Novel Pyrazole Derivatives Effectively Inhibit Osteoclastogenesis, a Potential Target for Treating Osteoporosis

Article abstract of DOI:10.1021/jm502014h

As human beings live longer, age-related diseases such as osteoporosis will become more prevalent. Intolerant side effects and poor responses to current treatments are observed. Therefore, novel effective therapeutic agents are greatly needed. Here, pyrazole derivatives were designed and synthesized, and their osteoclastogenesis inhibitory effects both in vitro and in vivo were evaluated. The most promising compound 13 with a 2-(dimethylamino)ethyl group inhibited markedly in vitro osteoclastogenesis as well as the bone resorption activity of osteoclasts. Compound 13 affected osteoclasts early proliferation and differentiation more than later fusion and maturation stages. In ovariectomized (OVX) mice, compound 13 can inhibit the loss of trabecular bone volume, trabecular bone number, and trabecular thickness. Moreover, compound 13 can antagonize OVX-induced reduction of serum bone resorption marker and then compensatory increase of the bone formation marker. To sum up, compound 13 has high potential to be developed into a novel therapeutic agent for treating osteoporosis in the future.

Full text of DOI:10.1021/jm502014h

Article
pubs.acs.org/jmc
Novel Pyrazole Derivatives Effectively Inhibit Osteoclastogenesis, a
Potential Target for Treating Osteoporosis
Ting-Hao Kuo,† Tzu-Hung Lin,† Rong-Sen Yang,§ Sheng-Chu Kuo,∥ Wen-Mei Fu,*,†,‡
and Hsin-Yi Hung*,⊥,#
‡
†Pharmacological Institute and Drug Research Center, College of Medicine, National Taiwan University, Taipei, 100, Taiwan
§Department of Orthopedics, College of Medicine, National Taiwan University Hospital, Taipei 100, Taiwan
∥Graduate Institute of Pharmaceutical Chemistry, China Medical University, Taichung, 404, Taiwan
⊥Chinese Medicine Research and Development Center, China Medical University Hospital, Taichung, 404, Taiwan
#School of Pharmacy, National Cheng Kung University Hospital, College of Medicine, National Cheng Kung University, Tainan, 701,
Taiwan
S
* Supporting Information
ABSTRACT: As human beings live longer, age-related diseases such as osteoporosis will
become more prevalent. Intolerant side effects and poor responses to current treatments
are observed. Therefore, novel effective therapeutic agents are greatly needed. Here,
pyrazole derivatives were designed and synthesized, and their osteoclastogenesis
inhibitory effects both in vitro and in vivo were evaluated. The most promising compound
13 with a 2-(dimethylamino)ethyl group inhibited markedly in vitro osteoclastogenesis as
well as the bone resorption activity of osteoclasts. Compound 13 affected osteoclast’s
early proliferation and differentiation more than later fusion and maturation stages. In
ovariectomized (OVX) mice, compound 13 can inhibit the loss of trabecular bone
volume, trabecular bone number, and trabecular thickness. Moreover, compound 13 can
antagonize OVX-induced reduction of serum bone resorption marker and then
compensatory increase of the bone formation marker. To sum up, compound 13 has
high potential to be developed into a novel therapeutic agent for treating osteoporosis in
the future.
not only in bone formation but also in osteoclast maturation
and bone resorption. Osteoclast formation starts from a
monocyte progenitor and requires the presence of a receptor
activator of nuclear factor kappa-B ligand (RANKL) and a
macrophage colony-stimulating factor (M-CSF). Interaction
between RANKL expressed on an osteoblast and RANK
expressed on an osteoclast precursor activates RANKL−RANK
signaling to induce bone resorption via the NF-κB pathway.
However, M-CSF, which is released from osteoblasts, acts via
the colony-stimulating factor 1 receptor on the osteoclast
precursor and leads to the differentiation of osteoclasts from
monocyte/macrophage.7−9
Since osteoclastogenesis is critical for bone resorption, many
pharmacological agents aim to suppress RANKL-induced
osteoclastogenesis, such as hormone replacement therapy and
bisphosphonates like alendronate and denosumab, a human
monoclonal antibody against RANKL.10 However, there are
intolerable side effects and inadequate response to current
therapeutic agents. A new therapeutic direction was found by
the studies on a nitric oxide (NO) donor for the treatment of
osteoporosis. Clinical data were retrieved from women taking
INTRODUCTION
■
With the increase in aging populations, fracture rates
accompanying urbanization also increase. In the United States,
the population with an elevated risk of osteoporotic fracture
contains an estimated 19% of older men and 30% of older
women, which is similar in Europe and is projected to rise
worldwide.1−3 Remaining challenges in implementing therapy
for osteoporosis still occur, even though the diagnosis is made
and a decision is made regarding treatment, which largely result
from bad compliance, intolerable side effects, and inadequate
response to current therapeutic agents.4 Therefore, new
therapeutic agents for treating osteoporosis are still a huge
need.
