Modified salicylanilide and 3-phenyl-2 H -benzo[ e ][1,3]oxazine-2,4(3 H)-dione derivatives as novel inhibitors of osteoclast differentiation and bone resorption

Article abstract of DOI:10.1021/jm5007897

Inhibition of osteoclast formation is a potential strategy to prevent inflammatory bone resorption and to treat bone diseases. In the present work, the purpose was to discover modified salicylanilides and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione derivatives as potential antiosteoclastogenic agents. Their inhibitory effects on RANKL-induced osteoclastogenesis from RAW264.7 cells were evaluated by TRAP stain assay. The most potent compounds, 1d and 5d, suppressed RANKL-induced osteoclast formation and TRAP activity dose-dependently. The cytotoxicity assay on RAW264.7 cells suggested that the inhibition of osteoclastic bone resorption by these compounds did not result from their cytotoxicity. Moreover, both compounds downregulated RANKL-induced NF-κB and NFATc1 in the nucleus, suppressed the expression of osteoclastogenesis-related marker genes during osteoclastogenesis, and prevented osteoclastic bone resorption but did not impair osteoblast differentiation in MC3T3-E1. Therefore, these modified salicylanilides and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-diones could be potential lead compounds for the development of a new class of antiresorptive agents.

Full text of DOI:10.1021/jm5007897

Article
pubs.acs.org/jmc
Modified Salicylanilide and 3‑Phenyl‑2H‑benzo[e][1,3]oxazine-
2,4(3H)‑dione Derivatives as Novel Inhibitors of Osteoclast
Differentiation and Bone Resorption
Chun-Liang Chen,†,‡ Fei-Lan Liu,§ Chia-Chung Lee,†,‡ Tsung-Chih Chen,†,‡ Ahmed Atef Ahmed Ali,‡,∥
Huey-Kang Sytwu,‡,⊥ Deh-Ming Chang,*,‡,§ and Hsu-Shan Huang*,†,‡,#
†Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University,
Taipei 110, Taiwan ROC
‡Graduate Institute of Life Sciences, National Defense Medical Center, Taipei 114, Taiwan ROC
§Rheumatology/Immunology/Allergy, Taipei Veterans General Hospital, Taipei 112, Taiwan ROC
∥Taiwan International Graduate Program, Molecular and Cell Biology Program, Institute of Molecular Biology, Academia Sinica,
Taipei 115, Taiwan ROC
⊥Department and Graduate Institute of Microbiology and Immunology, National Defense Medical Center, Taipei 114, Taiwan ROC
#School of Pharmacy, National Defense Medical Center, Taipei 114, Taiwan ROC
S
* Supporting Information
ABSTRACT: Inhibition of osteoclast formation is a potential
strategy to prevent inflammatory bone resorption and to treat
bone diseases. In the present work, the purpose was to
discover modified salicylanilides and 3-phenyl-2H-benzo[e]-
[1,3]oxazine-2,4(3H)-dione derivatives as potential antiosteo-
clastogenic agents. Their inhibitory effects on RANKL-induced
osteoclastogenesis from RAW264.7 cells were evaluated by
TRAP stain assay. The most potent compounds, 1d and 5d,
suppressed RANKL-induced osteoclast formation and TRAP
activity dose-dependently. The cytotoxicity assay on
RAW264.7 cells suggested that the inhibition of osteoclastic
bone resorption by these compounds did not result from their
cytotoxicity. Moreover, both compounds downregulated
RANKL-induced NF-κB and NFATc1 in the nucleus, suppressed the expression of osteoclastogenesis-related marker genes
during osteoclastogenesis, and prevented osteoclastic bone resorption but did not impair osteoblast differentiation in MC3T3-E1.
Therefore, these modified salicylanilides and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-diones could be potential lead
compounds for the development of a new class of antiresorptive agents.
regulates the process of osteoclastogenesis and maintains the
survival of mature osteoclasts.8 When RANKL binds to the cell-
surface receptor RANK, the initiation of signal transduction
triggers downstream signaling cascades, including the NF-κB,
mitogen-activated protein kinase, c-fos, and nuclear factor of
activated T cells c1 (NFATc1) pathways that contribute to
RANKL-induced osteoclastogenesis.9,10 Among these tran-
scription factors, NFATc1 is a critical target for controlling
excessive osteoclastogenesis. Agents that can suppress NFATc1,
such as FK506 and cyclosporin A, have been approved for the
inhibition of osteoclastogenesis.11,12 On the basis of promoter
analyses, some osteoclast-specific genes, such as the tartrate-
resistant acid phosphatase (TRAP), cathepsin K, MMP-9, and
dendritic cell-specific transmembrane protein (DC-STAMP)
INTRODUCTION
■
Bone is a dynamic tissue that is constantly undergoing a repair
and renewal process termed bone remodeling.1 Bone-resorbing
cells (osteoclasts) together with bone-forming cells (osteo-
blasts) play the pivotal roles in bone remodeling.2 Generalized
bone loss with reduced mineral density, bone destruction, and
imbalance of bone remodeling are pathological hallmarks of
osteoporosis, inflammatory joint disease, and rheumatoid
arthritis (RA).3−5 Bone resorption is dependent on a cytokine
known as receptor activator of nuclear factor κB ligand
(RANKL), and RANKL inhibitors can prevent focal bone
loss that occurs in animal models of RA.6
Osteoclasts originate from the fusion of mononuclear
progenitors of the monocyte/macrophage hematopoietic
lineage that resorb bone in the presence of RANKL.2,7
RANKL is a member of the tumor necrosis factor (TNF)
superfamily produced mainly by the osteoblasts, which
Received: June 27, 2014
Published: September 9, 2014
© 2014 American Chemical Society
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genes, are directly regulated by NFATc1 and required for bone
resorptive activity of osteoclasts and cell−cell fusion during
osteoclast development.10,13−16 Further, NFATc1 is a critical
target gene of NF-κB in the early phase of osteoclastogenesis.12
The importance of NFATc1 in osteoclastogenesis is also
demonstrated by the in vitro observation that NFATc1−/−
embryonic stem cells exhibit a defect in osteoclastogenesis.12,14
Also, c-fos expression is essential for osteoclastogenesis in
response to RANKL stimulation, as c-fos-deficient mice were
found to develop osteopetrosis due to a lack of osteoclasts.17
Through these mechanisms of RANKL-induced osteoclasto-
genesis and bone resorption, the RANK signaling pathway
provides molecular targets for the development of therapeutics
to treat RA and other bone-related diseases.18 Thus, searching
for powerful small-molecule osteoclastogenesis inhibitors that
can suppress RANKL signaling and regulate the activity of
osteoclasts still holds great promise for the development of
antiresorptive agents.
Furthermore, synthetic analogues of bisphosphonates are
currently the most important and effective antiresorptive drugs,
but they have been shown to cause gastrointestinal tract side
effects, osteonecrosis of the jaw, and renal toxicity and to lead
to the suppression of bone turnover as well as a reduction in
bone quality after long-term treatment.19−21 Hence, numerous
osteoclastogenesis inhibitors have been developed, including
natural product and synthetic small molecules such as sodium
salicylate, paeonol, rosmarinic acid, biphenyl-containing de-
rivatives, and salicylanilides (Scheme 1).22−27 In addition,
salicylanilides were found to be inhibitors of interleukin
production and to show potent antitumor activity.28,29 As
part of an effort to profile potential osteoarthritis therapeutics,
we previously reported that a salicylanilide core scaffold
derivative (HS-Cf) inhibits TNF-α-induced interferon regu-
latory factor-1 in porcine chondrocytes.30 We also found that
the salicylanilide derivative NDMC101 (1a) inhibits RANKL-
induced osteoclast differentiation by suppressing NFATc1 and
NF-κB activity and ameliorates paw-swelling inflammation of
bones in collagen-induced arthritic mice.25 On the basis of
these findings, salicylanilide-derived small molecules could be
lead structures that warrant further optimization.
In the present work, we developed a series of novel synthetic
inhibitors of RANKL-induced osteoclastogenesis. We designed
and synthesized a series of small molecules containing various
core scaffolds such as salicylanilide analogues (series a, b, and
c), 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione ana-
logues (series d), and N-phenylbenzamide analogues (series
e). These compounds were classified as series a−e according to
the substituent at the 2-position of benzene ring A of the
salicylanilide core structure. For each series, there are six
different member compounds distinguished by the substituent
groups of benzene rings A and B (Scheme 1). Furthermore,
since some biphenyl-containing derivatives inhibit osteoclastic
bone resorption by inducing osteoclast apoptosis and inhibiting
osteoclast formation,26,27,31 we were particularly interested in
replacing the iodine atom in 5a and 6a with a phenyl group,
affording compounds 7a−12a. To characterize the biological
activities of these synthetic compounds, we evaluated their
effects on RANKL-induced osteoclastogenesis from osteoclast
precursor cells (RAW264.7) by the TRAP stain assay. Using
this screen, we identified 3-phenyl-2H-benzo[e][1,3]oxazine-
2,4(3H)-dione derivatives 1d and 5d, which can effectively
inhibit RANKL-induced osteoclastogenesis. On the basis of this
preliminary screening, we also constructed structure−activity
relationships (SARs) of these synthetic compounds according
to the inhibition of RANKL-induced osteoclastogenesis. Within
an in vitro screening research program to discover these
inhibitors, our results showed that hydroxyl or halogen groups
on salicylanilide and 3-phenyl-2H-benzo[e][1,3]oxazine-
2,4(3H)-dione derivatives could be the key elements related
to the inhibition activity.
Scheme 1. Chemical Structures of Osteoclast Inhibitors and
Classification of Our Synthetic Compounds
On the basis of these results, we considered the most potent
compounds 1d and 5d to inhibit RANKL-induced osteoclast
formation and antiresorptive effects by suppressing osteoclasto-
genesis-related genes, reducing NFATc1 levels, and blocking
NF-κB nuclear translocation. Furthermore, osteogenesis is a
vital process for the maintenance of skeleton and bone
formation in terms of bone development.32 Mature osteoblastic
cells exhibit their osteoblastic characteristics during osteo-
genesis, such as alkaline phosphatase (ALP) production and
deposition of extracellular matrix (ECM) on the substrate,
which are subsequently mineralized.33 Currently, the effects of
salicylanilides and 3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-
dione derivatives on osteogenesis are still unknown. Hence, we
investigated the effects of compounds 1a, 1d, and 5d on
osteoblastic differentiation using MC3T3-E1 mouse calvaria-
derived osteoprogenitor cells, which are widely used as a model
to study osteogenic development.34,35 We found that 1a, 1d,
and 5d showed no significant effects on osteoblastic differ-
entiation markers such as the ALP activity and mineral
deposition in MC3T3-E1 cells. This demonstrates that these
novel modified salicylanilides and 3-phenyl-2H-benzo[e][1,3]-
oxazine-2,4(3H)-dione derivatives could be inhibitors of
RANKL-induced osteoclastogenesis without affecting osteo-
blast differentiation. Thus, these structures could be promising
lead compounds that warrant further structure optimization
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a
Scheme 2. Synthesis of Modified Salicylanilide and 3-Phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione Derivatives
a
Reagents and conditions: (i) thionyl chloride, THF, reflux; (ii) aniline series, THF, reflux; (iii) iodomethane or 2,4-difluorobenzyl bromide, K2CO3,
acetone, reflux; (iv) methyl chloroformate, pyridine, reflux; (v) K2CO3, Pd(OAc)2, PPh3, toluene, reflux; (vi) aniline series, TEA, THF, room
temperature.
and SAR studies to present novel drugs for the treatment of
bone diseases.
benzoyl chloride or 4-methoxybenzoyl chloride. Then the
modified salicylanilides 1a−6a were cyclized and converted to
3-phenyl-2H-benzo[e][1,3]oxazine-2,4(3H)-dione derivatives
1d−6d by treatment with methyl chloroformate in pyridine.
