Synthesis and biological evaluation of heterocyclic ring-fused betulinic acid derivatives as novel inhibitors of osteoclast differentiation and bone resorption

Article abstract of DOI:10.1021/jm201540h

A series of betulinic acid (BA) derivatives were designed and synthesized by introducing various fused heterocyclic rings at C-2 and C-3 positions. Their inhibitory effects of RANKL-induced osteoclastogenesis were evaluated by using a cell-based tartrate-resistant acid phosphatase (TRAP) activity assay. To our delight, most of these compounds exhibited a dramatic increase in inhibitory potency, compared with BA. The most potent compound, 20, showed 66.9% inhibition even at the low concentration of 0.1 μM, which was about 200-fold more potent than the lead compound BA. What's more, the cytotoxicity assay on RAW264.7 suggested that the inhibition of 20 on osteoclast differentiation did not result from its cytotoxicity. The primary mechanistic study indicated that 20 could inhibit osteoclastogenesis-related marker gene expression levels of cathepsin K and TRAP. More importantly, 20 could attenuate bone loss of ovariectomy mouse in vivo. Therefore, these BA derivatives could be used as potential leads for the development of a new type of antiosteoporosis agent.

Full text of DOI:10.1021/jm201540h

Article
pubs.acs.org/jmc
Synthesis and Biological Evaluation of Heterocyclic Ring-Fused
Betulinic Acid Derivatives as Novel Inhibitors of Osteoclast
Differentiation and Bone Resorption
Jun Xu,†,∥ Zhenxi Li,§,∥ Jian Luo,§,∥ Fan Yang,† Ting Liu,† Mingyao Liu,*,§ Wen-Wei Qiu,*,‡
and Jie Tang*,†
†Shanghai Engineering Research Center for Molecular Therapeutics and New Drug Development, East China Normal University,
Shanghai 200062, China
‡Institute of Medicinal Chemistry and Department of Chemistry, East China Normal University, Shanghai 200062, China
§Shanghai Key Laboratory of Regulatory Biology, Institute of Biomedical Sciences and School of Life Sciences, East China Normal
University, Shanghai 200241, China
S
* Supporting Information
ABSTRACT: A series of betulinic acid (BA) derivatives were designed and synthesized by introducing various fused heterocyclic
rings at C-2 and C-3 positions. Their inhibitory effects of RANKL-induced osteoclastogenesis were evaluated by using a cell-
based tartrate-resistant acid phosphatase (TRAP) activity assay. To our delight, most of these compounds exhibited a dramatic
increase in inhibitory potency, compared with BA. The most potent compound, 20, showed 66.9% inhibition even at the low
concentration of 0.1 μM, which was about 200-fold more potent than the lead compound BA. What’s more, the cytotoxicity assay
on RAW264.7 suggested that the inhibition of 20 on osteoclast differentiation did not result from its cytotoxicity. The primary
mechanistic study indicated that 20 could inhibit osteoclastogenesis-related marker gene expression levels of cathepsin K and
TRAP. More importantly, 20 could attenuate bone loss of ovariectomy mouse in vivo. Therefore, these BA derivatives could be
used as potential leads for the development of a new type of antiosteoporosis agent.
treatment of associated pathologies. Estrogen loss in women is
associated with elevated bone resorption casused by a rise in
osteoclast numbers. Estrogen-replacement therapy (ERT) has
long been considered the first line therapy for preventing
postmenopausal osteoporosis in women.7 However, ERT
increases the risk of breast cancer, stroke, heart attack, and
clots.8 Because of these estrogen-like side effects, ERT is only
used for short durations (less than 3 years). Bisphosphonates
(BPs) are widely used agents in the management of
osteoporosis by prevention of excessive osteoclast-mediated
bone resorption, owing to their ability to inhibit osteoclast
activity.9 However, taking BPs can cause severe renal toxicity
and osteonecrosis of the jaw; moreover, oral administration of
INTRODUCTION
■
Osteoporosis is a serious public health problem, particularly
among postmenopausal women, which is characterized by
reduced bone mass and increased risk of fractures. Human bone
is a continuously renewed organ that maintains its quality
through a delicate balance between osteoclast-mediated bone
resorption and osteoblast-mediated bone formation.1 Abnor-
mally increased osteoclastic bone resorption plays a key role in
the pathogenesis of osteoporosis, which is associated with
postmenopause,2 advancing age,3 tumors,4 infections,5 and
inflammatory response.6 Efforts have been primarily concen-
trated on the development of drugs that block bone resorption
through decrease of the formation and activity of osteoclasts or
promotion of bone formation through increase of the formation
and activity of osteoblasts. Although various agents are available
for the treatment of osteoporosis, few are ideal for the
Received: November 15, 2011
Published: March 21, 2012
© 2012 American Chemical Society
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a
Scheme 1. Synthesis of Pyrazine and Thiazole Derivatives
a
(a) IBX, DMSO, rt, 0.5 h (90%); (b) sulfur, ethylenediamine, morpholine, reflux, 4 h (31%); (c) 5,5-dibromomeldrum’s acid, Et2O, rt, 12 h; (d)
thiourea or thioacetamide, EtOH, reflux, 3 h (57% for 4 and 55% for 5 over two steps).
BPs is complicated by poor bioavailability and poor gastro-
intestinal tolerability.10 Parathyroid hormone (PTH), a
polypeptide, was noted to stimulate bone formation by
increasing osteoblast numbers and activity.11 However, the
recommended duration of PTH therapy is relatively short (2
years in the United States and 18 months in Europe) because of
its increased incidence of osteosarcoma.12 Thus, the develop-
ment of new alternative agents for the treatment of
osteoporosis is in considerable demand.
Nowadays, a great variety of antiosteoporosis drugs are
available; however, the most relatively successful treatments of
osteoporosis are primarily based on antiresorptive agents by
inhibition of osteoclast differentiation.13 In recent years,
researchers have clearly uncovered that the receptor activator
of NF-κB (RANK), its ligand (RANKL), and the decoy
receptor osteoprotegerin (OPG) are key regulators of
osteoclastic bone resorption in vitro and in vivo.14 When the
key cytokine RANKL (secreted by osteoblastic cells) binds to
RANK (expressed on the surface of osteoclasts and osteoclast
precursors), it promotes the differentiation of osteoclasts
precursors into full mature, multinucleated, and functional
osteoclasts. The antiosteoporosis drugs development has been
improved greatly by discovery of RANK/RANKL/OPG
signaling pathway in recent decades.
Natural products play a major role in drug discovery, and
nearly half of the new drugs introduced into the market in the
past 2 decades are natural products or their derivatives.15 In
searching for new types of antiosteoclast formation chemical
entities, researchers have found that some natural compounds,
especially triterpenoids, could inhibit osteoclastogenesis, such
as acetyl-11-keto-β-boswellic acid (AKBA) from ayurvedic
therapeutic plant Boswellia serrata,16 25-acetylcimigenol xylo-
pyranoside (ACCX) from black cohosh,17 oleanolic acid
derivatives,18 and ganoderic acid DM.19 Recently, we also
found maslinic acid, a natural pentacyclic triterpene acid that
can suppress osteoclastogenesis and prevent ovariectomy-
induced bone loss by regulating RANKL-mediated NF-kB
and MAPK signaling pathways.20 Betulinic acid (BA) is also a
natural pentacyclic triterpenoid, which is found in many plant
species and is particularly abundant in plants of the genus
Sambucus, for example, Sambucus williamsii Hance (SWH). In
China, SWH is a folk medicine with a long history of being
used for treatment of bone fractures and joint diseases, and the
present study investigates that SWH extract can increase bone
mass and bone stress in ovariectomized rats by increasing the
OPG/RANKL mRNA ratio.21 BA has also been reported to
exhibit a variety of biological activities, such as anti-
inflammatory,22 anti-HIV,23 anticancer,24 and antioxidant
activities, etc.25 Recently, Chiou et al. also reported that BA
increases alkaline phosphatase (ALP) activity and calcium
nodule formatiom. Furthermore, it increases the OPG/RANKL
ratio to repress bone catabolism without obvious effect on
osteoblastogenesis.26
Considering the positive effects of SWH and BA on bone
fracture and bone loss, BA is very likely to exhibit inhibitory
effects on osteoclast differentiation. To our best knowledge, the
inhibitory effects of BA on osteoclast cells have not been
investigated to date. Thus, we initially screened the activity of
BA in osteoclastogenesis and we were pleased to find that BA is
a moderate osteoclast differentiation inhibitor (IC50 ≈ 20 μM).
