hydroxide monohydrate
(
LiOH•H O) saponification and boron tribromide (BBr ) demethylation [45]. Under treatment
2 3
with thionyl chloride, 4 was converted to the corresponding acyl chloride (5) and then used
without further purification. Unpurified 5 was reacted with various anilines to afford the
desired products (6a-6l) in 30–51% yields. The byproducts were separated by the differences
of physical chemistry properties which were purified by using recrystallization and
chromatography. The purity of all synthetic compounds was more than 95%. The structures of
1
13
all new synthesized compounds were elucidated with H NMR, C NMR, high resolution
mass (HRMS), and Fourier-transform infrared (FT-IR) spectroscopies and these results are
presented in the experimental part. In addition, the structure of 6g was confirmed by using
5

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single-crystal X-ray crystallography in Fig. 3.
3
. Pharmacological results and discussion
3
.1. Effects of the synthesized compounds on RANKL-induced osteoclastogenesis
The inhibitory activities of all synthesized compounds on RANKL-induced
TRAP-positive MNCs in RAW264.7 cells were screened by using TRAP-staining activity
assay. As illustrated in Table 1, our synthesized compounds could decrease the percentage of
MNCs at a concentration of 5 µM. Among these new compounds, the percent inhibition of
MNCs values for compounds 6d, 6f, and 6i at 5 µM are higher than 55%; the values are 67.32
±
4.38%, 59.21 ± 2.28%, and 71.32 ± 3.39%, respectively. These compounds could reduce the
numbers of TRAP-positive MNCs at 5 µM (Fig. 4). Compared with our previous study,
NDMC101 showed very poor inhibitory effect on TRAP staining MNCs at 5 µM [40].
Besides, the cell viabilities of these new compounds at the screening concentration were also
examined to investigate whether the inhibitory effects of our new compounds on
osteoclastogenesis are attributed to the cellular toxicity. Most of our synthesized compounds
unaffected cell viability of RAW264.7 cells (> 85%, Table 1). These observations prove that
their inhibitory activities were not due to their cytotoxic effects.
In addition, compounds 6d, 6f, 6i, and NDMC101 were further selected and evaluated
their inhibition on RANKL-induced osteoclastogenesis by using TRAP activity assay, which
is a more quantitative analysis method for osteoclast activity than TRAP-staining assay. IC50
values of these compounds are ranked in an order of 6i (2.95 ± 0.64 µM) > 6d (3.66 ± 0.08
µM) > 6f (4.57 ± 0.24 µM) > NDMC101 (Table 2). On the basis of these screening results,
the SARs of these synthetic compounds were briefly summarized as follows. Amongst
compounds bearing mono-substituted at the aniline moiety, we explored that substituents at
R position showed relatively higher activities than those at R or R positions, especially for
2
1
3
3
-cyano aniline. Amongst compounds bearing the difluoro groups at the aniline moiety, we
6

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explored that 2,5-difluoro analogs showed relatively higher activities than 2,4-difluoro
analogs and 3,4-difluoro analogs. Based on the preliminary SARs, it was confirmed that
2
’,4’-difluorophenyl moiety on salicylanilides could modulate the inhibitory activity for most
of these newly synthesized compounds showed higher inhibitory activity than NDMC101,
especially for 6d and 6i. Thus, the most potent compounds, 6d and 6i, were selected for
further mechanistic studies in the RANKL/RANK signaling pathway.
3
.2. Effects of 6d and 6i on osteoclastogenesis-related marker genes during the
RANKL-induced osteoclast development
Osteoclast differentiation is associated with up-regulation of osteoclastogenesis-related
marker genes such as NFATc1, c-fos, TRAP, cathepsin K, MMP-9, and DC-STAMP in
response to binding RANKL to RANK [46, 47]. In order to study whether compounds 6d and
6
i would regulate the genes expressions during the RANKL-induced osteoclastogenesis, we
assessed the inhibitory effects of 6d and 6i on the expression levels of NFATc1, c-fos, TRAP,
cathepsin K, MMP-9, and DC-STAMP genes by using reverse transcription polymerase chain
reaction (RT-PCR) assay. Compared with the expression levels without RANKL treatment,
the gene expression levels were increased in response to RANKL stimulation. However, the
gene expression levels were dramatically suppressed after treatment with 6d and 6i in a
dose-dependent manner (Fig. 5A). By further analyzing PCR data, the expression levels of
TRAP, cathepsin K, c-fos, NFATc1, and MMP-9 were significantly decreased after treatment
with 6d and 6i at concentrations of 2.5 and 5 µM (Fig. 5B). These results revealed that 6d and
6
i could suppress RANKL-induced mRNA expression levels in response to inhibiting
RANKL-RANK signaling pathway.
3
.3. Effects of 6d and 6i on RANKL-induced NF-κB and NFATc1 expression levels in
nucleus
Some major transcriptional factors can be activated and they are responsible for
7