This reaction was based on a literature report describing the
cyclization reaction of salicylanilides.36 Furthermore, the
biphenyl-containing derivatives 7a−12a were prepared by
palladium-catalyzed Suzuki−Miyaura coupling reactions be-
tween various 2,4-disubstituted boronic acids and iodine-atom-
containing salicylanilide 5a or 6a. The purities of all final
compounds (>95%) were determined by HPLC. The structures
CHEMISTRY
■
The synthetic routes to obtain the target modified salicylanilide
derivatives by condensation reactions of salicylic acid and 2,4-
disubstituted anilines are outlined in Scheme 2. Commercially
available salicylic acid (1) or 2-hydroxy-4-methoxybenzoic acid
(2) was transformed into the corresponding acid chloride using
excess thionyl chloride, and the acid chloride was reacted with
the substituted anilines to give the corresponding compounds
1a−6a. In order to identify the role of the hydroxyl group in
the activity of salicylanilide, 2-methoxybenzanilide (1b−6b), 2-
(2,4-difluorobenzyl)oxybenzanilide (1c−6c), and N-phenyl-
benzamide (1e−6e) derivatives were prepared. Compounds
1b−6b and 1c−6c were obtained from the reaction of 1a−6a
with iodomethane or 2,4-difluorobenzyl bromide through
nucleophilic substitution. The N-phenylbenzamide derivatives
1e−6e were obtained from the aniline series by reaction with
of the synthesized compounds were determined by 1H and 13
C
NMR spectroscopy and high-resolution mass spectrometry
(HRMS).
RESULTS AND DISCUSSION
■
Effects of the Synthetic Compounds on RANKL-
Induced Osteoclast Differentiation. After the construction
of a series of synthetic compounds, we evaluated their
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inhibitory activities against RANKL-induced osteoclast differ-
entiation from RAW264.7 cells. As shown in Table 1, several
analogues caused more than 50% inhibition of RANKL-induced
osteoclastogenesis at 10 μM as determined by the TRAP assay
using 1a as a positive control. According to previous studies,
the starting materials (salicylic acid and 2-hydroxy-4-methox-
ybenzoic acid) revealed inhibition of venom-induced inflam-
mation or osteoclast differentiation,22,37 but they showed less
inhibition against RANKL-induced osteoclast differentiation at
10 μM. Herein, the SAR study was focused on the effects of the
side chains on the aromatic ring. Among our synthetic
salicylanilide derivatives (1a−6a), it was noted that the
osteoclast inhibitory activities of the 4-iodo-substituted (5a,
6a) and 4-chloro-substituted (1a, 2a) compounds were more
potent than those of the 2-fluoro-substituted (3a, 4a)
compounds. These observations revealed that the halogen
substituent (inhibitory activities: Cl and I > F) at the 4-position
on benzene ring B of the salicylanilide may modulate the
inhibition of osteoclastogenesis. We also found that the
inhibition of osteoclastogenesis was increased by introducing
the methoxy substituent group at the 4-position of the benzene
ring A compared with the corresponding H-substituted
compounds (inhibitory activities: 2a > 1a; 4a > 3a; 6a > 5a)
(Table 1). In addition, replacement of the 2-hydroxyl group of
the salicylanilide with a methoxy group (1b−6b) or a 2-(2,4-
difluorobenzyl)oxy group (1c−6c) led to the loss of
osteoclastogenesis inhibitory activity. This suggests that the 2-
hydroxyl moiety of salicylanilide derivatives could be a key
pharmacophore for inhibition of osteoclast formation. We also
observed that the absence of the hydroxyl group at the 2-
position of the N-phenylbenzamide derivatives (1e−6e) led to
loss of potency against RANKL-induced osteoclastogenesis. In
addition, there are some reports indicating that biphenyl-
containing derivatives can inhibit osteoclastic bone resorption
by inducing osteoclast apoptosis and inhibiting osteoclast
formation.26,27 However, our compounds with a biphenyl
structure (7a−12a) were less potent than 1a at a concentration
of 10 μM. Next, the antiosteoclastogenic effects of 3-phenyl-
2H-benzo[e][1,3]oxazine-2,4(3H)-dione derivatives (1d−6d)
were evaluated. Among them, compounds 1d and 5d were the
most potent inhibitors among our synthetic compounds against
RANKL-induced osteoclast formation. Hence, 1d and 5d were
selected as potential structures for further pharmacological
evaluations. The SARs and the key pharmacophore of our
synthetic compounds for osteoclastogenesis inhibition are
described in Scheme 3.
In order to confirm that the antiosteoclastogenic effects of
compounds are not attributable to cellular toxicity, we
investigated the effect of the compounds on cell viability
using RAW264.7 osteoclast progenitor cells by performing the
MTT assay (Table 1). The results showed that all of the
compounds had slight or no effect on cell viability of
RAW264.7 cells at a concentration of 10 μM [survival rate
>85% except for 2b (52.9%), 3a (76.1%), 8a (64.5%), and 10a
(44.5%)]. Furthermore, the concentrations of compounds
required to inhibit cell viability by 50% of the mean (CC50)
were determined. Interestingly, all of the synthetic analogues
had higher CC50 values than 1a (23.2 μM) except for 2b (13.6
μM). These results suggest that the antiosteoclastogenic
activities of these compounds are not due to their cytotoxicities
toward osteoclast precursor cells.
Inhibitory Effects of 1d and 5d against RANKL-
Induced Osteoclast Differentiation. Compounds 1d and
5d were the most potent inhibitors among all of the tested
compounds. Both compounds reduced the number of red
TRAP-positive osteoclasts and decreased the size of multi-
nucleated osteoclasts significantly in a dose-dependent manner
(Figure 1). Compounds 1d (Figure 2A) and 5d (Figure 2E)
also showed no effect on the cell viability of osteoclast
progenitor cells at 5 and 10 μM (Figure 2B,F). Furthermore, in
Table 1. Effects of Compounds on Cell Viability and
RANKL-Induced Osteoclast Differentiation from RAW264.7
Cells
a
b
cell viability
osteoclastogenesis
survival rate at 10 μM
TRAP+ MNCs at 10 μM
compd
(%)
CC50 (μM)
(%)
c
1
99.8 12.2
99.4 7.6
93.0 3.1
101.8 4.5
107.2 4.5
97.9 2.0
105.2 7.4
103.0 2.8
52.9 2.8
107.3 4.4
100.5 2.4
105.5 1.2
76.1 6.3
108.5 4.0
101.1 3.2
88.8 0.6
105.2 9.4
89.7 4.0
102.5 9.3
106.0 8.8
90.9 5.4
101.8 4.9
96.7 5.0
95.4 5.4
85.0 4.2
91.4 3.4
105.2 3.4
94.4 4.8
95.0 1.7
104.2 9.3
103.2 9.0
91.4 9.2
102.5 7.6
64.5 7.3
90.9 3.1
44.5 6.6
105.1 5.7
102.5 6.0
>40
99.8 7.5
79.6 8.3
49.4 5.4
86.0 9.9
74.6 6.2
19.7 4.9
77.9 6.4
23.8 3.4
60.2 3.6
68.7 4.6
36.0 1.8
100.4 4.0
85.2 11.5
89.1 5.1
97.6 6.0
82.5 8.4
75.2 8.1
74.9 7.4
93.0 7.7
66.2 8.1
67.5 5.0
73.3 8.1
70.6 13.9
106.6 6.7
85.5 8.5
1.8 2.4
c
2
>40
1a
1b
1c
1d
1e
2a
2b
2c
2d
2e
3a
3b
3c
3d
3e
4a
4b
4c
4d
4e
5a
5b
5c
5d
5e
6a
6b
6c
6d
6e
7a
8a
9a
10a
11a
12a
23.2 0.6
>40
>40
26.5 1.0
>40
32.3 4.9
13.6 1.2
>40
>40
>40
38.4 2.7
>40
>40
>40
>40
>40
>40
>40
>40
>40
23.3 0.3
>40
32.6 3.8
23.6 0.4
>40
76.8 10.7
39.4 9.3
92.4 9.8
51.3 6.6
44.4 9.4
89.3 6.9
100.1 6.2
50.1 11.5
84.8 7.4
60.6 5.8
91.2 7.9
92.5 12.1
27.6 2.0
>40
36.8 5.9
30.1 0.9
32.8 7.3
28.6 3.1
26.3 3.7
27.2 3.8
d
N.T.
29.8 5.0
>40
a
CC50 is the concentration of the tested compound required to inhibit
b
cell viability by 50% of the mean (N = 3). To induce differentiation of
murine RAW 264.7 cells, the cells were cultured in α-MEM medium
containing 10% FBS and 100 ng/mL RANKL with or without the
tested compound (10 μM) for 4 days. TRAP-positive multinucleated
cells (TRAP+ MNCs) having five or more nuclei were considered as
osteoclasts. RANKL-induced osteoclast differentiation is shown as
relative activity (% of the compound-treated group relative to the
c
RANKL-treated group). Salicylic acid (1), 2-hydroxy-4-methoxyben-
d
zoic acid (2). N.T. = not tested.
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Scheme 3. SARs and the Key Pharmacophore for Osteoclastogenesis Inhibition
1d and 5d on RANKL-induced TRAP activity were
determined. Both compounds were found to dose-dependently
suppress RANKL-induced TRAP activity (Figure 2D,H) with
IC50 values of 10.6 and 7.3 μM, respectively (Table 2). On the
basis of these results, we conclude that our new 3-phenyl-2H-
benzo[e][1,3]oxazine-2,4(3H)-dione derivatives 1d and 5d are
potent inhibitors of RANKL-induced osteoclastogenesis.