Therefore, we considered BA as a lead compound for further
development of novel antiosteoporosis agents. It is well-known
that pentacyclic triterpenes including BA are too hydrophobic
to have reasonable water solubility and related pharmacokinetic
properties. Thus, in order to find more potent osteoclasto-
genesis inhibitors with better water solubility, we designed and
synthesized a series of heterocyclic ring-fused BA derivatives at
C-2 and C-3 and biological activities were evaluated in this
report. The inhibition of RANKL-induced osteoclast differ-
entiation in RAW264.7 cells of BA derivatives were tested in
vitro. Results showed that compound 20 was the most potent
inhibitor. The effects on cytotoxicity and reduction of marker
genes on osteoclasts precursor of 20 were then investigated.
The osteoprotective effect of the most promising BA derivative
20 was also evaluated by bone antiresoption in ovariectomized
rats in vivo.
RESULTS AND DISCUSSION
■
Synthesis of Inhibitors. A series of BA derivatives with
heterocyclic rings (pyrazine, pyrimidine, indole, thiazole,
isoxazolone, isoxazole, thiadiazole, and pyrazole) fused at C-2
and C-3 positions were synthesized (Scheme 1−3) based on
our previous research.27
The synthesis of pyrazine and thiazole derivatives is outlined
in Scheme 1. Oxidation of BA with 2-iodoxybenzoic acid (IBX)
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a
Scheme 2. Synthesis of 5(4H)-Isoxazolone and Isoxazole Derivatives
a
The X shown is a double bond unless otherwise stated. Reagents and conditions: (a) H2 (1 atm), Pd/C, THF, MeOH, rt, 12 h (94%); (b)
RCOOEt (R = −CF3, −H, −COOEt), MeONa, THF, rt, 5 h (7, 9, 10, 11) or RCOOEt (R = −OEt), NaH, THF, 60 °C, 5 h (8); (c) NH2OH·HCl,
EtOH, reflux, 3 h (50% for 12, 75% for 13, and 70% for 14 and 15 over two steps); (d) LiOH, EtOH, rt, 15 h (80%).
a
Scheme 3. Synthesis of Pyrazole Derivatives
a
The X shown is a double bond unless otherwise stated. Reagents and conditions: (a) NH2NH2·HCl, EtOH, reflux, 3 h (52% for 17, 50% for 18,
70% for 20, 70% for 21, and 62% for 22 over two steps); (b) PhNHNH2·HCl, EtOH, reflux, 3 h (73%); (c) LiOH, EtOH, rt, 15 h (81%).
gave intermediate 1, which was then converted to pyrazine
derivative 2 in the presence of ethylenediamine and sulfur in
morpholine.28 Thiazole derivatives of 4 and 5 were obtained by
bromination of 1 with 5,5-dibromomeldrum’s acid, followed by
heterocyclization with thiourea or thioacetamide in EtOH.
The synthesis of 5(4H)-isoxazolone and isoxazole derivatives
is outlined in Scheme 2. Hydrogenation of 1 with Pd/C under
H2 afforded 6. Intermediates 7, 8, 9, 10, 11 were obtained by
Claisen condensation of 1 or 6 with corresponding esters in the
presence of MeONa. 5(4H)-Isoxazolone derivative 12 was
prepared by reaction of 8 with hydroxylamine hydrochloride in
EtOH. Treatment of 9, 10, and 11 with hydroxylamine
hydrochloride in EtOH gave isoxazole derivatives 13, 14, and
15, respectively. Hydrolysis of 15 with LiOH in EtOH afforded
16.
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Chart 1. Single-Crystal X-ray Diffraction Analysis of 20
a
Scheme 4. Synthesis of 1,2,3-Thiadiazole, Pyrimidine, and Methyl Esterification Derivatives
a
Reagents and conditions: (a) CH3I, K2CO3, DMF, 40 °C, 5 h (90%); (b) NH2NHCONH2.HCl, Et3N, THF, rt, 15 h; (c) SOCl2, reflux, 4 h (45%
over two steps); (d) LiI, DMF, 140 °C, 16 h, 71%; (e) HCOOEt, MeONa, THF, rt, 5 h; (f) NH2NH2·HCl, EtOH, reflux, 3 h (66%); (g)
NH2OH·HCl, EtOH, reflux, 3 h (64%); (h) N,N-dimethylformamide dimethyl acetal, toluene, reflux, 12 h; (i) ethanimidamide hydrochloride or
guanidine hydrochloride, NaOMe, EtOH, reflux, 3 h (44% for 32 and 48% for 33 over two steps); (j) LiI, anhydrous DMF, reflux, 48 h (52% for 34)
or LiI, n-octylamine, anhydrous DMF, reflux 48 h (65% for 35).
The synthesis of pyrazole derivatives is outlined in Scheme 3.
Pyrazole derivatives 17, 18, 20, 21, and 22 were synthesized by
condensation of 7, 8, 9, 10, and 11 with hydrazine
hydrochloride in EtOH, respectively. Compound 19 was
obtained by reaction of 9 with phenylhydrazine hydrochloride.
Compound 23 was prepared in a manner similar to that for 17,
by hydrolyzing with LiOH in EtOH. The structure and
configuration of 20 were proven by single-crystal X-ray
diffraction analysis29 (Chart 1).
The synthesis of 1,2,3-thiadiazole, pyrimidine, and methyl
esterification derivatives is outlined in Scheme 4. Compound
24 was prepared by methyl esterification of 1 with MeI.
Reaction of 24 with semicarbazide hydrochloride produced 25.
1,2,3-Thiadiazole derivative 27 was obtained by reaction of 25
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d
Table 1. Inhibitory Effects of BA and Its Derivatives against RANKL-Induced Osteoclast Differentiation on RAW.264.7 Cells
a
b
c
d
Inhibition (%) = 100% − osteoclast formation (%) of each tested compound.30 X is single bond. R is methyl group. Unless otherwise indicated,
the X and R are a double bond and H atom, respectively.
with SOCl2, then demethylation of C-28 methyl ester with LiI
in DMF. Claisen condensation of 24 with HCOOEt produced
28, which was further reacted with hydrazine hydrochloride or
hydroxylamine hydrochloride to produce 29 or 30, respectively.
Compounds 32 and 33 were obtained by reaction of 24 with
N,N-dimethylformamide dimethyl acetal in toluene and then
condensation with ethanimidamide hydrochloride and guan-
dine hydrochloride, respectively. Demethylation of C-28 methyl
ester of compound 32 with LiI in refluxing DMF gave 34.
According to our previous report,27 the amino group in the
guanidine ring was methylated by the MeI generated from the
demethylation of C-28 methyl ester of compound 33. To avoid
this undesired reaction, n-octylamine was added as a MeI
scavenger, and thus, compound 35 was synthesized successfully.
Screening Betulinic Acid and Its Derivatives by
Osteoclast Differentiation Assay. The inhibitory effects of
all the heterocyclic ring-fused betulinic acid derivatives were
evaluated against RANKL-induced osteoclast differentiation on
RAW.264.7 cells. RAW264.7 is a mouse monocytic cell line that
is used as a standard osteoclast differentiation model. Normally,
it will take 3 days for RAW264.7 cells to differentiate into
mature osteoclasts with RANKL stimulation. The mature
osteoclasts are characterized as tartrate-resistant acid phospha-
tase (TRAP) positive (red) multinucleated cells. First,
RAW264.7 cells were treated with 50 ng/mL RANKL along
with the treatment of each tested compound at various
concentrations of 5, 10, 20 μM (Table 1). For the compounds
with almost full inhibition at 5 μM, further tests at lower
concentrations of 0.1, 0.5, 1.0 μM were performed (Table 2).
TRAP-positive multinucleated cells containing three or more
nuclei were counted as osteoclasts. As shown in Table 1, the
inhibitory effect of these heterocyclic ring-fused derivatives was
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derivative 20. Its inhibition was 66.9% even at the low
concentration of 0.1 μM (IC50 = 0.09 μM), which was about
200-fold more potent than the lead compound BA (IC50 ≈ 20
μM, Supporting Information Figure 1). Figure 1 showed that
20 significantly reduced the number of mature osteoclast in
RAW264.7 cells, which was confirmed by TRAP staining.
