Development of 1-Amino-4-(phenylamino)anthraquinone-2-sulfonate Sodium Derivatives as a New Class of Inhibitors of RANKL-Induced Osteoclastogenesis

Article abstract of DOI:10.1002/ardp.201500475

A series of 1-amino-4-(phenylamino)anthraquinone-2-sulfonate sodium derivatives was synthesized and evaluated for osteoclast inhibition using a TRAP-staining assay. Among them, two compounds, LCCY-13 and LCCY-15, dose-dependently suppressed receptor activator of nuclear factor-κB ligand (RANKL)-induced osteoclast formation. Moreover, the cytotoxicity assay on RAW264.7 cells suggested that the inhibition of osteoclastic bone resorption by these compounds was not a result of their cytotoxicity. Further, the inhibitory activities of compounds LCCY-13 and LCCY-15 were further confirmed by including specific inhibition of NFATc1 expression levels in nuclei using an immunofluorescent analysis. In addition, LCCY-13 and LCCY-15 also significantly attenuated the bone resorption activity of osteoclasts according to a pit formation assay. Thus, a new class of 1-amino-4-(phenylamino)anthraquinone-2-sulfonate sodium compounds might be considered as an essential lead structure for the further development of anti-resorptive agents.

Full text of DOI:10.1002/ardp.201500475

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 342–355
Archiv der Pharmazie
Full Paper
Development of 1-Amino-4-(phenylamino)anthraquinone-2-
sulfonate Sodium Derivatives as a New Class of Inhibitors of
RANKL-Induced Osteoclastogenesis
Chia-Chung Lee1,2,3†, Chun-Liang Chen1,2,3†, Fei-Lan Liu3, Chung-Yu Chiou1,2, Tsung-Chih Chen1,2
,
Cheng-Chi Wu3,4, Wei-Hsin Sun5, Deh-Ming Chang3,4ꢀ, and Hsu-Shan Huang1,2,4
1
Graduate Institute of Cancer Biology and Drug Discovery, College of Medical Science and Technology,
Taipei Medical University, Taipei, Taiwan
School of Pharmacy, National Defense Medical Center, Taipei, Taiwan
Rheumatology/Immunology/Allergy, Taipei Veterans General Hospital, Taipei, Taiwan
Graduate Institute of Life Sciences, National Defense Medical Center, Taipei, Taiwan
Department of Life Sciences, National Central University, Jhongli City, Taiwan
2
3
4
5
A series of 1-amino-4-(phenylamino)anthraquinone-2-sulfonate sodium derivatives was synthesized
and evaluated for osteoclast inhibition using a TRAP-staining assay. Among them, two compounds,
LCCY-13 and LCCY-15, dose-dependently suppressed receptor activator of nuclear factor-kB ligand
(RANKL)-induced osteoclast formation. Moreover, the cytotoxicity assay on RAW264.7 cells suggested
that the inhibition of osteoclastic bone resorption by these compounds was not a result of their
cytotoxicity. Further, the inhibitory activities of compounds LCCY-13 and LCCY-15 were further
confirmed by including specific inhibition of NFATc1 expression levels in nuclei using an immunofluo-
rescent analysis. In addition, LCCY-13 and LCCY-15 also significantly attenuated the bone resorption
activity of osteoclasts according to a pit formation assay. Thus, a new class of 1-amino-4-(phenyl-
amino)anthraquinone-2-sulfonate sodium compounds might be considered as an essential lead
structure for the further development of anti-resorptive agents.
Keywords: Anthraquinone derivatives / Osteoclastogenesis / RANKL / TRAP-staining assay
Received: January 3, 2016; Revised: March 20, 2016; Accepted: March 23, 2016
DOI 10.1002/ardp.201500475
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
pathogenesis of osteoporosis [2], periodontal diseases, and
rheumatoid arthritis (RA) [3, 4]. The receptor activator of the
Introduction
Bone remodeling is a continual renewal and repair process,
which is mediated by the balance between bone-resorbing
osteoclast cells and bone-forming osteoblast cells [1]. Accu-
mulating evidence has demonstrated that abnormally in-
creased osteoclastic bone resorption plays a pivotal role in the
nuclear factor (NF)-kB ligand (RANKL) is a member of the
tumor necrosis factor (TNF) family which is essential for
osteoclast differentiation, activation, and survival [5, 6]. When
the RANKL binds to its receptor, RANK, on the surface of
osteoclast precursor cells, it promotes activation of intracel-
lular signaling pathways, including the transcriptional factors,
NF-kB [7–9] and nuclear factor of activated T cells c1
Correspondence: Prof. Hsu-Shan Huang, Graduate Institute of
Cancer Biology and Drug Discovery, College of Medical Science
and Technology, Taipei Medical University, Taipei 110, Taiwan,
R.O.C.
ꢀAdditional correspondence: Prof. Deh-Ming Chang,
E-mail: ming0503@ms3.hinet.net
E-mail: huanghs99@tmu.edu.tw
Fax: þ886-2-6638-7537
†These two authors contributed equally to this work.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
342

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 342–355
New Class of Inhibitors of RANKL-Induced Osteoclastogenesis
Archiv der Pharmazie
(NFATc1) [10–12]. Notably, NFATc1 is a key transcriptional
showed that GoSlo-SR-5-69 was a potent opener of the BK
channel that can modulate the influx of Ca2þ
concentrations [26].
factor, which is not only activated by NF-kB but also by Ca2þ
/
calmodulin-regulated phosphatase calcineurin [12]. Further,
calcium signaling activates the NFATc1 transcriptional factor,
which triggers its auto-amplification and induces transcrip-
tion of osteoclast-specific genes, such as NFATc1 [10, 11] and
tartrate-resistant acid phosphatase (TRAP) [13] for osteoclast
differentiation.
Dye ligands are considered important alternatives to
natural counterparts for binding different types of protein
structures because these ligands can interact with active sites
of many proteins by mimicking the structures of the
substrates, cofactors, or binding agents for those
proteins [14–17]. Bioactive anthraquinone derivatives
(Fig. 1) are some of the most widely used reactive dyes in
drug discovery that were found to have a diverse range of
biological activities such as anti-inflammatory [18], anti-
tumor [19, 20], and anti-viral activities [17]. Among these
anthraquinone dye ligands, it is noteworthy to mention that
anilinoanthraquinone derivatives were found to act as
antagonists of P2 purinergic receptors [21–24], inhibitors of
In a previous study, P2 purinergic receptors were considered
to be potential drug targets for the development of anti-
resorptive agents aimed at treating osteoporosis and other
bone-related diseases [29]. Moreover, accumulating studies
have demonstrated that intracellular Ca2þ signaling plays a
crucial role in RANKL-induced osteoclast differentiation [30,
31]. Through activation of RANKL-RANK signaling pathways,
Ca2þ oscillations result in Ca2þ/calcineurin-dependent de-
phosphorylation and activation of NFATc1, which can be
translocated to nuclei and induce transcription of osteoclasto-
genesis-related genes. Thus, how to maintain the basal Ca2þ
concentrations and regulate NFATc1 activity in the RANKL-
induced osteoclastogenesis have also become potential drug
targets for developing anti-resorptive agents. As these
anilinoanthraquinone analogs can act as antagonists of
nucleotide receptors and/or modulate calcium signaling
cascades, we herein applied their pharmacological character-
istics and biological activities to design a new class of RANKL-
induced osteoclastogenesis inhibitors. Based on these
findings, we hypothesized that the scaffold of 1-amino-4-
(phenylamino)anthraquinone-2-sulfonate sodium can be
considered a lead structure for developing potential osteo-
clastogenesis inhibitors.
nucleotidases [25], and activators of large conductance Ca2þ
-
activated Kþ channels [26–28]. For example, the anilinoan-
thraquinone derivatives, Acid Blue 25 (AB-25) and PSB-716,
were identified as potent P2Y2 antagonists and/or inhibitors
of ecto-50-nucleotidases [24, 25]. Further, a recent study
Figure 1. Bioactive structures of 1-amino-4-(phenylamino)anthraquinone-2-sulfonate sodium derivatives.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
343

