5-Lipoxygenase inhibitors suppress RANKL-induced osteoclast formation via NFATc1 expression

Article abstract of DOI:10.1016/j.bmc.2015.09.025

5-Lipoxygenase synthesizes leukotrienes from arachidonic acid. We developed three novel 5-LO inhibitors having a benzoxazole scaffold as a potential anti-osteoclastogenics. They significantly suppressed RANKL-induced osteoclast formation in mouse bone marrow-derived macrophages. Furthermore, one compound, K7, inhibited the bone resorptive activity of osteoclasts. The anti-osteoclastogenic effect of K7 was mainly attributable to reduction in the expression of NFATc1, an essential transcription factor for osteoclast differentiation. K7 inhibited osteoclast formation via ERK and p38 MAPK, as well as NF-κB signaling pathways. K7 reduced lipopolysaccharide (LPS)-induced osteoclast formation in vivo, corroborating the in vitro data. Thus, K7 exerted an inhibitory effect on osteoclast formation in vitro and in vivo, properties that make it a potential candidate for the treatment of bone diseases associated with excessive bone resorption.

Full text of DOI:10.1016/j.bmc.2015.09.025

Bioorganic & Medicinal Chemistry 23 (2015) 7069–7078
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
5-Lipoxygenase inhibitors suppress RANKL-induced osteoclast
formation via NFATc1 expression
Ju-Hee Kang a, Zheng Ting a, Mi-ran Moon a, Jung-Seon Sim a, Jung-Min Lee a, Kyung-Eun Doh c,
Sunhye Hong c, Minghua Cui c, Sun Choi c, Hyeun Wook Chang b, Hea-Young Park Choo c, , Mijung Yim a,
⇑
⇑
a College of Pharmacy, Sookmyung Women’s University, Seoul 140-742, Republic of Korea
b College of Pharmacy, Yeungnam University, Gyeongsan 712-749, Republic of Korea
c College of Pharmacy, Graduate School of Pharmaceutical Sciences, and Global Top 5 Research Program, Ewha Womans University, Seoul 120-750, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 5 July 2015
Revised 14 September 2015
Accepted 15 September 2015
Available online 15 September 2015
5-Lipoxygenase synthesizes leukotrienes from arachidonic acid. We developed three novel 5-LO inhibi-
tors having a benzoxazole scaffold as a potential anti-osteoclastogenics. They significantly suppressed
RANKL-induced osteoclast formation in mouse bone marrow-derived macrophages. Furthermore, one
compound, K7, inhibited the bone resorptive activity of osteoclasts. The anti-osteoclastogenic effect of
K7 was mainly attributable to reduction in the expression of NFATc1, an essential transcription factor
for osteoclast differentiation. K7 inhibited osteoclast formation via ERK and p38 MAPK, as well as
Keywords:
5-Lipoxygenase
Benzoxazole scaffold
RANKL-induced osteoclast formation
NFATc1
NF-jB signaling pathways. K7 reduced lipopolysaccharide (LPS)-induced osteoclast formation in vivo,
corroborating the in vitro data. Thus, K7 exerted an inhibitory effect on osteoclast formation in vitro
and in vivo, properties that make it a potential candidate for the treatment of bone diseases associated
with excessive bone resorption.
Ó 2015 Elsevier Ltd. All rights reserved.
Lipopolysaccharide (LPS)-induced osteoclast
formation
1. Introduction
Osteoclast differentiation is primarily regulated by two cytoki-
nes: macrophage-colony stimulating factor (M-CSF) and receptor
Osteoclasts are multinucleated cells derived from the
monocyte-macrophage lineage of hematopoietic precursors.1
Osteoclasts are bone-resorbing cells and increased numbers of
osteoclasts can lead to the development of diseases characterized
by bone loss, such as osteoporosis, rheumatoid arthritis, Paget’s
disease, periodontal disease, osteosarcoma, and cancer bone
metastasis.2–5 Pharmaceutical inhibition of osteoclast differentiation
is considered a promising therapeutic strategy for the prevention
of bone loss-associated disorders and related fractures.
activator of nuclear factor-jB ligand (RANKL). The binding of
M-CSF to its receptor, M-CSFR on the surface of osteoclast precur-
sor induces surface expression of RANK (a receptor of RANKL). The
binding of RANKL to RANK triggers activation of signaling mole-
cules, including mitogen-activated protein kinases (MAPKs) and
NF-jB that then evoke downstream activation of c-Fos and nuclear
factor of activated T cells c1 (NFATc1).6 c-Fos is an essential factor
for induction of NFATc1, a master transcription factor of osteoclast-
specific genes and therefore of osteoclast differentiation.7
5-Lipoxygenase (5-LO) is a crucial enzyme of the arachidonic
acid (AA) cascade catalyzing the formation of bioactive leuko-
trienes (LTs). 5-LO catalyzes the conversion of AA to 5(S)-hydroper-
oxyeicosatetraenoic acid (5-HPETE) and leukotriene (LT)A4.8,9 The
intermediate LTA4 can be further converted to LTB4 by LTA4 hydro-
lase10 or to LTC4 by LTC4 synthase.11 5-LO-derived AA metabolites
represent potent mediators of inflammatory reactions. In recent
years, accumulating evidence indicates that the 5-LO pathway
plays a role in the development of allergic diseases, such as
Abbreviations: 5-LO, 5-lipoxygenase; LTs, leukotrienes; BMMs, bone marrow-
derived macrophages; BMMCs, bone marrow-derived mast cells; LPS, lipopolysac-
charide; M-CSF, macrophage-colony stimulating factor; RANK, a receptor of RANKL;
RANKL, receptor activator of nuclear factor-jB ligand; ERK, extracellular signal-
regulated kinases; MAPKs, mitogen-activated protein kinases; NFATc1, nuclear
factor of activated T cells c1; TRAP, tartrate-resistant acid phosphatase; TRAP+,
tartrate-resistant acid phosphatase positive; MNCs, multinucleated cells; DC-
STAMP, dendritic cell-specific transmembrane protein; Atp6vod2, D2 isoform of
vacuolar (H+) ATPase (v-ATPase) Vo domain; CTR, calcitonin receptor.
asthma,12 and
a variety of inflammatory disorders, such as
rheumatoid arthritis.13 Furthermore, the 5-LO pathway also plays
a key role in bone metabolism. Genetic loss of 5-LO activity alters
bone morphology,14 and increases cortical bone thickness.15
⇑
Corresponding authors.
