Discovery of novel benzopyranyl tetracycles that act as inhibitors of osteoclastogenesis induced by receptor activator of NF-κB ligand

Article abstract of DOI:10.1021/jm1011269

A novel benzopyran-fused molecular framework 7ai was discovered as a specific inhibitor of RANKL-induced osteoclastogenesis using a cell-based TRAP activity assay from drug-like small-molecule libraries constructed by diversity-oriented synthesis. Its inhibitory activity was confirmed by in vitro evaluations including specific inhibition of RANKL-induced ERK phosphorylation and NF-κB transcriptional activation. 7ai can serve as a specific small-molecule modulator for mechanistic studies of RANKL-induced osteoclast differentiation as well as a potential lead for the development of antiresorptive drugs.

Full text of DOI:10.1021/jm1011269

8760 J. Med. Chem. 2010, 53, 8760–8764
DOI: 10.1021/jm1011269
Discovery of Novel Benzopyranyl Tetracycles that Act as Inhibitors of Osteoclastogenesis
Induced by Receptor Activator of NF-KB Ligand
Mingyan Zhu,† Myung Hee Kim,§ Sanghee Lee,† Su Jung Bae,§ Seong Hwan Kim,*,§ and Seung Bum Park*,†,‡
†Department of Chemistry, and ‡Department of Biophysics and Chemical Biology, Seoul National University, Seoul 151-747, Korea, and
§Laboratory of Chemical Genomics, Pharmacology Research Center, Bio-organic Science Division, Korea Research Institute of Chemical
Technology, Daejeon 305-600, Korea
Received June 16, 2010
A novel benzopyran-fused molecular framework 7ai was discovered as a specific inhibitor of RANKL-
induced osteoclastogenesis using a cell-based TRAP activity assay from drug-like small-molecule
libraries constructed by diversity-oriented synthesis. Its inhibitory activity was confirmed by in vitro
evaluations including specific inhibition of RANKL-induced ERK phosphorylation and NF-κB
transcriptional activation. 7ai can serve as a specific small-molecule modulator for mechanistic studies
of RANKL-induced osteoclast differentiation as well as a potential lead for the development of
antiresorptive drugs.
Introduction
development of new therapeutic agents for metabolic bone
diseases. The mechanistic understanding of the differentia-
tion, fusion, activation, and function of osteoclasts leads to
the identification of various potentialtargetsandstrategies for
the discovery of antiresorptive drugs. Inhibition of the osteo-
clastogenesis is one of the potential therapeutic approaches to
bone diseases, particularly to pathological bone loss.7 Several
naturally occurring small molecules have been reported to
inhibit the differentiation and functionalization of osteoclasts.8
With the increasing study of osteoclast, the discovery of novel
small-molecule osteoclastogenesis inhibitors can provide valu-
able insights into osteoclast biology and provide potential lead
compounds for development of antiresorptive agents.
In this paper, we report a novel synthetic small-molecule
inhibitor of RANKL-induced osteoclastogenesis that was iden-
tified by cell-based high-throughput screening of drug-like small-
molecule libraries constructed by diversity-oriented synthesis
(DOS). We previously reported a series of DOS pathways of
novel heterocyclic core skeletons by the creative recombination
of privileged substructural motifs such as benzopyran, pyrim-
idine, pyrazolopyrimidine, and benzodiazepine.9 We have
successfully constructed natural product-like small-molecule
libraries with over 2000 members containing more than 30
unique core skeletons.10 To identify small-molecule inhibitors
of RANKL-induced osteoclastogenesis, we performed a cell-
based tartrate-resistant acid phosphatase (TRAP) activity
assay. When treated with RANKL, RAW264.7 (mouse leu-
kemic monocyte/macrophage), a type of osteoclast progenitor
cells, differentiated into TRAP-positive (TRAPþ) multinu-
cleated osteoclasts.11 By this high-throughput screening, we
identified benzopyran-fused tetracyclic molecular frameworks
7, which can effectively inhibit RANKL-induced osteoclasto-
genesis.