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promoting osteoclastic genes expressions by a mechanism dependent on RANKL-RANK
interaction [46, 47]. For example, NF-κB and NFATc1 are essential for the osteoclast
differentiation and formation [23, 24]. In addition, it has been reported that NF-κB is an
upstream transcriptional factor which can modulate NFATc1 expression. In the inactive state,
NF-κB is retained in the cytoplasm. When being activated, NF-κB can translocate from the
cytoplasm to the nucleus. Thus, we firstly examined the effects of compounds 6d and 6i on
RANKL-stimulated the nuclear expression levels of NF-κB in RAW264.7 cells by using
NF-κB activity assay. This assay is used to analyze the NF-κB activity on enzyme-linked
immunosorbent assay (ELISA) plates. Compared with the NF-κB expression levels in nucleus
without RANKL stimulation, the nuclear accumulation of NF-κB was markedly increased
after treatment with RANKL (as shown in Fig. 6). However, the elevated levels of nuclear
NF-κB were reduced by treatment with 6d and 6i.
In order to confirm whether compounds 6d and 6i would suppress the nuclear protein
levels of NF-κB and NFATc1, we further analyzed the transcriptional factors such as NF-κB
and NFATc1 in nuclear extracts by using Western Blotting assay. The data are expressed as the
ratios of the protein levels of NF-κB and NFATc1 normalized to the level of TATA-box
binding protein (TBP). Compared with the expression levels without RANKL treatment, the
expression levels of NF-κB and NFATc1 in nuclear extracts were increased by treatment with
RANKL (Fig. 7). However, the elevated levels of NF-κB and NFATc1 in nuclear extracts
were reduced by treatment with 6d and 6i. These results further confirmed that 6d and 6i
might suppress the RANKL-induced NF-κB activation and NFATc1 expressions in nucleus.
By performing these biological results, the mechanistic studies revealed that
5
-(2',4'-difluorophenyl)-salicylanilide derivatives 6d and 6i not only reduced the
RANKL-induced NF-κB activation and NFATc1 expression in nucleus, but also suppressed
NFATc1-regulated mRNA expressions in the RANKL-RANK signaling pathway.
8

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3
.4. Effects of 6d and 6i on RANKL-induced bone-resorbing activity of osteoclasts
Since bone resorption is involved in the characteristic feature of functional osteoclasts,
RANKL can induce bone-resorbing activity of mature osteoclasts through RANKL-RANK
interaction. To investigate whether compounds 6d and 6i would attenuate the bone-resorbing
activity of mature osteoclasts, the pit formation assay was used for evaluating the effects of
6
d and 6i on dentine slices in this study. Thus, RAW264.7 cells were seeded onto dentine
slices and then differentiated into mature osteoclasts in response to RANKL stimulation. By
pit formation assay, the resorbing pits were formed on the dentine slices by mature osteoclasts.
However, the percentages of resorbed area/total area were significantly reduced by treatment
with 6d (80.55 ± 2.11%) and 6i (59.29 ± 1.07%), respectively (Fig. 8A and 8B). Based on
these encouraging results, we further confirmed that 6d and 6i could reduce osteoclast
formation and ameliorate RANKL-induced bone-resorbing activity of mature osteoclasts in a
dose-dependent manner (as shown in supplementary information).
4
. Conclusions
In this study, we synthesized a series of 5-(2',4'-difluorophenyl)-salicylanilides and
evaluated their inhibitory effects on RANKL-induced osteoclastogenesis. The general SARs
were established through TRAP-staining assay. These SARs was outlined as follows: (a)
compounds bearing mono-substituted groups at R position had higher activities than those at
2
R or R positions at the aniline moiety, especially for 3-cyano aniline; (b) compounds bearing
1
3
di-substituted groups at the aniline moiety revealed that difluoro groups showed relatively
higher inhibitory activities, especially for 2,5-difluoro analogs. Among these new compounds,
6
d and 6i exhibited potent inhibitory activity toward RANKL-induced osteoclastogenesis.
Their inhibitory effects of 6d and 6i were further evaluated and confirmed by inhibiting
NF-κB transcriptional activation and suppressing NFATc1-regulated
osteoclastogenesis-related genes. In addition, the pit formation assay showed that 6d and 6i
9

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could significantly ameliorate bone-resorbing activity of mature osteoclasts. These biological
results revealed the core structure of 5-(2',4'-difluorophenyl)-salicylanilides that can be
developed as potent anti-resorptive agents for the treatment of osteopenic disorders
characterized by excessive osteoclast activation.
5
. Materials and methods
5
.1. Chemistry
Unless otherwise stated, all materials used were commercially available. Chemical
reagents and solvents were purchased from Aldrich and Merck without further purification.
Reactions requiring anhydrous conditions were performed in oven-dried glassware and cooled
under nitrogen atmosphere. Melting points were determined by a Büchi B-545 melting point
apparatus and are uncorrected. The infrared (IR) spectra of all new compounds were recorded
in KBr discs on Thermo Scientific Nicolet iS5 FT-IR spectrometer. Analytical thin layer
1
chromatography was performed with E. Merck silica gel 60 F . H Nuclear magnetic
2
54
1
3
resonance (NMR) and C NMR spectra were recorded on Varian Gemini 300 (300 MHz) and
Agilent 400 MR DD2 (400 MHz). value is presented in parts per million (ppm) relative to
δ
TMS as an internal standard (0 ppm). Multiplicities were recorded as singlet (s), doublet (d),
triplet (t), quartet (q), and double of doublet (dd). Coupling constants (J) are expressed in Hz.
High resolution mass spectra (HRMS) were measured by Finnigan MAT-95XL (high
resolution electron impact ionization, HREI) and Finnigan MAT-95S (high resolution
electrospray ionization, HRESI). Spectral data are recorded as m/z values. The purity of all
synthetic compounds was analyzed by reverse-phase high performance liquid
chromatography (HPLC) analysis to be >95%. HPLC spectra for all compounds were
acquired using a Hitachi L-2000 series system with UV detector (L-2400, Hitachi). The
retention time (t ) is expressed in min at UV detection of 208 nm. By using HPLC analysis,
R
chromatography performed on a C18 reverse-phase column (XBridge Shield RP18 Column,
1
0