Compound 20 Has Little Cytotoxicity on Osteoclasts
within the Effective Concentrations. Compound 20 is the
most potent inhibitor among these tested BA derivatives. To
examine whether the impaired osteoclastogenesis in the
presence of 20 is due to the decrease in viability of the
precursor cells, we investigated the cytotoxicity of this
compound upon osteoclast precursors RAW264.7 by standard
MTS assay (Figure 2). Compound 20 did not show any
Table 2. Inhibitory Effects of Potent Activity of BA
Derivatives against RANKL-Induced Osteoclast
Differentiation on RAW.264.7 Cells
a
inhibition (%)
compd
0.1 μM
0.5 μM
1.0 μM
5
35.4 9.5
58.3 5.6
66.8 0.6
0
73.7 4.0
78.9 0.4
25.4 8.0
85.1 1.5
87.6 1.5
0
13
14
20
21
22
35
0
0
66.9 0.4
72.5 2.9
82.3 3.0
0
55.4 1.3
0
0
4.9 0.6
14.5 2.7
a
Inhibition (%) = 100% − osteoclast formation (%) of each tested
compound.
much stronger than that of their lead compound BA except the
C-28 methyl ester compounds (29, 30, 32, and 33). Although
methyl esterification at C-28 position led to a significant
decrease in the inhibitory activity (29, 30, 32, 33 vs 20, 13, 34,
35), hydrogenation of isopropenyl at the C-19 position had
only minimal effect on inhibition (13 vs 14 and 20 vs 21 in
Tables 1 and 2). As seen from Tables 1 and 2, the five-
membered heterocyclic ring-fused derivatives had more potent
effects than the corresponding six-membered analogues in most
cases (5, 13, 20 vs 2, 34, 35). The inhibitory potency decreased
if the substituents were introduced into the five-membered
heterocyclic rings (13 and 20 vs 12, 15, 16 and 19, 17, 18, 22,
23). Further, we can see that the inhibition decreased more as
hydrophilic substituent groups were introduced into the five-
membered heterocyclic rings than that with hydrophobic
substituent groups (4 vs 5; 16, 18 vs 15, 17; 23 vs 22). The
inhibitory effects of some derivatives were almost 100% at 5
μM as shown in Table 1. Therefore, we tested these
compounds at 0.1, 0.5, and 1.0 μM further. The results in
Table 2 showed that the inhibition of 14, 22, and 35 decreased
dramatically, although they inhibited RANKL-induced osteo-
clastogenesis completely at 5 μM. To our delight, compounds
5, 13, 20, and 21 still possessed potent antiosteoclastogenesis
effects even at low concentrations, especially the pyrazole
Figure 2. Cytotoxic effect of compound 20 on precursor osteoclasts.
RAW264.7 cells (3000/well) were plated on 96-well plates. Cells were
incubated with the indicated doses of 20. After incubation for 72 h at
37 °C, the percentage of cell survival was determined with the MTS/
CellTiter 96 aqueous assay (Promega, Madison, WI). The data are the
mean of three experiments carried out in triplicate. The bar indicates
the SD.
cytotoxic effects at the concentration (0.1 μM) of about half the
inhibition (66.9%) of osteoclastogenesis, and only slight effects
on cell viability were observed at high concentrations of 5 and
10 μM (the concentration of compound 20 reduction of cell
viability to 50% is 35.34 μM). The results suggested that the
effects of 20 on osteoclast differentiation were not from its
cytotoxicity. The selectivity index was calculated as the ratio of
Figure 1. Compound 20 inhibits RANKL-induced osteoclastogenesis in a dose-dependent manner. RAW 264.7 cells (3 × 103 cells/well) were
treated with or without RANKL (50 ng/mL), followed by addition of the indicated concentrations of 20 for 3 days and stained for TRAP expression.
(Top) Photographs of cells (original magnification 100×). (Bottom) TRAP positive multinucleated (>3 nuclei) osteoclasts were counted. The data
are the mean of three experiments carried out in triplicate. The bar indicates the SD.
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the concentration of the compound that reduced cell viability
to 50% to the concentration of the compound needed to inhibit
the osteoclast differentiation effect to 50% of the control value.
The selectivity index of compound 20 was 35.34/0.09 = 392.67.
Compound 20 Suppresses the Osteoclastogenesis-
Related Marker Gene Expression. The TRAP enzyme is
abundantly expressed by osteoclasts, and studies on TRAP
knockout mice showed disturbed endochondral ossification
with decreased resorptive activity of osteoclasts.31 Cathepsin K
is highly expressed in osteoclasts near the ruffled border
membrane,32 and it has been shown to participate in osteoclast-
mediated degradation of the subosteoclastic collagenous bone
matrix.33 Increased levels of cathepsin K mRNA have been
observed under conditions of enhanced bone resorption.34
Osteoclast function is executed directly by the expression of a
large number of related marker genes, such as TRAP and
cathepsin K. We investigated whether 20 can regulate
osteoclastogenesis-related marker gene expression by quantita-
tive real-time PCR. Our results (Figure 3 and Supporting
Figure 4. Compound 20 prevented ovariectomy-induced bone loss by
suppressing osteoclast activity in vivo. (A) Representative von Kossa
stained sections of lumbar vertebrae from sham-operated mice, OVX
mice, and OVX + 20 (10 mg/kg) mice. (B) Bone value/total value
(BV/TV), trabecular number (Tb.N), and trabecular space (Tb.Sp)
were analyzed. (C) Representative osteoclast TRAP stained sections of
the proximal tibia from sham-operated mice, OVX mice, and OVX +
20 (10 mg/kg) mice.
sham controls, suggesting that ovariectomy induces osteoclasto-
genesis. With treatment of OVX-mice by 20, the OVX-induced
osteoclast activity (OVX + 20 vs OVX mice) was dramatically
decreased, suggesting that 20 can inhibit ovariectomy-induced
osteoclast activity in vivo.
Figure 3. Compound 20 inhibits the expression of osteoclast mark
genes. RAW264.7 cells were pretreated with or without 5 μM 20 for 6
h in the presence of 50 ng/mL RANKL for the indicated time periods.
Total RNA was isolated and subjected to quantitative real-time PCR
for the expression levels of TRAP and cathepsin K. The data are the
mean of three experiments carried out in triplicate. The bar indicates
the SD.
CONCLUSIONS
■
We synthesized over 20 heterocyclic ring-fused betulinic acid
derivatives and evaluated their inhibition on RANKL-induced
osteoclast formation in preosteoclast RAW264.7 cells. Among
them, compounds 5, 20, and 21 exhibited potent inhibitory
activity on RANKL-induced osteoclast formation by TRAP
assay. Especially 20 (IC50 = 0.09 μM) showed about 200-fold
more potency than lead compound BA. The cell viability test of
20 on RAW264.7 showed very low cytotoxic effects. The
primary mechanism study indicated that 20 could inhibit
osteoclastogenesis-related marker gene expression levels of
cathepsin K and TRAP during osteoclastogenesis. The
experiments of ovariectomy mouse model also showed that
20 could inhibit ovariectomy-induced osteoclast activity in vivo.
Further SAR analysis and mechanism studies of these potential
leads are ongoing.
Information Figure 2) indicated that the expression of TRAP
and cathepsin K was up-regulated with RANKL stimulation,
while 20 could inhibit RANKL-induced expression levels of the
two genes during osteoclastogenesis in a dose- and time-
dependent manner, which was consistent with our previous
observation that 20 could suppress osteoclast differentiation.
Compound 20 Prevents OVX-Induced Bone Loss by
Suppressing Osteoclast Activity in Vivo. Activation of
osteoclasts plays a key role in bone loss, including menopausal
osteoporosis, in which estrogen deficiency enhances the genesis
and activity of osteoclasts and results in an unbalanced increase
in bone resorption. To examine whether compound 20 inhibits
osteoclast in vivo, we used the ovariectomy mouse model to
mimic menopause-induced bone loss in women. Spine is one of
the most severely affected sites by osteoporosis. Figure 4A and
Figure 4B showed an obvious decrease in bone volume and
trabecular number and an increase in trabecular space at the
lumbar spine in OVX mice compared with sham-operated
controls. Treatment of OVX mice with 20 (10 mg/kg)
significantly prevented the OVX-induced bone loss, as
measured by different parameters (Figure 4A,B). To investigate
whether 20 would prevent bone loss through inhibiting
osteoclastogenesis activity in vivo, TRAP staining was
performed on the proximal tibia section of each group. The
staining results (Figure 4 C) showed that the activity and area
of osteoclasts in OVX mouse notably increased compared with
In conclusion, we report BA heterocyclic derivatives as a
series of new chemical entities for the first time. Especially 20,
which exhibited potent inhibition of osteoclastogenesis both in
vitro and in vivo, could be used not only as a chemical tool for
the study of osteoporosis biology but also as a promising lead
for the development of a new class of antiosteoporosis agents.