Bones are constantly renewed by the delicate balance
maintained between bone resorption by osteoclasts and bone
formation by osteoblasts.1 Osteoclasts are multinucleated
cells derived from hematopoietic stem cells of monocyte/
macrophage lineage and are closely related to macrophages.2
Two critical factors supplied mainly by osteoblasts, the
receptor activator of NF-κB ligand (RANKLa) and macro-
phage-colony stimulating factor (M-CSF), are essential for
the formation of osteoclast precursors and for osteoclastogen-
esis in bones. In particular, RANKL is a key factor in the
regulation process of osteoclastogenesis and maintaining the
survival of mature osteoclasts.1,3 The binding of RANKL to
its receptor RANK triggers the activation of the signaling
molecules involved in osteoclastogenesis and of transcription
factors for the regulating genes required for the differentiation
of osteoclasts and their bone resorptive activity.4 The abnor-
mal increase in the number and activity of osteoclasts due to
aging or in the condition of diseases such as osteoporosis,
rheumatoid arthritis, and cancer with bone metastasis leads to
increased bone resorption.5 Current therapeutic approaches
to bone diseases mainly focus on the inhibition of bone
resorption. Bisphosphonates, synthetic analogues of pyro-
phosphates, are currently the most important and effective
antiresorptive drugs available,6 but they have been shown to
have gastrointestinal tract side effects, and long-term treat-
ment with bisphosphonate leads to suppression of bone turn-
over, reducing the bone quality. It is insistent need for the
*To whom correspondence should be addressed. For S.B.P.: phone,
(þ)82-2-880-9090; fax, (þ)82-2-884-4025; E-mail, sbpark@snu.ac.kr.
For S.H.K.: phone, (þ)82-42-860-7687; fax, (þ)82-42-861-0307; E-mail,
hwan@krict.re.kr.
aAbbreviations: RANKL, receptor activator of NF-κB ligand; M-CSF,
macrophage-colony stimulating factor; DOS, diversity-oriented synthesis;
TRAP, tartrate-resistant acid phosphatase; CCK-8, cell counting kit-8;
BMM, bone marrow monocyte; NFATc1, nuclear factor of activated T
cells.
On the basis of this preliminary screening, we constructed a
focused library containing 24 analogues for studying the
structure-activity relationship of the molecular framework
7 to enhance their efficacy (Scheme 1). The analogues were
r
pubs.acs.org/jmc
Published on Web 11/29/2010
2010 American Chemical Society

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8761
Scheme 1. Synthetic Scheme of the Novel Benzopyran-Fused
Tetracyclic Molecular Framework 7a
a Reagents and conditions: (a) ketone, pyrrolidine, EtOH, reflux;
(b) CuBr2, EtOAc/CHCl3/ MeOH, reflux; (c) NaBH4, EtOH, 40 °C;
(d) p-TsOH, toluene, 80 °C; (e) vinyl boronic acid dibutyl ester, Na2CO3,
Pd(PPh3)4, EtOH/toluene/H2O, 70 °C; (f) 4-substituted 1,2,4-triazoline-
3,5-dione derivatives, toluene, rt.
synthesizedvia the following series ofreactions: (1) cyclization
of substituted o-hydroxyacetophenone 1 with different
ketones to afford 2, (2) monobromination of 2 to afford 3, (3)
carbonyl reduction of 3 and subsequent dehydration under
acidic condition yields vinylbromide-embedded benzopyranyl
intermediates 5, (4) palladium-mediated Suzuki cross-coupling
of 5 with vinyl boronate to afford dienes 6, (5) aza-Diels-Alder
reaction of 6 with 4-substituted-1,2,4-triazoline-3,5-diones
(commercial available or in-house synthesized, see Supporting
Information Scheme S1) to obtain the desired molecular frame-
work 7. The exact structure of 7 was determined by NMR
analysis as well as X-ray crystallography (Figure 1A).