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1
30Å, 5 µm, 4.6 mm × 250 mm, Waters). A flow rate of 1.0 mL/min was used with a mobile
phase gradient of 0-20% H O in MeOH.
2
5
.2. X-ray crystallography
A single crystal of suitable size for X-ray diffractometry was selected under a
microscope and mounted on the tip of a glass fibre, which was positioned on a copper pin.
Crystallographic assay was performed as described in the reported protocol [48]. The X-ray
data for 6g was collected with a Bruker Kappa CCD diffractometer, employing
graphite-monochromated Mo-Kα radiation at 200 K and the θ–2θ scan mode. The space group
for 6g was determined on the basis of systematic absences and intensity statistics, and the
structure of 6g was solved by direct methods using SIR92 or SIR97 and refined with
SHELXL-97.
5
.3. Methyl 5-iodo-2-methoxybenzoate (2)
-Iodosalicylic acid (2.51 g, 9.47 mmol) was dissolved in acetone (50 mL), and
5
potassium carbonate (5.24 g, 37.86 mmol) and methyl iodide (5.38 g, 37.86 mmol) were
added. The mixture was refluxed for 48 h and then cooled at room temperature. The reaction
was filtered through celite, and the filtrate was removed in vacuo and the residue was
extraction with ethyl acetate (3 × 15 mL), washed with 10% NaHCO (15 mL), H O (2 × 15
3
2
mL), brine (10 mL), and then dried over anhydrous MgSO . The resulting residue was
4
purified by flash chromatography (ethyl acetate/n-hexane 1:2) to obtain 2 as white solid (yield
1
8
7
3%); Mp: 53-54 °C (EtOAc); H NMR (400 MHz, DMSO-d ): δ ppm 7.88 (d, J = 3 Hz, 1H),
6
1
3
.83 (dd, J = 11, 3 Hz, 1H), 6.99 (d, J = 11 Hz, 1H), 3.79 (s, 3H), 3.77 (s, 3H); C NMR (100
MHz, DMSO-d ): δ ppm 52.12, 55.98, 82.12, 115.35, 122.32, 138.37, 141.62, 157.88, 164.71.
6
+
+
+
HRMS (ESI) m/z：calcd [M] , 291.9596 (C H IO ), found [M+H] : 292.9683.
9
9
3
5
.4. Methyl 2',4'-difluoro-4-methoxy-[1,1'-biphenyl]-3-carboxylate (3)
11

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To a mixture of potassium carbonate (2.07 g, 15 mmol), palladium (II) acetate (0.06 g,
.25 mmol), triphenylphosphine (0.21 g, 0.75 mmol) and 2,4-difluorophenyl boronic acid,
0
(1.03 g 6.5 mmol) in toluene (25 mL) was added dropwise with constant stirring to a solution
of 2 (1.48 g, 5 mmol). The reaction mixture was refluxed for 24 h. After cooling to room
temperature, the solution was filtered through celite and then concentrated in vacuo. The
crude was purified by flash chromatography (ethyl acetate/n-hexane 1:2) to obtain 3 as a
1
white solid (yield 76%). Mp: 209-210 °C (EtOAc); H NMR (400 MHz, DMSO-d ): δ ppm
6
7
.77 (t, J = 2 Hz, 1H), 7.69 (dt, J = 11, 2.5 Hz, 1H), 7.54-7.61 (m, 1H), 7.35 (td, J = 10.5, 3
13
Hz, 1H), 7.26 (d, J = 11 Hz, 1H), 7.13 (td, J = 11, 3 Hz, 1H), 3.86 (s, 3H), 3.79 (s, 3H); C
NMR (100 MHz, DMSO-d ) 52.01, 55.99, 104.49, 112.07, 112.97, 120.18, 123.55, 125.96,
6
+
+
1
30.78, 131.62, 133.66; HRMS (ESI) m/z：calcd [M] , 278.0755 (C H F O ), found
15 12 2 3
+
[
M+H] : 279.0838.
.5. 2',4'-Difluoro-4-hydroxy-[1,1'-biphenyl]-3-carboxylic acid (4) [45]
To a solution of 3 (1.39 g, 5 mmol) in dichloromethane (50 mL) at -78 °C was added
5
dropwise boron tribromide (5.62 mL, 32.94 mmol) for 5-10 min. The reaction mixture was
stirred overnight at room temperature. Upon completion, the reaction was quenched with H O
2
(30 mL), extracted with dichloromethane (3 × 30 mL), brine (10 mL), dried over anhydrous
MgSO , and then concentrated in vacuo. The residue was carried onto the next step without
4
further purification. To a solution of residue in tetrahydrofuran/methanol/water (1:1:1) was
added lithium hydroxide monohydrate (1.42 g, 15 mmol). The reaction was stirred at room
temperature upon completion. And then, the reaction was acidified with 30% HCl (aq)
,
extracted with ethyl acetate (3 × 25 mL), dried over MgSO , and then concentrated in vacuo.
4
The residue was purified by flash chromatography (CH Cl , 1% MeOH, 0.2% acetic acid) to
2
2
1
obtain 3 as a white solid (yield 43%). Mp: 213-214 °C (EtOAc); H NMR (400 MHz,
DMSO-d ): δ ppm 7.04 (d, J = 8.7 Hz, 1H), 7.14 (td, J = 10.45, 2.55 Hz, 1H), 7.30 (td, J =
6
1
2