Effects of 1d and 5d on the Expression of NFATc1-
Mediated Genes and Osteoclastogenesis-Related
Marker Genes during RANKL-Induced Osteoclast Devel-
opment. Osteoclast formation is executed directly by the
upregulation of specific genes such as NFATc1 and c-fos in
response to the RANKL−RANK interaction.6 Herein we
examined the effects of 1d and 5d on RANKL-induced
regulation of NFATc1 and c-fos expression for 24 or 48 h using
gapdh as an internal control (Figure 3). Increased levels of
NFATc1 and c-fos mRNA were observed after RANKL
stimulation, but expression of both NFATc1 and c-fos were
significantly decreased by treatment with 1d and 5d. The
expression of DC-STAMP is rapidly induced by RANKL,
regulated by NFATc1, and related to cell−cell fusion during
osteoclast development.15,16 Our results revealed that the DC-
STAMP mRNA level increased in the presence of RANKL and
decreased upon treatment with 1d and 5d (Figure 3).
Moreover, the TRAP enzyme is abundantly expressed by
osteoclasts and involved in bone resorption.38 Cathepsin K is a
matrix-degrading enzyme that degrades type-I collagen and
collagenous bone matrix.39,40 MMP-9 is one of the matrix
metalloproteases that initiates bone degradation by removing
the bone-lining collagen and involves in the invasion of
osteoclasts.41,42 As shown in Figure 3, the mRNA expression
levels of TRAP, cathepsin K, and MMP-9 increased in response
to RANKL, but expressions of these mRNA were significantly
suppressed by 1d and 5d in a concentration-dependent
manner. On the basis of these results, we conclude that
compounds 1d and 5d may inhibit osteoclast differentiation
through inhibition of RANKL-induced NFATc1 and c-fos gene
expression.
Figure 1. Effects of compounds 1d and 5d on RANKL-induced
osteoclast differentiation from RAW264.7 cells. RAW264.7 cells were
cultured with the tested compounds in the presence of RANKL (100
ng/mL) for 4 days. Cells were then fixed and stained for TRAP. The
TRAP-positive multinucleated cells containing more than five nuclei
were visualized by light microscopy. [Salicylic acid (1); 2-hydroxy-4-
methoxybenzoic acid (2).]
comparison with the RANKL-treated group, compound 1d
decreased the percentage of TRAP-positive multinucleated cells
generated (percentage of the 1d-treated group relative to the
RANKL-treated group), with 74.6% at 5 μM and 45.9% at 7.5
μM (Figure 2C). Also, TRAP-positive multinucleated cells were
significantly reduced after treatment with compound 5d
(Figure 2G) (74.1% at 2.5 μM, 49.1% at 5 μM, and 29.1% at
7.5 μM). In addition, the IC50 values of the compounds on
RANKL-induced osteoclastogenesis from RAW264.7 cells were
calculated. The IC50 values of 1d and 5d for TRAP-positive
multinucleated osteoclast formation were 7.1 and 4.9 μM,
respectively (Table 2). To test whether the compounds
inhibited the activity of osteoclasts, the inhibitory effects of
Effects of 1d and 5d on RANKL-Induced NF-κB and
NFATc1 Levels in the Nucleus. Activation of the tran-
scription factor NF-κB is important in osteoclastogenesis and
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activated RAW264.7 cells. As shown in Figure 4, NF-κB and
NFATc1 in nuclear extracts were increased after 24 h of
treatment with RANKL compared with the levels in RAW264.7
cells without RANKL treatment. Data are expressed as the ratio
of the protein level of NF-κB or NFATc1 normalized to TATA-
box binding protein (TBP). The elevated levels of nuclear NF-
κB and NFATc1 were reduced by 1d and 5d in a dose-
dependent manner. These results suggest that compounds 1d
and 5d may suppress RANKL-induced NF-κB and NFATc1
activation during osteoclastogenesis from macrophages.
Effects of 1d and 5d on RANKL-Induced Bone
Resorption in RAW264.7 Cells. To examine the effects of
compounds 1d and 5d on osteoclastic bone resorption, we used
a pit formation assay to investigate the function of osteoclasts.
As shown in Figure 5, resorption pits were formed on the bone
slice by osteoclasts after stimulation with RANKL. A dose-
dependent attenuation of resorbed areas in the examined slices
was observed after treatment with various concentrations of
compounds 1d and 5d. Furthermore, compounds 1d and 5d
significantly decreased the percentage of resorbed area relative
to the total area at concentrations of 2.5, 5, 7.5, and 10 μM.
These results indicate that both compounds prevent osteo-
clastic bone resorption by inhibiting osteoclastogenesis.
Compounds 1a, 1d, and 5d Did Not Affect Osteo-
blastic Differentiation of MC3T3-E1 Osteoprogenitor
Cells. We investigated the effects of compounds 1a, 1d, and 5d
on osteoblastic differentiation using the MC3T3-E1 cell line. As
shown in Figure 6, differentiated osteoblasts exhibited ALP
activity and ECM deposition in the presence of β-glycerol
phosphate and ascorbic acid for 14 days. Compounds 1a, 1d,
and 5d did not affect the ALP activity and ECM deposition
from MC3T3-E1 cells at a concentration of 7.5 μM. These
results indicate that compounds 1a, 1d, and 5d may not impair
osteogenesis at concentrations higher than that needed to
inhibit osteoclast formation.
Figure 2. Inhibition of RANKL-induced osteoclast differentiation by
compounds 1d and 5d. (A, E) Chemical structures of 1d and 5d. (B,
F) Effects of compounds 1d and 5d (5−40 μM) on cell viability of
RAW264.7 cells by MTT assay. (C, G) Compounds 1d and 5d
reduced RANKL-induced osteoclast differentiation in a dose-depend-
ent manner. TRAP+ MNCs having five or more nuclei were considered
as osteoclasts. (D, H) TRAP activities were assessed for compounds
1d and 5d on osteoclastic activity: *, p < 0.05 and **, p < 0.01
compared with the RANKL-treated group.
CONCLUSIONS
■
In summary, we synthesized series of salicylanilide analogues
(series a, b, and c), 3-phenyl-2H-benzo[e][1,3]oxazine-
2,4(3H)-dione analogues (series d), and N-phenylbenzamide
analogues (series e) and then evaluated their inhibition of
RANKL-induced osteoclastogenesis in preosteoclast RAW264.7
cells. We also studied the SARs of these compounds based on
the inhibition of RANKL-induced osteoclast formation. The 2-
hydroxyl group of salicylanilide could be the key element
related to the activity for inhibition of RANKL-induced
osteoclastogenesis. The halogen substituent at the 4-position
on benzene ring B of the salicylanilide core may play an
important role in the inhibition of osteoclastogenesis, in which
the inhibition decreased in the order Cl, I > F, biphenyl group.
Furthermore, the new heterocyclic ring-fused compounds 1d
and 5d exhibited more potent inhibition activities than the
other compounds. Both 1d and 5d suppressed RANKL-
induced osteoclast differentiation, TRAP activity, and bone
resorption in a dose-dependent manner. Moreover, compounds
1d and 5d showed no significant cytotoxic effects on the cell
viability of RAW264.7 cells at 10 μM. Our mechanistic
experiments indicated that 1d and 5d can inhibit the expression
of NFATc1 and c-fos genes as well as osteoclastogenesis-related
marker genes (including DC-STAMP, TRAP, cathepsin K, and
MMP-9) during osteoclastogenesis. The blockade of osteoclas-
togenesis by 1d and 5d might be mediated by attenuation of
RANKL-induced NF-κB translocation. We have provided the
Table 2. IC50 Values of Compounds for RANKL-Induced
Osteoclastogenesis from RAW264.7 Cells
a
IC50 (μM)
compd
TRAP+ MNCs
TRAP activity
1a
1d
5d
10.0 0.7
7.1 0.5
4.9 0.5
12.5 1.3
10.6 0.7
7.3 0.4
a
IC50 is the concentration of tested compound required to inhibit
TRAP+ MNCs or TRAP activity by 50% of the mean.
activation of mature osteoclasts.43,44 The accumulated evidence
has suggested that NF-κB is an upstream transcription factor
regulating NFATc1 expression.12,45 NFATc1 is a key tran-
scription factor involved in osteoclastogenesis and osteoclast
formation.46,47 Therefore, to investigate the molecular mech-
anism of 1d- and 5d-mediated inhibition of osteoclast
differentiation and activation, we performed Western blots to
examine NF-κB and NFATc1 in nuclear extracts from RANKL-
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Figure 3. Effects of compounds 1d and 5d on osteoclastogenic mRNA expression. RAW264.7 cells were pretreated with or without the tested
compounds for 1 h and then supplemented with RANKL (100 ng/mL) for the indicated time points. Total RNA was isolated with TRIzol, and 2 μg
of each total RNA was used to transcribe the cDNA. The cDNA was amplified by PCR from mouse-specific primers. Results are expressed as mean
SD of three independent experiments. *, p < 0.05 and **, p < 0.01 compared with the RANKL-treated group.
1
determined using a Buchi 545 melting point apparatus. H NMR and
̈
first evidence that salicylanilides and 3-phenyl-2H-benzo[e]-
[1,3]oxazine-2,4(3H)-dione derivatives inhibit RANKL-in-
duced osteoclastogenesis but do not affect osteogenesis.
Therefore, compounds 1d and 5d may improve bone health
through reducing osteoclast formation and function. Overall,
our study indicates that this novel 3-phenyl-2H-benzo[e][1,3]-
oxazine-2,4(3H)-dione structure offers an attractive starting
point for further optimization and represents a promising lead
for the development of a new class of antiresorptive agents.
13C NMR spectra were recorded on an Agilent 400 MR DD2 (400
MHz) apparatus. Mass spectra were obtained using Finnigan MAT-
95XL (high-resolution electron impact ionization, HREI) and
Finnigan MAT 95S (high-resolution electrospray ionization, HRESI)
instruments. The purities of all compounds were analyzed on a C18
reversed-phase column (XBridge BEH Shield RP18 Column, 130 Å, 5
μm, 4.6 mm × 250 mm, Waters) by HPLC (model l-2000, HITACHI)
with UV detection (model l-2400, HITACHI); each compound was
dissolved in MeOH, and the mobile phase was water and MeOH. A
preliminary evaluation of the UV spectra was carried out by
spectrophotometric analysis to determine the value of λmax for each
compound. The purities of the synthetic compounds for biological
evaluation were greater than 95%. Reagents and solvents were
EXPERIMENTAL SECTION
■
Chemistry. All of the reactions were monitored using TLC plates
(silica gel F254 plates, Merck). The melting points of compounds were
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Figure 4. Effects of compounds 1d and 5d on RANKL-induced NF-κB
and NFATc1 in nuclear extracts. RAW264.7 cells were pretreated with
different concentrations of 1d and 5d for 1 h prior to treatment with
RANKL (100 ng/mL) for 24 h. Nuclear extracts were analyzed by
Western blot using antibodies against NF-κB and NFATc1. TBP was
evaluated as an internal control.