After the construction of a 24-member focused library of 7,
we evaluated their inhibitory activities against RANKL-
induced osteoclastogenesis. RAW264.7 cells were treated with
100 ng/mL of RANKL along with the treatment of each
analogue of 7 at a concentration of 10 μM.12 As shown in
Table 1, several analogues of 7 could cause more than 50%
inhibition of RANKL-induced osteoclastogenesis, which was
determined by measuring the cellular TRAP activity. Their
antiosteoclastogenic effects were not attributed to the cellular
toxicity, which was confirmed by the cell viability test using
CCK-8 assay toward RAW264.7 osteoclast progenitor cells
(Table 2). We also confirmed that the olefin moiety on the
tetracyclic molecular framework 7 is an essential element for
the inhibitory activity of these analogues because we observed
a significantly low inhibitory activity in the case of saturated
analogue 7s which was obtained by catalytic hydrogenation of
7a with Pd/C in the presence of atmospheric hydrogen gas
(Supporting Information Figure S3). In addition, the inhibi-
tory activities of analogues of 7 with various substituents
at the R1, R2, andR3 positions on the benzene rings revealed that
the phenolic hydroxyl moiety at the R2 position is essential for
the inhibitory activity. Further, the replacement of this hy-
droxyl group with other functional groups led to a significant
decrease in the inhibitory activity (entries 1-8, Table 1),
indicating that the inhibitory mechanism of 7a may involve
specific hydrogen-bonding interactions with the hydroxyl
moiety at the R2 position. The introduction of the cyclopentyl
spiropyran moiety (7i) in place of the dimethyl moiety (7a) at
the R position increased the inhibitory activity, but 7i also
exhibited cellular cytotoxicity against osteoclast precursor
cells; therefore, 7i was not considered for further biological
evaluation (Table 2). On the other hand, the introduction of
a piperidinyl moiety at the R position (7j, 7k) led to a loss
of their inhibitory activity. The significant reduction in the
Figure 1. Compound 7ai inhibit RANKL-induced osteoclastogen-
esis: (A) compound 7ai, a novel inhibitor of RANKL-induced osteo-
clastogenesis and its X-ray crystal structure; (B) inhibitory efficacy of
7ai on RANKL-induced osteoclastogenesis in RAW264.7 and BMM
cells; (C) 7ai reduced the TRAP activity in a dose-dependent manner. *,
P < 0.05; **, P < 0.01; ***, P < 0.001.
inhibitory activities upon the introduction of alkyl substitu-
ents at the R4 position emphasized that the presence of a
phenyl moiety at R4 is essential for maximum inhibitory
activity (entries 12-14, Table 1). The introduction of various
substituents on the phenyl moiety of 7a revealed that most of
the substitutions at the R4 position are not favorable to their
inhibitory activity, but we confirmed that the presence of
methyl and tert-butyl moieties at the para position on the
phenyl ring (7ai and 7aii, respectively) conserved the inhibitory
activity of these compounds, similar to that of 7a, by considering
the inhibitory activity (IC50 values) and their cytotoxicity
toward osteoclast precursor cells (Table 2). On the basis of
these results, we identified 7ai as the best small-molecule
inhibitor of RANKL-induced osteoclastogenesis. As shown in
Figure 1B, 7ai can significantly reduce the number of multi-
nucleated mature osteoclasts both in RAW264.7 cells and in
murine bone marrow monocyte (BMM; primary osteoclast
precursor) cells as confirmed by TRAP staining. 7ai caused a
dose-dependent inhibitory activity against RANKL-induced
osteoclastogenesis with an IC50 value of approximately 7 μM
in both RAW264.7 and BMM cells (Figure 1C).

8762 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Zhu et al.