ACCEPTED MANUSCRIPT
1
0.35, 2.71 Hz, 1H), 7.49-7.58 (m, 1H), 7.64 (dt, J = 8.4, 3 Hz, 1H), 7.90 (t, J = 2.1 Hz, 1H);
1
3
C NMR (100 MHz, DMSO-d ) : δ ppm 104.49, 112.07, 113.24, 117.62, 123.76. 125.15,
6
+
1
30.33, 131.54, 135.84, 157.81, 160.29, 160.78, 162.76; HRMS (ESI) m/z：calcd [M] ,
+
2
50.0442 (C H F O ), found [M+H] : 251.0518.
1
3
8
2
3
5
.6. N-(4-Chloro-2-fluorophenyl)-2',4'-difluoro-4-hydroxybiphenyl-3-carboxamide (6a)
To a solution of 4 (0.9 g, 4 mmol) in anhydrous tetrahydrofuran (30 mL) was added
dropwise thionyl chloride (1 mL, 14 mmol), and the mixture was refluxed under nitrogen
atmosphere for 8 h. After cooling to room temperature, the mixture was steamed to give the
intermediate by Dean-Stark apparatus. The residue was carried onto the next step without
further purification and it was directly reacted with 4-chloro-2-fluoroaniline (0.5 mL, 4 mmol)
in anhydrous tetrahydrofuran (30 mL) for 14 h. After removal of tetrahydrofuran, the reaction
mixture was washed with ethyl acetate/n-hexane and the crude product was extraction with
ethyl acetate (3 × 25 mL), washed with 10% NaHCO (15 mL), H O (3 × 25 mL), brine (10
3
2
mL), and then dried over anhydrous MgSO . The organic layer was collected, dried over
4
anhydrous MgSO , and then concentrated in vacuo. The crude product was washed and
4
purified by crystallization from hot ethanol to obtain 6a as white powder (yield 30%). Mp:
-1
1
2
45-246 °C (EtOH); IR (KBr, νmax cm ): 1637 (CO), 3127 (NH), 3300 (OH); H NMR (300
MHz, DMSO-d ): δ ppm 7.12 (d, J = 6.0 Hz, 1H), 7.19 (td, J = 9.0, 1.8 Hz), 7.31-7.40 (m,
6
2
H), 7.55-7.64 (m, 3H), 8.14 (t, J = 1.5 Hz, 1H), 8.24 (t, J = 8.7 Hz, 1H), 10.77 (s, 1H), 12.19
1
3
(
s, 1H); C NMR (75 MHz, DMSO-d ): δ ppm 104.51, 112.07, 116.20, 117.66, 123.92,
6
1
1
24.04, 125.41, 128.15, 130.49, 131.58, 134.23, 151.85, 154.31, 157.78, 160.26, 162.71,
+
64.25; HPLC purity: >95%, t = 9.07 min; HRMS (EI) m/z：calcd [M] , 377.0430
R
+
(
C H ClF NO ), found, 377.0433.
1
9
11
3
2
5
.7. N-(2,4-difluorophenyl)-2',4'-difluoro-4-hydroxybiphenyl-3-carboxamide (6b)
1
3

ACCEPTED MANUSCRIPT
The title compound was synthesized from 2,4-difluoroaniline (0.4 mL, 4 mmol) in a
similar manner as described for 6a. The pure compound was obtained as white powder (yield
-1
4
2%). Mp: 233-234 °C (EtOH); IR (KBr, νmax cm ): 1652 (CO), 3127 (NH), 3301 (OH);
1
H NMR (400 MHz, DMSO-d ): δ ppm 7.10-7.23 (m, 3H), 7.33-7.45 (m, 2H), 7.55-7.64 (m,
6
1
3
2
1
1
H), 8.06-8.15 (m, 2H), 10.64 (s, 1H), 12.16 (s, 1H); C NMR (75 MHz, DMSO-d ): δ ppm
6
04.63, 104.93, 111.32, 112.50, 117.90, 122.90, 124.41, 125.53, 125.98, 130.63, 132.05,
34.66, 153.09, 155.56, 157.69, 158.06, 160.68, 163.15, 165.26; HPLC purity: >95%; t =
R
+
+
6
.45 min; HRMS (EI) m/z：calcd [M] , 361.0726 (C H F NO ), found, 361.0730.
19 11 4 2
5
.8. N-(3,4-difluorophenyl)-2',4'-difluoro-4-hydroxy-[1,1'-biphenyl]-3-carboxamide (6c)
The title compound was synthesized from 3,4-difluoroaniline (0.4 mL, 4 mmol) in a
similar manner as described for 6a. The pure compound was obtained as beige powder (yield
-1
6
1%). Mp: 231-232 °C (EtOH); IR (KBr, νmax cm ): 1636 (CO), 3139 (NH), 3304 (OH);
1
H NMR (300 MHz, DMSO-d ): δ ppm 7. 09 (d, J = 8.7 Hz, 1H), 7.20 (td, J = 8.4, 3 Hz, 1H),
6
7
.45-7.49 (m, 3H), 7.56-7.64 (m, 2H), 7.87-7.93 (m, 1H), 8.01 (t, J = 0.9 Hz, 1H), 10.55 (s,
1
3
1
H), 11.70 (s, 1H); C NMR (75 MHz, DMSO-d ): δ ppm 104.86, 110.16, 112.29, 117.59,
6
1
17.67, 118.17, 124.25, 127.55, 131.89, 134.14, 135.39, 144.89, 147.61, 150.34, 157.73,
58.01, 160.47, 162.92, 166.27; HPLC purity: >95%; t = 7.72 min; HRMS (EI) m/z：calcd
1
R
+
+
[
M] , 361.0726 (C H F NO ), found, 361.0724.
19 11 4 2
5
.9. N-(2,5-difluorophenyl)-2',4'-difluoro-4-hydroxy-[1,1'-biphenyl]-3-carboxamide (6d)
The title compound was synthesized from 2,5-difluoroaniline (0.4 mL, 4 mmol) in a
similar manner as described for 6a. The pure compound was obtained as white powder (yield
-1
3
7%). Mp: 213-214 °C (EtOH); IR (KBr, νmax cm ): 1655 (CO), 3124 (NH), 3227 (OH);
1
H NMR (300 MHz, DMSO-d ): δ ppm 6.99-7.05 (m, 1H), 7.12-7.22 (m, 2H), 7.33-7.44 (m,
6
1
4