E-mail addresses: hypark@ewha.ac.kr (H.-Y. Park Choo), myim@soomyung.ac.kr
(M. Yim).
http://dx.doi.org/10.1016/j.bmc.2015.09.025
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

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J.-H. Kang et al. / Bioorg. Med. Chem. 23 (2015) 7069–7078
Leukotrienes produced via 5-LO can stimulate osteoclast formation
and activity, suggesting that the increased cortical thickness found
in 5-LO knockout mice may be associated with reduced osteoclast
number and activity.16–18 Thus 5-LO inhibitors are of therapeutic
value for the treatment or prevention of bone related-diseases.
The 5-LO protein consists of a C-terminal catalytic domain and
an N-terminal C2-like b-barrel domain.19 The catalytic domain
contains a non-heme iron in the active site that acts as an electron
acceptor or donor during catalysis.20 During enzyme activation, the
Reaction of 3a–c and 4-cyanophenol under K2CO3 produced 4a–c,
and the following reaction with NH2OH gave rise to the final com-
pounds 5a–c (Scheme 1).
5-LO inhibition was measured as the formation of LTC4 in the
bone marrow-derived mast cells as explained in a previous
study.24 The ability of compounds 5a, 5b and 5c to inhibit 5-LO
(IC50) were 1.72, 4.10, and 4.90 lM respectively.
2.2. Molecular modeling
iron is oxidized from the ferrous state (Fe2+) to the ferric (Fe3+
)
form, thus gaining entry in the catalytic cycle. The C2-like domain
has a regulatory function. Most 5-LO inhibitors bind to the catalytic
site of the enzyme and exhibit varying efficiencies of competitive
inhibition.21 Zileuton, N-[1-(1-benzothien-2-yl)ethyl]-N-hydrox-
yurea, is the first and only approved 5-LO inhibitor to date. How-
ever, it only partially inhibits the synthesis of leukotrienes at
clinical dose and the therapeutic index is limited by side-effects.22
Currently, there is no drug available that completely inhibits the
actions of all the 5-LO-induced mediators. Therefore, there is a
need for the development of new inhibitors with a pharmacologi-
cal profile of high potency of enzyme inhibition.
Recently we reported the design and synthesis of a series of
benzoxazoles and benzothiazoles as 5-LO inhibitors.23,24 In our
continuing effort, we synthesized the novel three compounds
having a benzoxazole scaffold, investigated as a potential anti-
osteoclastogenics using mouse bone marrow-derived macrophages
(BMMs) in this study. Furthermore, we selected one compound
K7, evaluated the pharmacological effect of K7 on osteoclast
formation, in vitro and in vivo. Our findings suggested that K7
could be used as a new therapeutic agent against bone lytic
diseases involving increased osteoclastogenesis.
To investigate the binding interactions of most active com-
pound 5a (K7) in 5-LO, we performed the docking study using
the human X-ray crystal structure (PDB ID: 3V99).25 As shown in
Figure 1, the 4-ethylaniline group nicely fitted into the bottom hole
at the active site, forming the hydrophobic interactions with
Phe555, Phe610, and Tyr660. The benzene ring of the benzoxazole
group participated in hydrophobic interactions with Phe555,
Leu607, and Phe610. The amide NH was able to form hydrogen
bonding with Ala672. The phenoxy phenyl ring made the
p–p
stacking with Phe177 and also involved in the hydrophobic inter-
actions with Phe177 and Ala410 in the upper region of the binding
site. Furthermore, the nitrogen of carbamimidoyl amine formed
hydrogen bonding with Ala410.
2.3. Inhibition of RANKL-induced osteoclast formation and
bone resorption in vitro
To check the effect of 5a–c on RANKL-induced osteoclast forma-
tion, we used a mouse bone marrow-derived macrophages (BMMs)
culture system. BMMs can differentiate into tartrate-resistant acid
phosphatase positive (TRAP+) multinucleated osteoclasts in the
presence of M-CSF and RANKL. BMMs were cultured with various
concentrations of three compounds. TRAP+ multinucleated cells
2. Results
(MNCs) containing more than 3 or 5 nuclei, >100
were counted as mature osteoclasts. IC50 of 5a, 5b and 5c were
1.31, 0.56, and 0.58 M, respectively, which is more effective than
zileuton (>20 M). 5a (K7) suppressed the RANKL-induced
osteoclast formation in a dose-dependent manner, with complete
inhibition at 10 M (Fig. 2A). K7 did not affect cell proliferation,
lm in diameter,
2.1. Synthesis of (5-(4-(N0-hydroxycarbaimidoyl)-phenoxy)-N-
(2-(4-substituted phenylamino)benzo[d]oxazole-5-yl)pentana-
mide (5a–c) and their 5-LO inhibition
l
l
l
Chemical synthesis of benzoxazole derivatives is delineated in
Scheme 1. The synthesis of starting compounds was reported in
previous study.24 Briefly, 5-nitro-2-aminophenol was reacted with
ethylphenyl isothiocyanate to give the corresponding thiourea,
then benzoxazoles (1a–c) were prepared by KO2 catalyzed ring clo-
sure. The nitro compounds 1a–c were reduced to amino com-
pounds 2a–c by 5% Pd/C and hydrogen. Coupling of amino group
and 5-bromovaleric acid by PyBOP resulted in the amides 3a–c.
suggesting that the anti-osteoclastogenic effect of K7 was not
attributable to cellular proliferation (Fig. 2B). Bone resorption is a
unique function of osteoclasts, generating resorption pits on
bone.26 To examine whether the effect of K7 on osteoclast
formation extended to osteoclastic function, we performed an
in vitro resorption pit assay using the dentin slice. Many resorption
pits were generated in wells with RANKL-treated osteoclasts. In
H
H2N
O2N
Br
N
N
O
N
O
N
O
NH
NH
NH
a
O
b
3 a-c
1 a-c
2 a-c
c
H
N
O
N
H
N
NH
O
N
O
H2N
O
O
d
NH
O
N
NC
HO
5a : R=Et (K7)
b : R=H
c : R=OCH3
4 a-c
Scheme 1. Reagents and conditions: (a) 5% Pd/C, H2, CH3OH, room temperature, 24 h, (b) 5-bromovaleric acid, PyBOP, iPr2NEt, DMF, room temperature, 16 h,
(c) 4-cyanophenol, K2CO3, DMF, 60 °C, 5 h, (d) CH3OH, NH2OH 50% in water, 40 °C, 16 h.