EXPERIMENTAL SECTION
■
General. All reagents and chemicals were purchased from
commercial suppliers and used without further purification unless
otherwise stated. When needed, the reactions were carried out in
flame-dried or oven-dried glassware under a positive pressure of dry
N2. Flash column chromatography was performed on silica gel
(QinDao, 200−300 mesh) using the indicated eluents. Thin-layer
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chromatography was carried out on silica gel plates (QinDao) with a
layer thickness of 0.25 mm. Melting points were determined using the
MEL-TEMP 3.0 apparatus and are uncorrected. 1H (400 and 500
MHz) and 13C (100 and 125 MHz) NMR spectra were recorded on a
JEOL-400 and Bruker AM-500 spectrometer with CDCl3 or DMSO-d6
as solvent and tetramethylsilane (TMS) as the internal standard unless
otherwise stated. All chemical shift values were reported in units of δ
(ppm). The following abbreviations were used to indicate the peak
multiplicity: s = singlet; d = doublet; t = triplet; m = multiplet; br =
broad. High-resolution mass data were obtained on a Bruker
micrOTOF-Q II spectrometer. Purity of all final analogues for
biological testing was confirmed to be >95% as determined by HPLC
analysis (for data, see Supporting Information). HPLC analysis was
conducted according to the following method with the retention time
expressed in min at UV detection of 210, 230, and 254 nm. For HPLC
method, an Agilent 1200 series HPLC instrument was used, with
chromatography performed on a ZORBAX 150 mm × 4.6 mm, 5 μm
C18 column with mobile phase gradient of 0−10% H2O in MeOH,
with a flow rate of 1.0 mL/min.
3H), 0.81 (s, 3H). 13C NMR (DMSO-d6, 100 MHz) δ: 177.3, 165.2,
151.3, 150.3, 112.4, 109.6, 55.4, 52.1, 48.4, 48.4, 46.5, 41.9, 40.1, 38.3,
38.0, 37.6, 36.5, 36.2, 32.9, 31.5, 30.1, 30.0, 29.2, 25.0, 21.9, 20.8, 19.0,
18.9, 15.9, 15.3, 14.2. ESI-HRMS (m/z) [M + H]+ calcd for
C31H47N2O2S, 511.3358, found 511.3353.
Compound 5. To a solution of crude compound 3 (497 mg, 0.93
mmol) in ethanol (10 mL) was added thioacetamide (150 mg, 2
mmol) at 23 °C. The reaction mixture was heated under reflux for 3 h.
After cooling, the mixture was concentrated. The residue was purified
by flash chromatography (petroleum/AcOEt, 5/1 v/v) to afford 5
1
(280 mg, 55% over two steps) as a white solid, mp 261−264 °C. H
NMR (CDCl3, 400 MHz) δ: 4.74 (s, 1H), 4.61 (s, 1H), 3.06−3.03 (m,
1H), 2.74−2.70 (m, 1H), 2.63 (s, 3H), 2.28−2.23 (m, 3H), 2.21−2.15
(m, 1H), 2.00−1.98 (m, 2H), 1.75−1.70 (s, 4H), 1.66−1.35 (m, 13H),
1.33−1.27 (m, 5H), 1.18 (s, 3H), 1.00 (s, 3H), 0.97 (s, 3H), 0.78 (s,
3H). 13C NMR (CDCl3,100 Hz) δ: 181.0, 163.3, 156.2, 150.7, 126.0,
109.6, 56.4, 52.7, 49.8, 49.1, 49.0, 47.4, 46.8, 42.4, 40.7, 39.0, 38.5,
38.2, 37.0, 33.4, 32.1, 30.5, 30.3, 29.8, 25.4, 22.3, 19.4, 19.3, 18.6, 16.0,
15.7, 14.5. ESI-HRMS (m/z) [M + H]+ calcd for C32H48NO2S,
510.3406, found 510.3428.
Compound 1. A solution of BA (2.28 g, 5 mmol) in THF (10 mL)
was added dropwise to a stirring solution of IBX (2.1 g, 7.5 mmol) in
DMSO (30 mL) at 23 °C. The reaction mixture was stirred for 2 h at
23 °C and then diluted with AcOEt (30 mL) and washed with brine
(50 mL). The organic layer was dried over anhydrous Na2SO4 and
concentrated in vacuo. The residue was purified by flash
chromatography (petroleum ether/AcOEt, 8/1 v/v) to give 1 (2.05
Compound 6. To a solution of betulonic acid 1 (600 mg, 1.32
mmol) in THF (10 mL) and MeOH (5 mL) was added 10% Pd on
carbon (60 mg) at 23 °C. The mixture was subjected to 1 atm of H2
and was stirred for 12 h at 23 °C. The mixture was filtered, and the
filtrate was concentrated to give 6 (570 mg, 94%) as a white solid, mp
1
250−252 °C. H NMR (CDCl3, 400 MHz) δ: 2.62−2.32 (m, 2H),
1
g, 90%) as a white solid, mp 251−253 °C. H NMR (CDCl3, 400
2.30−2.15 (m, 3H), 2.02−1.12 (m, 22H), 1.05 (s, 3H), 0.99 (s, 3H),
0.94 (s, 6H), 0.91 (s, 3H), 0.83 (d, J = 6.4 Hz, 3H), 0.73 (d, J = 6.0
Hz, 3H).
MHz): δ 4.70 (s, 1H), 4.57 (s, 1H), 3.01−2.96 (m, 1H), 2.46−2.37
(m, 2H), 2.27−2.16 (m, 2H), 1.96−1.86 (m, 3H), 1.66 (s, 3H), 1.62−
1.57 (m, 2H), 1.46−1.16 (m, 16H), 1.03 (s, 3H), 0.98 (s, 3H), 0.95 (s,
3H), 0.93 (s, 3H), 0.89 (s, 3H). 13C NMR (CDCl3, 100 MHz): δ
218.3, 182.6, 150.2, 109.7, 56.4, 54.8, 49.8, 49.1, 47.3, 46.8, 42.4, 40.6,
39.5, 38.4, 37.0, 36.8, 34.0, 33.5, 32.0, 30.5, 29.6, 26.6, 25.4, 21.3, 20.9,
19.6, 19.3, 15.9, 15.7, 14.6.
Compound 12. To a solution of 1 (227 mg, 0.5 mmol) in THF
(10 mL) was added NaH (100 mg, 60% in mineral oil, 2.5 mmol)
under N2 at 23 °C. The reaction mixture was stirred for 0.5 h at 23 °C,
and then diethyl carbonate (295 mg, 2.5 mmol) was added dropwise
to the mixture. Then the mixture was heated to 60 °C and kept at this
temperature for 5 h. After cooling, the reaction mixture was poured
into 1 N HCl (30 mL) and extracted with AcOEt (30 mL × 3). The
organic layer was isolated and washed with brine, dried with anhydrous
Na2SO4, and concentrated to afford crude product 8 as an oil, which
was used without further purification.
Compound 2. To a solution of 1 (455 mg, 1 mmol) in morpholine
(5 mL) was added sulfur (300 mg, 9.4 mmol) and ethylenediamine
(0.33 mL, 5 mmol) at 23 °C. The reaction mixture was heated under
reflux for 4 h. After cooling, the reaction mixture was poured into
water and extracted with AcOEt (30 mL × 3). The organic layer was
washed with brine, dried over anhydrous Na2SO4, and concentrated in
vacuo. The residure was purified by flash chromatography (petroleum
ether/AcOEt, 5/1 v/v) and then crystallized from methanol to give 2
To a solution of 8 in ethanol (6 mL) was added hydroxylamine
hydrochloride (69 mg, 1 mmol) at 23 °C. The reaction mixture was
heat to 80 °C and kept at this temperature for 3 h. After cooling, the
reaction mixture was concentrated. The residue was purified by flash
chromatography (MeOH/DCM, 5/95 v/v) to afford 12 (125 mg, 50%
1
(150 mg, 31%) as a white solid, mp 245−247 °C. H NMR (CDCl3,
400 MHz) δ: 8.37 (d, J = 2.1 Hz, 1H), 8.24 (d, J = 2.2 Hz, 1H), 4.70
(s, 1H), 4.57 (s, 1H), 3.00−2.96 (m, 2H), 2.41−2.37 (m, 1H), 2.26−
2.20 (m, 2H), 1.98−1.90 (m, 2H), 1.72−1.69 (m, 1H), 1.65 (s, 3H),
1.55−1.33 (m, 13H) 1.23 (s, 3H), 1.20 (s, 3H), 1.19−1.00 (m, 3H),
0.96 (s, 3H), 0.95 (s, 3H), 0.74 (s, 3H). 13C NMR (CDCl3,100 MHz):
δ 181.6, 159.7, 150.7, 150.3, 142.3, 142.2, 109.7, 56.4, 53.0, 49.2, 48.7,
48.4, 46.9, 42.5, 40.5, 39.4, 38.4, 37.0, 36.7, 33.3, 32.1, 31.4, 30.6, 29.7,
25.4, 23.9, 21.3, 20.0, 19.4, 16.0, 15.6, 14.6. ESI-HRMS (m/z): [M +
H]+ calcd for C32H47N2O2, 491.3638, found 491.3634.