ACCEPTED MANUSCRIPT
1
3
2
H), 7.55-7.65 (m, 2H), 8.14-8.23 (m, 2H), 10.89 (s, 1H), 12.22 (s, 1H); C NMR (75 MHz,
DMSO-d ): δ ppm 104.58, 108.98, 110.61, 112.17, 116.23, 117.80, 123.97, 125.87, 127.51,
6
1
30.84, 131.61, 134.35, 149.01, 156.57, 157.83, 157.90, 160.32, 162.78, 163.89; HPLC purity:
+
+
>
95%; t = 7.80 min; HRMS (EI) m/z：calcd [M] , 361.0726 (C H F NO ), found,
R 19 11 4 2
3
61.0731.
5
.10.
2',4'-Difluoro-4-hydroxy-N-(2-(trifluoromethyl)phenyl)-[1,1'-biphenyl]-3-carboxa
mide (6e)
The title compound was synthesized from 2-(trifluoromethyl)aniline (0.5 mL, 4 mmol) in
a similar manner as described for 6a. The pure compound was obtained as beige powder
-1
(
yield 36%). Mp: 180-181 °C (EtOH); IR (KBr, νmax cm ): 1636 (CO), 3132 (NH), 3327
1
(
OH); H NMR (300 MHz, DMSO-d ): δ ppm 7.11-7.22 (m, 2H), 7.32-7.43 (m, 2H),
6
1
3
7
.54-7.65 (m, 2H), 7.70-7.78 (m, 2H), 8.18-8.20 (m, 2H), 10.81(s, 1H), 12.19 (s, 1H); C
NMR (75 MHz, DMSO-d ): δ ppm 104.52, 111.70, 117.56, 121.05, 123.89, 124.01, 125.38,
6
1
1
25.71, 126.22, 126.33, 130.77, 131.58, 133.31, 134.29, 135.32, 156.81, 157.80, 160.27,
+
62.73, 164.42; HPLC purity: >95%; t = 5.44 min; HRMS (EI) m/z：calcd [M] , 393.0788
R
+
(
C H F NO ), found, 393.0784.
2
0
12
5
2
5
.11.
2',4'-Difluoro-4-hydroxy-N-(3-(trifluoromethyl)phenyl)-[1,1'-biphenyl]-3-carboxa
mide (6f)
The title compound was synthesized from 3-(trifluoromethyl)aniline (0.5 mL, 4 mmol) in
a similar manner as described for 6a. The pure compound was obtained as beige powder
-
1
(
(
yield 32%). Mp: 202-203 °C (EtOH); IR (KBr, νmax cm ): 1638 (CO), 3152 (NH), 3324
1
OH); H NMR (300 MHz, DMSO-d ): δ ppm 7.10 (d, J = 8.7 Hz, 1H), 7.20 (td, J = 9, 2.7 Hz,
6
1
H), 7.37 (td, J = 11.1, 2.4 Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.57-7.65 (m, 3H), 7.95 (d, J =
1
3
8
.1 Hz, 1H), 8.05 (t, J = 1.2 Hz, 1H), 8.21 (s, 1H), 10.66 (s, 1H), 11.70 (s, 1H); C NMR (75
1
5

ACCEPTED MANUSCRIPT
MHz, DMSO-d ): δ ppm 104.51, 112.04, 116.83, 117.53, 118.39, 120.44, 124.09, 124.11,
6
1
1
24.33, 125.12, 129.46, 129.44, 129.99, 131.39, 133.94, 157.79, 157.80, 160.25, 162.71,
+
66.43; HPLC purity: >95%; t = 7.27 min; HRMS (EI) m/z：calcd [M] , 393.0788
R
+
(
C H F NO ), found, 393.0787.
2
0
12
5
2
5
.12.
2',4'-Difluoro-4-hydroxy-N-(4-(trifluoromethyl)phenyl)-[1,1'-biphenyl]-3-carboxa
mide (6g)
The title compound was synthesized from 4-(trifluoromethyl)aniline (0.5 mL, 4 mmol) in
a similar manner as described for 6a. The pure compound was obtained as white powder
-
1
(
(
yield 44%). Mp: 227-228 °C (EtOH); IR (KBr, νmax cm ): 1637 (CO), 3086 (NH), 3320
1
OH); H NMR (300 MHz, DMSO-d ): δ ppm 7.10 (d, J = 8.4 Hz, 1H), 7.18 (td, J = 13.7, 1.5
6
Hz, 1H), 7.36 (td, J = 9.3, 2.7 Hz, 1H), 7.74 (d, J = 9.0 Hz, 2H), 7.95 (d, J = 8.4 Hz, 2H), 8.03
1
3
(
s, 1H), 10.67 (s, 1H), 11.66 (s, 1H); C NMR (75 MHz, DMSO-d ): δ ppm 104.53, 112.09,
6
117.53, 118.74, 120.59, 123.05, 124.09, 124.39, 125.31, 126.09, 129.68, 131.72, 133.98,
1
57.51, 157.86, 160.32, 162.77, 166.27; HPLC purity: >95%; t = 7.87 min; HRMS (EI) m/z：
R
+
+
calcd [M] , 393.0788 (C H F NO ), found, 393.0791.
20
12
5
2
5
.13.
N-(3-ethynylphenyl)-2',4'-difluoro-4-hydroxy-[1,1'-biphenyl]-3-carboxamide (6h)
The title compound was synthesized from 3-ethynylaniline (0.45 mL, 4 mmol) in a
similar manner as described for 6a. The pure compound was obtained as grey powder (yield
-1
3
8%). Mp: 215-216 °C (EtOH); IR (KBr, νmax cm ): 1644 (CO), 2227 (CC triple bond), 3094
1
(
NH), 3301 (OH); H NMR (300 MHz, DMSO-d ): δ ppm 7.09 (d, J = 8.7 Hz, 1H), 7.17-7.26
6
(m, 2H), 7.33-7.41 (m, 2H), 7.57-7.65 (m, 2H), 7.70-7.73 (m, 1H), 7.89 (t, J = 1.8 Hz, 1H),
1
3
8
.05 (t, J = 1.2 Hz, 1H), 10.48 (s, 1H), 11.79 (s, 1H); C NMR (75 MHz, DMSO-d ): δ ppm
6
8
0.43, 83.09, 104.30, 112.09, 117.67, 121.77, 123.72, 123.80, 124.12, 125.18, 127.37, 129.06,
1
6