Figure 6. Effects of compounds 1a, 1d, and 5d on osteoblast
differentiation from MC3T3-E1 cells. Cells were treated with the
tested compounds in the presence of β-glycerol phosphate (10 mM)
and ascorbic acid (50 μg/mL) for 14 days, and then the effects of the
compounds on ALP activity and ECM calcification were evaluated by
the ALP or Alizarin red S staining methods. Furthermore, the ECM
calcification was quantified by the amount of cell-bound Alizarin red S.
Alizarin red S was released by incubation in 10% cetylpyridinium
chloride and then detected by measuring the absorbance at 540 nm.
Data are presented as the mean
experiments.
SD of three independent
anhydrous acetone (10 mL), potassium carbonate (0.69 g, 5 mmol),
and compound 1a−6a (2 mmol), and the mixture was refluxed for 8 h.
After cooling to room temperature, the reaction mixture was
concentrated in vacuo. The residue was extracted with ethyl acetate,
dried over anhydrous magnesium sulfate, concentrated, and recrystal-
lized with hot methanol.
General Procedure III: Preparation of Compounds 1c−6c. To a
solution of compound 1a−6a (2 mmol) in anhydrous acetone (10
mL) was added potassium carbonate (0.69 g, 5 mmol) and 2,4-
difluorobenzyl bromide (0.56 mL, 4.4 mmol), and the mixture was
refluxed for 6 h. After cooling to room temperature, the reaction
mixture was concentrated in vacuo. The residue was extracted with
ethyl acetate, dried over anhydrous magnesium sulfate, concentrated,
and recrystallized with hot methanol.
General Procedure IV: Preparation of Compounds 1d−6d.
Methyl chloroformate (0.3 mL, 3.6 mmol) was added dropwise to a
stirred solution of 1a−6a (1 mmol) in dry pyridine (8 mL) at 0 °C,
and then the mixture was refluxed for 2 h. After 16 h of stirring at
room temperature, the pH of the reaction mixture was adjusted to 6
with 1 N HCl(aq). The resulting white mixture was cooled in an ice
bath to obtain a solid compound. The product was filtered and
recrystallized from hot ethanol.
General Procedure V: Preparation of Compounds 7a−12a. A
mixture of potassium carbonate (1.04 g, 7.5 mmol), palladium(II)
acetate (0.034 g, 0.15 mmol), triphenylphosphine (0.131 g, 0.5 mmol),
and boronic acid (2,4-difluorophenylboronic acid, 4-chloro-2-fluo-
rophenylboronic acid, or 2,4-dichlorophenylboronic acid) (3.5 mmol)
in toluene (20 mL) was added dropwise with constant stirring to a
solution of compound 5a (0.89 g, 2.5 mmol) or 6a (0.97 g, 2.5 mmol)
in toluene (10 mL). The reaction mixture was refluxed for 24 h. After
cooling to room temperature, the solution was filtered through Celite
and concentrated in vacuo. The crude product was purified using a
flash chromatography column with hexane and dichloromethane to
give the desired compound as a white solid.
Figure 5. Effects of 1d and 5d on resorption pit formation. RAW264.7
cells were cultured on bone slices with various concentrations of
compounds 1d and 5d in the presence of RANKL (100 ng/mL) for 4
days. After incubation, bone slices were subjected to 0.1% toluidine
blue to visualize the resorption pits. Resorbed lacunae on the bone
slices were visualized using light microscopy. The ratios of resorbed
area relative to the total area were calculated using ImageJ software. *,
p < 0.05 and **, p < 0.01 compared with RANKL-treated group.
purchased from Merck and Sigma-Aldrich and used without further
purification.
General Procedure I: Preparation of Compounds 1a−6a. Thionyl
chloride (2.5 mL, 34 mmol) was added to a solution of salicylic acid
(1) (1.38 g, 10 mmol) or 2-hydroxy-4-methoxybenzoic acid (2) (1.68
g, 10 mmol) in THF (50 mL) dropwise with stirring. The mixture was
refluxed for 3 h and then steam-distilled using a Dean−Stark
apparatus, and the produced 2-hydroxybenzoyl chloride or 2-
hydroxy-4-methoxybenzoyl chloride was collected. This benzoyl
chloride residue was directly refluxed with 4-chloro-2-fluorobenzen-
amine (1.1 mL, 10 mmol), 2,4-difluoroaniline (1.0 mL, 10 mmol), or
2-fluoro-4-iodoaniline (2.37 g, 10 mmol) in THF (50 mL) for 12 h.
The resulting solution was concentrated and extracted with ethyl
acetate. The organic phase was dried over anhydrous magnesium
sulfate and concentrated in vacuo. The crude product was crystallized
from warm dichloromethane to give the desired compound.
General Procedure II: Preparation of Compounds 1b−6b.
Iodomethane (0.28 mL, 4.4 mmol) was added to the mixture of
General Procedure VI: Preparation of Compounds 1e−6e. To a
solution of aniline (4-chloro-2-fluoroaniline, 2,4-difluoroaniline, or 2-
fluoro-4-iodoaniline) (10 mmol) in tetrahydrofuran (40 mL) was
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+
added benzoyl chloride (3) (1.16 mL, 10 mmol) or 4-methoxybenzoyl
chloride (4) (1.35 mL, 10 mmol) with a basic catalyst (TEA, 1.5 mL).
The reaction mixture was refluxed for 16 h, concentrated in vacuo, and
washed with methanol. The desired product was recrystallized from
hot dichloromethane.
HRMS (ESI) m/z: calcd for C15H14ClFNO3 [M + H]+ 310.0646,
found 310.0655.
N-(4-Chloro-2-fluorophenyl)-2-((2,4-difluorobenzyl)oxy)-4-me-
thoxybenzamide (2c). The pure compound was obtained as a yellow
powder (yield 53%). Mp 138−139 °C. 1H NMR (400 MHz, CDCl3):
δ 3.87 (s, 3H), 5.24 (s, 2H), 6.63−6.69 (m, 2H), 6.88−6.95 (m, 2H),
6.99−7.11 (m, 2H), 7.45 (q, J = 7.5 Hz, 1H), 8.25 (d, J = 8.8 Hz, 1H),
8.53 (t, J = 8.6 Hz, 1H), 9.92 (s, 1H). 13C NMR (100 MHz, CDCl3): δ
55.62, 64.78, 64.82, 99.62, 104.07, 104.33, 104.58, 106.32, 111.74,
111.77, 111.95, 111.99, 114.38, 115.13, 115.36, 117.87, 117.91, 118.02,
118.06, 122.31, 122.33, 124.57, 124.60, 125.98, 126.08, 127.76, 127.86,
132.02, 132.06, 132.11, 132.16, 134.41, 150.76, 153.22, 157.50, 160.10,
160.22, 162.25, 162.37, 162.60, 162.72, 162.92, 163.95, 164.75, 164.87.
N-(4-Chloro-2-fluorophenyl)-2-hydroxybenzamide (1a).25 The
pure compound was obtained as a white powder (yield 46%). Mp
187−188 °C. 1H NMR (400 MHz, CDCl3): δ 6.95 (t, J = 7.6 Hz, 1H),
7.05 (d, J = 8.4 Hz, 1H), 7.19−7.22 (m, 2H), 7.48 (t, J = 7.8 Hz, 1H),
7.52 (d, J = 8.0 Hz, 1H), 8.14 (br, 1H), 8.30 (t, J = 8.8 Hz, 1H), 11.67
(s, 1H). 13C NMR (100 MHz, CDCl3): δ 114.17, 115.81, 116.03,
119.06, 119.24, 123.07, 123.08, 124.15, 124.25, 124.97, 125.00, 125.50,
129.86, 129.96, 135.18, 151.46, 153.92, 161.83, 168.12. HRMS (EI)
+
HRMS (ESI) m/z: calcd for C21H16ClF3NO3 [M + H]+ 422.0771,
+
m/z: calcd for C13H9ClFNO2 [M]+ 265.0306, found 265.0305.
found 422.0778.
N-(4-Chloro-2-fluorophenyl)-2-methoxybenzamide (1b). The
pure compound was obtained as a white powder (yield 81%). Mp
114−115 °C. 1H NMR (400 MHz, CDCl3): δ 4.07 (s, 3H), 7.04 (d, J
= 8.4 Hz, 1H), 7.12−7.16 (m, 3H), 7.49−7.54 (m, 1H), 8.28 (dd, J =
8.0 Hz, J = 1.6 Hz, 1H), 8.58 (t, J = 9.0 Hz, 1H), 10.34 (s, 1H). 13C
NMR (100 MHz, CDCl3): δ 56.20, 111.56, 115.25, 115.48, 121.09,
121.66, 122.32, 122.34, 124.75, 124.79, 126.07, 126.17, 128.04, 128.14,
132.47, 133.67, 150.98, 153.44, 157.35, 163.14. HRMS (EI) m/z: calcd
3-(4-Chloro-2-fluorophenyl)-7-methoxy-2H-benzo[e][1,3]-
oxazine-2,4(3H)-dione (2d). The pure compound was obtained as a
white powder (yield 41%). Mp 206−207 °C. H NMR (400 MHz,
1
CDCl3): δ 3.93 (s, 3H), 6.78 (s, 1H), 6.94 (d, J = 8.8 Hz, 1H), 7.29−
7.31 (m, 3H), 8.01 (d, J = 8.8 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 8.20
(d, J = 8.4 Hz, 1H), 8.26−8.34 (m, 2H). 13C NMR (100 MHz,
CDCl3): δ 56.18, 100.35, 106.58, 113.81, 117.51, 117.74, 120.60,
120.74, 125.28, 125.32, 129.88, 130.87, 136.38, 136.48, 147.21, 154.64,
156.45, 158.99, 159.37, 166.51. HRMS (ESI) m/z: calcd for
+
for C14H11ClFNO2 [M]+ 279.0462, found 279.0458.
N-(4-Chloro-2-fluorophenyl)-2-((2,4-difluorobenzyl)oxy)-
+
C15H10ClFNO4 [M + H]+ 322.0282, found 322.0295.
benzamide (1c). The pure compound was obtained as a white powder
1
N-(4-Chloro-2-fluorophenyl)-4-methoxybenzamide (2e). The
pure compound was obtained as a white powder (yield 88%). Mp
158−159 °C. 1H NMR (400 MHz, CDCl3): δ 3.87 (s, 3H), 6.97 (d, J
= 8.8 Hz, 2H), 7.13−7.16 (m, 2H), 7.84 (d, J = 8.8 Hz, 2H), 7.96 (s,
1H), 8.40 (d, J = 8.8 Hz, 1H), 8.44 (t, J = 8.6 Hz, 1H). 13C NMR (100
MHz, CDCl3): δ 55.46, 114.06, 115.45, 115.68, 122.36, 124.81,
124.84, 125.44, 125.54, 126.25, 128.56, 128.66, 128.98, 151.03, 153.48,
(yield 51%). Mp 125−126 °C. H NMR (400 MHz, CDCl3): δ 5.28
(s, 2H), 6.87−6.95 (m, 2H), 7.02 (dd, J = 10.8 Hz, J = 2.4 Hz, 1H),
7.10−7.19 (m, 3H), 7.42−7.56 (m, 2H), 8.30 (dd, J = 7.6 Hz, J = 1.6
Hz, 1H), 8.53 (t, J = 8.8 Hz, 1H), 10.04 (s, 1H). 13C NMR (100 MHz,
CDCl3): δ 64.92, 64.96, 104.08, 104.33, 104.58, 111.69, 111.73,
111.91, 111.94, 112.68, 115.21, 115.44, 118.05, 118.09, 118.20, 118.23,
121.58, 122.15, 122.45, 122.47, 124.62, 124.66, 125.78, 125.88, 128.15,
128.25, 131.93, 131.97, 132.03, 132.08, 132.78, 133.67, 150.87, 153.33,
156.24, 160.10, 160.22, 162.25, 162.37, 162.60, 162.72, 163.02, 164.75,
+
162.80, 164.84. HRMS (ESI) m/z: calcd for C14H12ClFNO2 [M +
H]+ 280.0541, found 280.0534.