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 342–355
C.-C. Lee et al.
Archiv der Pharmazie
Therefore, we developed a series of anilinoanthraquinone
derivatives as a new class of osteoclastogenesis inhibitors in
the present work. In order to investigate the inhibitory effects
of the synthesized compounds on RANKL-induced osteoclas-
togenesis, a TRAP-staining assay was used as a screening tool
in our study. On the basis of these screening results, we
constructed the structure–activity relationships (SARs) of all
synthesized compounds according to the inhibitory results of
RANKL-induced osteoclastogenesis. Among our synthesized
compounds, we found that compounds LCCY-13 and LCCY-15
were the most active derivatives, which effectively inhibited
RANKL-induced osteoclastogenesis. In addition, the inhibitory
effects of LCCY-13 and LCCY-15 on RANKL-induced osteo-
clastogenesis were further investigated through suppressing
the RANKL-induced nuclear NFATc1 expression and amelio-
rating bone-resorbing activity. Detailed descriptions of the
synthesis procedure and biological evaluation of our synthe-
sized compounds are presented as follows.
prepare the anilinoanthraquinone derivatives. This reaction
was achieved through the Ullman coupling reaction using a
phosphate buffer solution (Na3PO4 (aq) and Na2HPO4 (aq), pH
6–7), which possibly increased the nucleophilicity of the
amino group and the rate of the chemical reaction. The
synthesized compounds were obtained using the Ullman
coupling reaction of bromaminic acid (1) with anilines in the
presence of a phosphate buffer using copper powder as a
catalyst in water at 120°C for 12 h under a mini-cleave
apparatus. Subsequently, the reaction mixture was cooled to
room temperature and filtered, and the aqueous solution was
reduced under reduced pressure. The residue was washed
with dichloromethane and water, and the crude residue was
purified using recrystallization (dichloromethane/ethanol
¼ 2:1 to 1:2) to obtain the compounds. The structures of all
synthesized compounds were elucidated with their spectro-
scopic characterizations using 1H nuclear magnetic resonance
(NMR), 13C NMR, and high-resolution mass spectra (HRMS) in
the Experimental section.
Biological activity
Effects of synthetic compounds on RANKL-induced
osteoclastogenesis
Results and discussion
Chemistry
In the present work, the synthesis procedure of the targeted
compounds is outlined in Scheme 1. Compared to previous
studies, this study provided a different reactive apparatus to
Inhibitory activities of all synthetic compounds on the RANKL-
induced TRAP-positive MNCs in RAW264.7 cells were screened
at a concentration of 30 mM using a TRAP-staining assay. As
Scheme 1. Synthesis of 1-amino-4-(phenylamino)anthraquinone-2-sulfonate sodium derivatives. Reagents and conditions:
(i) Anilines, sodium phosphate buffer (pH 6–7), copper powder, 100–120°C, 12–14 h.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
344

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 342–355
New Class of Inhibitors of RANKL-Induced Osteoclastogenesis
Archiv der Pharmazie
illustrated in Table 1, the percent inhibition of MNC values for
compounds LCCY-01 (AB-25), LCCY-13, and LCCY-15 at 30 mM
were all >50%; the percentage values of MNCs were
47.91 ꢁ 0.34%, 41.78 ꢁ 3.59%, and 45.92 ꢁ 2.11%, respec-
tively. In this study, the SARs of our synthetic compounds
focused on the inhibitory effects of side chains on the
aromatic ring D. The SARs of our synthetic compounds for
osteoclastogenesis inhibition are outlined in Fig. 2. Among
our synthetic derivatives, it was noted that the inhibitory
activities of osteoclastogenesis at the meta-substituted
compounds were more active than at the ortho-substituted
and/or para-substituted compounds, especially for LCCY-13.
These observations revealed that the methoxy substituent
(inhibitory activities, OCH3 > OH > Cl) at the meta-position on
the aromatic ring D of 1-amino-4-(phenylamino)anthraqui-
none-2-sulfonate sodium might modulate the inhibition of
osteoclastogenesis. Among our synthesized compounds,
compounds LCCY-13 and LCCY-15 were the most active
Table 1. Effects of 1-amino-4-(phenylamino)anthraquinone-2-sulfonate sodium derivatives on RANKL-induced
osteoclastogenesis (TRAPþ MNCs) and cell viability in RAW264.7 cells.
Substituents
Osteoclastogenesis (%)a)
TRAP-positive MNCs
Compound
R2
R3
R4
R5
CC50 (mM)b)
LCCY-01 (AB-25)
LCCY-02
LCCY-03
LCCY-04
LCCY-05
LCCY-06
LCCY-07
LCCY-08
LCCY-09
LCCY-10 (GoSlo-SR-5-6)
LCCY-11
LCCY-12
LCCY-13
LCCY-14
LCCY-15
LCCY-16
LCCY-17
LCCY-18
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
NO2
NH2
CH3
OH
CN
H
H
H
H
H
H
H
47.91 ꢁ 0.34
57.32 ꢁ 2.71
92.22 ꢁ 6.87
93.01 ꢁ 0.22
91.38 ꢁ 6.73
ND
>100
>100
>100
87.58 ꢁ 4.61
94.28 ꢁ 4.11
50.96 ꢁ 8.88
77.58 ꢁ 6.22
>100
72.42 ꢁ 0.66
86.92 ꢁ 2.34
92.49 ꢁ 1.03
95.02 ꢁ 2.13
87.78 ꢁ 0.88
61.38 ꢁ 3.17
41.78 ꢁ 3.59
54.92 ꢁ 3.12
45.92 ꢁ 2.11
89.76 ꢁ 0.56
92.30 ꢁ 2.79
89.01 ꢁ 3.15
87.98 ꢁ 4.35
85.40 ꢁ 4.92
92.95 ꢁ 3.08
82.61 ꢁ 3.30
93.27 ꢁ 5.59
80.91 ꢁ 6.72
95.61 ꢁ 3.26
See above structure
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
CF3
CH3
CN
OCH3
OH
OCH3 OCH3
Cl
F
Cl
CH3
CN
CF3
F
F
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
F
94.35 ꢁ 7.06
>100
76.84 ꢁ 2.92
87.52 ꢁ 5.79
>100
57.59 ꢁ 2.82
>100
F
F
92.69 ꢁ 0.72
94.21 ꢁ 8.28
>100
95.39 ꢁ 10.26
89.33 ꢁ 3.15
89.06 ꢁ 1.78
>100
H
H
H
H
H
H
H
H
LCCY-19
LCCY-20
LCCY-21
LCCY-22
LCCY-23
LCCY-24
LCCY-25
Cl
Cl
F
>100
>100
>100
F
a) RAW 264.7 cells were cultured in the maintaining media and RANKL (100 ng/mL) with or without the tested compound (30 mM)
for 4 days. TRAP-positive multinucleated cells (MNCs) were counted as osteoclasts (three or more than three nuclei). RANKL-
induced osteoclast differentiation is shown as relative activity (% of RANKL-treated with compounds/RANKL-treated without
compounds as vehicle).
b) CC50 is the concentration of tested compounds (mM) required to inhibit cell viability by 50% of the mean. All representative
data are the means ꢁ SD of experiments performed at least three times independently.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
345