J.-H. Kang et al. / Bioorg. Med. Chem. 23 (2015) 7069–7078
7071
Figure 1. Docking result of K7 in the human 5-lipoxygenase. (A) Predicted binding interactions of K7 at the active site of h5-LO. The key interacting residues are labeled and
shown as capped-stick with their carbon atoms in white color. The ferrous ion (Fe2+) is displayed as a ball in red-orange color. The secondary structure of h5-LO is colored
with gray. K7 is displayed in ball-and-stick with the carbon atoms in magenta color. The van der Waals surface of K7 is colored by the lipophilic potential. Hydrogen bonds are
shown in black dashed lines. (B) The Fast Connolly surface of h5-LO and the van der Waals surface of K7. Molecular surface of h5-LO was created by MOLCAD and presented
with the lipophilic potential. The surface of h5-LO is Z-clipped for clarity, and that of K7 is in magenta color. (C) Van der Waals surface of the docked K7 is colored by the
lipophilic potential.
Figure 2. K7 inhibits RANKL-induced osteoclast formation and bone resorption. (A) BMMs were cultured with RANKL (100 ng/mL) and M-CSF (30 ng/mL) in the absence or
presence of K7 at the indicated concentrations for 4 days. Cells were fixed and stained for TRAP and TRAP+ MNCs containing more than 3 or 5 nuclei were counted. (B) BMMs
were cultured with or without K7 (10 lM) and M-CSF for 4 days, and the cell number was assessed by MTT assay. (C) BMMs were differentiated on dentine slices with M-CSF
(30 ng/mL) and RANKL (100 ng/mL) for 4 days, and then K7 (10 lM) was treated for 2 days in the presence of M-CSF and RANKL. The remaining cells were removed and
stained with toluidine blue. The resorbed pit numbers were counted. Veh, vehicle; R, RANKL; N.S., not significant (*, #p <0.05 vs Veh). Data are expressed as mean SD of three
independent experiments.
contrast, the treatment of K7 strongly inhibited formation
of resorption pits by the RANKL-treated osteoclasts (Fig. 2C).
Together, these results suggested that K7 exerted inhibitory effects
on osteoclast formation and function, which eventually translated
to reduced bone resorption.
2.4. Osteoclastogenic genes expression
RANKL induces many target genes responsible for osteoclast
differentiation and function.27 For example, osteoclasts fuse via
dendritic cell-specific transmembrane protein (DC-STAMP) and

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J.-H. Kang et al. / Bioorg. Med. Chem. 23 (2015) 7069–7078
the D2 isoform of vacuolar (H+) ATPase (v-ATPase) Vo domain
(Atp6v0d2).28 Furthermore, osteoclasts recognize and physically
attach to bone via am- and b3-integrin.29 After attaching to the
bone, osteoclasts dissolve the bone matrix by acidification, fol-
lowed by the secretion of proteolytic enzymes such as cathepsin
K.30 On the other hand, calcitonin receptor (CTR) on osteoclasts
regulates calcium homeostasis and conserves bone mass.31 To con-
firm the inhibitory effect of K7, we investigated the expression of
marker genes required for osteoclast differentiation and bone
resorption by RT-PCR. As shown in Figure 3, K7 significantly
decreased the RANKL-induced expression of DC-STAMP, Atp6v0d2,
am- and b3-integrin, cathepsin K and CTR.
active form of NFATc1 in BMMs using a retroviral infection system.
NFATc1-transduced BMMs were cultured with M-CSF and RANKL,
in the absence or presence of K7 for 4 days. As shown in Figure 4B,
NFATc1 overexpression completely reversed the inhibition of
osteoclast formation in the presence of K7. These data indicated
that the inhibitory effect of K7 on osteoclast differentiation was
due mainly to reduction in NFATc1 expression.
It is well known that RANKL stimulates multiple intracellular
signaling cascades including MAPK pathways (ERK, SAPK/JNK,
and p38) and the NF-j
B pathway.32–35 These signaling cascades
lead to the induction NFATc1 and subsequent osteoclast differenti-
ation.36 To define the molecular mechanism of the inhibitory
effects of K7 on osteoclastogenesis, we next examined the effects
of K7 on the early signaling pathways induced by RANKL in BMMs.
As shown in Figure 4C and D, activation of ERK and p38 MAPK was
observed after RANKL treatment, which was suppressed on
pretreatment with K7. These results suggested the involvement
of ERK and p38 MAP kinase pathways in the anti-osteoclastogenic
2.5. Downregulation of RANKL-induced NFATc1 expression via
MAPK and NF-jB pathways
NFATc1 plays a pivotal role in RANKL-induced up-regulation of
marker genes associated with osteoclast differentiation and activa-
tion.6 Thus, we investigated the effect of K7 on RANKL-induced
expression of NFATc1. K7 completely blocked the induction
of NFATc1 by RANKL (Fig. 4A). To investigate whether down-
regulation of NFATc1 protein levels is involved in the anti-
osteoclastogenic effects of K7, we overexpressed a constitutively
effect of K7. We also examined its effect on the NF-
pathway. Upon RANKL stimulation, NF- B is activated through
IKK activation and the subsequent phosphorylation and
degradation of I
B.37 RANKL stimulation led to the degradation
of I B, and K7 inhibited this process (Fig. 4E). Taken together, K7
jB signaling
j
j
j
Figure 3. K7 suppresses mRNA expression of osteoclastogenic genes induced by RANKL. BMMs were cultured for 4 days in the absence or presence of the 10 lM of K7 with
RANKL (100 ng/mL) and M-CSF (30 ng/mL). Total RNA was then isolated from the cells and cDNA templates were prepared. mRNA expression was determined by RT-PCR
using specific primers designed for each gene. Veh, vehicle; R, RANKL (*p <0.05). Data are expressed as mean SD of three independent experiments.