The homeostasis of bone metabolism consists of a
continuously balanced remodeling process by two opposed
forces: bone resorption and bone formation. Osteoclasts
derived from hematopoietic stem cells play an important role
in bone resorption, and osteoblasts derived from mesenchymal
stem cells mediate the main part in bone formation or the so-
called bone mineralization. Imbalance actions between
osteoclasts and osteoblasts will cause lots of metabolic bone
disorders, such as osteoporosis resulting from increased
osteoclast activity and decreased osteoblast activity.5,6 To
date, many studies have suggested that osteoblasts play a role
Received: December 29, 2014
© XXXX American Chemical Society
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a
Scheme 1. Synthesis of Pyrazole Derivatives 2−7 and 9−13
a
Reagent and conditions: (a) PDC, CH2Cl2; (b) H3C-PPh3Br, tBuOK, THF; (c) (i) BH3·SMe2, THF, 0 °C, N2; (ii) NaOH, H2O2, 0 °C→ r.t.; (d)
NaH, CH3I, CH2Cl2; (e) Ac2O, reflux for 6, t-BuOH, (COCl)2, CH2Cl2 for 7; (f) EDCI, DMAP, MeOH for 9, tBuOH for 10; (g) EDCI, HOBt,
NEM, CH3NHOCH3; (h) (i) (COCl)2, CH2Cl2, N2; (ii) HOCH2CH2N(CH3)2, CH2Cl2; (i) CH3MgBr, CH2Cl2.
a
Scheme 2. Synthesis of Pyrazole Derivatives 14−15
a
Reagent and conditions: (a) (i) FeCl3, CH2Cl2, ClCH2CH2Cl, reflux; (ii) PhCH2NHNH2·2HCl, NaOAc, MeOH; (iii) Pb(OAc)4, BF3·Et2O,
CH2Cl2, 0 °C; (b) KOH, MeOH.
organic nitrates, and the results showed nitrite use was
associated with increased bone mineral density (BMD) and
lower risk of hip fracture.11,12 3-(5′-Hydroxymethyl-2′-furyl)-1-
benzyl indazole is well-known for the NO-dependent soluble
guanylyl cyclase (sGC) activator and subsequently increasing
cyclic guanosine monophosphate (cGMP) formation.13 In-
spired by the studies above, we thus aim to discover a potential
osteoclastogenesis inhibitor by screening a series of small
molecules with the core structure of 3-(5′-hydroxymethyl-2′-
furyl)-1-benzyl indazole to study the structure−activity relation-
ship, action mechanism, and in vivo effects.
RESULTS AND DISCUSSION
■
Chemistry. The relationship between pyrazole structure
and antiosteoporosis activity is largely undefined. Therefore,
diversified pyrazole derivatives were designed and synthesized.
As shown in Scheme 1, 3-(5′-hydroxymethyl-2′-furyl)-1-benzyl
indazole (1) or 5′-carboxylic acid derivative (8) (courtesy of
Yungshin Pharm Ind. Co. Ltd.) were used as starting materials.
B
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Figure 1. Inhibition of osteoclastogenesis by pyrazole derivatives. (A) Bone marrow-derived preosteoclasts from male rats were seeded in 24 wells.
Different derivatives were added in the presence of 50 ng/mL RANKL and 20 ng/mL M-CSF for 5 days. TRAP staining was then performed. Nuclei
greater than 3 were counted as osteoclasts. The representative images are shown in B. Data are presented as the mean SEM (N = 6), *, p < 0.05 as
compared with the Ctrl (control).
Pyridium dichromate oxidation was applied, and 5′-carbalde-
hyde derivative 2 was obtained.14 The Wittig reaction using
methyltriphenylphosphonium bromide and potassium tert-
butoxide as base was given 5′-vinyl derivative 3. Hydro-
boration−oxidation was applied and 5′-hydroxyethyl derivative
4 was obtained. Methoxymethyl derivative 5 was obtained by
treating 1 with sodium hydride and iodomethane.15 Ester-
ification with acetic anhydride or oxalyl chloride and tert-
butanol was given 6 or 7, respectively.16 Reverse ester linkage
derivatives were synthesized using N-(3-(dimethylamino)-
propyl)-N′-ethylcarbodiimide hydrochloride (EDCI), DMAP,
and methanol for 9 or tert-butanol for 10.15,17 Weinreb amide
derivative 11 was easily obtained in high yield using EDCI, 1-
hydroxybenzotriazole hydrate (HOBt), and N,O-dimethylhy-
droxylamin. 5′-Ethanone derivative 12 was obtained via
methylmagnesium bromide substitution. Compound 13 was
synthesized from compound 8. Under inert atmosphere, oxalyl
chloride (10 equiv) was added to compound 8 (1 equiv) in
anhydrous dichloromethane at 0 °C to form an acyl chloride
intermediate. Excess oxalyl chloride was evaporated, and N,N-
dimethylethanolamine (3 equiv) was then added to the reaction
mixture to give compound 13.
in Scheme 2. Benzyl chloride and methyl 3-methyl-2-furoate
were used as starting materials and underwent Friedel−Crafts
acylation using an iron(III) chloride as catalyst. The
corresponding ketone went through a condensation reaction
with benzylhydrazine. Finally, cyclization was done by treating
Pb(OAc)4 and then BF3·Et2O to yield methyl 5-(1′-benzyl-1H-
indazol-3′-yl)-3-methylfuran-2-carboxylate (14).18 Carboxylic
acid derivative 15 was simply obtained by potassium hydroxide
reflux with 14.