Table 1. Inhibitory Effects of Synthetic Analogues with Molecular Framework 7 on RANKL-Induced Osteoclastogenesis Measured by %TRAP
Activity
entry
compd
R
R1
R2
R3
R4
%TRAP activitya
1
7a
7b
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
hydro
hydro
hydro
hydro
hydro
hydroxy
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydroxy
hydro
hydro
hydro
hydro
methoxy
methoxy
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
hydro
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
phenyl
43 ( 1.5
104 ( 2.2
108 ( 5.2
99 ( 4.1
100 ( 1.4
69 ( 7.3
121 ( 7.8
107 ( 2.7
30 ( 2.8
113 ( 0.3
99 ( 5.2
113 ( 4.1
95 ( 4.7
98 ( 4.8
50 ( 3.7
53 ( 4.8
93 ( 5.3
86 ( 3.5
68 ( 5.6
101 ( 0.7
105 ( 4.3
112 ( 6.0
95 ( 3.6
83 ( 3.2
2
3
7c
7d
methoxy
methoxy
hydro
4
5
7e
6
7f
7g
methoxy
fluoro
7
8
7h
7i
methylamino
hydroxy
hydroxy
hydroxy
hydroxy
hydroxy
hydroxy
hydroxy
hydroxy
hydroxy
hydroxy
hydroxy
hydroxy
hydroxy
hydroxy
hydroxy
hydroxy
9
cyclopentylb
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
7j
7k
N-acetyl-40-piperidylb
N-propyl-40-piperidylb
7l
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
cyclohexyl
methyl
benzyl
7m
7n
7ai
7aii
7aiii
7aiv
7av
7avi
7avii
7aviii
7aix
7ax
p-methylphenyl
p-tert-butylphenyl
p-methoxyphenyl
m-methoxyphenyl
p-fluorophenyl
o-iodophenyl
m-nitrophenyl
p-chlorophenyl
o-chlorohenyl
p-bromohenyl
a Relative TRAP activity of individual compounds at 10 μM in the presence of 100 ng/mL RANKL; 100% refers the difference of TRAP activity
between the treatment of 100 ng/mL RANKL and vehicle. The screening was carried out in triplicate and the number here were the mean value
containing standard derivations. b R is connected to the quaternary carbon through spiropyranyl connection.
Table 2. IC50 of Compounds on RANKL-Induced Osteoclastogenesis
and Cell Viability
analogues, suggesting that the p38 phosphorylation can be
blocked by the treatment with analogues of 7 due to its
structural similarity, but the inhibitory effect of 7b on p38
phosphorylation is insufficient to obstruct the RANKL-
induced formation of TRAP-positive multinucleated osteo-
clasts. This led us to conclude that the interference with ERK
activation may be responsible for the inhibitory effects of 7ai
and 7a; therefore, the potential target protein of our anti-
osteoclastic agents may lie the upstream of ERK phosphor-
ylation. Moreover, NF-κB is activated and translocated into
the nucleus upontreatmentwithRANKL, which results inthe
expression of target genes for osteoclast differentiation. The
involvement of NF-κB in the inhibitory effect of 7a or 7ai on
the osteoclastogenesis was confirmed by NF-κB luciferase
activity assay (Figure 2B); the RANKL-induced transcrip-
tional activation of NF-κB was dramatically inhibited by the
treatment of either 7a or 7ai in a dose-dependent manner but
not by 7b.
We then monitored the expression levels of the biomarkers
and specific transcription factors involved in osteoclastogen-
esis. The key biomarkers of osteoclastogenesis are the follow-
ing: MMP-9, one of main matrix metalloproteases involved in
the invasive activity of osteoclast,15 c-Src, intracellular signal
transduction molecule required for osteoclast activation,16 as
well as TRAP.17 As shown in Figure 3A, the expression levels
of these osteoclastogenesis biomarkers were significantly
reduced upon treatment with our antiosteoclastic agents 7ai and
7a at 10 μM. However, the treatment with structurally similar
inactive analogue 7b at the same concentration resulted in a
drastic difference in the expression levels. Transcription
factors also play a critical role in RANKL-induced osteo-
clast development. For example, NFATc1 (nuclear factor of
activated T cells, calcineurin-dependent 1, also known as
NFAT2) and c-fos (a member of the AP-1 protein family,
IC50 (μM)
RAW264.7
cell viabilitya
compd
BMM
day 1
day 4
7a
7i
7.07 ( 0.30
5.09 ( 0.97
7.97 ( 0.57
6.94 ( 0.10
8.08 ( 0.30
N/D
6.13 ( 1.13
6.05 ( 1.28
7.05 ( 0.80
6.77 ( 0.61
11.94 ( 5.06
22.20 ( 1.18
N/D
85.88
77.79
100.33
79.67
99.02
N/D
73.97
45.96
86.86
73.43
80.72
N/D
7ai
7aii
7av
7f
7b
N/D
N/D
N/D
a Cell viability was measured by CCK-8 assay in RAW264.7 cells
upon treatment with individual compounds at 30 μM final concentra-
tions for 1 day and 4 days. Cell viability represents the percentage of the
control. N/D: Not determined due to its low inhibitory activity.