J.-H. Kang et al. / Bioorg. Med. Chem. 23 (2015) 7069–7078
7073
Figure 4. K7 inhibits RANKL-induced NFATc1 expression and signaling pathways. (A) BMMs were pretreated with K7 (10
lM) for 30 min, and then stimulated with 200 ng/mL
RANKL for 24 h in the presence of M-CSF (30 ng/mL). Cell lysates were the subjected to Western blot analysis with NFATc1 antibody. (B) BMMs were infected through the
retrovirus packaging system. After puromycin selection, cells were collected and cultured for 4 days with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the presence or
absence of K7 (10
30 min, and then treated with or without 200 ng/mL of RANKL for 15 min (for ERK, p38, and I
antibodies. Antibodies specific for ERK, p38, I
B or b-actin were used to normalize the cell extracts, respectively. Veh, vehicle; R, RANKL; N.S., not significant (*p <0.05). Data
l
M). Cells were fixed and stained for TRAP. TRAP+ MNCs were counted. (C–E) BMMs were preincubated in the absence or presence of 10
lM of K7 for
jB). Cell lysates were then subjected to Western blot analysis with the indicated
j
are expressed as mean SD of three independent experiments.
inhibited RANKL-induced expression of NFATc1, which is attributable
to the modulation of ERK, p38 MAPK, and NF-jB signaling pathways.
or without K7. TRAP staining of whole calvariae showed that LPS
markedly increased osteoclast numbers. In contrast, treatment
with K7 significantly decreased the number of osteoclasts in LPS-
injected calvarial bone (Fig. 5B and C). Taken together, these data
indicated that K7 has an inhibitory effect on LPS-induced osteo-
clastogenesis in vivo.
2.6. Prevention of LPS-induced osteoclastogenesis in vivo
We evaluated the in vivo effect of K7 on osteoclast formation
using the lipopolysaccharide (LPS)-challenged mouse model. LPS
stimulates bone loss by increasing the number of osteoclasts
in vivo.38 Firstly, we examined the effect of K7 on LPS-induced
osteoclast formation in a culture system. As shown in Figure 5A,
K7 suppressed LPS-induced osteoclast formation in vitro. Next,
we injected LPS into the supra calvarial region of mice treated with
3. Discussion
5-LO converts AA into LTA4, which is used to synthesize LTB4
and the cysteinyl leukotrienes.39,40 These metabolites of 5-LO are
lipid signaling molecules that affect many physiological processes,

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J.-H. Kang et al. / Bioorg. Med. Chem. 23 (2015) 7069–7078
NF-jB signaling pathways. RANKL-induced expression of the tran-
scription factor NFATc1, known to play a critical role in osteoclast
development, was downregulated by K7. Because the exogenous
expression of NFATc1 completely rescued the suppression of osteo-
clastogenesis by K7, we speculated that the inhibitory effect of K7
on osteoclastogenesis attributes to the suppression of NFATc1
expression. During osteoclastogenesis, NFATc1 translocates from
the cytoplasm to the nucleus to initiate transcription. In the
nucleus, NFATc1 works together with other transcription factors,
such as AP-1, PU.1, CREB, and microphthalmia-associated tran-
scription factor (MITF). Accumulating evidence indicates that
NFATc1 regulates the expression of osteoclast genes, including
DC-STAMP, Atp6v0d2, am
- and b3-integrin, cathepsin K and
CTR.43–45 Consistent with this, K7 also targets these molecules to
regulate osteoclast differentiation.
LPS has been widely recognized as a key factor in the develop-
ment of inflammatory bone loss.37 Injection of LPS in mice evoked
inflammatory bone loss by activation and differentiation of osteo-
clasts.46,47 We verified that K7 totally inhibited LPS-induced osteo-
clast formation in vivo in mouse calvaria, which was consistent
with its in vitro effect. These data indicated that K7 is a potential
drug for osteoclast-mediated resorptive bone diseases.
The structure of K7 is related to both the compounds DW 1350
and zileuton. DW 1350, N-hydroxy-4-{5-[4-(5-isopropyl-2-
methyl-1,3-thiazol-4-yl)phenoxy]pentoxy}benzamidine,
is
a
reported LTB4 receptor antagonist for the prevention and treat-
ment of osteoporosis.48,49 DW 1350 showed inhibitory effects
against osteoclast in terms of differentiation, formation, fusion
and bone absorption. Zileuton, a 5-LO inhibitor, inhibits leuko-
trienes (LTB4, LTC4, LTD4, and LTE4) formation from AA
(IC50 = 0.19
tory effect on osteoclasts (IC50 = >20
though the production of LTB4 would be affected by this drug.
Our compound K7 successfully inhibited 5-LO (IC50 = l.72 M)
and suppressed RANKL-induced osteoclast formation (IC50 = 1.31
M). The docking study of K7 using the human X-ray crystal struc-
l
M) (Fig. 6). However, zileuton did not have an inhibi-
lM) in our experiment, even
l
l
ture shown in Figure 1, showed similar pattern to the reported
binding of zileuton. The 4-ethylaniline group nicely fitted into
the bottom hole at the active site, and the amide NH was able to
form hydrogen bonding with Ala672. In addition to zileuton simi-
lar parts, K7 has N-hydroxy benzamidine moiety connected to pen-
toxy linker to amide NH. Therefore it is suggested that N-hydroxy
benzamidine moiety connected to pentoxy or butoxy linker
would be required for the inhibitory effect against osteoclast
differentiation.