ACCEPTED MANUSCRIPT
1
29.27, 131.63, 133.89, 138.36, 157.57, 158.05, 160.21, 162.74, 166.40; HPLC purity: >95%;
+
+
t = 6.37 min; HRMS (EI) m/z：calcd [M-H] , 348.0836 (C H F NO ), found, 348.0835.
R
21 12
2
2
5
.14.
N-(3-cyanophenyl)-2',4'-difluoro-4-hydroxy-[1,1'-biphenyl]-3-carboxamide (6i)
The title compound was synthesized from 3-aminobenzonitrile (0.47 g, 4 mmol) in a
similar manner as described for 6a. The pure compound was obtained as beige powder (yield
-1
5
1%). Mp: 229-230 °C (EtOH); IR (KBr, νmax cm ): 1651 (CO), 2231 (CN triple bond), 3101
1
(
NH), 3363 (OH); H NMR (300 MHz, DMSO-d ): δ ppm 7.10 (d, J = 8.4 Hz, 1H), 7.16-7.22
6
(m, 1H), 7.35 (td, J = 10.35, 2.7 Hz, 1H) 7.55-7.63 (m, 4H), 7.96-8.00 (m, 1H), 8.02 (t, J =
1
3
0
.9 Hz, 1H), 8.21 (t, J = 0.9 Hz, 1H), 10.63 (s, 1H), 11.66 (s, 1H, OH); C NMR (75 MHz,
DMSO-d ): δ ppm 104.53, 111.60, 112.08, 117.49, 118.59, 123.51, 124.06, 125.28, 127.64,
6
1
29.57, 130.26, 131.67, 133.99, 139.14, 157.49, 157.80, 160.27, 162.49, 162.72, 166.23;
+
+
2
HPLC purity: >95%; t = 6.92 min; HRMS (EI) m/z：calcd [M] , 350.0867 (C H F N O ),
R
20 12
2
2
found, 350.0857.
5
.15.
N-(4-cyanophenyl)-2',4'-difluoro-4-hydroxy-[1,1'-biphenyl]-3-carboxamide (6j)
The title compound was synthesized from 4-aminobenzonitrile (0.47 g, 4 mmol) in a
similar manner as described for 6a. The pure compound was obtained as yellowish powder
-1
(
yield 54%). Mp: 189-190 °C (EtOH); IR (KBr, νmax cm ): 1651 (CO), 2224 (CN triple bond),
1
3
076 (NH), 3351 (OH); H NMR (300 MHz, DMSO-d ): δ ppm 7.19 (td, J = 8.4, 0.9 Hz, 1H),
6
7
.35 (td, J = 10.2, 2.4 Hz, 1H), 7.55-7.63 (m, 2H), 7.81-7.84 (m, 2H), 7.91-7.94 (m, 1H), 7.98
1
3
(
t, J = 1.2 Hz, 1H), 10.70 (s, 1H), 11.55 (s, 1H); C NMR (75 MHz, DMSO-d ): δ ppm
6
1
1
04.51, 112.07, 117.40, 119.10, 120.47, 124.03, 125.25, 129.72, 131.65, 133.26, 133.85,
42.69, 157.08, 157.73, 160.25, 162.58, 162.76, 166.09; HPLC purity: >95%; t = 6.84 min;
R
+
+
HRMS (EI) m/z：calcd [M] , 350.0867 (C H F N O ), found, 350.0872.
2
0
12
2
2
2
1
7

ACCEPTED MANUSCRIPT
5
.16.
6k)
N-(3,4-dimethoxyphenyl)-2',4'-difluoro-4-hydroxy-[1,1'-biphenyl]-3-carboxamide
(
The title compound was synthesized from 3,4-dimethoxyaniline (0.61 g, 4 mmol) in a
similar manner as described for 6a. The pure compound was obtained as beige powder (yield
-1
1
3
4%). Mp: 186-187 °C (EtOH); IR (KBr, νmax cm ): 1680 (CO), 3145 (NH), 3363 (OH); H
NMR (300 MHz, DMSO-d ): δ ppm 3.75 (s, 3H), 3.76 (s, 3H), 6.95 (d, J = 9 Hz), 7.07 (d, J =
6
6
.6 Hz, 1H), 7.17-7.25 (m, 2H), 7.31-7.39 (m, 2H), 7.56-7.66 (m, 2H), 8.11 (s, 1H), 10.33 (s,
1
3
1
H), 12.12 (s, 1H); C NMR (75 MHz, DMSO-d ): δ ppm 55.47, 55.67, 104.48, 106.15,
6
117.79, 112.02, 113.24, 117.48, 124.19, 125.04, 128.97, 131.31, 131.77, 133.91, 145.69,
1
48.48, 157.72, 158.56, 160.26, 162.64, 166.26; HPLC purity: >95%; t = 7.61 min; HRMS
R
+
+
(
EI) m/z：calcd [M] , 385.1126 (C H F NO ), found, 385.1124.
21 17 2 4
5
.17.
6l)
2',4'-Difluoro-4-hydroxy-N-(3-methoxyphenyl)-[1,1'-biphenyl]-3-carboxamide
(
The title compound was synthesized from 3-methoxyaniline (0.46 g, 4 mmol) in a
similar manner as described for 6a. The pure compound was obtained as light orange powder
-
1
(
(
yield 32%). Mp: 187-188 °C (EtOH); IR (KBr, νmax cm ): 1633 (CO), 3183 (NH), 3329
1
OH); H NMR (300 MHz, DMSO-d ): δ ppm 3.76 (s, 3H), 6.70-6.74 (m, 1H), 7.08 (d, J =
6
8
.4 Hz, 1H), 7.16-7.39 (m, 4H), 7.57-7.65 (m, 2H), 8.07 (s, 1H), 10.39 (s, 1H), 11.90 (s, 1H);
1
3
C NMR (75 MHz, DMSO-d ): δ ppm 55.08, 104.48, 106.15, 117.79, 112.02, 113.24, 117.48,
6
1
1
24.19, 125.04, 128.97, 131.31, 131.77, 133.91, 145.69, 148.48, 157.72, 158.56, 160.26,
+
62.64, 166.26; HPLC purity: >95%; t = 5.89 min; HRMS (EI) m/z：calcd [M] , 355.1020
R
+
(
C H F NO ), found, 355.1014.
2
0
15
2
3
5
.2. Biological materials and methods
1
8