Inhibition of Osteoclastogenesis by Pyrazole Deriva-
tives. In order to quickly obtain a structure−activity relation-
ship, diversified pyrazole derivatives including ester, acid,
aldehyde, alcohol, alkene, and amide were chosen for screening
osteoclastogenesis inhibitory activity. Each derivative was added
to bone marrow-derived preosteoclasts (from male rats) to
examine the inhibitory activity on osteoclastogenesis in 24
wells. Different testing compounds at 10 μM were added in the
presence of RANKL (50 ng/mL) and M-CSF (20 ng/mL) for
5 days. After 5 days, tartrate resistant acid phosphatase-stain
(TRAP-stain) was used to confirm osteoclast formation. Both
TRAP-positive and nuclei ≥3 cells were counted. Compounds
4, 6, 7, 8, 11, and 12 were found to reduce 20−25% of
osteoclast formation compared with that of the vehicle (Ctrl)
(Figure 1A), and the representative images are shown in Figure
1B. However, compound 13 markedly inhibited osteoclast
formation over 90% (Figure 1A and B). Taken together, these
3-(5′-Hydroxymethyl-2′-furyl)-1-benzyl indazole was easily
metabolized by liver microsomal enzymes (unpublished data).
Therefore, methyl substitution on the 4′ position was designed
to reduce the first-pass effect. The synthetic procedure is shown
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Figure 2. Inhibition of osteoclastogenesis by compound 13. Bone marrow-derived preosteoclasts from male rats were seeded in 24 wells. (A)
Compound 13 was added in different days in the presence of 50 ng/mL RANKL and 20 ng/mL M-CSF. Note that compound 13 (10 μM) markedly
inhibited osteoclastogenesis when incubated on days 1−5 (N = 3). The representative images are shown in B. (C) Compound 13 inhibited
osteoclastogenesis in a concentration-dependent manner (N = 3). (D) Compared with alendronate (ALN), compound 13 had similar inhibitory
action at 10 μM on osteoclastogenesis (N = 4). Data are presented as the mean SEM, *, p < 0.05 as compared with the Ctrl (control).
results suggest that the derivatives with a core structure of 3-
(5′-hydroxymethyl-2′-furyl)-1-benzylindazole can inhibit osteo-
clastogenesis. Compared with compounds 6, 7, 9, and 10, the
order of the ester bond seems to be important for activity.
From compounds 1 and 4, we can see that one more carbon
contributes some effects to the activity. Moreover, a longer
chain with more heteroatoms such as 4 and 13 seems to exert
more potent inhibitory activity. Among all of the compounds,
compound 13 with the 2-(dimethylamino)ethyl group was the
most potent.
and compound 13 was added on day 3. After 5 days, the result
showed that compound 13 inhibited two phases but exerted
much stronger effects in early phase than in late phase (Figure
2A), and the representative images are shown in Figure 2B.
Compound 13 exerted concentration-dependent inhibition of
osteoclastogenesis (Figure 2C). In addition, compound 13
exerted inhibitory activity in osteoclastogenesis similar to that
of alendronate (ALN) at 10 μM (Figure 2D). It was also found
that compound 13 at 10 μM had no cytotoxicity in primary
preosteoclasts (Figure S1, Supporting Information).
Inhibition of Osteoclastogenesis and the Bone
Resorption Activity of Osteoclasts by Compound 13.
To our current knowledge, two phases are involved in
osteoclast formation: the early phase of proliferation and
differentiation and the late phase of fusion and maturation.8,19
Therefore, compound 13 was chosen to study the mechanism
of the inhibition of osteoclastogenesis. In the presence of 50
ng/mL RANKL and 20 ng/mL M-CSF for 5 days, day 1 to day
3 was defined as the early phase, and compound 13 was added
on day 1. However, day 3 to day 5 was defined as the late phase,
It is well known that mature osteoclasts play an important
role in bone resorption. As compound 13 markedly inhibits
osteoclastogenesis, we next examined whether compound 13
inhibited the function of osteoclasts. Preosteoclasts were seeded
in Osteo Assay Plate (Corning, USA) in the presence of 50 ng/
mL RANKL and 20 ng/mL M-CSF for the resorption assay.
After 5 days of culture, compound 13 was then added for
another 3 days. It was found that compound 13 inhibited the
resorption area of osteoclasts over 75% (Figure 3A and B).
There was no significant difference in osteoclastic number
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Figure 3. Inhibition of bone resorption activity of osteoclasts by compound 13. Bone marrow-derived preosteoclasts were seeded in Osteo Assay
Plate 24 wells (Corning, USA) in the presence of 50 ng/mL RANKL and 20 ng/mL M-CSF for 5 days. RANKL and M-CSF were then removed, and
compound 13 (10 μM) was added for another 3 days. (A) Compared with the control, compound 13 significantly inhibited the resorption activity of
osteoclasts (N = 4). The representative images are shown in B. (C) There was no significant difference in osteoclast number on day 8 between the
control and compound 13 (N = 4). The representative images are shown in D. Data are presented as the mean SEM, *, p < 0.05 as compared with
the Ctrl (control).