1
over two steps) as a white solid, mp 243−245 °C. H NMR (CDCl3,
400 MHz) δ: 12.1 (brs, 2H), 4.71 (s, 1H), 4.58 (s, 1H), 2.98−2.93 (m,
1H), 2.30−2.27 (m, 1H), 2.14−2.08 (m, 2H), 1.82−1.78 (m, 2H),
1.68 (s, 3H), 1.63−1.18 (m, 15H), 1.17 (s, 3H), 1.14−1.12 (m, 1H),
1.06 (s, 3H), 1.04−1.02 (m, 1H), 0.96 (s, 3H), 0.91 (s, 3H), 0.74 (s,
3H). 13C NMR (CD3OD, 100 MHz) δ: 177.3, 171.9, 169.7, 150.3,
109.7, 108.0, 55.5, 52.1, 48.5, 48.3, 46.6, 42.1, 40.2, 37.7(2C), 36.4,
34.2, 34.0, 32.8, 31.7, 30.2, 29.4, 28.8, 25.1, 21.5, 21.0, 19.1, 18.2, 15.9,
15.4, 14.4. ESI-HRMS (m/z) [M + H]+ calcd for C31H45NNaO4,
518.3246, found 518.3274.
Compound 3. To a solution of 1 (455 mg, 1 mmol) in Et2O (10
mL) was added 5,5-dibromo-2,2-dimethyl-4,6-dioxy-1,3-dioxane (160
mg, 0.53 mmol) at 23 °C. The reaction mixture was stirred for 12 h at
23 °C and then poured into water and extracted with Et2O (30 mL ×
3). The organic layer was washed with brine, dried over anhydrous
Na2SO4, and concentrated in vacuo to give a crude product 3 (497 mg,
0.93 mmol). The crude product 3 was used without further
purification.
Compound 13. To a solution of 9 (prepared with the same
procedure and scale as that of 13) in ethanol (6 mL) was added
hydroxylamine hydrochloride (69 mg, 1 mmol). The reaction mixture
was heated under reflux for 3 h. After cooling, the mixture was
concentrated. And the residue was purified by silica gel chromatog-
raphy (petroleum ether/AcOEt, 4/1 v/v) to give 13 (181 mg, 75.4%
over two steps) as a white solid, mp 249−251 °C. 1H NMR (DMSO-
d6, 400 MHz) δ: 7.99 (s, 1H), 4.77 (s, 1H), 4.65 (s, 1H), 3.08−3.02
(m, 1H), 2.51−2.47 (m, 1H), 2.33−2.26 (m, 3H), 2.21−2.00 (m, 3H),
1.98−1.94 (m, 1H), 1.80 (m, 3H), 1.76−1.50 (m, 14H), 1.29−1.10
(m, 6H), 1.02 (s, 3H), 1.09 (s, 3H), 0.82 (s, 3H). 13C NMR (CDCl3,
100 MHz) δ: 182.7, 173.3, 150.6, 150.5, 110.0, 109.8, 56.4, 53.0, 49.2,
48.7, 48.4, 46.9, 42.4, 40.5, 39.4, 38.4, 37.0, 36.7, 33.3, 32.1, 31.4, 30.6,
29.7, 25.4, 23.9, 21.3, 20.0, 19.4, 16.0, 15.7, 14.6. ESI-HRMS (m/z) [M
+ Na]+ calcd for C31H45NNaO3, 502.3297, found 502.3273.
Compound 4. To a solution of crude compound 3 (497 mg, 0.93
mmol) in ethanol (10 mL) was added thiourea (152 mg, 2 mmol) at
23 °C. The reaction mixture was heated under reflux for 3 h. After
cooling, the mixture was concentrated. The residue was purified by
flash chromatography (petroleum ether/AcOEt, 2/1 v/v) to give 4
1
(291 mg, 57% over two steps) as a white solid, mp 262−264 °C. H
NMR (DMSO-d6, 400 MHz) δ: 12.10 (brs, 1H), 6.60 (s, 2H), 4.70 (s,
1H), 4.57 (s, 1H), 2.96 (m, 1H), 2.45−2.41 (m, 1H), 2.31−2.25 (m,
1H), 2.14−2.12 (m, 1H), 2.05−2.01 (m, 1H), 1.82−1.80 (m, 2H),
1.65 (s, 3H), 1.58−1.10 (m, 19H), 1.00 (s, 3H), 0.96 (s, 3H), 0.92 (s,
3129
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Compound 14. To a solution of 6 (229 mg, 0.5 mmol) and
NaOMe (135 mg, 2.5 mmol) in THF (10 mL) was added dropwise
ethyl formate (185 mg, 2.5 mmol) at 23 °C under N2 . The reaction
mixture was stirred at 23 °C for 15 h and then poured into 1 N HCl
(40 mL) and extracted with AcOEt (30 mL × 3). The organic layer
was washed with brine, dried with anhydrous Na2SO4, and
concentrated to afford 10 as a colorless oil, which was used without
further purification.
To a solution of 7 in ethanol (6 mL) was added hydrazine
dihydrochloride (105 mg, 1.0 mmol) at 23 °C. The reaction mixture
was heated under reflux for 3 h. After cooling, the mixture was
concentrated. The residue was purified by silica gel chromatography
(petroleum ether/AcOEt, 2/1 v/v) to give 17 as a white solid (142 mg
1
52% over two steps), mp 256−258 °C. H NMR (DMSO-d6, 500
MHz) δ: 13.2 (brs, 1 H), 12.1 (brs, 1 H), 4.70 (s, 1H), 4.57 (s, 1H),
2.97 (m, 1H), 2.58 (d, J = 15.2 Hz, 1H), 2.28 (m, 1H), 2.13 (d, J = 10
Hz, 1H), 1.99 (d, J = 15.2 Hz, 1H), 1.81 (m, 2H), 1.68 (s, 4H), 1.61−
1.27 (m, 13H), 1.24 (m, 5H), 1.12 (s, 3H), 0.98 (s, 3H), 0.94 (s, 3H),
0.78 (s, 3H). 13C NMR (DMSO-d6, 125 MHz) δ: 177.4, 150.4, 148.0,
124.0, 121.3, 110.4, 109.7, 55.4, 52.2, 48.5, 48.3, 46.8, 42.1, 39.0, 37.9,
37.6, 36.3, 35.3, 34.2, 32.8, 31.6, 30.1, 29.3, 28.4, 25.1, 24.7, 20.9, 18.9,
18.3, 15.8, 15.3, 14.2. ESI-HRMS (m/z) [M + Na]+ calcd for
C32H45F3N2NaO2, 569.3331, found 569.3359.
To a solution of 10 in ethanol (6 mL) was added hydroxylamine
hydrochloride (69 mg, 1 mmol) at 23 °C . The reaction mixture was
heated under reflux for 3 h. After cooling, the mixture was
concentrated. The residue was purified by silica gel chromatography
(petroleum ether/AcOEt, 4/1 v/v) to give 14 (169 mg, 70% over two
1
steps) as a white solid, mp 257−259 °C. H NMR (DMSO-d6, 400
MHz) δ: 7.98 (s, 1H), 2.50−2.47 (m, 1H), 2.29−2.22 (m, 3H), 1.97−
1.93 (m, 1H), 1.90−1.18 (m, 22H), 1.17 (s, 3H), 0.98 (s, 6H), 0.86 (d,
J = 6.8 Hz, 3H), 0.80 (s, 3H), 0.76 (d, J = 6.8 Hz, 3H). 13C NMR
(CDCl3, 100 MHz) δ: 182.9, 173.1, 150.3, 108.9, 56.8, 53.4, 48.8, 48.6,
44.1, 42.5, 40.6, 38.8, 38.2, 37.3, 35.7, 34.7, 33.3, 31.9, 29.7, 29.7, 28.6,
26.7, 22.9, 22.6, 21.3, 21.0, 18.6, 15.9, 15.7, 14.6, 14.5. ESI-HRMS (m/
z) [M + Na]+ calcd for C31H47NNaO3, 504.3454, found 504.3462.
Compound 15. Diethyl oxalate (394 mg, 2.7 mmol) was added
dropwise to a solution of 1 (228 mg, 0.5 mmol) and NaOMe (135 mg,
2.5 mmol) in THF (10 mL) under N2. The reaction mixture was
stirred for 12 h at room temperature and then poured into 1 N HCl
(40 mL) and extracted with AcOEt (30 mL × 3). The organic layer
was washed with brine, dried with anhydrous Na2SO4, and
concentrated to afford crude product 11 as a colorless oil, which
was used without further purification.