ACCEPTED MANUSCRIPT
RAW264.7 murine monocyte/macrophage cells were obtained from American Type
Culture Collection (ATCC, Rockville, MD, USA); Receptor activator of nuclear factor
kappa-B ligand (RANKL, Peprotech, Inc., London, UK); Specific monoclonal primary
antibodies NF-κB (Clone code: E379, Epitomics, Inc., CA, USA); NFATc1 (Clone code: 7A6,
Santa Cruz Biotechnology, Santa Cruz Inc., CA, USA); TATA box-binding protein (Clone
code: 1TBP18, Abcam Inc., Cambridge, MA, USA); NE-PER nuclear and cytoplasmic
extraction reagents (Thermo Fisher Scientific, USA); Unless otherwise stated, all materials
and assay kits were used from commercially available.
5
.2.1. Cell culture
RAW264.7 cells were obtained from American Type Culture Collection (ATCC,
Rockville, MD, USA). These cells were cultured with Dulbecco's Modified Eagle Medium
DMEM, Gibco BRL, NY, USA) and supplemented with 10% heat-inactivated fetal bovine
serum (FBS) in 95% air, 5% CO humid atmosphere at 37 °C.
(
2
5
.2.2. Cell viability assay [40, 41]
RAW264.7 cells were seeded into a 96-well culture plate at a density of 1 × 10 per well
4
with 200 µL medium (DMEM) supplemented with 10% FBS and treated with a series of
synthesized compounds for 24 h. After 24 h, 96-well plate were washed three times with
phosphate-buffered saline (3 × PBS) and then treated with medium containing 500 µg/mL of
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reagent for 1 h at 37 °C.
Cells were then washed with PBS and solubilized in 100 µL of DMSO. MTT reagent was
designed to yield a formazan by metabolic reduction of viable cells. Absorbance was
measured at 540 nm by using an ELISA reader.
5
.2.3. Tartrate-resistant acid phosphatase (TRAP) staining assay [40, 41]
3
RAW264.7 cells were seeded into 96-well culture plate at a density of 1 × 10 per well
4
and/or seeded into 48-well plate at a density of 2 × 10 cells per well with alpha-Minimum
1
9

ACCEPTED MANUSCRIPT
Essential Medium (α-MEM, Gibco BRL, NY, USA) containing 10% FBS, 2 mM L-glutamate,
1
00 ng/mL of recombinant soluble murine RANKL (Peprotech, London, UK) in the presence
with or without tested compounds. Cell cultures were incubated with 95% air, 5% CO at 37
2
°
C for 4 days. Cell culture media (containing the test compounds) were changed on day 2.
After 4 days, cells were washed with PBS and fixed with solution (acetone/citrate
solution/37% formaldehyde, 65:25:8) for 30 min. And then, cells were incubated with
light-protected incubator at 37 °C for 1 h by using the Leukocyte Acid Phosphatase Assay kit
(Cat.387A, Sigma). Cells were washed three times with distilled water and TRAP-positive
multinucleated cells containing three or more than three nuclei (N > 3) were counted under a
light microscope and photographed.
5
.2.4. Tartrate-resistant acid phosphatase (TRAP) activity assay [41]
RAW264.7 cells were seeded into 48-well plate at a density of 2 × 10 cells per well with
4
α-MEM medium containing 10% FBS, 2 mM L-glutamate, 100 ng/mL of recombinant
soluble murine RANKL in the presence with or without tested compounds. For the
measurement of TRAP activity, the cells were washed with phosphate-buffered saline (PBS)
on 4 day, and then 100 µL of lysis solution (0.1% Triton X-100 in 50 mM Tris-HCl, pH = 7.2)
was added to each well for 15 min under ice-bath. Subsequently, the 100 µL of substrate
solution (2 mg/mL 4-nitrophenyl phosphate in 0.09 M citrate buffer, Sigma Chemical Co.)
was added to each well and incubated for 15 min at 37 °C. The reaction was stopped by
adding 200 µL of 0.1 N NaOH (aq) and transferred 100 µL mixtures into 96-well plates.
Absorbance was measured at 405 nm by using an ELISA reader. The intracellular protein
level was measured at 562 nm by using a BCA protein assay kit (Pierce, USA).
5
.2.5. Reverse transcription polymerase chain reaction (RT-PCR) analysis [40, 41]
5
RAW264.7 cells were seeded into 6-cm dish at a density of 1 × 10 cells per well with
DMEM medium containing 10% FBS. After 1 day, cells were starved for 24 h by replacing
2
0

ACCEPTED MANUSCRIPT
DMEM medium to α-MEM medium, 100 ng/mL of recombinant soluble murine RANKL in
the presence with or without the test compounds for 24 h. Total RNA was isolated from
cultured cells with Trizol reagent (Invitrogen). The RNA (2 µg) was reversibly transcribed
with SuperScript II reverse transcriptase (Invitrogen) and oligo-(dT) primer. The cDNA was
1
5
amplified using mouse-specific primer (Table 3). The PCR was performed in 25 µL of 10 mM
Tris-HCl (pH = 8.8), 1.5 mM MgCl , 50 mM KCl, 0.1% Triton X-100, 1 unit DNA
2
polymerase (Protech), 200 µM dNTP and 10 µM primer. PCR products were separated on
1
.5% agarose gels, and the bands were visualized by ethidium bromide staining (EtBr). The
optical densities for each gene were normalized to the corresponding values for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH).
5
.2.6. Western Blotting assay [40, 41]
RAW264.7 cells were seeded into 6-cm dish at a density of 1 × 10 cells per well with
5
DMEM medium containing 10% FBS for 24 h. And then, the cells were pretreated with the
test compounds in α-MEM medium containing 10% FBS for 1 h prior to treatment with
RANKL (100 ng/mL) for 24 h. After 1 day, the cells were washed three times with PBS.
Nuclear extracts were collected by NE-PER Nuclear and Cytoplasmic Extraction Kit (Cat.
7
8833, Thermo Scientific, USA) according to the manufacturer's instructions. Then,
equivalent amounts of nuclear protein extracts (18 µg) protein were loaded for 12%
SDS-PAGE gels and transferred to immobilon polyvinyldifuoride members. Non-specific
binding was blocked by soaking members in the TBS buffer containing 5% BSA for 1 h. The
blots were probed with specific monoclonal primary antibodies (mAb, 1:1000), such as
NF-κB (Clone code: E379, Epitomics, Inc., CA, USA), NFATc1 (Clone code: 7A6, Santa
Cruz Biotechnology, Santa Cruz, CA, USA), and TATA box-binding protein (TBP, Clone code:
1
TBP18, Abcam Inc., Cambridge, MA, USA) for 12 h at 4 °C. After further washing by TBST,
the blots were subsequently incubated with a secondary antibody coupled to
2
1