Figure 4. Inhibition of RANKL-induced NF-κB activation in RAW264.7 cells by compound 13. RAW264.7 cells were treated with RANKL (50 ng/
mL) for several time intervals. Western blot analysis revealed that treatment with RANKL increased the phosphorylation of IKK and P65 beginning
from 5 min. Pretreatment with compound 13 (10 μM) markedly inhibited RANKL-induced phosphorylation of IKK and P65.
between the control and compound 13 on day 8 (Figure 3C
and D). These results indicate that compound 13 also exerted
direct bone resorption inhibitory activity.
Moreover, we also used bone marrow-derived preosteoclasts
from male ICR mice to examine the effect of compound 13 on
osteoclastogenesis. As shown in Figure S2 (Supporting
Information), compound 13 (10 μM) also exerted marked
inhibition on osteoclastogenesis. We also examined the effect of
pyrazole compounds on bone nodule formation of osteoblasts.
Compound 13 did not exert inhibitory action on osteoblasts
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Figure 5. Compound 13 inhibits ovariectomy (OVX)-induced bone loss in tibia. Female mice were ovariectomized. Compound 13 (Cmpd 13) was
administered at 10 mg/kg (s.c.), 5 days/week. Tibiae were removed from mice on day 30 and fixed in 4% paraformaldehyde, and μCT analysis was
then performed. Note that compound 13 antagonized OVX-induced reduction of bone volume (A) and the decrease of trabecular bone number (B)
and trabecular thickness (C). Radiographs are shown in D. (E) H&E staining was performed. Note that OVX reduced the trabecular number, which
was antagonized by compound 13 treatment. Data are presented as the mean SEM (N = 9 ~ 10), *, p < 0.05 as compared with Sham. #, p < 0.05
as compared with the OVX group.
(Figure S3A, Supporting Information). Furthermore, com-
pound 13 had no effect on the cell viability of osteoblasts
(Figure S3B, Supporting Information). To sum up, compound
13 significantly inhibited osteoclastogenesis and the bone
resorption activity of osteoclasts and had no effect on the bone
nodule formation of osteoblasts.
Compound 13 Inhibits RANKL-Induced NF-κB Activa-
tion. Since the NF-κB pathway plays an important role in
RANKL-RANK signaling, we then examined the potential
mechanisms of compound 13 on the NF-κB signaling pathway.
Since we have found that compound 13 also inhibited
osteoclast formation in macrophage-like RAW 264.7 cells
(data not shown), we then used RAW cells to investigate the
effect of compound 13 on NF-κB signaling. RAW cells were
treated with RANKL at 50 ng/mL to activate NF-κB signaling.
RANKL rapidly phosphorylated the inhibitor of κB kinase (Ikk)
and also P65. As shown in Figure 4, significant phosphorylation
of IKK and P65 was observed within 15 min following the
treatment of RANKL. Compound 13 at 10 μM significantly
inhibited RANKL-induced NF-κB activation. Therefore, the
decrease of the RANKL-induced NF-κB pathway may be
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involved in the inhibitory action of compound 13 in
osteoclastogenesis.
Compound 13 Inhibits Osteoporosis in Ovariectom-
ized OVX Mice. Ovariectomy (OVX) in female rodents is a
well-established animal model in osteoporosis. OVX mice were
characterized by remarkably decreased trabecular bone volume
relative to the tissue volume (BV/TV %), trabecular bone
number (Tb.N), and trabecular thickness (Tb.Th) in tibiae
compared with those sham-operated (Sham) mice.19 Com-
pound 13 was evaluated for its potency against osteoporosis in
vivo using bilateral ovariectomy and sham-operated female ICR
mice (12 weeks-old). Two days after surgery, the mice were
subcutaneously injected with compound 13 (10 mg/kg, 5 days/
week) or vehicle for 30 days. After 30 days, tibiae were taken
out from mice and fixed in 4% paraformaldehyde and
microcomputed tomography (μ-CT) analysis was then
performed. BV/TV %, Tb.N, and Tb.Th were used as the
osteoporosis parameters. Our findings revealed that BV/TV,
trabecular bone number, and trabecular thickness were more
significantly reduced, which were antagonized by compound 13
treatment (Figure 5A−D). H&E staining was also performed. It
was also found that the trabecular bone decreased in OVX
mice, which was antagonized by compound 13 treatment
(Figure 5E).
Compound 13 Antagonizes OVX-Induced Change of
Serum Bone Resorption and Bone Formation Markers.
Ovariectomized and sham-operated female ICR mice were
sacrificed on day 30, and blood samples were collected for
analysis. A RatLaps EIA kit was used to detect the serum level
of C-terminal telopeptides of type-I collagen (CTx), a bone
resorption marker (Figure 6A). A Rat-MID osteocalcin EIA kit
was used to detect the serum level of osteocalcin, a bone
formation marker (Figure 6B). It was found that ovariectomy
increased both serum markers of CTx and osteocalcin, which
can be significantly antagonized by compound 13.