Depending on their actions at the ferric iron, which is at the
center of the 5-LO active site, 5-LO inhibitors are conventionally
classified into three categories: redox inhibitor, iron ligand inhibi-
tor, and non-redox inhibitor.50 During the process of enzyme acti-
vation, lipid peroxide converts inactive 5-LO with ferrous iron into
active 5-LO with ferric iron. Redox inhibitors reduce ferric iron to
the inactive ferrous iron. Iron ligand inhibitors have binding affin-
ity to the ferric iron and block the binding ability of substrates
without changing the iron state. Non-redox inhibitors compete
with substrates for binding to 5-LO.51 Given that redox and non-re-
dox inhibitors may have qualitatively and quantitatively different
effects on the products of 5-LO catalyzed reactions, we could not
exclude the possibility that K7 may act on 5-LO differently from
zileuton. The mechanism by which K7 inhibits 5-LO needs further
investigation.
Figure 5. K7 inhibits LPS-induced osteoclast formation in vitro and in vivo. (A)
BMMs were pre-incubated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) for 36 h
and then treated with M-CSF (30 ng/mL) and LPS (100 ng/mL) in the absence or
presence of K7 (10 l
M) for 4 days. TRAP+ cells that had more than 3 nuclei were
counted. (B) Calvarias of mice that received vehicle, LPS or LPS plus K7 (5 mg/kg)
were subjected to TRAP staining. (C) TRAP+ stained areas in calvarias were
quantified by using the image
J program. Results are representative of three
independent sets of similar experiments. Veh, vehicle (*p <0.05).
including inflammation and allergy.41,14 Recently 5-LO was sug-
gested to be a direct regulator of bone metabolism. Furthermore,
we have previously provided mechanistic insight by demonstrat-
ing that pharmacological blockade of 5-LO inhibits RANKL-induced
osteoclastogenesis and bone loss.42 Thus 5-LO inhibitors are con-
sidered as good therapeutic agents for bone-related diseases.
In this study, we showed that K7, a novel 5-LO inhibitor, pre-
vented RANKL-induced osteoclastogenesis. It is well known that
RANKL-induced activation of the NF-jB and MAPK pathways is
In conclusion, K7 may be suitable for therapeutics in diseases
characterized by excessive osteoclast-mediated bone resorption
such as rheumatoid arthritis, osteoporosis or cancer-induced focal
bone loss.
required for osteoclastogenesis.32–35 Further experiments sug-
gested that the molecular mechanisms underlying the anti-
osteoclastogenic effects of K7 involved the ERK, p38 MAPK, and

J.-H. Kang et al. / Bioorg. Med. Chem. 23 (2015) 7069–7078
7075
H
O
N
N
O
NH
H2N
HO
O
N
K7
O
O
HO
O
H2N
HO
N
S
NH2
N
N
S
CH3
DW1350
Zileuton
Figure 6. The structure of DW 1350 and zileuton which are related to K7.
7.021 (d, J = 8.4 Hz, 1H), 6.942 (d, J = 9.2 Hz, 2H), 6.732
(d, J = 2 Hz, 1H), 6.418 (dd, J = 8.4, 2.4 Hz, 1H), 3.789 (s, 3H);
HR-FABMS Calcd for C14H14N3O2 (M++H): 256.1081, Found:
256.1084.
4. Experimental
4.1. Materials
Antibodies against ERK, phospho-ERK, IjB, b-actin, phospho-
4.3. Synthesis of N-(2-(4-substituted phenylamino)benzo[d]
oxazol-5-yl)-5-bromo-pentanamide (3a–c)
p38, p38, c-Fos and NFATc1 were purchased from Cell Signaling
Technology (Beverly, MA, USA). All other reagents were from
Sigma–Aldrich (St. Louis, MO, USA). ICR mice were obtained from
Samtako Inc. (Seoul, Korea). Mice were maintained in the animal
facility of the Sookmyung Women’s University on a 12:12-h
light–dark cycle, and were allowed food and water ad libitum. All
experiments were performed in accordance with institutional
guidelines approved by the Sookmyung Women’s University Ani-
mal Care and Use Committee.
Compound 2a–c (0.31 mmol), 5-bromovaleric acid (0.051 g,
0.28 mmol), and PyBOP (0.163 g, 0.31 mmol) in DMF(8 mL) were
added to diisopropyl ethylamine (0.073 g, 0.57 mmol) in ice bath
and the mixture was stirred for 16 h at room temperature. Ethyl
acetate 8 mL was added and the mixture was washed with 10%
HCl, saturated NaHCO3 and NaCl solution. The organic layer was
dried over MgSO4 and the solvent was removed in vacuo. Column
chromatography (EtOAc/hexane = 2:1) gave compound 3a–c.
4.2. Synthesis of 5-(4-(N0-hydroxycarbamidoyl)-phenoxy)-N-(2-
(4-substituted-phenylamino)benzo[d]oxazol-5-yl)pentanamide
(2a–c)
4.3.1. N-(2-(4-Ethylphenylamino)benzo[d]oxazol-5-yl)-5-bromo-
pentanamide (3a)
(0.054 g, 46%): Pale brown powder, mp 196–198 °C; 1H NMR
(400 MHz, Acetone-d6) d 9.360 (s, 1H), 9.152 (s, 1H), 7.871 (s,
1H), 7.755 (d, J = 8.4, 2H), 7.343 (d, J = 8.8, 1H), 7.266 (d, J = 8.4,
1H), 7.230 (d, J = 8.8, 2H), 3.546 (t, J = 6.6, 2H), 2.628 (q, J = 7.6,
2H), 2.428 (t, J = 7.2, 2H), 1.989–1.919 (m, 2H), 1.899–1.803 (m,
2H), 1.218 (t, J = 7.6, 3H); HR-FABMS Calcd for C20H23BrN3O2
(M++H): 416.0974, Found: 416.0969.
The starting compounds 1a–c were prepared as reported.24 The
compound 1a–c (0.35 mmol) in 10 mL methanol was added 5% Pd/
C and hydrogen, and stirred for 24 h at room temperature. The
reaction mixture was filtered in celite, and the solvent was
removed in vacuo to give 2a–c.