N-(2,4-Difluorophenyl)-2-hydroxybenzamide (3a).48 The pure
compound was obtained as a white powder (yield 56%). Mp 186−
187 °C. 1H NMR (400 MHz, CDCl3): δ 6.95 (t, J = 8.6 Hz, 3H), 7.05
(d, J = 8.4 Hz, 1H), 7.48 (t, J = 7.8 Hz, 1H), 7.53 (d, J = 8.0 Hz, 1H),
8.06 (s, 1H), 8.22−8.25 (m, 1H), 11.74 (s, 1H). 1H NMR (400 MHz,
DMSO-d6): δ 6.96−7.01 (m, 2H), 7.10−7.14 (m, 1H), 7.35−7.41 (m,
1H), 7.42−7.47 (m, 1H), 8.00 (d, J = 7.6 Hz, 1H), 8.08−8.14 (m,
1H), 8.06 (s, 1H), 8.22−8.25 (m, 1H), 10.60 (s, 1H), 11.92 (s, 1H).
13C NMR (100 MHz, DMSO-d6): δ 103.87, 104.12, 104.14, 104.38,
111.18, 111.21, 111.39, 111.43, 117.15, 119.52, 122.52, 122.56, 122.64,
122.67, 124.97, 124.99, 125.06, 125.09, 129.97, 133.98, 152.55, 152.68,
155.01, 155.14, 157.28, 157.39, 157.50, 159.71, 159.82, 165.13. HRMS
+
164.87. HRMS (ESI) m/z: calcd for C20H14ClF3NO2 [M + H]+
392.0665, found 392.0673.
3-(4-Chloro-2-fluorophenyl)-2H-benzo[e][1,3]oxazine-2,4(3H)-
dione (1d). The pure compound was obtained as a white powder
(yield 52%). Mp 180−181 °C. 1H NMR (400 MHz, CDCl3): δ 7.30−
7.32 (m, 3H), 7.37 (d, J = 8.4 Hz, 1H), 7.43 (t, J = 7.6 Hz, 1H), 7.78
(t, J = 7.8 Hz, 1H), 8.13 (d, J = 7.6 Hz, 1H). 13C NMR (100 MHz,
CDCl3): δ 113.79, 116.73, 117.54, 117.77, 120.46, 120.60, 125.33,
125.36, 125.84, 128.50, 130.74, 136.51, 136.61, 136.84, 146.89, 152.74,
+
156.37, 158.91, 159.74. HRMS (ESI) m/z: calcd for C14H8ClFNO3
[M + H]+ 292.0177, found 292.0181.
N-(4-Chloro-2-fluorophenyl)benzamide (1e). The pure compound
1
+
(ESI) m/z: calcd for C13H10F2NO2 [M + H]+ 250.0680, found
was obtained as a white powder (yield 95%). Mp 129−130 °C. H
NMR (400 MHz, CDCl3): δ 7.15−7.19 (m, 2H), 7.51 (d, J = 7.4 Hz,
2H), 7.57−7.60 (m, 1H), 7.88 (d, J = 7.6 Hz, 2H), 8.03 (s, 1H), 8.44
(t, J = 8.6 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 115.53, 115.76,
122.40, 122.41, 124.88, 124.92, 125.25, 125.35, 127.05, 128.92, 129.01,
132.32, 134.13, 151.06, 153.52, 165.37. HRMS (ESI) m/z: calcd for
C13H10ClFNO+ [M + H]+ 250.0435, found 250.0431.
250.0660.
N-(2,4-Difluorophenyl)-2-methoxybenzamide (3b). The pure
compound was obtained as a white powder (yield 37%). Mp 87−88
°C. 1H NMR (400 MHz, CDCl3): δ 4.07 (s, 3H), 6.87−6.92 (m, 2H),
7.05 (d, J = 8.4 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 7.50−7.54 (m, 1H),
8.29 (dd, J = 7.8 Hz, J = 1.8 Hz, 1H), 8.53−8.59 (m, 1H), 10.25 (s,
1H). 13C NMR (100 MHz, CDCl3): δ 56.19, 103.07, 103.31, 103.34,
103.57, 110. 98, 111.02, 111.20, 111.23, 111.53, 121.15, 121.64,
122.53, 122.56, 122.62, 122.65, 123.57, 123.68, 132.46, 133.58, 151.21,
151.33, 153.66, 153.77, 156.90, 157.01, 157.34, 159.34, 159.46, 163.12.
N-(4-Chloro-2-fluorophenyl)-2-hydroxy-4-methoxybenzamide
(2a). The pure compound was obtained as a white powder (yield
41%). Mp 189−190 °C. 1H NMR (400 MHz, CDCl3): δ 3.84 (s, 3H),
6.48−6.51 (m, 2H), 7.17−7.20 (m, 2H), 7.41 (d, J = 9.6 Hz, 1H), 7.93
(br, 1H), 8.27 (t, J = 8.8 Hz, 1H), 12.09 (s, 1H). 13C NMR (100 MHz,
CDCl3): δ 55.55, 101.79, 106.98, 107.77, 115.73, 115.96, 122.98,
124.37, 124.47, 124.91, 124.95, 126.78, 129.45, 129.55, 151.38, 153.84,
+
HRMS (ESI) m/z: calcd for C14H12F2NO2 [M + H]+ 264.0836,
found 264.0827.
2-((2,4-Difluorobenzyl)oxy)-N-(2,4-difluorophenyl)benzamide
(3c). The pure compound was obtained as a white powder (yield
41%). Mp 135−136 °C. 1H NMR (400 MHz, CDCl3): δ 5.29 (s, 2H),
6.75−6.80 (m, 1H), 6.84−6.95 (m, 3H), 7.13−7.19 (m, 2H), 7.43−
7.54 (m, 2H), 8.30 (dd, J = 7.8 Hz, J = 1.8 Hz, 1H), 8.48−8.54 (m,
1H), 9.97 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 64.87, 64.90,
103.00, 103.24, 103.27, 103.50, 104.07, 104.33, 104.58, 110.86, 110.90,
111.08, 111.11, 111.69, 111.73, 111.90, 111.94, 112.64, 118.09, 118.12,
118.24, 118.27, 121.64, 122.12, 122.66, 122.68, 122.75, 122.77, 123.24,
123.27, 123.34, 123.38, 131.88, 131.94, 131.98, 132.03, 132.75, 133.56,
151.10, 151.22, 153.56, 153.67, 156.21, 156.91, 157.02, 159.35, 159.47,
+
164.35, 165.09, 168.02. HRMS (ESI) m/z: calcd for C14H12ClFNO3
[M + H]+ 296.0490, found 296.0491.
N-(4-Chloro-2-fluorophenyl)-2,4-dimethoxybenzamide (2b). The
pure compound was obtained as a white powder (yield 83%). Mp
1
155−156 °C. H NMR (400 MHz, CDCl3): δ 3.86 (s, 3H), 4.02 (s,
3H), 6.51 (d, J = 1.6 Hz, 1H), 6.64 (dd, J = 8.8 Hz, J = 2.0 Hz, 1H),
7.10−7.14 (m, 2H), 8.22 (d, J = 8.8 Hz, 1H), 8.57 (t, J = 8.8 Hz, 1H),
10.19 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 55.55, 56.13, 98.59,
105.75, 114.00, 115.14, 115.37, 122.12, 122.14, 124.66, 124.70, 126.25,
126.35, 127.62, 127.72, 134.09, 150.84, 153.30, 158.66, 163.01, 164.04.
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160.06, 160.19, 162.21, 162.33, 162.57, 162.69, 162.99, 164.71, 164.83.
CDCl3): δ 55.48, 103.28, 103.51, 103.54, 103.77, 111.16, 111.19,
111.37, 111.41, 114.07, 122.77, 122.80, 122.86, 122.89, 122.99, 123.03,
126.40, 128.97, 151.38, 151.50, 153.83, 153.95, 157.18, 157.30, 159.63,
+
HRMS (ESI) m/z: calcd for C20H14F4NO2 [M + H]+ 376.0961,
found 376.0955.
+
3-(2,4-Difluorophenyl)-2H-benzo[e][1,3]oxazine-2,4(3H)-dione
159.75, 162.75, 164.92. HRMS (ESI) m/z: calcd for C14H12F2NO2
[M + H]+ 264.0836, found 264.0826.
(3d). The pure compound was obtained as a white powder (yield
1
35%). Mp 182−183 °C. H NMR (400 MHz, CDCl3): δ 7.01−7.06
N-(2-Fluoro-4-iodophenyl)-2-hydroxybenzamide (5a). The pure
compound was obtained as a white powder (yield 67%). Mp 196−197
(m, 2H), 7.32−7.39 (m, 1H), 7.43 (d, J = 8.4 Hz, 1H), 7.43 (t, J = 7.6
Hz, 1H), 7.76−7.81 (m, 1H), 8.14 (dd, J = 8.0 Hz, J = 1.6 Hz, 1H).
13C NMR (100 MHz, CDCl3): δ 105.08, 105.32, 105.35, 105.58,
112.08, 112.12, 112.31, 112.35, 113.84, 116.73, 125.84, 128.53, 130.88,
130.90, 130.98, 131.00, 136.82, 147.08, 152.76, 156.81, 156.93, 159.34,
159.47, 159.90, 161.96, 162.07, 164.47, 164.59. HRMS (ESI) m/z:
1
°C. H NMR (400 MHz, CDCl3): δ 6.93−6.97 (m, 1H), 7.04 (d, J =
8.4 Hz, 1H), 7.46−7.54 (m, 4H), 8.10−8.15 (m, 2H), 11.66 (s, 1H).
13C NMR (100 MHz, CDCl3): δ 86.62, 86.70, 114.21, 119.07, 119.25,
123.59, 124.12, 124.34, 125.43, 125.50, 133.94, 133.96, 135.20, 151.17,
+
153.65, 161.84, 168.09. HRMS (ESI) m/z: calcd for C13H10FINO2
+
calcd for C14H8F2NO3 [M + H]+ 276.0472, found 276.0454.