Figure 6. Compound 13 antagonizes OVX-induced change of serum
levels of bone resorption and bone formation markers. Female mice
were ovariectomized. Mice were then sacrificed 4 weeks after
ovariectomy, and the serum was collected. (A) Determination of C-
terminal telopeptides of type-I collagen (CTx) in serum using a
RatLaps EIA kit (N = 8). (B) Determination of osteocalcin in serum
using a Rat-MID osteocalcin EIA kit (N = 9). Note that OVX
increased both serum markers of CTx and osteocalcin, which was
antagonized by compound 13 (Cmpd 13) treatment. Data are
presented as the mean SEM, *, p < 0.05 as compared with Sham. #,
p < 0.05 as compared with the OVX group.
visualized under UV 254−366 nm. The following adsorbant was used
for column chromatography: silica gel 60 (Merck, particle size 0.063−
0.200 mm). NMR spectra were obtained on a Bruker Avance III 500
FT-NMR spectrometer in CDCl3. High resolution MS spectra were
measured either in the instruments center of National Chung Hsing
University (JEOL JMS-700 spectrometer) or National Tsing Hua
University (FINNIGAN, MAT-95XL HRMS). All tested compounds
have above 95% purity by HPLC using a Shimadza LC-20AT system
and a photodiode array detector SPD-M-20A. The NMR spectra of all
known compounds have NMR data identical to the reported
compounds.
1-Benzyl-3-(5′-ethenylfuran-2′-yl)indazole (3). To the solution of
methyltriphosphonium bromide (714 mg, 2 mmol) in anhydrous THF
(4 mL), t-BuOK (225 mg, 2 mmol) was added, and the solution was
turned to yellow. After 10 min, 5-(1-benzyl-1H-indazol-3-yl)furan-2-
carbaldehyde (300 mg, 1 mmol) in anhydrous THF (2 mL) was added
to the previous solution. After the reaction was completed, saturated
ammonia chloride solution was added to stop the reaction. THF was
evaporated, and the mixture was partitioned with CH2Cl2, washed with
brine, and dried over MgSO4. The desired compound was obtained by
flash chromatography using a hexane−EtOAc system (37% yield,
orange oil). 1H NMR (200 Hz, CDCl3) δ 8.13(d, J = 8 Hz, 1H), 7.15−
7.40 (m, 8H), 6.90 (d, J = 3.4 Hz, 1H), 6.59 (dd, J = 11.0, 17.0 Hz,
1H), 6.41 (d, J = 3.4 Hz, 1H), 5.81 (d, J = 17.0 Hz, 1H), 5.62 (s, 2H),
5.22 (d, J = 11.0 Hz, 1H). HRMS [ESI]+ calculated for C20H16N2O
300.1263; found [M − 1]+ 299.0688.
CONCLUSIONS
■
With the aging of the population, diseases related with aging
such as osteoporosis will become more and more critical.
Patients suffer intolerable side effects and poor responses to
current treatments. Therefore, novel and effective therapeutic
agents are in great need. Here, a series of pyrazole derivatives
were designed, synthesized, and evaluated for their osteoclasto-
genesis inhibitory effects both in vitro and in vivo. The most
promising compound 13 with the 2-(dimethylamino)ethyl
group showed significant in vitro osteoclastogenesis inhibitory
effects and retainment of BV/TV, trabecular bone number, and
trabecular thickness in OVX mice. Moreover, compound 13
affected osteoclast proliferation and differentiation more than
later fusion and maturation stages. In addition, compound 13
also exerted inhibitory action on the bone resorption activity of
osteoclasts. From serum analysis in OVX mice, compound 13
can antagonize OVX-induced reduction of serum bone
resorption markers and then compensatory increase of bone
formation markers. To sum up, compound 13 has high
potential to be developed as a novel therapeutic agent for
treating osteoporosis in the future.
EXPERIMENTAL SECTION
1-Benzyl-3-(5′-ethoxyfuran-2′-yl)-1H-indazole (4). Hydrobora-
tion−oxidation was performed to obtain the desired compound. To
the solution containing 3 (243 mg, 0.81 mmol) in anhydrous THF (6
mL) at 0 °C under inert atmosphere, 0.81 mL of borane dimethyl
sulfide complex solution (2 M in THF) was added. After 3 h, sodium
■
Chemistry. All of the solvents and reagents were obtained
commercially and used without further purification. The progress of
all of the reactions was monitored by TLC on precoated silica gel 60
F254 plates of thickness 0.25 mm (Merck). The chromatograms were
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hydroxide solution [421 mg in EtOH/H2O (65/32)] followed by
hydrogen peroxide (30% wt, 2 mL) was added, and the reaction was
stopped after 4 h. The mixture was partitioned with water and
dichloromethane and dried over MgSO4. The desired compound was
obtained by flash chromatography using a hexane−EtOAc system (7%
yield, colorless amorphous). 1H NMR (200 Hz, CDCl3) δ 8.01 (d, J =
8.0 Hz, 1H), 7.14−7.33 (m, 8H), 6.83 (d, J = 3.2 Hz, 1H), 6.28 (d, J =
3.2 Hz, 1H), 5.62 (s, 2H), 3.97 (t, J = 6.2 Hz, 2H), 3.03 (t, J = 6.2 Hz,
2H). HRMS [ESI]+ calculated for C20H18N2O2 318.1368; found [M +
1]+ 319.1441.
tert-Butyl 5-(1-benzyl-1H-indazol-3-yl)furan-2-carboxylate (10).