4.2.1. 5-(4-(N0-Hydroxycarbamidoyl)-phenoxy)-N-(2-(4-ethyl-phe-
nylamino)benzo[d]oxazol-5-yl)pentanamide (2a)
(0.079 g, 88%): Grey solid, mp 143–145 °C; 1H NMR (400 MHz,
Acetone-d6) d 9.170 (s, 1H), 7.737 (d, J = 8.4, 2H), 7.205 (d, J = 8.4,
2H), 7.036 (d, J = 8.8, 2H), 6.752 (s, 1H), 6.439 (d, J = 8.4, 1H),
4.443 (d, J = 10.4, 1H), 2.615 (q, J = 7.6, 2H), 1.210 (t, J = 7.6, 3H);
4.3.2. N-(2-(Phenylamino)benzo[d]oxazol-5-yl)-5-bromo-
pentanamide (3b)
(0.385 g, 62%): White powder, mp 166–168 °C; 1H NMR
(400 MHz, Acetone-d6) d 9.464 (br s, 1H), 9.165 (br s, 1H), 7.891–
7.846 (m, 3H), 7.405–7.346 (m, 3H), 7.282 (d, J = 8.8 Hz, 1H),
7.061 (t, J = 7.4 Hz, 1H), 3.547 (t, J = 6.6, 2H), 2.430 (t, J = 7.4, 2H),
1.974–1.920 (m, 2H), 1.879–1.822 (m, 2H); HR-FABMS Calcd for
HR-FABMS Calcd for
254.1292.
C
15H16N3O (M++H): 254.1293, Found:
4.2.2. 5-(4-(N0-Hydroxycarbamidoyl)-phenoxy)-N-(2-(phenyla-
mino)benzo[d]oxazol-5-yl)pentanamide (2b)
C
18H19BrN3O2 (M++H): 388.0655, Found: 388.0648.
(0.361 g, 91%): Brown powder, mp 161–163 °C; 1H NMR
(400 MHz, Acetone-d6) d 9.271 (br s, 1H), 7.841 (d, J = 7.6 Hz, 2H),
7.357 (t, J = 7.6 Hz, 2H), 7.064–7.006 (m, 2H), 7.770 (d, J = 0.8 Hz,
1H), 6.456 (dd, J = 8.6, 2.2 Hz, 1H); HR-FABMS Calcd for
4.3.3. N-(2-(4-Methoxyphenylamino)benzo[d]oxazol-5-yl)-5-
bromo-pentanamide (3c)
(0.055 g, 55%): White powder, mp 179–180 °C; 1H NMR
(400 MHz, Acetone-d6) d 9.266 (br s, 1H), 9.145 (br s, 1H), 7.844
(d, J = 1.6 Hz, 1H), 7.755 (d, J = 9.2 Hz, 2H), 7.330 (dd, J = 8.4,
2.5 Hz, 1H), 7.250 (d, J = 8.4 Hz, 1H), 6.965 (d, J = 9.2 Hz, 2H),
3.800 (s, 3H), 3.545 (t, J = 6.4 Hz, 2H), 2.425 (t, J = 7.2 Hz, 2H),
1.972–1.918 (m, 2H), 1.875–1.819 (m, 2H); HR-FABMS Calcd for
C
13H12N3O (M++H): 226.0975, Found: 226.0978.
4.2.3. 5-(4-(N0-Hydroxycarbamidoyl)-phenoxy)-N-(2-(4-meth-
oxy-phenylamino)benzo[d]oxazol-5-yl)pentanamide (2c)
(0.067 g, 14%): Brown powder, mp 156–158 °C; 1H NMR
(400 MHz, Acetone-d6) d 9.060 (br s, 1H), 7.737 (d, J = 9.2 Hz, 2H),
C
19H21BrN3O3 (M++H): 418.0761, Found: 418.0756.

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J.-H. Kang et al. / Bioorg. Med. Chem. 23 (2015) 7069–7078
4.4. Synthesis of 5-(4-cyanophenoxy)-N-(2-(4-substituted phe-
nylamino)benzo[d]-oxazol-5-yl)pentanamide (4a–c)
(br s, 1H), 7.901 (d, J = 2 Hz, 1H), 7.859 (d, J = 7.6, 2H), 7.643 (d,
J = 8.8 Hz, 2H), 7.404–7.353 (m, 3H), 7.280 (d, J = 8.8, 1H), 7.061 (tt,
J = 7.4, 0.8 Hz, 1H), 6.930 (d, J = 9.2 Hz, 2H), 5.373 (br s, 1H), 4.070
(t, J = 5.6 Hz, 2H), 2.470 (t, J = 7 Hz, 2H), 1.894–1.872 (m, 4H);
HR-FABMS Calcd for C25H25N5O4 (M++H): 460.1985, Found: 460.1988.
4-Cyanophenol (0.024 g, 0.20 mmol) in DMF 3 mL and K2CO3
(0.032 g, 0.23 mmol were stirred at 50–55 °C for 30 min. Then
compound 3a–c (0.15 mmol) in DMF 4 mL was added and stirred
at 60 °C for 5 h. After the reaction mixture was cooled to rt, Ethyl
acetate 7 mL was added and washed with water, and saturated
NaCl solution. The organic layer was dried over MgSO4 and the sol-
vent was removed in vacuo. Washing with ether gave 4a–c.
4.5.3. 5-(4-N0-Hydroxycarbamimidoyl)-phenoxy)-N-(2-(4-meth-
oxy-phenylamino)benzo[d]oxazol-5-yl)pentanamide (5c)
(0.008 g, 30%) white powder, mp 200–202 °C; 1H NMR (400 MHz,
Acetone-d6) d 9.259 (s, 1H), 9.148 (s, 1H), 8.686 (s, 1H), 7.856
(s, 1H), 7.754 (d, J = 8.8 Hz, 2H), 7.642 (d, J = 8.8 Hz, 2H), 7.337 (d,
J = 8.4 Hz, 1H), 7.247 (d, J = 8.8 Hz, 1H), 6.965 (d, J = 9.2 Hz, 2H),
6.929 (d, J = 9.2 Hz, 2H), 5.362 (br s, 2H), 4.070 (t, J = 6.2 Hz, 2H),
3.800 (s, 3H), 2.463 (t, J = 7.2 Hz, 2H), 1.905–1.870 (m, 4H);
HR-FABMS Calcd for C26H27N5O5 (M++H): 490.2090, Found: 490.2095.