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 342–355
C.-C. Lee et al.
Archiv der Pharmazie
treatment with compound LCCY-15 (97.11 ꢁ 2.69% at
20 mM, 71.71 ꢁ 2.31% at 30 mM, and 23.34 ꢁ 2.91% at
40 mM). Moreover, neither compound, LCCY-13 (Fig. 4c)
or LCCY-15 (Fig. 4f), showed an effect on cell viability of the
osteoclast progenitor cells in a dose-dependent manner.
Based on these results, we concluded that the skeleton of 1-
amino-4-(phenylamino)anthraquinone-2-sulfonate sodium
is an attractive scaffold for the inhibition of RANKL-induced
osteoclastogenesis, especially for compounds LCCY-13 and
LCCY-15.
Effects of LCCY-13 and LCCY-15 on RANKL-induced
nuclear translocation and expression of NFATc1
Figure 2. The structure–activity relationships of 1-amino-4-
(phenylamino)anthraquinone-2-sulfonate sodium analogs for
the osteoclastogenesis inhibition.
Binding the RANKL to RANK activates several transcription
factors responsible for promoting osteoclastic gene expres-
sion, especially NFATc1. It is one of the key transcription
factors involved in osteoclast differentiation by treatment
with the RANKL. In an inactive state, NFATc1 is retained in the
cytoplasm. When activated, NFATc1 is translocated from the
cytoplasm to nuclei. Thus, we examined the effects of LCCY-13
and LCCY-15 on the nuclear translocation of NFATc1 in
RANKL-activated RAW264.7 cells by immunofluorescent
staining (Fig. 5a and b). Upon RANKL stimulation, nuclear
accumulation of NFATc1 was markedly increased by RANKL
stimulation for 24 h compared to levels in cells without RANKL
treatment. This increasing level of nuclear NFATc1 was
reduced by compounds LCCY-13 and LCCY-15. Hence, com-
pounds LCCY-13 and LCCY-15 could reduce osteoclast
formation by suppressing RANKL-induced nuclear transloca-
tion and expression of NFATc1.
inhibitors of RANKL-induced osteoclast formation. In order to
confirm that the inhibitory effects of compounds were not
attributed to cellular toxicity, we further investigated the
effects of compounds on cell viability using an MTT assay
(Table 1). Results showed that no compounds had any effect
on the cell viability of RAW264.7 cells at a concentration of
30 mM (survival rate > 85%). In addition, the concentrations of
compounds required to inhibit cell viability by 50% of the
mean were determined and recognized as the CC50. Interest-
ingly, all synthetic analogs showed higher CC50 values except
for LCCY-06 (50.96 ꢁ 8.88% mM). These results suggest that
the anti-osteoclastogenic activities of these compounds were
not due to their cytotoxicities toward osteoclast precursor
cells. Both compounds reduced the number of TRAPþ
osteoclasts and significantly decreased the size of multinucle-
ated osteoclasts in a dose-dependent manner (Fig. 3) There-
fore, the most active compounds LCCY-13 and LCCY-15 were
selected for further inhibitory studies of the RANKL/RANK
signaling pathway.
Effects of LCCY-13 and LCCY-15 on RANKL-induced bone
resorption
To investigate whether these compounds, LCCY-13 and
LCCY-15, can attenuate the bone-resorbing activity of
osteoclasts, a pit formation assay was used to evaluate the
effects of these compounds on dentine slices in this study.
Thus, RAW264.7 cells were seeded onto dentine slices and
then differentiated into osteoclasts in response to RANKL
stimulation. Using the pit formation assay, resorbing pits
were formed on the dentine slices by osteoclasts. However,
percentages of the resorbed area/total area were signifi-
cantly reduced by treatment with LCCY-13 (63.59 ꢁ 2.46%)
and LCCY-15 (54.53 ꢁ 2.79%; Fig. 6a and b). Based on these
encouraging results, we further confirmed that LCCY-13 and
LCCY-15 could reduce osteoclast formation and ameliorate
RANKL-induced bone-resorbing activity of osteoclasts in a
dose-dependent manner (as shown in the Supporting
Information).
Inhibitory effects of LCCY-13 and LCCY-15 against RANKL-
induced osteoclast differentiation
Among all tested compounds, compounds LCCY-13 and
LCCY-15 were the most active osteoclast inhibitors. Com-
pounds LCCY-13 and LCCY-15 not only reduced the number
of TRAPþ MNCs but also significantly decreased the size of
MNCs in a dose-dependent manner (Fig. 3). Furthermore,
the IC50 values of compounds on RANKL-induced osteo-
clastogenesis from RAW264.7 cells were calculated. IC50
values of LCCY-13 and LCCY-15 for TRAP-positive MNCs
were 32.18 ꢁ 0.44 (Fig. 4a) and 34.47 ꢁ 0.47 mM (Fig. 4d),
respectively. Further, in a comparison with the RANKL-
treated group, compound LCCY-13 decreased the percent-
age of TRAP-positive MNCs (the percentages of LCCY-13-
treated group/RANKL-treated group) generated with
95.87 ꢁ 3.42% at 20 mM, 81.99 ꢁ 5.55% at 30 mM, and
27.28 ꢁ 1.87% at 40 mM (Fig. 4b). As shown in Fig. 4e,
TRAP-positive MNCs were also significantly reduced after
Conclusion
With continued interest in the scaffold of 1-amino-4-(phenyl-
amino)anthraquinone-2-sulfonate sodium, a new class of
1-amino-4-(phenylamino)anthraquinone-2-sulfonate sodium
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
346

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 342–355
New Class of Inhibitors of RANKL-Induced Osteoclastogenesis
Archiv der Pharmazie
Figure 3. Effects of LCCY-13 and LCCY-15 on RANKL-induced osteoclast differentiation from RAW264.7 cells using TRAP-staining
assay. Control was cultured without compounds in the absence of RANKL. Vehicle was cultured without the tested compounds and
RAW264.7 cells were cultured with LCCY-13 and LCCY-15 in the presence of RANKL (100 ng/mL). After 4 days, the mature osteoclasts
were fixed and stained by TRAP. The TRAP-positive MNCs containing more than three nuclei (N > 3) were visualized by light
microscopy.
derivatives was developed as potential anti-resorptive agents,
and their inhibitory activities on RANKL-induced osteoclast
Experimental
Chemistry
formation were evaluated. Based on our biological results, this
study indicated that compounds LCCY-13 and LCCY-15 had
higher inhibitoryactivities toward RANKL-inducedosteoclasto-
genesis. The proposed mechanism of this activity is shown in
Fig. 7. TheinhibitoryactivitiesofLCCY-13andLCCY-15mightbe
affected by suppressing RANKL-induced nuclear translocation
and expression of NFATc1. In addition, a pit formation assay
showed that compounds LCCY-13 and LCCY-15 could reduce
the bone-resorbing activity of osteoclasts. This study provides a
new class of 1-amino-4-(phenylamino)anthraquinone-2-sulfo-
nate sodium derivatives as a potent scaffold for the develop-
ment of anti-resorptive agents.
Unless otherwise stated, all materials used were commer-
cially available. Chemical reagents and solvents were
purchased from Aldrich and Merck without further purifica-
€
tion. Melting points were determined with a Buchi B-545
melting point apparatus and are uncorrected. Analytical
thin-layer chromatography was performed with E. Merck
silica gel 60 F254. 1H NMR and 13C NMR spectra were recorded
on an Agilent 400 MR DD2 (400 MHz). d values are presented
in parts per million (ppm) relative to tetramethylsilane (TMS)
as an internal standard (0 ppm). Multiplicities were recorded
as singlet (s), doublet (d), triplet (t), quartet (q), and double
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
347

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 342–355
C.-C. Lee et al.
Archiv der Pharmazie
Figure 4. Inhibition of the RANKL-induced osteoclast differentiation by compounds LCCY-13 and LCCY-15: (a, d) Chemical structure
of LCCY-13 or LCCY-15; (b, e) compound LCCY-13 or LCCY-15 reduced the RANKL-induced osteoclast differentiation in a dose-
dependent manner. TRAPþ MNCs having three or more nuclei were considered as osteoclasts; (c, f) effects of compound LCCY-13 or
LCCY-15 (20–40 mM) on cell viability of RAW264.7 cells by MTT assay. The data represent the means ꢁ SD of more than three cultures
(n > 3).
of doublet (dd). Coupling constants (J) are expressed in Hz.
The reverse-phase high-performance liquid chro-
General procedure for the synthesis of 1-amino-4-
(phenylamino)anthraquinone-2-sulfonate sodium
derivatives (LCCY-01–25)
A buffer solution of a phosphate solution (Na3PO4 (aq) and
Na2HPO4 (aq)), bromaminic acid sodium salt (1.61 g, 4 mmol),
a catalytic amount (30 mg) of copper powder, and the
appropriate aniline (32 mmol) were added to 25 mL
matographic (HPLC) spectra for all compounds were ac-
quired using a Hitachi L-2000 series system with a UV
detector (L-2400, Hitachi). The retention time (tR) was
expressed in min at 254 nm. Chromatography was performed
on a C18 reverse-phase column (XBridge Shield RP18 column,
̊
€
130A, 5 mm, 4.6 mm ꢂ 250 mm, Waters). The HRMS were
Tinyclave reaction vials (Buchi). The mixtures were capped
measured with a Finnigan MAT-95S (high-resolution electro-
spray ionization, HRESI). Spectral data are recorded as m/z
values.
and heated to 100–120°C for 12–14 h. The reaction mixture
was then cooled to room temperature, filtered, and
extracted with ethyl acetate (3 ꢂ 50 mL). The combined
organic layers were dried over anhydrous Na2SO4 and
concentrated under reduced pressure. The crude product
Please also see the Supporting Information for the InChI
codes of the tested compounds.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
348