[M + H]+ 357.9740, found 357.9729.
N-(2,4-Difluorophenyl)benzamide (3e). The pure compound was
N-(2-Fluoro-4-iodophenyl)-2-methoxybenzamide (5b). The pure
compound was obtained as a white powder (yield 34%). Mp 183−184
°C. 1H NMR (400 MHz, CDCl3): δ 4.07 (s, 3H), 7.04 (d, J = 8.4 Hz,
1H), 7.14 (t, J = 7.6 Hz, 1H), 7.44−7.54 (m, 3H), 8.27 (dd, J = 7.8 Hz,
J = 1.8 Hz, 1H), 8.40 (d, J = 8.4 Hz, 1H), 10.36 (s, 1H). 13C NMR
(100 MHz, CDCl3): δ 56.21, 84.64, 84.72, 111.54, 121.06, 121.66,
123.04, 123.05, 123.54, 123.76, 127.28, 127.37, 132.47, 133.72, 133.77,
133.81, 150.81, 153.28, 157.33, 163.16. HRMS (ESI) m/z: calcd for
1
obtained as a white powder (yield 91%). Mp 120−121 °C. H NMR
(400 MHz, CDCl3): δ 6.88−6.93 (m, 2H), 7.47−7.51 (m, 2H), 7.55−
7.59 (m, 1H), 7.87 (d, J = 7.2 Hz, 1H), 8.00 (s, 1H), 8.33−8.39 (m,
1H). 13C NMR (100 MHz, CDCl3): δ 103.32, 103.56, 103.59, 103.83,
111.14, 111.18, 111.36, 111.40, 123.01, 123.03, 123.09, 123.12, 127.04,
128.84, 132.17, 151.55, 151.67, 154.00, 154.11, 157.38, 157.49, 159.83,
159.94, 165.49. HRMS (ESI) m/z: calcd for C13H10F2NO+ [M + H]+
234.0730, found 234.0727.
+
C14H12FINO2 [M + H]+ 371.9897, found 371.9906.
N-(2,4-Difluorophenyl)-2-hydroxy-4-methoxybenzamide (4a).
The pure compound was obtained as a white powder (yield 30%).
Mp 181−182 °C. 1H NMR (400 MHz, CDCl3): δ 3.84 (s, 3H), 6.49−
6.51 (m, 2H), 6.93 (t, J = 8.6 Hz, 2H), 7.42 (d, J = 8.8 Hz, 1H), 7.85
(s, 1H), 8.19−8.25 (m, 1H), 12.14 (s, 1H). 13C NMR (100 MHz,
CDCl3): δ 55.54, 101.78, 103.57, 103.81, 103.84, 104.07, 106.98,
107.69, 111.30, 111.34, 111.52, 111.56, 123.66, 123.76, 123.77, 126.76,
151.93, 152.05, 154.39, 154.51, 157.73, 157.85, 160.20, 160.31, 164.31,
165.01, 168.09. HRMS (ESI) m/z: calcd for C14H12F2NO3+ [M + H]+
280.0785, found 280.0777.
2-((2,4-Difluorobenzyl)oxy)-N-(2-fluoro-4-iodophenyl)benzamide
(5c). The pure compound was obtained as a white powder (yield
60%). Mp 135−136 °C. 1H NMR (400 MHz, CDCl3): δ 5.28 (s, 2H),
6.87−6.95 (m, 2H), 7.13−7.19 (m, 2H), 7.33 (dd, J = 10.2 Hz, J = 1.8
Hz, 1H), 7.42−7.48 (m, 2H), 7.50−7.55 (m, 1H), 8.29 (dd, J = 7.8
Hz, J = 1.8 Hz, 1H), 8.35 (t, J = 8.4 Hz, 1H), 10.06 (s, 1H). 13C NMR
(100 MHz, CDCl3): δ 64.90, 64.94, 84.80, 84.87, 104.09, 104.34,
104.60, 111.70, 111.74, 111.92, 111.96, 112.64, 117.99, 118.04, 118.14,
118.18, 121.55, 122.15, 123.16, 123.51, 123.73, 127.00, 127.10, 131.96,
132.00, 132.05, 132.10, 132.79, 133.64, 133.68, 133.71, 150.69, 153.17,
156.23, 160.11, 160.23, 162.25, 162.36, 162.61, 162.73, 163.03, 164.74,
N-(2,4-Difluorophenyl)-2,4-dimethoxybenzamide (4b). The pure
compound was obtained as a white powder (yield 33%). Mp 144−145
−
164.86. HRMS (ESI) m/z: calcd for C20H12F3INO2 [M − H]−
1
°C. H NMR (400 MHz, CDCl3): δ 3.87 (s, 3H), 4.03 (s, 3H), 6.53
481.9865, found 481.9884.
(s, 1H), 6.65 (d, J = 8.8 Hz, 1H), 6.88−6.91 (m, 2H), 8.24 (d, J = 8.8
Hz, 1H), 8.51−8.57 (m, 1H), 10.11 (s, 1H). 13C NMR (100 MHz,
CDCl3): δ 55.58, 56.15, 98.66, 102.99, 103.23, 103.26, 103.49, 105.71,
110.90, 110.94, 111.11, 111.15, 114.12, 122.39, 122.42, 122.48, 122.51,
134.10, 151.11, 151.22, 153.55, 153.67, 156.70, 156.82, 158.68, 159.14,
3-(2-Fluoro-4-iodophenyl)-2H-benzo[e][1,3]oxazine-2,4(3H)-
dione (5d). The pure compound was obtained as a white powder
(yield 41%). Mp 183−184 °C. 1H NMR (400 MHz, CDCl3): δ 7.01−
7.11 (m, 1H), 7.37 (dd, J = 8.2 Hz, J = 0.6 Hz, 1H), 7.41−7.45 (m,
1H), 7.63−7.67 (m, 2H), 7.76−7.81 (m, 1H), 8.14 (dd, J = 7.8 Hz, J =
1.4 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 95.01, 95.09, 113.81,
116.75, 125.87, 126.17, 126.39, 128.54, 131.28, 134.29, 134.33, 136.86,
152.76, 156.07, 158.63, 159.67. HRMS (ESI) m/z: calcd for
+
159.26, 163.03, 163.97. HRMS (ESI) m/z: calcd for C15H14F2NO3
[M + H]+ 294.0942, found 294.0938.
2-((2,4-Difluorobenzyl)oxy)-N-(2,4-difluorophenyl)-4-methoxy-
benzamide (4c). The pure compound was obtained as a white powder
+
1
C14H8FINO3 [M + H]+ 383.9533, found 383.9558.
(yield 28%). Mp 132−133 °C. H NMR (400 MHz, CDCl3): δ 3.88
N-(2-Fluoro-4-iodophenyl)benzamide (5e). The pure compound
(s, 3H), 5.25 (s, 2H), 6.63 (s, 1H), 6.68 (d, J = 8.8 Hz, 1H), 6.73−6.79
(m, 1H), 6.83−6.95 (m, 3H), 7.46 (q, J = 7.6 Hz, 1H), 8.26 (d, J = 8.8
Hz, 1H), 8.47−8.53 (m, 1H), 9.84 (s, 1H). 13C NMR (100 MHz,
CDCl3): δ 55.62, 64.80, 99.65, 102.93, 103.16, 103.19, 103.43, 104.08,
104.33, 104.58, 106.29, 110.82, 110.84, 111.02, 111.05, 111.74, 111.78,
111.95, 111.99, 114.50, 117.96, 118.00, 118.11, 122.55, 122.57, 122.63,
122.66, 123.46, 123.50, 123.56, 123.60, 131.95, 132.00, 132.06, 132.10,
134.39, 151.01, 151.13, 153.47, 153.58, 156.73, 156.84, 157.49, 159.17,
159.28, 160.07, 160.20, 162.23, 162.35, 162.58, 162.70, 162.90, 163.87,
164.73, 164.85. HRMS (ESI) m/z: calcd for C21H16F4NO3+ [M + H]+
406.1066, found 406.1067.
1
was obtained as a white powder (yield 61%). Mp 153−154 °C. H
NMR (400 MHz, CDCl3): δ 7.47−7.53 (m, 4H), 7.57−7.61 (m, 1H),
7.88 (d, J = 8.0 Hz, 2H), 8.02 (s, 1H), 8.27 (t, J = 8.4 Hz, 1H). 13C
NMR (100 MHz, CDCl3): δ 85.57, 85.65, 123.01, 123.02, 123.83,
124.05, 127.05, 128.92, 132.33, 133.85, 133.89, 134.13, 150.83, 153.30,
165.32. HRMS (ESI) m/z: calcd for C13H10FINO+ [M + H]+
341.9791, found 341.9798.
N-(2-Fluoro-4-iodophenyl)-2-hydroxy-4-methoxybenzamide
(6a). The pure compound was obtained as a white powder (yield
40%). Mp 214−215 °C. 1H NMR (400 MHz, CDCl3): δ 3.85 (s, 3H),
6.49−6.51 (m, 2H), 7.41 (d, J = 8.8 Hz, 1H), 7.49−7.52 (m, 2H), 7.94
(s, 1H), 8.11 (t, J = 8.4 Hz, 1H), 12.08 (s, 1H). 13C NMR (100 MHz,
CDCl3): δ 51.59, 86.13, 86.21, 101.80, 107.02, 107.78, 123.50, 124.03,
124.24, 125.65, 125.75, 126.76, 133.87, 133.91, 151.09, 153.57, 164.38,
165.11, 167.98. HRMS (ESI) m/z: calcd for C14H12FINO3+ [M + H]+
387.9846, found 387.9862.
3-(2,4-Difluorophenyl)-7-methoxy-2H-benzo[e][1,3]oxazine-
2,4(3H)-dione (4d). The pure compound was obtained as a white
powder (yield 51%). Mp 142−143 °C. 1H NMR (400 MHz, CDCl3):
δ 3.93 (s, 3H), 6.78 (s, 1H), 6.94 (d, J = 8.8 Hz, 1H), 7.03 (m, 2H),
7.31−7.36 (m, 1H), 8.02 (d, J = 8.8 Hz, 1H). 13C NMR (100 MHz,
CDCl3): δ 55.16, 100.33, 105.02, 105.25, 105.28, 105.51, 106.63,
112.00, 112.04, 112.23, 112.26, 113.78, 129.87, 130.98, 130.99, 131.08,
147.38, 154.64, 156.87, 156.99, 159.40, 159.50, 161.89, 162.00, 164.40,
164.51, 166.48. HRMS (ESI) m/z: calcd for C15H10F2NO4+ [M + H]+
306.0578, found 306.0560.