After identifying novel small-molecule inhibitors of
RANKL-induced osteoclastogenesis, we examined whether
7ai and 7a could induce any perturbations in the signaling
cascades that were monitored by immunoblot analysis. Upon
treatment with RANKL, the intracellular receptor RANK
recruits adaptor molecules such as TRAF6 and activates
multiple downstream signaling pathways.13 NF-κB and
MAPKs (JNK, p38, and ERK) are involved in the early
signaling event induced by RANKL.1,14 In this study,
RAW264.7 cells were treated with RANKL in the presence
of three analogues of 7 (7a, 7ai, and 7b), and the cell lysates
were analyzed by Western blotting. We selected 7b as a
negative control because it is structurally almost identical to
activecompound 7aexcept for the absence of hydroxyl moiety
at R2 position. As shown in Figure 2A, our novel antiosteo-
clastic agents 7ai and 7a can selectively inhibit the RANKL-
induced activation of ERK phosphorylation but not in the
case of the inactive analogue 7b. However, the RANKL-
induced phosphorylation of p38 was inhibited by these three

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24 8763
Figure 2. Inhibitory effects on RANKL-induced early signaling pathways upon treatment with analogues of 7 in RAW264.7 cells. (A) Western
blot analysis of RANKL-induced activation of MAPK signaling pathways in RAW264.7 cells; actin was used as the internal control for
cytosolic fractions. (B) NF-κB luciferase activity assay data to confirm the inhibitory effect of RANKL-induced NF-κB activation upon
treatment with analogues of 7 in RAW264.7 cells.
structure-activity relationship study established 7ai as a pro-
mising antiosteoclastic agent. Its inhibitory activity toward
RANKL-induced osteoclast differentiation was confirmed by
in vitro evaluations including specific inhibitions of ERK
phosphorylation, NF-κB activation, and mRNA expression
of key osteoclast biomarkers and transcription factors involved
in RANKL-induced osteoclastogenesis. Even though the
further in vivo evaluation of 7ai is currently underway to
confirm the specific inhibition of RANKL-induced osteoclastic
bone resorption, this novel molecular framework can be used as
a specific small-molecule modulator for mechanistic studies of
RANKL-induced osteoclastogenesis as well as potential lead
compounds for the development of antiresorptive drugs. Since
we determined that the inhibition of signaling process on ERK
phosphorylation and NF-κB activation is the major cause of the
inhibitory activity toward RANKL-induced osteoclastogenesis,
it is important to identify the molecular target of this antiosteo-
clastic agent; studies for target identification will be reported in
due course.
Experimental Section
Cell Culture and RANKL-Induced Osteoclast Differentiation.
All the materials for the cell culture were purchased from
HyClone (UT, USA). Osteoclast generation was achieved using
either RAW264.7 cells or primary cultures of mouse BMMs.