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 342–355
New Class of Inhibitors of RANKL-Induced Osteoclastogenesis
Archiv der Pharmazie
Figure 5. Effects of LCCY-13 (a) and LCCY-15 (b) on RANKL-induced nuclear translocation of NFATc1 by using immunofluorescent
analysis.RAW264.7cellswereculturedwithorwithoutRANKL(100 ng/mL).LCCY-13(a)orLCCY-15(b)wasaddedtosomeofthecultures
treated with RANKL. After 24 h, the cells were stained with anti-NFATc1 antibody (green) and DAPI (nuclear staining, red). The nuclear
localization of NFATc1 was confirmed in merged images of RAW264.7 cells treated with RANKL (yellow).
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
349

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 342–355
C.-C. Lee et al.
Archiv der Pharmazie
Figure 6. Effects of LCCY-13 and LCCY-15 on the resorption pit formation. (a) RAW264.7 cells were cultured on bone slice with
various concentrations of compound LCCY-13 or LCCY-15 in the presence of RANKL (100 ng/mL) for 4 days. (b) The resorbed lacunae
on the bone slices were visualized by using light microscopy. Data represent the means ꢁ SD of more than six slices.
Figure 7. Proposed mechanism for the inhibition of RANKL-induced osteoclastogenesis and bone resorption by compound LCCY-13
or LCCY-15.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
350

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 342–355
New Class of Inhibitors of RANKL-Induced Osteoclastogenesis
Archiv der Pharmazie
was purified by crystallization from dichloromethane and
ethanol (dichloromethane/ethanol ¼ 2:1 to 1:2) to afford
compounds LCCY-01–25.
123.04, 124.04, 126.35, 126.46, 130.65, 133.20, 133.27, 133.52,
134.02, 134.53, 136.83, 141.96, 143.14, 144.61, 182.15, 182.56;
HRMS (ESI) m/z: calcd. [MꢃNa]ꢃ, 407.0711 (C21H15N2O5Sꢃ);
found, 407.0707.
Sodium 1-amino-9,10-dioxo-4-phenylamino-9,10-
dihydroanthracene 2-sulfonate (LCCY-01)
Sodium 1-amino-4-(4-hydroxyphenylamino)-9,10-dioxo-
9,10-dihydroanthracene 2-sulfonate (LCCY-06)
Yield: 25%; m.p.: >300°C (EtOH) [32]; Rf: 0.57 (EA/EtOH/
1
CH2Cl2 ¼ 2:1:1); H NMR (400 MHz, DMSO-d6): d (ppm) 7.20 (t,
Yield: 28%; m.p.: >300°C (EtOH) [32]; Rf: 0.53 (EA/EtOH/
CH2Cl2 ¼ 2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 6.84 (d,
J ¼ 8.4, 2H), 7.10 (d, J ¼ 8.4, 2H), 7.80–7.84 (m, 3H), 8.24–8.29
(m, 4H), 9.55 (bs, 2H), 12.05 (s, 1H); 13C NMR (100 MHz, DMSO-
d6): d (ppm) 109.31, 110.34, 116.39, 122.93, 126.26, 126.64,
126.95, 130.27, 133.29, 134.08, 134.46, 134.95, 143.12, 143.42,
144.33, 155.69, 182.00; HRMS (ESI) m/z: calcd. [MꢃNa]ꢃ,
409.0518 (C20H13N2NaO6Sꢃ); found, 409.0500.
J ¼ 7.6 Hz, 1H), 7.28 (d, J ¼ 7.6 Hz, 2H), 7.39–7.48 (m, 2H), 7.82–
7.86 (m, 2H), 8.02 (s, 1H), 8.25–8.28 (m, 2H), 10.12 (br, 2H), 12.05
(s, 1H); 13C NMR (100 MHz, DMSO-d6): d (ppm) 109.60, 111.85,
123.13, 123.51, 124.92, 126.70, 126.98, 130.19, 133.27, 133.66,
133.96, 134.53, 139.65, 141.18, 143.01, 144.72, 182.26, 182.90;
HRMS (ESI) m/z: calcd. [MꢃNa]ꢃ, 393.0565 (C20H13N2O5Sꢃ);
found: 393.0545.
Sodium 1-amino-4-(4-chlorophenylamino)-9,10-dioxo-
9,10-dihydroanthracene 2-sulfonate (LCCY-02)
Sodium 1-amino-4-(4-cyanophenylamino)-9,10-dioxo-
9,10-dihydroanthracene 2-sulfonate (LCCY-07)
Yield: 67%; m.p.: >300°C (EtOH) [32]; Rf: 0.65 (EA/EtOH/
CH2Cl2 ¼ 2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 7.30 (d,
J ¼ 6.9 Hz, 2H), 7.47 (d, J ¼ 8.7 Hz, 2H), 7.83–7.86 (m, 2H), 7.96
(s, 1H), 8.22–8.27 (m, 2H), 10.01 (s, 2H), 11.86 (s, 1H); 13C NMR
(100 MHz, DMSO-d6): d (ppm) 109.76, 112.52, 123.09, 124.91,
126.45, 126.97, 128.48, 130.01, 133.48, 133.80, 133.85, 134.50,
138.89, 140.39, 142.84, 144.80, 182.39, 183.22; HRMS (ESI) m/z:
calcd. [MꢃNa]ꢃ, 427.0174 (C20H12ClN2O5Sꢃ); found, 426.9712.
Yield: 30%; m.p.: >300°C (EtOH); Rf: 0.71 (EA/EtOH/CH2Cl2 ¼
2:1:1); 1H NMR (400 MHz, DMSO-d6):
d (ppm) 7.35 (d,
J ¼ 8.8 Hz, 2H), 7.79 (d, J ¼ 8.4 Hz, 2H), 7.85–7.89 (m, 2H),
8.09 (s, 1H), 8.20–8.27 (m, 2H), 9.96 (br, 2H), 11.49 (s, 1H);
13C NMR (100 MHz, DMSO-d6): d (ppm) 103.97, 110.51, 115.91,
119.73, 120.34, 124.62, 126.53, 126.96, 133.54, 133.62, 134.22,
134.29, 134.43, 136.46, 142.00, 145.44, 145.53, 182.85, 184.20;
HRMS (ESI) m/z: calcd. [MꢃNa]ꢃ, 418.0498 (C21H12N3O5Sꢃ);
found, 418.0498.
Sodium 1-amino-4-(4-nitrophenylamino)-9,10-dioxo-9,10-
dihydroanthracene 2-sulfonate (LCCY-03)
Sodium 1-amino-4-(4-biphenylamino)-9,10-dioxo-9,10-
dihydroanthracene 2-sulfonate (LCCY-08)
Yield: 78%; m.p.: >300°C (EtOH); Rf: 0.71 (EA/EtOH/CH2Cl2
¼ 2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 7.32 (d,
J ¼ 9.2 Hz, 2H), 7.85–7.89 (m, 2H), 8.11 (s, 1H), 8.19–8.26 (m,
4H), 9.90 (bs, 2H), 11.30 (s, 1H); 13C NMR (100 MHz, DMSO-d6):
d (ppm) 110.90, 117.77, 118.58, 125.75, 126.56, 126.69, 126.96,
133.54, 133.66, 134.36, 134.40, 134.77, 141.00, 141.51, 145.91,
148.22, 183.12, 184.41; HRMS (ESI) m/z: calcd. [MꢃNa]ꢃ,
438.0522 (C20H12N3O7Sꢃ); found, 438.0396.
Yield: 54%; m.p.: >300°C (EtOH) [33]; Rf: 0.70 (EA/EtOH/
CH2Cl2 ¼ 2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 7.35–
7.39 (m, 3H), 7.46–7.48 (m, 2H), 7.71 (d, J ¼ 7.2 Hz, 2H), 7.75 (d,
J ¼ 8.4 Hz, 2H), 7.83–7.88 (m, 2H), 8.10 (s, 1H), 8.26–8.29 (m,
2H), 12.08 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d (ppm)
109.77, 112.28, 123.38, 123.47, 126.29, 126.41, 126.47, 128.26,
129.27, 129.42, 133.29, 133.69, 133.83, 134.47, 136.24, 139.19,
139.78, 140.63, 142.79, 144.79, 182.32, 183.00; HRMS (ESI) m/z:
calcd. [MꢃNa]ꢃ, 469.0873 (C26H17N2O5Sꢃ); found, 469.0873.
Sodium 1-amino-4-(4-aminophenylamino)-9,10-dioxo-
9,10-dihydroanthracene 2-sulfonate (LCCY-04)
Yield: 53%; m.p.: >300°C (EtOH) [32]; Rf: 0.43 (EA/EtOH/
CH2Cl2 ¼ 2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 5.22 (s,
2H), 6.63 (d, J ¼ 8.7 Hz, 2H), 6.94 (d, J ¼ 8.7 Hz, 2H), 7.80–7.83
(m, 3H), 8.25–8.26 (m, 2H), 10.11 (s, 2H), 12.10 (s, 1H); 13C NMR
(100 MHz, DMSO-d6): d (ppm) 114.67, 114.81, 123.17, 126.20,
126.36, 126.65, 128.06, 132.93, 133.04, 133.22, 133.87, 134.50,
134.89, 143.38, 144.41, 146.97, 181.47, 181.81; HRMS (ESI) m/z:
calcd. [MꢃNa]ꢃ, 408.0705 (C20H14N3O5Sꢃ); found, 408.0654.
Sodium 1-amino-4-(2-chlorophenylamino)-9,10-dioxo-
9,10-dihydroanthracene 2-sulfonate (LCCY-09)
Yield: 50%; m.p.: >300°C (EtOH) [32]; Rf: 0.71 (EA/EtOH/
CH2Cl2 ¼ 2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 7.19–
7.25 (m, 2H), 7.36 (t, J ¼ 2 Hz, 1H), 7.43 (t, J ¼ 8 Hz, 1H), 7.83–
7.87 (m, 2H), 8.00 (s, 1H), 8.23–8.28 (m, 2H), 10.08 (br, 2H),
11.79 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d (ppm) 109.89,
113.17, 121.28, 122.39, 123.39, 124.09, 126.46, 126.52, 131.61,
133.40, 133.82, 133.92, 134.42, 134.51, 139.63, 141.80, 142.71,
144.94, 182.49, 183.48; HRMS (ESI) m/z: calcd. [MꢃNa]ꢃ,
427.0182 (C20H12ClN2O5Sꢃ); found, 427.0155.
Sodium 1-amino-4-[(4-methylphenyl)amino]-9,10-dioxo-
9,10-dihydroanthracene-2-sulfonate (LCCY-05)
Yield: 27%; m.p.: >300°C (EtOH) [23]; Rf: 0.65 (EA/EtOH/
1
CH2Cl2 ¼ 2:1:1); H NMR (400 MHz, DMSO-d6): d (ppm) 2.33 (s,
Sodium 1-amino-4-(3-(trifluoromethyl)phenylamino)-
9,10-dioxo-9,10-dihydroanthracene 2-sulfonate (LCCY-10)
Yield: 21%; m.p.: >300°C (EtOH) [28]; Rf: 0.63 (EA/EtOH/
CH2Cl2 ¼ 2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 7.47 (d,
3H), 7.17 (d, J ¼ 8.4 Hz, 2H), 7.26 (d, J ¼ 8.1 Hz, 2H), 7.82–7.85 (m,
2H), 7.95 (s, 1H), 8.24–8.29 (m, 2H), 10.18 (br, 2H), 12.06 (s, 1H);
13C NMR (100 MHz, DMSO-d6): d (ppm) 20.97, 109.48, 112.26,
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
351