4.4.1. 5-(4-Cyanophenoxy)-N-(2-(4-ethyl phenylamino)benzo
[d]-oxazol-5-yl)pentanamide (4a)
(0.036 g, 52%): Pale yellow powder, mp 200–203 °C; 1H NMR
(400 MHz, Acetone-d6) d 9.358 (s, 1H), 9.151 (s, 1H), 7.874 (s,
1H), 7.755 (d, J = 8.4, 2H), 7.691 (d, J = 8.8, 2H), 7.339 (d, J = 8.6,
1H), 7.264 (d, J = 8.4, 1H), 7.231 (d, J = 8.4, 2H), 7.122 (d, J = 9.2,
2H), 4.167 (t, J = 6.0, 2H), 2.627 (q, J = 7.6, 2H), 2.473 (t, J = 7.0,
2H), 1.907–1.876 (m, 4H), 1.218 (t, J = 7.6, 3H); HR-FABMS Calcd
for C27H27N4O3 (M++H): 455.2083, Found: 455.2080.
4.6. Determination of 5-LO product LTC4 formation
Mouse bone marrow-derived mast cells (BMMC) were obtained
from male mice and cultured for up to 4 weeks in 50% enriched
medium (RPMI containing 2 mM
fer, 2 mg/mL sodium bicarbonate, 100 units/mL penicillin G,
100 g/mL streptomycin sulfate, and 0.25 g/mL amphotericin B)
supplemented with 10% fetal bovine serum (FBS) with IL-3 (Sigma
I4144, 2 ng/mL). Three weeks after the culture, more than 98% of
BMMC was found in the cells as assessed by staining method with
toluidine blue.
L-glutamine, 25 mM HEPES buf-
4.4.2. 5-(4-Cyanophenoxy)-N-(2-(phenylamino)benzo[d]-oxaz-
ol-5-yl)pentanamide (4b)
l
l
(0.259 g, 63%): white powder, mp 181–182 °C; 1H NMR
(400 MHz, Acetone-d6) d 9.464 (br s, 1H), 9.167 (br s, 1H), 7.893
(d, J = 2 Hz, 1H) 7.872–7.847 (m, 2H), 7.692 (d, J = 8.8 Hz, 2H),
7.405–7.343 (m, 3H), 7.281 (d, J = 8.8, 1H), 7.122 (d, J = 8.8, 2H),
7.061 (t, J = 7.4 Hz, 1H), 4.168 (t, J = 6 Hz, 2H), 2.476 (t, J = 6.8 Hz,
2H), 1.909–1.892 (m, 4H); HR-FABMS Calcd for
C
25H23N4O3
4.6.1. Determination of LTC4
(M++H): 427.1765, Found: 427.1761.
BMMC were suspended in the enriched medium at a cell den-
sity of 1 ꢀ 106 cells/mL, and were then incubated in a humidified
5% CO2 incubator with or without sample in DMSO (final DMSO
concentration was <0.5%) for 30 min at 37 °C. After the stimulation
with stem cell factor (SCF, Sigma S9915, 100 ng/mL) for 20 min, the
LTC4 release in supernatants was measured by an enzyme
immunoassay kit (Cayman Chemical, Ann Arbor, MI, USA) accord-
ing to the manufacturer’s instructions. All the experiments were
carried out in triplicate and the inhibition of LTC4 release was
determined by calculating % reduction of LTC4 release.
4.4.3. 5-(4-Cyanophenoxy)-N-(2-(4-methoxyphenylamino)
benzo[d]-oxazol-5-yl)pentanamide (4c)
(0.029 g, 48%): white powder, mp 213–215 °C; 1H NMR
(400 MHz, Acetone-d6) d 9.263 (br s, 1H), 9.146 (br s, 1H), 7.849
(s, 1H), 7.755 (d, J = 9.2 Hz, 2H), 7.691 (d, J = 8.8 Hz, 2H), 7.329 (d,
J = 8.4 Hz, 1H), 7.248 (d, J = 8.4 Hz, 1H), 7.122 (d, J = 9.2 Hz, 2H),
6.965 (d, J = 8.8 Hz, 2H), 4.167 (t, J = 6.2 Hz, 2H), 3.800 (s, 3H),
2.470 (t, J = 7 Hz, 2H), 1.905–1.874 (m, 4H); HR-FABMS Calcd for
C
26H25N4O4 (M++H): 457.1870, Found: 457.1871.
4.7. Molecular modeling
4.5. Synthesis of 5-(4-N0-hydroxycarbamimidoyl)-phenoxy)-N-
(2-(4-ethyl-phenylamino)benzo[d]oxazol-5-yl)pentanamide
(5a–c)
The 3-dimensional structure of the K7 was generated using
Concord and energy minimized with MMFF94s force field and
MMFF94 charge until the rms of Powell gradient was
0.05 kcal molꢁ1 Aꢁ1 in SYBYL-X 2.0 (Tripos Int., St. Louis, MO,
USA). Flexible docking study on the human 5-LO crystal structure
(PDB ID: 3V99) was carried out using GOLD v.5.2 (Cambridge Crys-
tallographic Data Centre, Cambridge, UK), which uses a genetic
algorithm (GA) and allows for full ligand flexibility.25 The binding
site was specified as 10 Å around the arachidonic acid complexed
in PDB 3V99. The coordination geometry of metal ion Fe2+ was
set to be octahedral in GOLD.52 K7 was docked with the GoldScore
scoring function, and the other parameters remained as default. All
the computation calculations were undertaken on an IntelÒ XeonTM
Quad-core 2.5 GHz workstation with Linux Cent OS release 5.5.
Compound 4a–c (0.07 mmol) in 5 mL methanol was added to
hydroxylamine 50% solution in water (0.54 mL, 0.35 mmol), and
refluxed for 16 h. After the reaction mixture was cooled to 40 °C,
water was added slowly to give the crystal. Washing with ether
gave 5a–c.