Mouse monocyte/macrophage RAW264.7 cells were purchased
from the American Type Culture Collection (VA, USA) and
maintained at 37 °C in Dulbecco’s Modified Eagle’s Medium
(DMEM) supplemented with 10% fetal bovine serum (FBS) and
1ꢀ antibiotic-antimycotic (Gibco, CA, USA) in a humidified
atmosphere with 5% CO2. The medium was changed every
3 days. For osteoclast differentiation, RAW264.7 cells were
suspended in R-minimal essential medium (MEM) supplemen-
ted with 10% FBS in the presence of 100 ng/mL RANKL (R&D
Systems, MN, USA) and plated at a density of 1 ꢀ 103 cells/well
in a 96-well plate (on differentiation day 0). Multinucleated
osteoclasts were observed on differentiation day 4. For the
generation of BMM-derived osteoclasts, monocytes were iso-
lated from the femurs and tibiae of BALB/c mice (Central Lab
Animal, Korea), seeded and cultured in R-MEM with 10% FBS
and 10 ng/mL macrophage colony stimulating factor (M-CSF;
R&D Systems) for 1 day. The suspended cells were then
transferred into new plates and cultured in R-MEM with 10%
FBS and 30 ng/mL M-CSF for 3 days; adherent cells at this stage
were considered to be M-CSF-dependent BMMs and were used
as osteoclast precursors. Differentiation of BMMs into osteo-
clasts was achieved after plating the former into 96-well plates at
Figure 3. In vitro confirmation of the inhibitory efficacy of 7a and
7ai. (A) Real-time PCR analysis of mRNA expression levels of
specific biomarkers for osteoclastogenesis, such as TRAP, MMP-9,
c-Src, in RAW264.7 cells. (B) Real-time PCR analysis of mRNA
expression level of key transcription factors in osteoclastogenesis,
such as NFATc1, c-fos, and fra2.
such as c-fos, fosB, fra1, fra2) activate gene transcription in
osteoclast differentiation, which can be strongly induced by
RANKL.8 In particular, NFATc1 is supposed to be one of the
key target genes of NF-κB in the early phase of osteoclasto-
genesis. By real-time PCR analysis, we confirmed that the
RANKL-induced mRNA expression of c-fos, NFATc1, and
fra2 was dramatically down-regulated upon treatment with
7ai and 7a but not upon treatment with inactive analogue 7b
(Figure 3B).
Conclusion
In summary, we identified a novel benzopyran-fused molec-
ular framework 7 that acts as an inhibitor of RANKL-induced
osteoclastogenesis; this was done by using a cell-based TRAP
activity assay from drug-like small molecule libraries constructed
by diversity-oriented synthesis. The subsequent systematic

8764 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 24
Zhu et al.
a density of 3 ꢀ 105 cells/well in R-MEM with 10% FBS, 100 ng/
mL RANKL, and 30 ng/mL M-CSF (on differentiation day 0).
Multinucleated osteoclasts were observed from differentiation
day 6 onward.
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TRAP Staining and Activity Assay. To identify compound
with the potential to inhibit the RANKL-induced osteoclasto-
genesis, RAW264.7 cells were suspended in R-MEM with 10%
FBS and 100 ng/mL RANKL and then plated in a 96-well plate
at a density of 1 ꢀ 103 cells/well. After 24 h, cells were treated
with compound or vehicle (DMSO). On differentiation day 4,
cells were fixed with 10% formalin for 10 min and ethanol/
acetone (1:1) for 1 min and then stained with a leukocyte acid
phosphatase kit 387-A (Sigma, MO, USA). Images of the cells
were captured by a microscope with DP Controller (Olympus
Optical, Japan). To measure the TRAP activity, osteoclasts
were fixed with 10% formalin for 10 min and 95% ethanol for
1 min, and then 100 μL of citrate buffer (50 mM, pH 4.6) containing
10 mM sodium tartrate and 5 mM p-nitrophenylphosphate (Sigma,
MO, USA) was added to the fixed cells-containing wells of a 96-well
plate. After incubation for 1 h, the enzyme reaction mixtures in the
wells were transferred to new plates containing 100 μL of 0.1 N
NaOH. Absorbance was measured at 410 nm using a Wallac
EnVision microplate reader (PerkinElmer, Finland). The experi-
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Acknowledgment. This work was supported by (1) the Na-
tional Research Foundation of Korea (NRF), (2) the WCU
program through the NRF funded by the Korean Ministry of
Education, Science and Technology (MEST), and (3) the Korea
Healthcare Technology R&D Project, Ministry of Health, Wel-
fare and Family Affairs, Korea (A084242 for S.H.K.). M.Z. and
S.L. are grateful for the fellowship award of the BK21 Program
and Seoul Science Fellowship award.
Supporting Information Available: All experimental proce-
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pounds, and procedures for biological studies (pdf and cif).
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