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 342–355
C.-C. Lee et al.
Archiv der Pharmazie
J ¼ 7.6 Hz, 1H), 7.56 (d, J ¼ 8 Hz, 1H), 7.62–7.66 (m, 2H), 7.83–
7.89 (m, 2H), 8.00 (s, 1H), 8.23–8.28 (m, 2H), 10.01 (br, 2H),
11.80 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d (ppm) d 110.02,
113.56, 118.93, 118.97, 123.30, 126.15, 126.45, 126.52, 131.08,
131.17, 133.42, 133.48, 133.94, 134.49, 139.27, 141.21, 142.64,
145.01, 182.56, 183.61; HRMS (ESI) m/z: calcd. [MꢃNa]ꢃ,
461.0430 (C21H12F3N2O5Sꢃ); found, 461.0425.
Sodium 1-amino-4-[(3,4-dimethoxyphenyl)amino]-9,10-
dioxo-9,10-dihydroanthracene-2-sulfonate (LCCY-15)
Yield: 12%; m.p.: >300°C (EtOH); Rf: 0.58 (EA/EtOH/CH2Cl2 ¼
2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 3.77 (s, 3H), 3.79
(s, 3H), 6.82 (dd, J ¼ 8.8 Hz, 1.6 Hz, 1H), 6.93 (d, J ¼ 2.4 Hz, 1H),
7.02 (d, J ¼ 8.8 Hz, 1H), 7.81–7.85 (m, 2H), 7.96 (s, 1H), 8.26–
8.29 (m, 2H), 10.15 (br, 2H), 12.01 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): d (ppm) 56.04, 56.15, 109.23, 109.38, 110.83,
112.87, 116.38, 123.28, 126.30, 126.44, 132.38, 133.13, 133.40,
134.06, 134.52, 142.63, 143.18, 144.55, 146.82, 149.89, 182.06,
182.26; HRMS (ESI) m/z: calcd. [MꢃNa]ꢃ, 453.0881
(C22H17N2O7Sꢃ); found, 453.0762.
Sodium 1-amino-4-(3-methylphenylamino)-9,10-dioxo-
9,10-dihydroanthracene 2-sulfonate (LCCY-11)
Yield: 43%; m.p.: >300°C (EtOH) [23]; Rf: 0.68 (EA/EtOH/
CH2Cl2 ¼ 2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 2.33 (s,
3H), 7.01–7.10 (m, 3H), 7.32 (t, J ¼ 7.6 Hz), 7.83–7.86 (m, 2H),
8.00 (s, 1H), 8.25–8.28 (m, 2H), 9.63 (br, 2H), 12.04 (s, 1H);
13C NMR (100 MHz, DMSO-d6): d (ppm) 21.26, 109.57, 111.69,
120.03, 120.63, 123.28, 124.22, 125.74, 126.38, 128.51, 130.06,
133.23, 133.60, 133.97, 134.52, 139.71, 141.40, 143.00, 144.67,
182.22, 182.80; HRMS (ESI) m/z: calcd. [MꢃNa]ꢃ, 407.0708
(C21H15N2O5Sꢃ); found, 407.0702.
Sodium 1-amino-4-(3-chloro-4-fluorophenylamino)-9,10-
dioxo-9,10-dihydroanthracene 2-sulfonate (LCCY-16)
Yield: 74%; m.p.: >300°C (EtOH) [23]; Rf: 0.57 (EA/EtOH/
CH2Cl2 ¼ 2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 7.31–
0
0
7.32 (m, 1H, Ar5 –H), 7.48 (t, J ¼ 9 Hz, 2H, Ar2 –H), 7.55 (dd,
0
J ¼ 8.8 Hz, 3.2 Hz, 1H, Ar6 –H), 7.83–7.87 (m, 2H, Ar6, 7–H), 7.88
(s, 1H, Ar3–H), 8.22–8.27 (m, 2H, Ar5, 8–H), 10.02 (s, 2H,
Ar1–NH2), 11.86 (s, 1H, Ar4–NH–Ar); 13C NMR (100 MHz, DMSO-
d6): d (ppm) 109.79, 112.63, 120.75, 124.13, 124.20, 125.47,
126.41, 126.50, 133.27, 133.36, 133.80, 133.84, 134.49, 137.40,
140.45, 142.84, 153.37, 155.80, 182.43, 183.30; HRMS (ESI)
m/z: calcd. [MꢃNa]ꢃ, 445.0078 (C20H11ClFN2O5Sꢃ); found,
445.0494.
Sodium 1-amino-4-(3-cyanophenylamino)-9,10-dioxo-
9,10-dihydroanthracene 2-sulfonate (LCCY-12)
Yield: 31%; m.p.: >300°C (EtOH); Rf: 0.66 (EA/EtOH/CH2Cl2 ¼
2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 7.57–7.61
(m, 3H), 7.74 (s, 1H), 7.85–7.88 (m, 2H), 7.97 (s, 1H), 8.23–
8.28 (m, 2H), 10.02 (br, 2H), 11.72 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): d (ppm) 110.03, 112.96, 113.75, 118.96, 123.27,
125.56, 126.47, 126.53, 127.13, 127.50, 131.33, 133.44, 133.75,
134.01, 134.50, 138.96, 141.38, 145.08, 182.57, 183.69; HRMS
(ESI) m/z: calcd. [MꢃNa]ꢃ, 456.0310 (C21H12N3O5Sꢃ); found,
456.0302.
Sodium 1-amino-4-[(3,4-difluorophenyl)amino]-9,10-
dioxo-9,10-dihydroanthracene-2-sulfonate (LCCY-17)
Yield: 31%; m.p.: >300°C (EtOH); Rf: 0.61 (EA/EtOH/CH2Cl2 ¼
2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 7.12–7.14 (m,
1H), 7.40–7.53 (m, 2H), 7.84–7.88 (m, 2H), 7.92 (s, 1H), 8.24–
8.28 (m, 2H), 10.02 (br, 2H), 11.78 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): d (ppm) 109.78, 112.64, 112.73, 112.92, 118.56,
118.74, 120.07, 120.10, 123.00, 126.41, 126.51, 133.36, 133.82,
133.84, 134.51, 140.35, 142.92, 144.84, 182.42, 183.33; HRMS
(ESI) m/z: calcd. [MꢃNa]ꢃ, 429.0372 (C20H11F2N2O5Sꢃ); found,
429.0362.
Sodium 1-amino-4-(3-methoxyphenylamino)-9,10-dioxo-
9,10-dihydroanthracene 2-sulfonate (LCCY-13)
Yield: 33%; m.p.: >300°C (EtOH); Rf: 0.51 (EA/EtOH/CH2Cl2
¼ 2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 3.78 (s, 3H),
6.76 (dd, J ¼ 8.4 Hz, 1.2 Hz, 1H), 6.83–6.86 (m, 2H), 7.33
(t, J ¼ 8 Hz, 2H), 7.83–7.86 (m, 2H), 8.08 (s, 1H), 8.25–8.28
(m, 2H), 10.05 (br, 2H), 12.00 (s, 1H); 13C NMR (100 MHz,
DMSO-d6): d (ppm) 55.64, 108.65, 109.66, 110.60, 112.09,
115.36, 123.56, 126.39, 126.47, 130.87, 133.29, 133.70, 133.90,
134.49, 140.82, 140.96, 142.79, 144.72, 160.78, 182.29, 182.99;
HRMS (ESI) m/z: calcd. [MꢃNa]ꢃ, 423.0663 (C21H15N2O6Sꢃ);
found, 423.0651.
Sodium 1-amino-4-(2-chlorophenylamino)-9,10-dioxo-
9,10-dihydroanthracene 2-sulfonate (LCCY-18)
Yield: 78%; m.p.: >300°C (EtOH) [32]; Rf: 0.71(EA/EtOH/
CH2Cl2 ¼ 2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 7.20 (t,
J ¼ 7.4 Hz, 1H), 7.39–7.46 (m, 2H), 7.62 (d, J ¼ 8 Hz, 1H), 7.82–
7.88 (m, 2H), 7.91 (s, 1H), 8.25–8.28 (m, 2H), 10.01 (bs, 2H),
11.96 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d (ppm) 109.93,
113.21, 123.10, 124.03, 125.79, 126.53, 126.73, 128.62, 130.79,
133.42, 133.76, 133.98, 134.52, 137.00, 139.53, 142.64, 144.88,
182.50, 183.75; HRMS (ESI) m/z: calcd. [MꢃNa]ꢃ, 427.0167
(C20H11F2N2O5Sꢃ); found, 427.0161.
Sodium 1-amino-4-(3-hydroxyphenylamino)-9,10-dioxo-
9,10-dihydroanthracene 2-sulfonate (LCCY-14)
Yield: 27%; m.p.: >300°C (EtOH) [32]; Rf: 0.62 (EA/EtOH/
CH2Cl2 ¼ 2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 6.60 (d,
J ¼ 8 Hz, 1H), 6.66–6.92 (m, 2H), 7.21 (t, J ¼ 8 Hz, 2H), 7.83–7.86
(m, 2H), 8.03 (s, 1H), 8.25–8.28 (m, 2H), 9.68 (s, 1H), 10.12 (br,
2H), 11.99 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d (ppm)
108.65, 109.67, 110.60, 112.08, 115.36, 123.56, 126.40, 126.47,
130.89, 133.29, 133.70, 133.90, 134.50, 140.82, 140.96, 142.79,
144.73, 160.77, 182.29, 182.99; HRMS (ESI) m/z: calcd.
[MꢃNa]ꢃ, 409.0502 (C20H13N2O6Sꢃ); found, 409.0494.
Sodium 1-amino-4-(2-methylphenylamino)-9,10-dioxo-
9,10-dihydroanthracene 2-sulfonate (LCCY-19)
Yield: 23%; m.p.: >300°C (EtOH) [23]; Rf: 0.68 (EA/EtOH/
CH2Cl2 ¼ 2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 2.81 (s,
3H), 7.17 (td, J ¼ 7 Hz, 1.6 Hz, 1H), 7.25–7.29 (m, 2H), 7.38 (d,
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
352