To a solution of tert-BuOH (0.03 mL, 0.3 mmol), DCC (49 mg, 0.23
mmol), and DMAP (29 mg, 0.23 mmol) in dichloromethane,
compound 8 (50 mg, 0.16 mmol) was added and stirred overnight.
Water and 1 N HCl solution were added, and the mixture was
partitioned with dichloromethane three times, washed with brine, and
dried over MgSO4. The residue was subjected to flash chromatography
using a hexane−EA system (16% yield, colorless amorphous). 1H
NMR (400 Hz, CDCl3) δ 8.27 (d, J = 7.8 Hz, 1H), 7.19−7.35 (m,
9H), 6.98 (d, J = 7.6 Hz, 1H), 5.63 (s, 2H), 1.23 (s, 9H). HRMS
[ESI]+ calculated for C23H22N2O3 374.1630; found [M+1]+ 375.1704.
5-(1-Benzyl-1H-indazol-3-yl)-N-methoxy-N-methylfuran-2-car-
boxamide (11). To a solution of compound 8 (955 mg, 3 mmol) in 5
mL of anhydrous CH2Cl2, one drop of DMF was added to increase
solubility, and oxalyl chloride (2.54 mL, 30 mmol) was added slowly at
0 °C and inert atmosphere for 30 min. Excess oxalyl chloride was
removed. Anhydrous CH2Cl2 (10−15 mL) was added to the residue,
and 2-dimethylaminoethanol (1 mL, 10 mmol) was added and stirred
overnight. After the usual workup, the residue was subjected to flash
chromatography using a dichloromethane−methanol system (47%
yield, light yellow, oily). 1H NMR (200 Hz, CDCl3) δ 8.07 (d, J = 8.2
Hz,1H), 7.17−7.36 (m, 9H), 6.96 (d, J = 3.3 Hz, 1H), 5.62 (s, 2H),
3.53 (dd, J = 5.8, 12.0 Hz, 2H), 2.56 (t, J = 6.0 Hz, 2H), 2.32 (s, 6H).
HRMS [ESI]+ calculated for C23H23N3O3 389.1739; found [M]+
389.1731.
2-(Dimethylamino)ethyl 5-(1-benzyl-1H-indazol-3-yl)furan-2-
carboxylate (13). To a solution containing compound 8 (955 mg, 3
mmol) in CH2Cl2 (5 mL) and several drops of DMF and 1,1′-
carbonyldiimidazole (CDI, 648 mg, 4 mmol) were added slowly. After
CO2 release, N,O-dimethylhydroxylamine (244 mg, 4 mmol) was
added and stirred overnight. Water was added to stop the reaction.
After partitioning with dichloromethane and drying over MgSO4, the
residue was subjected to flash chromatography to get the desired
compound (25% yield, colorless, amorphous). 1H NMR (200 Hz,
CDCl3) δ 8.31 (d, J = 8.0 Hz, 1H), 7.20−7.34 (m, 9H), 7.00 (d, J =
3.6 Hz, 1H), 5.62 (s, 2H), 3.80 (s, 3H), 3.39 (s, 3H). HRMS [ESI]+
calculated for C21H19N3O3 361.1426; found [M]+ 361.1422.
Methyl 5-(1-benzyl-1H-indazol-3-yl)-3-methylfuran-2-carboxy-
late (14). To a solution of benzyl chloride (10 g, 71.4 mmol) in
CH2Cl2 (50 mL) and ClCH2CH2Cl (50 mL), methyl 3-methyl-2-
furoate (5 g, 35.7 mmol) and anhydrous ferric chloride (5 g, 30.8
mmol) were added. The reaction mixture was then heated under
refluxing for 2 h, cooled, and quenched with water to stop the reaction.
After partitioning with dichloromethane and drying over MgSO4, the
residue was subjected to flash chromatography to the corresponding
ketone (1.75 g, 20% yield). The intermediate (1.19 g, 4.87 mmol) was
refluxed with sodium acetate (804 mg, 9.79 mmol) and hydrazine·
2HCl (3.05 g, 15.64 mmol) in MeOH for 5 h. MeOH was removed by
a vacuole. Water was added, and then, the mixture was partitioned
with DCM. Silica gel was applied to get the imine intermediate (537
mg, 31% yield). The intermediate was dissolved in 50 mL of
dichloromethane and added to the solution containing lead
tetraacetate (2.0 g, 4.63 mmol) and boron trifluoride diethyl etherate
(8 mL) at 0 °C and allowed to go through the cyclization reaction for
30 min. When the reaction was completed, the reaction mixture was
poured into ice water to stop the reaction and then was extracted with
dichloromethane. The organic layer was recovered, washed with water,
5% sodium carbonate solution until neutral, dried over MgSO4, and
filtered. The solvent of the filtrate was evaporated under reduced
pressure, and the residue was purified by column chromatography
(silica gel, dichloromethane/n-hexane = 2:1) to give the final product
(177 mg, 33% yield, colorless amorphous). 1H NMR (200 Hz, CDCl3)
δ 7.98−8.02 (m, 2H), 7.2−7.43 (m, 8H), 5.47 (s, 2H), 3.90 (s, 3H),
2.30 (s, 3H). HRMS [ESI]+ calculated for C21H18N2O3 346.1317;
found [M + 1]+ 347.1391.