N-(2,4-Difluorophenyl)-4-methoxybenzamide (4e). The pure
compound was obtained as a white powder (yield 45%). Mp 134−
135 °C. 1H NMR (400 MHz, CDCl3): δ 3.88 (s, 3H), 6.88−7.00 (m,
4H), 7.84−7.86 (m, 2H), 8.36−8.42 (m, 1H). 13C NMR (100 MHz,
N-(2-Fluoro-4-iodophenyl)-2,4-dimethoxybenzamide (6b). The
pure compound was obtained as a white powder (yield 30%). Mp
1
118−119 °C. H NMR (400 MHz, CDCl3): δ 3.88 (s, 3H), 4.03 (s,
3H), 6.53 (d, J = 1.6 Hz, 1H), 6.65 (dd, J = 8.8 Hz, J = 2.0 Hz, 1H),
7.43−7.48 (m, 2H), 8.23 (d, J = 8.8 Hz, 1H), 8.39 (t, J = 8.4 Hz, 1H),
10.22 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 55.60, 56.19, 84.22,
84.30, 98.67, 105.79, 114.10, 122.95, 122.97, 123.46, 123.67, 127.50,
127.60, 133.73, 133.77, 134.18, 150.75, 153.23, 158.71, 163.06, 164.10.
8081
dx.doi.org/10.1021/jm5007897 | J. Med. Chem. 2014, 57, 8072−8085

Journal of Medicinal Chemistry
Article
+
HRMS (ESI) m/z: calcd for C15H14FINO3 [M + H]+ 402.0002,
found 402.0014.
3H), 8.05 (s, 1H), 8.41 (t, J = 8.4 Hz, 1H), 12.16 (s, 1H). 13C NMR
(100 MHz, CDCl3): δ 55.56, 101.84, 107.18, 107.75, 107.79, 115.36,
115.56, 115.60, 116.92, 117.18, 122.16, 123.54, 124.05, 124.27, 124.98,
125.01, 125.33, 125.44, 126.78, 126.83, 130.97, 131.02, 133.90, 133.94,
134.34, 134.45, 151.42, 153.84, 158.15, 160.66, 164.42, 165.10, 168.12.
2-((2,4-Difluorobenzyl)oxy)-N-(2-fluoro-4-iodophenyl)-4-methox-
ybenzamide (6c). The pure compound was obtained as a white
powder (yield 27%). Mp 152−153 °C. 1H NMR (400 MHz, CDCl3):
δ 3.87 (s, 3H), 5.24 (s, 2H), 6.63−6.68 (m, 2H), 6.87−6.95 (m, 2H),
7.30−7.32 (m, 1H), 7.41−7.46 (m, 2H), 8.24 (d, J = 8.8 Hz, 1H), 8.35
(t, J = 8.4 Hz, 1H), 9.93 (s, 1H). 13C NMR (100 MHz, CDCl3): δ
55.62, 64.79, 64.82, 84.35, 84.43, 99.61, 104.07, 104.32, 104.58,
106.33, 111.73, 111.77, 111.95, 111.98, 114.40, 117.85, 117.89, 118.00,
118.03, 123.06, 123.41, 123.63, 127.22, 127.32, 132.03, 132.08, 132.13,
132.18, 133.57, 133.61, 134.42, 150.62, 153.10, 157.50, 160.10, 160.23,
162.25, 162.37, 162.61, 162.73, 162.92, 163.98, 164.75, 164.87. HRMS
+
HRMS (ESI) m/z: calcd for C20H15ClF2NO3 [M + H]+ 390.0709,
found 390.0730.
N-(2′,4′-Dichloro-3-fluoro-[1,1′-biphenyl]-4-yl)-2-hydroxybenza-
mide (11a). The pure compound was obtained as a white powder
1
(yield 18%). Mp 208−209 °C. H NMR (400 MHz, CDCl3): δ 6.98
(t, J = 7.6 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 7.28−7.35 (m, 4H),
7.49−7.52 (m, 2H), 7.58 (d, J = 8.0 Hz, 1H), 8.26 (s, 1H), 8.43 (t, J =
8.2 Hz, 1H), 11.79 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 114.32,
116.09, 116.29, 119.08, 119.23, 121.95, 125.05, 125.15, 125.52, 125.79,
125.82, 127.36, 129.92, 131.91, 133.15, 134.28, 135.14, 135.36, 135.44,
137.14, 151.13, 153.56, 161.92, 168.24. HRMS (ESI) m/z: calcd for
+
(ESI) m/z: calcd for C21H16F3INO3 [M + H]+ 514.0127, found
514.0139.
3-(2-Fluoro-4-iodophenyl)-7-methoxy-2H-benzo[e][1,3]oxazine-
2,4(3H)-dione (6d). The pure compound was obtained as a white
powder (yield 69%). Mp 200−201 °C. 1H NMR (400 MHz, CDCl3):
δ 6.78 (d, J = 2.0 Hz, 1H), 6.94 (dd, J = 2.0 Hz, J = 9.0 Hz, 1H), 7.06−
7.10 (m, 1H), 7.62−7.64 (m, 2H), 8.02 (d, J = 8.8 Hz, 1H). 13C NMR
(100 MHz, CDCl3): δ 56.16, 94.82, 94.90, 100.34, 106.56, 113.78,
121.88, 122.01, 126.08, 126.30, 129.86, 131.39, 134.20, 134.24, 147.08,
154.62, 156.12, 158.69, 159.25, 166.48. HRMS (ESI) m/z: calcd for
+
C19H13Cl2FNO2 [M + H]+ 376.0307, found 376.0318.
N-(2′,4′-Dichloro-3-fluoro-[1,1′-biphenyl]-4-yl)-2-hydroxy-4-me-
thoxybenzamide (12a). The pure compound was obtained as a white
powder (yield 60%). Mp 223−224 °C. 1H NMR (400 MHz, CDCl3):
δ 3.88 (s, 3H), 6.54−6.56 (m, 2H), 7.28−7.36 (m, 4H), 7.48−7.50 (m,
1H), 7.54 (d, J = 2.0 Hz, 1H), 8.09 (s, 1H), 8.43 (t, J = 2.0 Hz, 1H),
12.21 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 55.56, 101.83, 107.17,
107.75, 116.02, 116.22, 121.87, 125.28, 125.38, 125.74, 125.78, 126.83,
127.34, 129.90, 131.92, 133.16, 134.22, 134.98, 135.06, 137.22, 151.07,
153.50, 164.40, 165.08, 168.13. HRMS (ESI) m/z: calcd for
+
C15H10FINO4 [M + H]+ 413.9639, found 413.9644.
N-(2-Fluoro-4-iodophenyl)-4-methoxybenzamide (6e). The pure
compound was obtained as a white powder (yield 72%). Mp 161−162
°C. 1H NMR (400 MHz, CDCl3): δ 3.87 (s, 3H), 6.98 (d, J = 8.8 Hz,
2H), 7.44−7.49 (m, 2H), 7.83 (d, J = 8.8 Hz, 2H), 7.96 (s, 1H), 8.24
(t, J = 8.4 Hz, 1H). 13C NMR (100 MHz, CDCl3): δ 55.48, 85.18,
85.26, 114.08, 122.97, 122.98, 123.75, 123.97, 129.00, 133.80, 133.84,
150.81, 153.28, 162.82, 164.81. HRMS (ESI) m/z: calcd for
+
C20H15Cl2FNO3 [M + H]+ 406.0413, found 406.0446.
RAW264.7 Cell Culture. The murine monocyte/macrophage cell
line RAW264.7 (purchased from ATCC, USA) was cultured in
DMEM (Gibco BRL) containing 10% heat-inactivated FBS. The cells
were grown at 37 °C in a humid atmosphere containing 5% CO2.
Cell Viability by MTT Assay. The tetrazolium reagent 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was
designed to yield a formazan product upon metabolic reduction by
viable cells.49 RAW264.7 cells (1 × 104 cells/well) were seeded in 96-
well plates with DMEM supplemented with 10% FBS and treated with
various concentrations of synthetic compounds for 24 h. After
compound treatment, the 96-well plates were washed with PBS three
times, and then MTT solution (100 μL, 0.5 mg/mL) in the medium
was added to each well for 1 h at 37 °C. The cells were then washed
with PBS and solubilized in 100 μL of DMSO per well. The
absorbances at 540 nm were determined with an ELISA microplate
reader. The effects of compounds on cell viability were shown as
relative activity (% of compound-treated group relative to the DMSO
control group).
RANKL-Induced Osteoclast Differentiation and Measure-
ments of TRAP Activity. RAW264.7 cells were seeded at a density of
1 × 103 cells/well in 96-well plates or 5 × 104 cells/well in 48-well
plates and cultured in α-MEM (Gibco BRL) containing 10% FBS, 2
mM L-glutamate, 100 ng/mL recombinant soluble murine RANKL
(Peprotec), and various concentrations of synthetic compounds for 4
days. Cell culture media were changed on day 2. For RANKL-induced
osteoclast differentiation, cells were fixed with the fixing solution
(65:25:8 acetone/citrate solution/37% formaldehyde) for 5 min.
Multinucleated osteoclasts were quantified by determining cells that
were TRAP-positive [stained by tartrate-resistant acid phosphatase kit
(Sigma-Aldrich, 387A-1KT)]. The red TRAP-positive multinucleated
cells (>5 nuclei/cell) were visualized by light microscopy and counted
as mature osteoclasts. The effects of compounds on RANKL-induced
osteoclast differentiation were shown as the percentage of TRAP-
positive multinucleated cells (% of the compound-treated group
relative to the RANKL-treated group).
+
C14H12FINO2 [M + H]+ 371.9897, found 371.9921.
2-Hydroxy-N-(2′,3,4′-trifluoro-[1,1′-biphenyl]-4-yl)benzamide
(7a). The pure compound was obtained as a white powder (yield
1
52%). Mp 194−195 °C. H NMR (400 MHz, CDCl3): δ 6.90−6.99
(m, 3H), 7.06 (d, J = 8.4 Hz, 1H), 7.33−7.44 (m, 3H), 7.46−7.50 (m,
1H), 7.56 (d, J = 8.0 Hz, 1H), 8.24 (s, 1H), 8.42 (t, J = 8.6 Hz, 1H),
11.79 (s, 1H). 13C NMR (100 MHz, CDCl3): δ 104.34, 104.59,
104.85, 111.71, 111.74, 111.92, 111.96, 114.33, 115.44, 115.47, 115.64,
115.68, 119.05, 119.21, 122.23, 124.82, 124.92, 125.04, 125.07, 125.10,
125.53, 131.06, 131.11, 131.15, 131.20, 132.11, 132.20, 135.11, 151.50,
153.92, 158.38, 158.50, 160.88, 161.00, 161.23, 161.34, 161.90, 163.72,
163.83, 168.21. HRMS (ESI) m/z: calcd for C19H13F3NO2+ [M + H]+
344.0898, found 344.0905.