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 342–355
New Class of Inhibitors of RANKL-Induced Osteoclastogenesis
Archiv der Pharmazie
J ¼ 8.4 Hz, 1H), 7.74 (s, 1H), 7.83–7.86 (m, 2H), 8.27–8.29 (m,
2H), 10.18 (br, 2H), 12.31 (s, 1H); 13C NMR (100 MHz, DMSO-
d6): d (ppm) 18.23, 109.47, 111.22, 122.99, 124.82, 125.79,
126.41, 126.49, 127.43, 131.68, 132.60, 133.21, 133.57, 134.04,
134.59, 138.00, 142.32, 143.26, 144.55, 182.14, 182.79; HRMS
(ESI) m/z: calcd. [MꢃNa]ꢃ, 407.0744 (C21H15N2O5Sꢃ); found,
427.0702.
Sodium 1-amino-4-(2-methyl-5-chlorophenylamino)-9,10-
dioxo-9,10-dihydroanthracene 2-sulfonate (LCCY-24)
Yield: 24%; m.p.: >300°C (EtOH); Rf: 0.64 (EA/EtOH/CH2Cl2 ¼
2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 2.26 (s, 3H), 7.20
(dd, J ¼ 8 Hz, 2 Hz, 1H), 7.31 (d, J ¼ 2 Hz, 1H), 7.39 (d, J ¼ 8 Hz,
1H), 7.73 (s, 1H), 7.84–7.87 (m, 2H), 8.26–8.29 (m, 2H), 10.15
(br, 2H), 11.86 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d (ppm)
17.742, 109.74, 112.27, 122.94, 123.50, 125.01, 126.45, 126.51,
131.19, 131.29, 133.02, 133.31, 133.79, 133.86, 134.54, 139.81,
140.95, 143.01, 144.70, 182.15, 182.56; HRMS (ESI) m/z: calcd.
[MꢃNa]ꢃ, 441.0325 (C21H14ClN2O5Sꢃ); found, 441.0317.
Sodium 1-amino-4-(2-cyanophenylamino)-9,10-dioxo-
9,10-dihydroanthracene 2-sulfonate (LCCY-20)
Yield: 25%; m.p.: >300°C (EtOH); Rf: 0.57 (EA/EtOH/CH2Cl2
¼ 2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 7.27 (t,
J ¼ 7.6 Hz, 1H), 7.47 (d, J ¼ 8.4 Hz, 1H), 7.69–7.74 (m, 1H),
7.83–7.91 (m, 3H), 7.96 (s, 1H), 8.24–8.28 (m, 2H), 9.97 (br, 2H),
12.01 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d (ppm) 105.04,
110.18, 114.64, 117.40, 122.21, 123.53, 124.33, 126.57, 126.61,
133.56, 134.27, 134.46, 134.89, 137.95, 142.42, 143.48, 145.31,
182.70, 184.51; HRMS (ESI) m/z: calcd. [MꢃNa]ꢃ, 418.0508
(C21H12N3O5Sꢃ); found, 418.0503.
Sodium 1-amino-4-((2,5-difluorophenyl)amino)-9,10-
dioxo-9,10-dihydroanthracene-2-sulfonate (LCCY-25)
Yield: 88%; m.p.: >300°C (EtOH); Rf: 0.64 (EA/EtOH/CH2Cl2 ¼
2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 7.01–7.06 (m,
1H), 7.29–7.34 (m, 1H), 7.39–7.46 (m, 1H), 7.82–7.90 (m, 3H),
8.24–8.27 (m, 2H), 9.93 (br, 2H), 11.73 (s, 1H); 13C NMR
(100 MHz, DMSO-d6): d (ppm) 110.05, 110.49, 110.75, 111.28,
111.36, 111.53, 111.60, 113.55, 117.58, 117.68, 117.80, 117.90,
122.94, 126.49, 129.17, 133.49, 133.54, 134.10, 134.39, 138.81,
142.42, 144.85, 150.21, 152.91, 157.45, 159.86, 182.62, 184.09;
HRMS (ESI) m/z: calcd. [MꢃNa]ꢃ,429.0374 (C20H11F2N2O5Sꢃ);
found, 429.0357.
Sodium 1-amino-4-(2-(trifluoromethyl)phenylamino)-
9,10-dioxo-9,10-dihydroanthracene 2-sulfonate (LCCY-21)
Yield: 43%; m.p.: >300°C (EtOH); Rf: 0.28 (EA/EtOH/CH2Cl2 ¼
2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 7.07 (t, J ¼ 7.2 Hz,
1H), 7.28 (d, J ¼ 8.4 Hz, 1H), 7.49–7.53 (m, 1H), 7.83–7.88 (m,
2H), 7.92–7.96 (m, 1H), 8.10 (s, 1H), 8.20–8.26 (m, 2H), 9.97 (br,
2H), 12.27 (s, 1H); 13C NMR (100 MHz, DMSO-d6): d (ppm)
110.56, 116.38, 120.59, 121.86, 125.60, 126.49, 132.30, 133.46,
133.59, 133.91, 133.98, 134.47, 136.62, 141.32, 142.41, 145.34,
168.36, 182.91, 184.18; HRMS (ESI) m/z: calcd. [MꢃNa]ꢃ,
461.0407 (C21H12F3N3O5Sꢃ); found, 461.0419.
Biological study
Cell cultures
RAW264.7 cells were obtained from American Type Culture
Collection (ATCC, Rockville, MD, USA). These cells were
cultured with Dulbecco’s modified Eagle medium (DMEM,
Gibco, Grand Island, NY, USA) and supplemented with 10%
heat-inactivated fetal bovine serum (FBS) in a 95% air and 5%
CO2 humid atmosphere at 37°C.
Sodium 1-amino-4-(2,4-difluorophenylamino)-9,10-dioxo-
9,10-dihydroanthracene 2-sulfonate (LCCY-22)
Yield: 77%; m.p.: >300°C (EtOH); Rf: 0.62 (EA/EtOH/CH2Cl2 ¼
2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 7.20 (td,
J ¼ 8.8 Hz, 1.6 Hz, 1H), 7.44–7.54 (m, 2H), 7.66 (d, J ¼ 1.6 Hz,
1H), 7.82–7.88 (m, 2H), 8.25–8.28 (m, 2H), 11.70 (s, 1H);
13C NMR (100 MHz, DMSO-d6): d (ppm) 105.57, 105.81, 109.70,
112.01, 112.49, 112.52, 112.71, 122.38, 126.45, 126.50, 127.91,
128.00, 133.37, 133.76, 133.88, 134.51, 141.24, 143.01, 144.56,
182.44, 183.53; HRMS (ESI) m/z: calcd. [MꢃNa]ꢃ, 429.0415
(C20H11F2N2NaO5Sꢃ); found, 429.0357.
Cell viability assay [34, 35]
RAW264.7 cells were seeded into a 96-well culture plate at a
density of 104 cells per well with 200 mL medium (DMEM)
supplemented with 10% FBS and treated with a series of
synthesized compounds for 24 h. After 24 h, the 96-well plate
was washed three times with phosphate-buffered saline (3ꢂ
PBS) and then treated with medium containing 500 mg/mL of
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) reagent for 1 h at 37°C. Cells were then washed with
PBS and solubilized in 100 mL of DMSO. The MTT reagent was
designed to yield formazan by metabolic reduction in viable
cells. Absorbance was measured at 540 nm using an enzyme-
linked immunosorbent assay (ELISA) reader.
Sodium 1-amino-4-(2-fluoro-4-chlorophenylamino)-9,10-
dioxo-9,10-dihydroanthracene 2-sulfonate (LCCY-23)
Yield: 69%; m.p.: >300°C (EtOH); Rf: 0.64 (EA/EtOH/CH2Cl2
¼ 2:1:1); 1H NMR (400 MHz, DMSO-d6): d (ppm) 7.36 (dd,
J ¼ 8.6 Hz, 1.6 Hz, 1H), 7.47 (t, J ¼ 8.8 Hz, 1H), 7.62 (dd,
J ¼ 10.6 Hz, 2 Hz, 1H), 7.78 (d, J ¼ 1.2 Hz, 1H), 7.84–7.88 (m,
2H), 8.24–8.28 (m, 2H), 9.96 (br, 2H), 11.73 (s, 1H); 13C NMR
(100 MHz, DMSO-d6): d (ppm) 109.85, 112.86, 117.35, 117.58,
122.66, 125.78, 125.81, 126.30, 126.50, 126.53, 126.93, 126.98,
127.04, 129.02, 129.12, 133.44, 133.71, 134.02, 134.51, 139.83,
142.85, 144.78, 154.42, 182.52, 183.83; HRMS (ESI) m/z: calcd.
[MꢃNa]ꢃ, 445.0096 (C20H11ClFN2O5Sꢃ); found, 445.0061.
TRAP staining assay [34, 35]
RAW264.7 cells were seeded into a 48-well plate at a density
of 2 ꢂ 104 cells per well with alpha-minimum essential
medium (a-MEM, Gibco) containing 10% FBS, 2 mM
L-glutamate, and 100 ng/mL of recombinant soluble murine
RANKL (Peprotech, London, UK) in the presence or absence of
tested compounds. Cell cultures were incubated in 95% air
and 5% CO2 at 37°C for 4 days. Cell culture media (containing
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
353