ACCEPTED MANUSCRIPT
horseradish-peroxidase (a goat anti-rabbit IgG, 1:10000) for 1 h. The immunoreactive bands
were visualized by using the Immobilon Western Chemiluminescent HRP Substrate kit
(
Millipore) and by exposing a clear Blue X-ray film (Thermo Fisher Scientific, USA) to the
membrane.
.2.7. NF-
RAW264.7 cells were seeded into 6-cm dish at a density of 1 × 10 cells per well with
5
κB activity assay
5
DMEM medium containing 10% FBS for 24 h. And then, the cells were pretreated with the
test compounds in α-MEM medium containing 10% FBS for 1 h prior to treatment with
RANKL (100 ng/mL) for 24 h. After 1 day, the cells were washed three times with PBS.
Nuclear extracts were collected by NE-PER Nuclear and Cytoplasmic Extraction Kit (Cat.
7
8833, Thermo Scientific, USA). NF-κB translocation was determined by using a Cayman
Chemical NF-κB (p65) kit (Cat. 10007889), a sensitive method for the detection of specific
transcription factor DNA binding activity in nuclear extracts. This assay is used to analyze the
NF-κB activity or transactivation potential, as NF-κB binds to the response element
pre-coated on specific ELISA plates, a primary antibody followed by a tagged secondary
antibody is then added. Absorbance is read at 450 nm which correlates with NF-κB activity.
5
.2.8. Resorbing activity of osteoclasts by bone resorption assay [40]
RAW264.7 cells were seeded into dentine slices (24-well plates, Corning) at a density of
4
1
× 10 cells per well with α-MEM medium (containing 10% FBS, 2 mM L-glutamate), 100
ng/mL of recombinant soluble murine RANKL in the presence with or without the test
compounds. All cultured cells were replenished every 2 days with fresh medium containing
tested chemicals. After 4 days, the wells were washed three times with PBS, left in 1M
ammonium hydroxide to remove the attached cells and stained with 0.1% toluidine blue
(Sigma Chemical Co.). In optical field on a slice, the ratios of the resorbed area to the total
area were measured by using the ImageJ software.
2
2

ACCEPTED MANUSCRIPT
Acknowledgments
We would like to thank the Chugai Pharmaceutical Co Ltd., Taiwan for their partial
assistance in supporting our research. The present study was also supported by the National
Science Council Grant (NSC 101-2113-M-0163), Ministry of Science and Technology
(MOST 102-2314-B-075-083-MY3), and Taipei Medical University (TMU 102-AE1-B32).
We also thank Ting-Shen Kuo for kindly providing the information of crystallography.
Supplementary data
Inhibitory effects of compounds on RANKL-induced osteoclastogenesis and
1
13
bone-resorbing activities of mature osteoclasts; spectroscopic characterizations ( H NMR, C
NMR and HRMS spectra) of compounds; purity of final compounds.
2
3

ACCEPTED MANUSCRIPT
References
[
[
(
[
[
[
[
1] N.A. Sims, J.H. Gooi, Semin. Cell Dev. Biol. 19 (2008) 444-451.
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Legends
Table 1. Effects of 5-(2',4'-difluorophenyl)-salicylanilide derivatives on RANKL-induced
a
osteoclastogenesis and cell viability in RAW264.7 cells. RAW 264.7 cells, the cells were
cultured in the α-MEM medium containing 10% FBS and RANKL (100 ng/mL) with or
without the tested compound (5 µM) for 4 days. TRAP-positive multinucleated cells (MNCs)
were counted as osteoclasts (three or more than three nuclei (N > 3). RANKL-induced
osteoclast differentiation is shown as relative activity (% of RANKL-treated with compounds/
RANKL-treated without compounds as vehicle). Inhibition (%) = 100% - MNCs (%) of
b
compounds. Cell viability of tested compounds were determined relative to the control at
5
µM by MTT assay. All representative data were performed the means ± SD at least three
times independently (n > 3).
Table 2. IC50 values of compounds for RANKL-induced osteoclast differentiation from
a
RAW264.7 cells. TRAP activity was determined with a commercially available kit and then
was normalized to cellular proteins determined by BCA protein assay; IC50 is the
concentration of the test compounds required to inhibit 50% intensity of RANKL-induced
osteoclastogenesis.
Table 3. Primers used for RT-PCR
Figure 1. Chemical structures of osteoclast inhibitors
Figure 2. Design and synthesis of 5-(2',4'-difluorophenyl)-salicylanilide analogs as potential
osteoclast inhibitors
Figure 3. X-ray structure of 6g
Figure 4. Effects of compounds 6d, 6f, and 6i on RANKL-induced osteoclastogenesis from
RAW264.7 cells by using TRAP-staining activity assay. Control was cultured without
compounds in the absence of RANKL. Vehicle was cultured without the tested compounds
2
8