4.5.1. 5-(4-N0-Hydroxycarbamimidoyl)-phenoxy)-N-(2-(4-ethyl-
phenylamino)benzo[d]oxazol-5-yl)pentanamide (5a) (K7)
(0.024 g, 69%): White powder, mp 191–196 °C; 1H NMR
(400 MHz, Acetone-d6) d 9.364 (s, 1H), 9.162 (s, 1H), 8.693 (s,
1H), 7.881 (s, 1H), 7.753 (d, J = 8.8, 2H), 7.643 (d, J = 9.2, 2H),
7.349 (d, J = 8.0, 1H), 7.263 (d, J = 8.4, 1H), 7.229 (d, J = 8.8, 2H),
6.929 (d, J = 9.2, 2H), 5.371 (s, 2H), 4.070 (t, J = 6.0, 2H), 2.627 (q,
J = 7.6, 2H), 2.467 (t, J = 7.2, 2H), 1.893–1.872 (m, 4H), 1.218 (t,
J = 7.6, 3H); HR-FABMS Calcd for C27H30N5O4 (M++H): 488.2298,
Found: 488.2299.
4.8. Cells and culture system
Bone marrow cells were obtained from the long bones of 4- to
6-week-old male mice. Bone marrow cells were cultured in
alpha-minimal essential medium (MEM) containing 10% FBS with
the presence of M-CSF (10 ng/mL, R&D systems, Inc., Minneapolis,
MN, USA) overnight. Non-adherent cells were harvested and cul-
tured with M-CSF (30 ng/mL) for 3 days to generate the bone mar-
row-derived macrophages (BMMs). After 3 days, adherent cells
4.5.2. 5-(4-N0-Hydroxycarbamimidoyl)-phenoxy)-N-(2-(phenyl-
amino)benzo[d]oxazol-5-yl)pentanamide (5b)
(0.50 g, 18%): White powder, mp 202–203 °C; 1H NMR
(400 MHz, Acetone-d6) d 9.467 (br s, 1H), 9.178 (br s, 1H), 8.695

J.-H. Kang et al. / Bioorg. Med. Chem. 23 (2015) 7069–7078
7077
were used as osteoclast precursors. All cells were cultured in
alpha-MEM with 10% FBS at 37 °C in 5% CO2 incubator. To examine
osteoclast formation, BMMs were treated with reagents in the
presence of M-CSF (30 ng/mL) and RANKL (100 ng/mL, PeproTech,
Rocky Hill, NJ, USA). Cells were fixed and stained for TRAP, a mar-
ker enzyme of osteoclasts. The number of TRAP+ cells with more
than 3 or 5 nuclei was counted under a light microscope.
(10 lM) was treated for 2 days in the presence of M-CSF and
RANKL. The cells were removed from the dentin slice by
wiping the surface, then slices were stained with toluidine blue
(1 lg/mL, J.T. Baker, UK). And the numbers of pit formed by bone
resorption on the dentin slices were counted.
4.9. In vivo experiment
4.8.1. MTT assay
To study the effects of K7 on LPS-induced osteoclast formation
in vivo, 6-week-old male mice were divided into three groups.
Mice were administered K7 (5 mg/kg, intraperitoneally (ip)) or
vehicle (corn oil/DMSO = 9:1) daily. After a day, mice were injected
subcutaneously with LPS (5 mg/kg) or vehicle (PBS) over calvarial
bone. The mice were sacrificed 6 days after LPS injection, and
whole calvariae were fixed in 4% paraformaldehyde and stained
for TRAP.
BMMs were cultured with M-CSF (30 ng/mL, R&D) and the
reagents in alpha-MEM medium for 4 days, MTT solution was then
added and incubated in the dark. After 5 h solubilization buffer
(10% SDS in 0.01 M HCl) was added and cultured overnight. The
proliferation was measured at an OD value of 570 nm.
4.8.2. RT-PCR analysis
Total RNA was extracted from BMMs by Easy-Blue (iNtRON
Biotechnology, Inc.). cDNA was synthesized from total RNA by
using RevertAidTM first strand cDNA synthesis Kit (Fermentas, EU)
and amplified using PCR. Primers for osteoclastogenic genes used
in this study are as follows: calcitonin receptor (CTR), 50-tttcaa-
gaaccttagctgccagag-30 (forward), 50-caaggcacggacaatgttgagaag-30
(reverse); cathepsin K, 50-cttccaatacgtgcagcaga-30 (forward),
50-acgcaccaatatcttgcacc-30 (reverse); b-actin, 50-tttgatgtcacgcac-
gatttcc-30 (forward), 50-tgtgatggtgggaatgggtcag-30 (reverse);
Atp6v0d2, 50-tcagatctcttcaaggctgtgctg-30 (forward), 50-gtgccaaat-
gagttcagagtgatg-30 (reverse); DC-STAMP, 50-tggaagttcacttgaaac-
tacgtg-30 (forward), 50-ctcggtttcccgtcagcctctctc-30 (reverse);
am-Integrin, 50-cctcagagagggagatgttcacac-30 (forward), 50-aactgc-
caagatgatcacccacac-30 (reverse) b3-Integrin, 50-gatgacatcgagcaggt-
gaaagag-30 (forward), 50-ccggtcatgaatggtgatgagtag-30 (reverse).
The PCR program was as follows: 32 (b3-Integrin), 30 (Atp6v0d2,
DC-STAMP), 28 (CTR, am-Integrin), 22 (Cathepsin k, b-actin) cycles,
after an initial denaturation step at 94 °C for 3 min, then denatura-
tion at 94 °C for 30 s, annealing at 60 (am-Integrin), 59 (Atp6v0d2),
58 (CTR, Cathepsin k, b3-Integrin, b-actin and DC-STAMP)°C for
45 s, and extension at 72 °C for 60 s, with a final extension at
72 °C for 10 min.
4.10. Statistical analysis
Data represent the means and the SD from three independent
experiments. Statistical analysis was performed by one-way
analysis of variance followed by the Student’s t-test. p-Value
<0.05 was considered statically significant.
Acknowledgments
This research was supported by Basic Science Research
program through the National Research Foundation of Korea
(NRF)
– South Korea funded by the Ministry of Education
(NRF-2013R1A1A2062631) (to M. Yim), and by Grant from
National Leading Research Lab (NLRL) program (2011-0028885)
funded by the Ministry of Science, ICT and Future Planning (MSIP)
and the National Research Foundation of Korea (NRF) – South
Korea (to S. Choi).
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