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 342–355
C.-C. Lee et al.
Archiv der Pharmazie
the test compounds) were changed on day 2. After 4 days,
cells were washed with PBS and fixed with a solution (acetone/
citrate solution/37% formaldehyde, 65:25:8) for 30 min. Then,
cells were incubated in a light-protected incubator at 37°C for
1 h using a Leukocyte Acid Phosphatase Assay kit (cat. no.
387A, Sigma). Cells were washed three times with distilled
water, and TRAP-positive multinucleated cells containing
three or more nuclei (N > 3) were counted under a light
microscope and photographed.
[4] K. Okamoto, H. Takayanagi, Int. Immunopharmacol.
2011, 11, 543–548.
[5] T. Suda, N. Takahashi, N. Udagawa, E. Jimi, M. T. Gillespie,
T. J. Martin, Endocr. Rev. 1999, 20, 345–357.
[6] B. R. Wong, R. Josien, S. Y. Lee, M. Vologodskaia,
R. M. Steinman, Y. Choi, J. Biol. Chem. 1998, 273,
28355–28359.
[7] B. G. Darnay, V. Haridas, J. Ni, P. A. Moore,
B. B. Aggarwal, J. Biol. Chem. 1998, 273, 20551–20555.
[8] B. G. Darnay, J. Ni, P. A. Moore, B. B. Aggarwal, J. Biol.
Chem. 1999, 274, 7724–7731.
NFATc1 immunofluorescent staining assay
RAW264.7 cells were seeded onto glass coverslips and then
incubated with 100 ng/mL RANKL in the presence or absence
of compounds. The distribution of NFATc1 protein 24 h after
stimulation was assessed according to previously published
protocols [36]. Coverslips were removed, washed in PBS, fixed
in 4% paraformaldehyde, permeabilized with 0.1% Triton X-
100, incubated with 5% BSA, and incubated with a specific
anti-NFATc1 monoclonal anti-body (1:50, Santa Cruz, USA)
overnight. Cells were washed in PBS, incubated for 2 h with
FITC-conjugated goat anti-mouse immunoglobulin G (IgG;
Jackson ImmunoResearch Laboratories. Inc.). After the
immunostaining procedures, cells were nuclear-stained with
DAPI (Sigma). Fluorescence was visualized using a Leica
fluorescence microscope.
[9] T. Yamashita, Z. Yao, F. Li, Q. Zhang, I. R. Badell,
E. M. Schwarz, S. Takeshita, E. F. Wagner, M. Noda,
K. Matsuo, L. Xing, B. F. Boyce, J. Biol. Chem. 2007, 282,
18245–18253.
[10] M. Asagiri, K. Sato, T. Usami, S. Ochi, H. Nishina,
H. Yoshida, I. Morita, E. F. Wagner, T. W. Mak,
E. Serfling, H. Takayanagi, J. Exp. Med. 2005, 202,
1261–1269.
[11] K. Matsuo, D. L. Galson, C. Zhao, L. Peng, C. Laplace,
K. Z. Wang, M. A. Bachler, H. Amano, H. Aburatani,
H. Ishikawa, E. F. Wagner, J. Biol. Chem. 2004, 279,
26475–26480.
[12] B. Zhou, R. Q. Cron, B. Wu, A. Genin, Z. Wang, S. Liu,
P. Robson, H. S. Baldwin, J. Biol. Chem. 2002, 277,
10704–10711.
Resorbing activity of osteoclasts by bone resorption
assay [34, 35]
[13] A. R. Hayman, S. J. Jones, A. Boyde, D. Foster,
W. H. Colledge, M. B. Carlton, M. J. Evans, T. M. Cox,
Development 1996, 122, 3151–3162.
[14] T. M. Ou, Y. J. Lu, J. H. Tan, Z. S. Huang, K. Y. Wong,
L. Q. Gu, ChemMedChem 2008, 3, 690–713.
[15] C. C. Lee, D. M. Chang, K. F. Huang, C. L. Chen, T. C. Chen,
Y. Lo, J. H. Guh, H. S. Huang, Bioorg. Med. Chem. 2013,
21, 7125–7133.
[16] H. S. Huang, K. F. Huang, C. L. Li, Y. Y. Huang,
Y. H. Chiang, F. C. Huang, J. J. Lin, Bioorg. Med. Chem.
2008, 16, 6976–6986.
[17] K. C. Hsu, H. C. Hung, J. T. Horng, M. Y. Fang, C. Y. Chang,
L. T. Li, I. J. Chen, Y. C. Chen, D. L. Chou, C. W. Chang,
H. P. Hsieh, J. M. Yang, J. T. Hsu, PLoS ONE 2013, 8,
e56704.
RAW264.7 cells were seeded into dentine slices (24-well plates,
Corning) at a density of 104 cells per well with a-MEM
(containing 10% FBS and 2 mM L-glutamate) and 100 ng/mL
of recombinant soluble murine RANKL in the presence or
absence of the test compounds. All cultured cells were
replenished every 2 days with fresh medium containing the
tested chemicals. After 4 days, wells were washed three times
with PBS, and left in 1 M ammonium hydroxide to remove the
attached cells. Inanoptical field ona slice, ratios ofthe resorbed
areatothetotalareaweremeasuredusingtheImageJsoftware.
The present study was supported by grants from the Ministry
of Science and Technology (MOST 102-2314-B-075-083-MY3
and MOST 104-2113-M-038-001), Veterans General Hospitals
University System of Taiwan Joint Research Program
(VGHUST104-G1-3-1), and Taipei Medical University Grant
(TMU 102-AE1-B32).
[18] M. Y. Park, H. J. Kwon, M. K. Sung, Biosci. Biotechnol.
Biochem. 2009, 73, 828–832.
[19] C. C. Lee, K. F. Huang, P. Y. Lin, F. C. Huang, C. L. Chen,
T. C. Chen, J. H. Guh, J. J. Lin, H. S. Huang, Eur. J. Med.
Chem. 2012, 47, 323–336.
The authors have declared no conflict of interest.
[20] C. C. Lee, K. F. Huang, D. M. Chang, J. J. Hsu, F. C. Huang,
K. N. Shih, C. L. Chen, T. C. Chen, R. H. Chen, J. J. Lin,
H. S. Huang, Eur. J. Med. Chem. 2012, 50, 102–112.
[21] M. Glanzel, R. Bultmann, K. Starke, A. W. Frahm, Eur. J.
Med. Chem. 2003, 38, 303–312.
References
[1] J. C. Crockett, M. J. Rogers, F. P. Coxon, L. J. Hocking,
M. H. Helfrich, J. Cell Sci. 2011, 124, 991–998.
[2] E. Seeman, Lancet 2002, 359, 1841–1850.
[3] I. B. McInnes, G. Schett, N. Engl. J. Med. 2011, 365,
2205–2219.
[22] M. Glanzel, R. Bultmann, K. Starke, A. W. Frahm, Eur. J.
Med. Chem. 2005, 40, 1262–1276.
[23] S. Weyler, Y. Baqi, P. Hillmann, M. Kaulich, A. M. Hunder,
I. A. Muller, C. E. Muller, Bioorg. Med. Chem. Lett. 2008,
18, 223–227.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
354