5-(1-Benzyl-1H-indazol-3-yl)-3-methylfuran-2-carboxylic Acid
(15). Compound 14 was refluxed in MeOH and KOH(aq) until it
disappeared. The solvent was evaporated, and 1 N HCl aqueous
solution was added. Dichloromethane and ethyl acetate were used for
1
partitioning. H NMR (200 Hz, CH3OH-d4) δ 8.04 (d, J = 3.2 Hz,
1H), 7.24−7.50 (m, 8H), 5.57 (s, 2H), 2.38 (s, 3H). HRMS [ESI]+
calculated for C20H16N2O3 332.1161; found [M]+ 332.1167.
Materials for Biological Studies. Recombinant mouse RANKL
(462-TEC: amino acids 158−317 expressed in Esherichia coli) and
mouse M-CSF were purchased from R&D System (Minneapolis, MN,
USA). RANKL and M-CSF were stored in −20 °C. Tartrate resistant
acid phosphatase (TRAP) staining kit, 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT), β-glycerophosphate, and L-
ascorbic acid were obtained from Sigma-Aldrich (St. Louis, MO,
USA). Alendronate was from Calbiochem. α-MEM was from Gibco
Invitrogen (Carlsbad, CA, USA). Fetal bovine serum was from
Biological Industries (Kibbutz Beit Haemek, Israel). A murine
monocytic cell line, RAW264.7, was obtained from American Type
Culture Collection (Manassas, VA). Rabbit polyclonal antibody for
IKKα/β, phosphor-IKKα/β, phosphor-NFκB p65, NFκB p65, and
goat anti-mouse or anti-rabbit secondary antibody conjugated with
horseradish peroxidase were from Santa Cruz Biotechnology (Santa
Cruz, CA, USA). Mouse monoclonal antibody for actin was from
Merck-Millipore (Bedford, MA, USA).
Osteoclastogenesis. Bone marrow cells derived from tibiae and
femurs of 8−10 week old male Sprague−Dawley rats or ICR mice
were cultured with α-MEM. After incubation for 24 h, the
hematopoietic cells (nonadherent cells) were collected and plated at
106 cells/well in 24 wells in the presence of RANKL (50 ng/mL), M-
CSF (20 ng/mL), and the testing compounds for 5 days. After 5 days,
the cells were fixed in PBS containing 4% paraformaldehyde.
Osteoclast formation was confirmed by TRAP staining. Cells were
then treated with the TRAP staining kit (70 μg/mL Fast Garnet GBC
base solution, 125 μg/mL naphthol AS-B1 phosphoric acid, 100 mM
acetate, and 6.7 mM tartrate) at 37 °C for 1 h. Both TRAP-positive
and nuclei ≥3 were counted as osteoclasts.
Resorption Assay. Bone marrow-derived preosteoclasts from male
rats were seeded at 106 cells/well in Osteo Assay Plate 24 wells
(Corning, USA) in the presence of 50 ng/mL RANKL and 20 ng/mL
M-CSF for 5 days. After 5-days’ culture, RANKL and M-CSF were
then removed, and the testing compound was added for another 3
days. The wells were washed with 1 N sodium hydroxide solution to
remove the cells. The resorbed areas on the wells were photographed
with an inverted microscope (Olympus 4J18950-DP70, Tokyo, Japan)
and were quantified using ImageJ software, version 1.48 (US National
Institutes of Health, Bethesda, MD, USA).
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium Bro-
mide (MTT) Assay. Bone marrow-derived preosteoclasts (non-
adherent cells) were seeded at 106 cells/well in 24 wells in the
presence of 20 ng/mL M-CSF for 3 days. After 3 days’ culture, the
testing compound was added for another 5 days. After washing, 200
μL of α-MEM containing 0.5 mg/mL of MTT was added to each well.
Cells were incubated at 37 °C for 60 min. The blue crystals formed in
viable cells were solubilized with 500 μL of DMSO. The absorbance of
each well was measured at 550 nm. Osteoblasts were plated in 24 wells
for 24 h and then added with different testing compounds for another
24 h. After washing, 200 μL of α-MEM containing 0.5 mg/mL of
MTT was added to each well. Cell viability was assayed as mentioned
above.
Bone Nodule Formation. Cranium cultured cells (preosteoblasts)
were seeded in 24 wells and cultured with α-MEM containing 10%
FBS. Ten millimolar β-glycerophosphate and 50 μg/mL L-ascorbic
acid were then added in the presence or absence of testing compounds
for 2 weeks to form the bone mineralized nodule. After 2 weeks, the
cells were fixed in ice-cold 75% (v/v) ethanol for 30 min and stained
H
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Journal of Medicinal Chemistry
Article
with 40 mM alizarin red solution (pH 4.2). The alizarin red-S in
samples was quantified by measuring absorbance at 550 nm and
calculated according to a standard curve.