Compound 18. To a solution of 8 (prepared with the same
procedure and scale as preparation of 12) in ethanol (6 mL) was
added hydrazine dihydrochloride (105 mg, 1 mmol) at 23 °C. The
reaction mixture was heated under reflux for 3 h. After cooling, the
mixture was concentrated. The residue was purified by silica gel
chromatography (petroleum ether/AcOEt, 1/1 v/v) to give 18 (124
1
mg, 50% over two steps) as a white solid, mp 243−245 °C. H NMR
(CD3OD, 500 MHz) δ: 4.70 (s, 1H), 4.58 (s, 1H), 2.99−2.94 (m,
1H), 2.37−2.26 (m, 2H), 2.13−2.11 (m, 1H), 1.81−1.80 (m, 2H),
1.66−1.65 (m, 4H), 1.61−1.23 (m, 14H), 1.15 (m, 6H), 1.04−1.02
(m, 4H), 0.96 (s, 3H), 0.91 (s, 3H), 0.84−0.82 (m, 1H), 0.72 (s, 3H).
13C NMR (CD3OD, 125 Hz) δ: 177.2, 158.6, 150.2, 146.7, 109.5, 95.6,
55.4, 52.7, 48.5, 48.4, 46.5, 42.0, 40.2, 38.2, 37.7, 36.3, 35.0, 33.0, 32.9,
31.6, 30.1, 29.3, 25.1, 22.9, 22.7, 20.9, 18.9, 18.4, 15.8, 15.3, 14.3. ESI-
HRMS (m/z) [M + H]+ calcd for C31H47N2O3, 495.3587, found
495.3602.
To a solution of 11 in ethanol (6 mL) was added hydroxylamine
hydrochloride (69 mg, 1.0 mmol) at 23 °C. The reaction mixture was
heated under reflux for 3 h. After cooling, the reaction mixture was
concentrated. The residue was purified by silica gel chromatography
(petroleum ether/AcOEt, 4/1 v/v) to give the desired compound 15
(193 mg, 70% over two steps) as a white solid, mp 297−299 °C (dec).
1H NMR (DMSO-d6, 500 MHz) δ: 4.72 (s, 1H), 4.59 (s, 1H), 4.22 (q,
J = 7.0 Hz, 2H), 2.99−2.88 (m, 2H), 2.32−2.26 (m, 1H), 2.14−2.12
(m, 1H), 1.99−1.95 (m, 1H), 1.82−1.79 (m, 2H), 1.67 (m, 4H),
1.57−1.22 (m, 19H), 1.11 (s, 3H), 0.97 (s, 3H), 0.92 (s, 3H), 0.70 (s,
3H). 13C NMR (CDCl3, 125 MHz) δ: 182.7, 176.1, 160.9, 154.2,
150.2, 110.9, 109.8, 61.5, 56.3, 52.9, 49.1, 48.9, 46.8, 42.4, 40.6, 38.5,
38.4, 36.9, 36.0, 35.0, 33.1, 32.0, 30.4, 29.6, 28.4, 25.3, 21.2, 20.9, 19.2,
18.4, 16.1, 15.6, 14.5, 14.0. ESI-HRMS (m/z) [M + Na]+ calcd for
C34H49NNaO5, 574.3508, found 574.3559.
Compound 16. To a solution of compound 15 (276 mg, 0.5
mmol) in ethanol (15 mL) was added LiOH·H2O (42 mg, 1 mmol) at
23 °C. The reaction mixture was stirred at room temperature for 12 h.
The reaction mixture was poured into 1 N HCl (40 mL) and extracted
with AcOEt (30 mL × 3). The organic layer was washed with brine,
dried over anhydrous Na2SO4, and concentrated. The residue was
purified by silica gel chromatography (petroleum ether/AcOEt, 2/1 v/
v) to give 16 (212 mg, 81%) as a white solid, mp 284−286 °C (dec).
1H NMR (DMSO-d6, 500 MHz) δ: 4.71 (s, 1H), 4.57 (s, 1H), 2.97−
2.95 (m, 1H), 2.72 (d, J = 16 Hz, 1H), 2.30−2.25 (m, 1H), 2.13−2.11
(m, 1H), 2.03 (d, J = 16 Hz, 1H), 1.81−1.78 (m, 2H), 1.69−1.64 (m,
4H), 1.58−1.53 (m, 3H), 1.45−1.41 (m, 6H), 1.34−1.28 (m, 4H),
1.25 (s, 3H), 1.23−1.16 (m, 1H), 1.14 (s, 3H), 1.04−1.02 (m, 1H),
0.96 (s, 3H), 0.92 (s, 3H), 0.74 (s, 3H). 13C NMR (DMSO-d6, 125
MHz) δ: 177.2, 175.2, 161.7, 154.8, 150.2, 110.4, 109.6, 55.4, 52.1,
48.5, 48.2, 46.6, 42.1, 40.2, 38.1, 37.7, 36.3, 35.6, 34.6, 32.8, 31.6, 30.1,
29.3, 28.3, 25.0, 21.1, 21.0, 18.9, 18.0, 16.0, 15.3, 14.3. ESI-HRMS (m/
z) [M − H]− calcd for C32H44NO5, 522.3219, found 522.3185.
Compound 17. To a solution of 1 (227 mg, 0.5 mmol) and
sodium methoxide (143 mg, 2.5 mmol) in THF (10 mL) was added
ethyl trifluoracetate (355 mg, 2.5 mmol) under N2 at 23 °C. The
mixture was stirred at 23 °C for 12 h. The reaction mixture was poured
into 1 N HCl (50 mL) and extracted with AcOEt (30 mL × 3). The
organic layer was washed with brine, dried with anhydrous Na2SO4,
and concentrated to give crude product 7 as a pale yellow oil, which
was used without further purification.
Compound 19. Ethyl formate (185 mg, 2.5 mmol) was added
dropwise to a solution of 1 (227 mg, 0.5 mmol) and NaOMe (135 mg,
2.5 mmol) in THF (10 mL) under N2 at 23 °C. The reaction mixture
was stirred for 15 h at 23 °C and then poured into 1 N HCl (40 mL)
and extracted with AcOEt (30 mL × 3). The organic layer was washed
with brine, dried with anhydrous Na2SO4, and concentrated to afford
crude product 9 as a yellow oil, which was used without further
purification.
To a solution of 9 in ethanol (6 mL) was added phenylhydrazine
hydrochloride (91 mg, 0.63 mmol) at 23 °C. The reaction mixture was
heated under reflux for 3 h. After cooling, the mixture was
concentrated. The residue was purified by flash chromatography
(petroleum ether/AcOEt, 5/1 v/v) to afford 19 (202 mg, 73% over
1
two steps) as a white solid, mp 279−281 °C. H NMR (DMSO-d6,
400 MHz) δ: 12.09 (brs, 1H), 7.49−7.48 (m, 3H), 7.36−7.35 (m,
2H), 7.26 (s, 1H), 4.71 (s, 1H), 4.58 (s, 1H), 2.99−2.96 (m, 1H),
2.62−2.59 (m, 1H), 2.33−2.27 (m, 1H), 2.14−2.12 (m, 1H), 2.03−
1.99 (m, 1H), 1.83−1.81 (m, 2H), 1.66 (s, 3H), 1.57−1.09 (m, 16H),
0.96 (s, 3H), 0.95 (s, 3H), 0.92 (s, 6H), 0.79 (s, 3H). 13C NMR
(CD3OD, 100 MHz) δ: 177.5, 150.5, 145.6, 142.4, 137.9, 129.2 (2C),
129.1, 128.7 (2C), 113.6, 109.8, 55.6, 54.2, 48.8, 48.6, 46.6, 42.2, 40.2,
37.9, 37.8, 36.8, 36.4, 34.3, 33.1, 31.7, 30.2, 29.4, 29.0, 25.2, 22.1, 21.1,
19.1, 18.7, 15.8, 15.4, 14.4. ESI-HRMS (m/z) [M + Na]+ calcd for
C37H50N2O2Na, 577.3770, found 577.3798.
Compound 20. To a solution of 9 (prepared with the same
procedure and scale as preparation of 19) in ethanol (6 mL) was
added hydrazine hydrochloride (105 mg, 1 mmol) at 23 °C. The
reaction mixture was heated under reflux for 3 h. After cooling, the
mixture was concentrated. The residue was purified by silica gel
chromatography (petroleum ether/AcOEt, 1/1 v/v) to give 20 (168
1
mg, 70% over two steps) as a yellow powder, mp 257−259 °C. H
NMR (DMSO-d6, 500 MHz) δ: 12.19 (brs, 2H), 7.10 (s, 1H), 4.70 (s,
1H), 4.57 (s, 1H), 3.00−2.94 (m, 1H), 2.53−2.50 (m, 1H), 2.31−2.26
(m, 1H), 2.14−2.12 (m, 1H), 1.91−1.88 (m, 1H), 1.84−1.78 (m, 2H),
1.65 (s, 3H), 1.62−1.19 (m, 18H), 1.08 (s, 3H), 0.96 (s, 3H), 0.92 (s,
3H), 0.85−0.83 (m, 1H), 0.70 (s, 3H). 13C NMR (DMSO-d6, 125
MHz) δ: 177.6, 150.5, 148.3, 133.5, 111.4, 109.8, 55.6, 53.2, 48.7, 48.6,
46.7, 42.1, 40.3, 38.3, 37.8, 36.4, 36.3, 33.1(2C), 31.8, 30.9, 30.2, 29.4,
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25.3, 23.6, 21.1, 19.0, 18.7, 15.8, 15.5, 14.4. ESI-HRMS (m/z) [M +
H]+ calcd for C31H47N2O2, 479.3638, found 479.3671.