RCHH HARM
A P
Arch. Pharm. Chem. Life Sci. 2016, 349, 342–355
New Class of Inhibitors of RANKL-Induced Osteoclastogenesis
Archiv der Pharmazie
[24] Y. Baqi, K. Atzler, M. Kose, M. Glanzel, C. E. Muller,
J. Med. Chem. 2009, 52, 3784–3793.
[29] S. J. Dixon, S. M. Sims, Drug Develop. Res. 2000, 49,
187–200.
[25] Y. Baqi, S. Y. Lee, J. Iqbal, P. Ripphausen, A. Lehr,
A. B. Scheiff, H. Zimmermann, J. Bajorath, C. E. Muller,
J. Med. Chem. 2010, 53, 2076–2086.
[26] S. Roy, R. J. Large, A. M. Akande, A. Kshatri, T. I. Webb,
C. Domene, G. P. Sergeant, N. G. McHale,
K. D. Thornbury, M. A. Hollywood, Eur. J. Med. Chem.
2014, 75, 426–437.
[27] S. Roy, A. Morayo Akande, R. J. Large, T. I. Webb,
C. Camarasu, G. P. Sergeant, N. G. McHale,
K. D. Thornbury, M. A. Hollywood, ChemMedChem
2012, 7, 1763–1769.
[28] T. I. Webb, A. S. Kshatri, R. J. Large, A. M. Akande, S. Roy,
G. P. Sergeant, N. G. McHale, K. D. Thornbury,
M. A. Hollywood, PNAS 2015, 112, 2064–2069.
[30] T. Wada, T. Nakashima, N. Hiroshi, J. M. Penninger,
Trends Mol. Med. 2006, 12, 17–25.
[31] H. Takayanagi, Ann. N. Y. Acad. Sci. 2007, 1116, 227–237.
[32] Y. Baqi, C. E. Muller, Org. Lett. 2007, 9, 1271–1274.
[33] Z. Liang, J. Ai, X. Ding, X. Peng, D. Zhang, R. Zhang,
Y. Wang, F. Liu, M. Zheng, H. Jiang, H. Liu, M. Geng,
C. Luo, ACS Med. Chem. Lett. 2013, 4, 408–413.
[34] C. C. Lee, F. L. Liu, C. L. Chen, T. C. Chen, D. M. Chang,
H. S. Huang, Eur. J. Med. Chem. 2015, 98, 115–126.
[35] C. C. Lee, F. L. Liu, C. L. Chen, T. C. Chen, F. C. Liu,
A. A. Ahmed Ali, D. M. Chang, H. S. Huang, Bioorg. Med.
Chem 2015, 23, 4522–4532.
[36] C. P. Cheng, H. S. Huang, Y. C. Hsu, M. J. Sheu,
D. M. Chang, J. Clin. Immunol. 2012, 32, 762–777.
ß 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com
355