2-Hydroxy-4-methoxy-N-(2′,3,4′-trifluoro-[1,1′-biphenyl]-4-yl)-
benzamide (8a). The pure compound was obtained as a white powder
1
(yield 17%). Mp 211−212 °C. H NMR (400 MHz, CDCl3): δ 3.85
(s, 3H), 6.51−6.53 (m, 2H), 6.90−6.99 (m, 2H), 7.32−7.47 (m, 4H),
8.04 (s, 1H), 8.40 (t, J = 8.2 Hz, 1H), 12.19 (s, 1H). 13C NMR (100
MHz, CDCl3): δ 55.55, 101.83, 104.32, 104.59, 104.84, 107.19,
107.72, 111.69, 111.72, 111.90, 111.93, 115.38, 115.42, 115.59, 115.63,
122.17, 125.04, 125.07, 125.17, 126.82, 131.06, 131.11, 131.15, 131.20,
131.75, 131.83, 151.44, 153.86, 158.39, 158.51, 160.89, 161.01, 161.20,
161.32, 163.69, 163.80, 164.40, 165.07, 168.12. HRMS (ESI) m/z:
+
calcd for C20H15F3NO3 [M + H]+ 374.1004, found 374.1029.
N-(4′-Chloro-2′,3-difluoro-[1,1′-biphenyl]-4-yl)-2-hydroxybenza-
mide (9a). The pure compound was obtained as a white powder (yield
1
22%). Mp 206−207 °C. H NMR (400 MHz, CDCl3): δ 6.97 (t, J =
7.6 Hz, 1H), 7.06 (d, J = 8.4 Hz, 1H), 7.20−7.24 (m, 2H), 7.36−7.40
(m, 3H), 7.49 (t, J = 7.8 Hz, 1H), 7.56 (d, J = 8.0 Hz, 1H), 8.25 (s,
1H), 8.43 (t, J = 8.6 Hz, 1H), 11.77 (s, 1H). 13C NMR (100 MHz,
CDCl3): δ 114.31, 115.41, 115.43, 115.62, 115.66, 116.93, 117.19,
119.07, 119.22, 122.22, 125.00, 125.03, 125.07, 125.19, 125.52, 125.76,
130.97, 131.01, 131.82, 131.91, 134.41, 134.52, 135.15, 151.46, 153.89,
158.13, 160.64, 161.92, 168.21. HRMS (ESI) m/z: calcd for
For measurement of the TRAP activity, the cells were washed with
PBS on day 4, and 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 on ice.
Subsequently, 100 μL of the substrate solution (2 mg/mL 4-
nitrophenyl phosphate in 0.09 M citrate buffer, Sigma-Aldrich) was
added to each well, and the wells were incubated for 15 min at 37 °C.
To stop the reaction, the reaction mixtures in the wells were
transferred to new plates containing NaOH(aq) (200 μL, 0.1 N).
C19H13ClF2NO2 [M + H]+ 360.0603, found 360.0621.
+
N-(4′-Chloro-2′,3-difluoro-[1,1′-biphenyl]-4-yl)-2-hydroxy-4-me-
thoxybenzamide (10a). The pure compound was obtained as a white
powder (yield 27%). Mp 233−234 °C. 1H NMR (400 MHz, CDCl3):
δ 3.86 (s, 3H), 6.51−6.53 (m, 2H), 7.19−7.23 (m, 2H), 7.34−7.47 (m,
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Table 3. Primers Used for RT-PCR
target gene
forward primer sequence (5′−3′)
reverse primer sequence (5′−3′)
NFATc1
c-fos
CCCTGACCACCGATAGCACTCT
GGTTTCAACGCCGACTACGAG
CCGCTGTGGACTATCTGCTGTA
AGGCAGCTAAATGCAGAGGGTA
AGGCCTCTACAGAGTCTTTG
ACGGCTACTTGCGGTTTCACTA
GTGAGGCCGGTGCTGAGTATGT
GGCTGCCTTCCGTCTCATAGTG
CTGACACGGTCTTCACCATTCC
TTCCCGTCAGCCTCTCTCAAT
CAGGCGTTGTTCTTATTCCGAG
CAGTCCAACAAGAAAGGACG
DC-STAMP
cathepsin K
MMP-9
TRAP
GTGTGGGCATACTTCTTTCCTGT
ACAGTCTTCTGGGTGGCAGTGAT
GAPDH
Absorbances were measured at 405 nm with the ELISA reader. The
effects of compounds on RANKL-induced TRAP activity from
osteoclasts were shown as relative activity (% of the compound-
treated group relative to the RANKL-treated group).
the absence of ascorbic acid. The cells were grown at 37 °C in a humid
atmosphere containing 5% CO2. For the determination of osteoblast
differentiation, the cells were seeded at a density of 2.5 × 104 cells/well
in a 24-well plate and cultured in α-MEM containing 10% FBS, 2 mM
L-glutamate, 10 mM β-glycerol phosphate (Sigma-Aldrich), 50 μg/mL
ascorbic acid (Sigma-Aldrich), and various concentrations of the tested
compounds for 14 days. Cell culture media were changed every 3 days.
After 14 days, ECM calcification and ALP staining of the osteoblastic
MC3T3-E1 cells were measured as described in the next section.
ALP Staining and Measurement of ECM Calcification. ALP
staining of the osteoblastic MC3T3-E1 cells was performed using the
ALP staining kit (Sigma-Aldrich 86R-1KT). After osteoblast differ-
entiation, cells were fixed with the fixing solution (65:25:8 acetone/
citrate solution/37% formaldehyde) for 10 min. The ALP staining of
cells was assayed using the enzymatic kit according to the
manufacturer’s instructions. The stained cells were washed extensively
with distilled water and imaged using a camera.
The Alizarin red S staining method was used to determine the ECM
calcification of osteoblasts in vitro. After culture protocol as described
above, cells were fixed with 4% paraformaldehyde for 30 min and
stained with 2% Alizarin red S (Sigma-Aldrich). Alizarin red S staining
for calcium precipitation was imaged using a camera. To quantify the
ECM calcium deposits in the cell matrix, the staining of cells was
eluted with 100 μL of 10% cetylpyridinium chloride and quantified by
measuring the absorbance at 540 nm. The absorbance was shown as
relative activity (% of the compound-treated group relative to the
DMSO control group).
RT-PCR Analysis. RAW264.7 cells were suspended in DMEM
containing 10% FBS and seeded onto 6 cm dishes at a density of 1 ×
105 cells/plate. After 1 day, cells were starved for 24 h by replacing the
DMEM with α-MEM and subsequently exposed to 100 ng/mL
RANKL and various concentrations of the tested compounds for
either 24 or 48 h. Total RNA was isolated from the cultured cells with
TRIzol reagent (Invitrogen). The RNA (2 μg each) was reversibly
transcribed using SuperScript II reverse transcriptase (Invitrogen) and
oligo-(dT)15 primer. The cDNA was amplified using mouse-specific
primer (Table 3). Each of the PCR experiments was performed in 25
μL of 10 mM Tris-HCl (pH 8.8), 1.5 mM MgCl2, 50 mM KCl, 0.1%
Triton X-100, 1 unit of DNA polymerase (Protech), 200 μM dNTP,
and 10 μM primer. The PCR products were separated on 1.5% agarose
gels, and the bands were visualized by ethidium bromide staining. The
optical densities for each gene were normalized to the corresponding
values for gapdh gene.
Western Blot Analysis. RAW264.7 cells were seeded at a density
of 1 × 106 cells in 6 cm culture plates containing DMEM with 10%
FBS and incubated for 24 h to become attached. Cells were pretreated
with different concentrations of compounds in αMEM containing 10%
FBS for 1 h prior to treatment with RANKL (100 ng/mL) for 24 h.
The cells were then harvested and washed with cold PBS. Nuclear
protein extracts from RAW264.7 cells were prepared using NE-PER
nuclear and cytoplasmic extraction reagents (Thermo Fisher
Scientific) according to the manufacturer’s instructions. Nuclear
protein extracts (18 μg) were electrophoresed on 12% SDS-PAGE
gels and transferred to Immobilon poly(vinyl difluoride) members.
Nonspecific binding was blocked by soaking the members in TBS
buffer containing 5% BSA for 1 h. The blots were probed with anti-
NFATc1 (clone 7A6, Santa Cruz Biotechnology), anti-NF-κB (clone
E379, Epitomics), or anti-TATA box-binding protein (clone 1TBP18,
Abcam) monoclonal antibodies (1:1000) for 12 h at 4 °C. After
further washing with TBST, the blots were subsequently incubated
with a secondary antibody coupled to horseradish peroxidase
(1:10000) for 1 h. The immunoreactive bands were visualized using
the Immobilon Western chemiluminescent HRP substrate kit
(Millipore) and exposing a clear blue X-ray film (Thermo Fisher
Scientific) to the membrane. The optical densities for each NF-κB or
NFATc1 protein were normalized to the corresponding values for
TBP.
ASSOCIATED CONTENT
* Supporting Information
■
S
Inhibitory effects of 1a against RANKL-induced osteoclast
differentiation and NMR spectra of compounds 1a, 1d, 5a, and
5d. This material is available free of charge via the Internet at
http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Authors
*D.M.C: Tel: 886-2-28757799. Fax: +886-2-77351333. E-mail:
ming0503@ms3.hinet.net.
■
*H.S.H: Tel: 886-2-27361661 ext 7525. Fax: 886-2-66387537.
E-mail: huanghs99@tmu.edu.tw.
Notes
Pit Formation Assay. To assess the effects of compounds on
RANKL-induced bone resorption, RAW264.7 cells were suspended in
α-MEM containing 10% FBS and seeded (1 × 104 cells/well) onto
dentine slices (24 well plates, Corning) in the presence or absence of
100 ng/mL RANKL and compounds. The media of all of the cultured
cells were replaced every 2 days with fresh media containing the test
chemicals. After 4 days of culture, the wells were washed three times
with PBS and filled with 1 M ammonium hydroxide to remove the
attached cells that were stained with 0.1% toluidine blue (Sigma-
Aldrich). The ratios of resorbed area to total area were measured in
the optical field of the slice using ImageJ software.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Orient Europharma Co. Ltd., Taiwan, for their
assistance in supporting our research. The present study was
also supported by the National Science Council (Grants 101-
2113-M-016-001 and 102-2314-B-016-059-MY3) and the Chi-
Mei Medical Center (Grant CMNDMC-10210).
ABBREVIATIONS USED
RA, rheumatoid arthritis; RANKL, receptor activator of nuclear
factor-κB ligand; TNF, tumor necrosis factor; NFATc1, nuclear
■
Osteoblast Differentiation of MC3T3-E1 Cells. The mouse
calvaria-origin cell line MC3T3-E1 (purchased from ATCC, USA) was
maintained in α-MEM (Gibco BRL) with 10% heat-inactivated FBS in
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factor of activated T cells c1; TRAP, tartrate-resistant acid
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