(112 mg, 1 mmmol) and TEA (1 mL) at 23 °C. The reaction mixture
was stirred at 23 °C for 12 h and then poured into H2O (50 mL) and
extracted with AcOEt (30 mL × 3). The organic layer was washed with
brine, dried over anhydrous Na2SO4, and concentrated to give crude
product 25, which was used without further purification.
The crude product 25 was dissolved in SOCl2 (10 mL), and the
reaction mixture was heated under reflux for 4 h. After cooling, the
mixture was concentrated. The residue was purified by silica gel
chromatography (petroleum ether/AcOEt, 5/1 v/v) to give 26 (115
mg, 45% over two steps) as a pale yellow solid. 1H NMR (CDCl3, 500
MHz) δ: 4.76 (s, 1H), 4.62 (s, 1H), 3.67 (s, 3H), 3.16−3.19 (m, 1H),
2.30−2.27 (m, 3H), 1.90 (m, 2H), 1.70−1.02 (m, 24H), 1.01 (s, 3H),
0.99 (s, 3H), 0.95 (s, 3H), 0.79 (s, 3H).
Compound 21. To a solution of 6 (457 mg, 1 mmol) and sodium
methoxide (270 mg, 5 mmol) in THF (10 mL) was added dropwise
ethyl formate (370 mg, 5 mmol) under N2 at 23 °C. The mixture was
stirred at 23 °C for 5 h. The reaction mixture was poured into 1 N
HCl (50 mL) and extracted with AcOEt (30 mL × 3). The organic
layer was washed with brine, dried with anhydrous Na2SO4, and
concentrated to give crude product 10 as a pale yellow oil, which was
used without further purification.
To a solution of 10 in ethanol (10 mL) was added hydrazine
dihydrochloride (210 mg, 2 mmol) at 23 °C. The reaction mixture was
heated under reflux for 3 h. After cooling, the mixture was
concentrated. The residue was purified by silica gel chromatography
(petroleum ether/AcOEt, 1/1 v/v) to give 21 (337 mg, 70% over two
steps) as a pale yellow solid, mp 259−261 °C. 1H NMR (CD3OD, 400
MHz) δ: 7.18 (s, 1H), 2.69−2.65 (m, 1H), 2.43−2.37 (m, 1H), 2.30−
2.23 (m, 2H), 2.01−1.98 (m, 1H), 1.86−1.81 (m, 2H), 1.73−1.33 (m,
13H), 1.33−1.20 (m, 7H), 1.17 (s, 3H), 1.03−1.02 (m, 6H), 0.88 (d, J
= 6.4 Hz, 3H), 0.82 (s, 3H), 0.79 (d, J = 6.8 Hz, 3H). 13C NMR
(DMSO-d6, 125 MHz) δ: 180.2, 151.0, 134.4, 113.6, 58.0, 55.0, 50.4,
50.0, 45.6, 43.8, 42.0, 39.8, 39.6, 38.6, 37.8, 34.9, 34.6, 33.3, 31.4, 31.1,
31.0, 28.5, 24.1, 23.8, 23.5, 22.8, 20.3, 16.5, 16.5, 15.2(2C). ESI-HRMS
(m/z) [M + H]+ calcd for C31H49N2O2, 481.3794, found 481.3793.
Compound 22. To a solution of 11 (prepared with the same
procedure and scale as preparation of 16) in ethanol (6 mL) was
added hydrazine dihydrochloride (105 mg, 1 mmol) at 23 °C. The
reaction mixture was heated under reflux for 3 h. After cooling, the
mixture was concentrated. The residue was purified by silica gel
chromatography (petroleum ether/AcOEt, 2/1 v/v) to give 22 (169
Compound 27. To a solution of 26 (115 mg, 0.23 mmol) in dry
DMF (15 mL) was added anhydrous lithium iodide (308 mg, 2.3
mmol) at 23 °C. The reaction mixture was heated under reflux for 48
h under N2. After cooling, the mixture was poured into H2O (40 mL)
and extracted with AcOEt (30 mL × 3). The organic layer was washed
with brine, dried over anhydrous Na2SO4, and concentrated. The
residue was purified by silica gel chromatography (petroleum ether/
AcOEt, 1/1 v/v) to give 27 (80 mg, 71%) as a brown solid, mp 255−
1
257 °C. H NMR (CDCl3, 500 MHz,) δ: 4.75 (s, 1H), 4.62 (s, 1H),
3.19−3.02 (m, 2H), 2.30−2.67 (m, 3H), 1.99−1.98 (m, 2H), 1.88−
1.04 (m, 25H), 1.00 (m, 6H), 0.80 (s, 3H). 13C NMR (CDCl3, 125
MHz) δ: 182.5, 165.6, 150.3, 145.5, 109.8, 56.3, 52.4, 49.0, 48.9, 46.8,
42.4, 40.7, 38.6, 38.2, 37.3, 36.9, 36.2, 33.1, 31.9, 31.1, 30.4, 29.6, 25.3,
23.4, 21.3, 19.3, 19.1, 16.0, 15.5, 14.5. ESI-HRMS (m/z) [M + Na]+
calcd for C30H44N2NaO2S, 519.3021, found 519.3042.
Compound 29. To a solution of 24 (235 mg, 0.5 mmol) and
sodium methoxide (135 mg, 2.5 mmol) in THF (10 mL) was added
ethyl formate (185 mg, 2.5 mmol) at 23 °C. The mixture was stirred at
23 °C for 5 h and then poured into 1 N HCl (50 mL) and extracted
with AcOEt (30 mL × 3). The organic layer was washed with brine,
dried with anhydrous Na2SO4, and concentrated to afford crude
product 28 as a colorless oil, which was used without further
purification.
1
mg, 61% over two steps) as a white powder, mp 282−284 °C. H
NMR (DMSO-d6, 500 MHz) δ: 13.13 (brs, 1H), 12.09 (brs, 1H), 4.72
(s, 1H), 4.59 (s, 1H), 4.22 (q, J = 6.5 Hz, 2H), 2.98−2.88 (m, 2H),
2.32−2.27 (m, 1H), 2.14−2.12 (m, 1H), 1.99−1.96 (m, 1H), 1.83−
1.81 (m, 2H), 1.67−1.66 (m, 4H), 1.62−1.01 (m, 26H), 0.98 (s, 3H),
0.93 (s, 3H), 0.79 (s, 3H). 13C NMR (DMSO-d6, 125 MHz) δ: 177.2,
170.0, 162.1, 150.3(2C), 115.4, 109.6, 59.5, 55.5, 52.4, 48.5, 48.4, 46.6,
42.1, 40.2, 37.9, 37.7, 37.0, 36.3, 32.9, 32.9, 31.7, 30.5, 30.1, 29.3, 25.1,
23.0, 20.9, 19.0, 18.4, 16.0, 15.3, 14.3(2C). ESI-HRMS (m/z) [M +
H]+ calcd for C34H51N2O4, 551.3849, found 551.3849.
To a solution of 28 in ethanol (6 mL) was added hydrazine
dihydrochloride (80 mg, 0.76 mmol) at 23 °C. The reaction mixture
was heated under reflux for 3 h. After cooling, the mixture was
concentrated. The residue was purified by silica gel chromatography
(petroleum ether/AcOEt, 2/1 v/v) to give 29 (162 mg, 66%) as a pale
Compound 23. To a solution of compound 22 (551 mg, 1 mmol)
in ethanol (15 mL) was added LiOH·H2O (84 mg 2 mmol) at 23 °C.