AUTHOR INFORMATION
■
Corresponding Authors
*(W.-M.F.) Tel: +886-2-23123456, ext. 88319. E-mail:
wenmei@ntu.edu.tw.
*(H.-Y.H.) Tel: +886-6-2353535, ext. 6803. E-mail:
z10308005@email.ncku.edu.tw.
Ovariectomy-Induced Osteoporosis. All protocols complied
with institutional guidelines and were approved by the Animal Care
Committees of Medical College, National Taiwan University. Twelve
week old female ICR mice were used in the animal study. Mice were
ovariectomized bilaterally under isoflurane anesthesia, and control
mice were sham-operated for comparison. Mice were all kept under
controlled conditions at room temperature (22 1 °C) and a 12 h
light−dark cycle. The body weights of mice were recorded weekly.
Compound 13 was administered (s.c.) at 10 mg/kg, 5 days/week for
30 days.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The investigation was supported by research grants from
Ministry of Science and Technology of Republic of China
(MOST 103-2320-B-006-046-MY2) awarded to H.-Y.H.
Western Blot. RAW264.7 cells were plated in 6 wells. After
reaching confluence, cells were incubated with compound 13 (10 μM)
for 2 h and treated with RANKL (50 ng/mL) for different time
intervals. Cells were then washed with cold PBS and lysed for 30 min
at 4 °C with lysis buffer as described previously.3 Equal protein (30
μg) was applied per lane, and electrophoresis was performed under
denaturing conditions on a 10% SDS gel and transferred to an
immobilon-P (PVDF) membrane (Merck-Millipore, Bedford, MA,
USA). The blots were blocked with 5% nonfat milk in TBS-T (0.5%
Tween 20 in 20 mM Tris and 137 mM NaCl) for 1 h at room
temperature and then probed with antibodies against specific antigens
(1:1,000) at 4 °C overnight. After three washes by TBS-T, the blots
were subsequently incubated with goat anti-rabbit or anti-mouse
peroxidase-conjugated secondary antibody (1:10,000) for 1 h at room
temperature. The blots were visualized by enhanced chemilumines-
cence using Amersham Hyperfilm ECL (GE Healthcare, Upland, CA,
USA) or Biospectrum Imaging System (UVP, Upland, CA, USA).
Microcomputed Tomography (μCT) Analysis. Tibiae were
removed from mice on day 30 and fixed by 4% paraformaldehyde, and
μCT analysis was then performed. Fixed bones were subjected to X-
ray microtomography by using Skyscan 1076 (SKYSCAN, Kontich,
Belgium). Scanning was done at 40 kV and 600 μA, and the rotation
step was 0.3° per image. The images were collected at a resolution of 9
μm/pixel for isolated tibiae. Reconstruction of sections was carried out
with a GPU-based scanner software (GPU-NRecon). Quantification of
trabecular bone morphometric indexes was performed 0.5−1.5 mm
distal to the growth plate of the proximal ends of tibia. The analysis
was performed by scanner software (CTAn). Trabecular morphology
was described by measuring bone volume fraction (bone volume/
tissue volume, BV/TV), trabecular number (Tb.N), and trabecular
thickness (Tb.Th). The 3D images were obtained with scanner
software (CTvox).
Bone Histomorphometry. Tibiae were removed from mice on
day 30 and fixed by 4% paraformaldehyde at 4 °C for 48 h. Tibiae were
then decalcified with 10% Na2EDTA at 4 °C for 14 days, dehydrated
in increasing concentrations of ethanol, and embedded in paraffin. The
serial histological sections were cut longitudinally (4 μm) and stained
with Meyer’s hematoxylin−eosin solution.
Measurement of the Serum Levels of Bone Resorption and
Bone Formation Markers. Mice were sacrificed on day 30 after
ovariectomy, and blood samples were collected for the preparation of
serum samples. Serum samples were prepared by centrifuging blood
(1500g for 15 min) with vacutainer tubes (Becton Dickinson, Franklin
lakes, NJ, USA). C-Terminal telopeptides of type-I collagen (CTx), a
bone resorption marker, in serum was determined by using a RatLaps
EIA kit, and osteocalcin, a bone formation marker, in serum was
evaluated by using a Rat-MID osteocalcin EIA kit. ELISA kits were all
from Immunodiagonstic Systems Ltd. (Boldon Colliery, Tyne & Wear,
United Kingdom).
ABBREVIATIONS USED
■
OVX, ovariectomized; RANKL, receptor activator of nuclear
factor kappa-B ligand; M-CSF, macrophage colony-stimulating
factor; RANK, receptor activator of nuclear factor kappa-B; NF-
κB, nuclear factor kappa-B; NO, nitric oxide; BMD, bone
mineral density; sGC, soluble guanylyl cyclase; cGMP, cyclic
guanosine monophosphate; TRAP-stain, tartrate resistant acid
phosphatase-stain; BV/TV %, trabecular bone volume relative
to the tissue volume; Tb.N, trabecular bone number; Tb.Th,
trabecular thickness; CTx, C-terminal telopeptides of type-I
collagen
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* Supporting Information
Compound 13 has no inhibition of osteoblast and MTT results.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/jm502014h.
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