The reaction mixture was stirred at 23 °C for 12 h and then poured
into 1 N HCl (40 mL) and extracted with AcOEt (30 mL × 3). The
organic layer was washed with brine, dried over anhydrous Na2SO4,
and concentrated. The residue was purified by silica gel chromatog-
raphy (petroleum ether/AcOEt, 1/1 v/v) to give 23 (422 mg, 81%) as
a white solid, mp 292−296 °C (dec). 1H NMR (DMSO-d6, 500 MHz)
δ: 4.72 (s, 1H), 4.58 (s, 1H), 2.99−2.92 (m, 2H), 2.33−2.28 (m, 1H),
2.15−2.13 (m, 1H), 1.99−1.95 (m, 1H), 1.83−1.82 (m, 2H), 1.67 (s,
3H), 1.62−1.01 (m, 24H), 0.97 (s, 3H), 0.94 (s, 3H), 0.72 (s, 3H).
13C NMR (DMSO-d6, 125 MHz) δ: 177.4, 163.4, 150.4, 150.3, 136.7,
115.7, 109.7, 55.6, 52.6, 48.6, 46.7, 42.1, 40.4, 37.9, 37.8, 37.2, 36.5,
33.2, 33.1, 33.0, 31.8, 30.7, 30.2, 29.4, 25.3, 23.4, 21.2, 19.1, 18.6, 16.2,
15.5, 14.4. ESI-HRMS (m/z) [M − H]− calcd for C32H45N2O4,
521.3379, found 521.3393.
Compound 24. To a suspension of 1 (455 mg, 1 mmol) and
K2CO3 (280 mg, 2.0 mmol) in DMF (10 mL) was added dropwise
CH3I (1 mL) at 23 °C. The reaction mixture was heated to 40 °C and
kept at this temperature for 5 h. The reaction mixture was poured into
H2O (50 mL) and extracted with AcOEt (30 mL × 3). The organic
layer was washed with brine, dried over anhydrous Na2SO4, and
concentrated. The residue was purified by silica gel chromatography
(petroleum ether/AcOEt, 10/1 v/v) to give 24 (421 mg, 90%) as a
white solid, mp 239−241 °C. 1H NMR (CDCl3, 400 MHz) δ: 4.73 (s,
1H), 4.60 (s, 1H), 3.67 (s, 3H), 3.06−3.03 (m, 1H), 2.48 (m, 1H),
2.41(m, 1H), 2.21 (m, 3H), 1.68 (s, 3H), 1.52−1.08 (m, 17H), 1.06
(s, 3H), 1.01 (s, 3H), 0.97 (s, 3H), 0.95 (s, 3H),0.91 (s, 3H).
Compound 26. To a solution of compound 24 (235 mg, 0.5
mmol) in dry THF (20 mL) was added semicarbazide hydrochloride
1
yellow solid, mp 249−251 °C. H NMR (CDCl3, 500 MHz) δ: 7.22
(s, 1H), 4.76 (s, 1H), 4.62 (s, 1H), 3.68 (s, 3H), 3.05−3.00 (m, 1H),
2.64−2.61 (m, 1H), 2.26−2.24 (m, 2H), 1.96−1.71 (m, 4H), 1.70 (s,
3H), 1.68−1.60 (m, 1H), 1.58−1.32 (m, 11H), 1.28−1.24 (m, 5H),
1.22−1.19 (m, 1H), 1.18 (s, 3H), 1.10−1.05 (m, 1H), 1.00 (s, 3H),
0.98 (s, 3H), 0.79 (s, 3H). 13C NMR (CDCl3, 125 MHz) δ: 176.7,
150.6 (2C), 135.2, 112.6, 109.6, 56.6, 53.4, 51.3, 49.4, 49.2, 46.9, 42.4,
40.7, 38.6, 38.3, 36.9, 36.6, 33.4 (2C), 32.1, 31.1, 30.6, 29.8, 25.6, 23.9,
21.4, 19.4, 19.1, 15.9, 15.6, 14.7. ESI-HRMS (m/z) [M + Na]+ calcd
for C32H48N2NaO2, 515.3613, found 515.3619.
Compound 30. To a solution of 28 (prepared with the same
procedure and scale as in preparation of 29) in ethanol (10 mL) was
added hydroxyamine hydrochloride (52 mg, 0.75 mmol) at 23 °C. The
mixture was heated under reflux for 3 h. After cooling, the mixture was
concentrated. The residue was purified by silica gel chromatography
(petroleum ether/AcOEt, 5/1 v/v) to give 30 (158 mg, 64%) as a
white solid, mp 243−245 °C. 1H NMR (CDCl3, 500 MHz) δ: 7.52 (s,
1H), 4.74 (s, 1H), 4.60 (s, 1H), 3.67 (s, 3H), 3.02−2.92 (m, 2H),
2.27−2.19 (m, 3H), 1.90−1.87 (m, 2H), 1.72−1.68 (m, 4H), 1.61−
1.14 (m, 15H), 1.12 (s, 3H), 1.04 (s, 6H), 0.95 (s, 3H), 0.91 (s, 3H).
13C NMR (CDCl3, 125 MHz) δ: 176.5, 172.9, 150.4, 150.2, 109.6,
108.8, 56.5, 53.5, 51.2, 49.3, 49.1, 46.8, 42.3, 40.6, 38.8, 38.2, 36.8,
35.7, 34.7, 33.2, 32.0, 30.5, 29.7, 28.6, 25.4, 21.3, 21.1, 19.3, 18.7, 16.0,
15.6, 14.6. ESI-HRMS (m/z) [M + H]+ calcd for C32H48NO3,
494.3629, found 494.3619.
Compound 32. To a solution of 24 (470 mg, 1.0 mmol) in
toluene (10 mL) was added N,N-dimethylformamide dimethyl acetal
(0.7 mL, 5.0 mmol) at 23 °C. The reaction mixture was heated under
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Journal of Medicinal Chemistry
Article
reflux for 12 h. After cooling, the mixture was concentrated to give
crude product 31, which was used without further purification.
To a solution of 31 and sodium methoxide (54 mg, 1.0 mmol) in
absolute ethanol (20 mL) was added acetamidine acetate (118 mg, 1.0
mmol) at 23 °C. The reaction mixture was heated under reflux for 3 h
under N2. After cooling, the mixture was concentrated. The residue
was purified by silica gel chromatography (petroleum ether/AcOEt,
10/1 v/v) to give 32 (230 mg, 44% over two steps) as a pale yellow
solid, mp 249−251 °C. 1H NMR (CDCl3, 500 MHz) δ: 8.16 (s, 1H),
4.76 (s, 1H), 4.62 (s, 1H), 3.68 (s, 3H), 3.01 (m, 1H), 2.69 (d, J = 16
Hz, 1H), 2.65 (s, 3H), 2.29 (m, 2H), 2.18 (d, J = 15.6 Hz, 1H), 1.89
(m, 2H), 1.79 (m, 1H), 1.69 (s, 3H), 1.61−1.28 (m, 22H), 1.27 (s,
3H), 1.22 (s, 3H), 1.14−1.00 (m, 3H), 0.98 (s, 3H), 0.91 (s, 3H), 0.75
(s, 3H). ESI-HRMS (m/z) [M + H]+ calcd for C34H51N2O2, 519.3945,
found 519.3948.
Compound 33. To a solution of 31 (prepared with the same
procedure and scale as preparation of 32) and sodium methoxide (54
mg, 1.0 mmol) in absolute ethanol (20 mL) was added guanidine
hydrochloride (96 mg, 1 mmol) at 23 °C. The reaction mixture was
heated under reflux for 3 h. After cooling, the reaction mixture was
concentrated. The residue was purified by silica gel chromatography
(DCM/MeOH, 20/1 v/v) to give 33 (249 mg, 48%), mp 251−253
°C. 1H NMR (CDCl3, 500 MHz) δ: 7.86 (s, 1H), 4.83 (brs, 2H), 4.75
(s, 1H), 4.61 (s, 1H), 3.68 (s, 3H), 3.02−2.99 (m, 1H), 2.60−2.56 (m,
1H), 2.26−2.24 (m, 2H), 2.09−2,04 (m, 2H), 1.89 (m, 3H), 1.76 (m,
3H), 1.67 (m, 3H), 1.61−1.14 (m, 14H), 1.14 (s, 3H), 0.99 (s, 3H),
0.98 (s, 3H), 0.75 (s, 3H). 13C NMR (CDCl3, 125 MHz) δ: 177.0,
174.0, 162.2, 158.7, 150.8, 117.5, 109.9, 56.8, 53.3, 51.4, 49.6, 49.0,
47.1, 42.6, 42.0, 40.7, 39.4, 38.5, 37.1, 36.5, 33.6, 32.3, 30.9, 30.7, 29.8,
25.7, 23.4, 21.6, 20.2, 19.5, 15.7, 15.5, 14.8. ESI-HRMS (m/z) [M +
H]+ calcd for C33H50N3O2, 520.3903, found 520.3907.
fetal bovine serum (FBS). Thereafter, the cell medium was changed
with same medium containing RANKL (50 ng/mL) and various
concentrations of tested compounds. After 3 days, cells were fixed and
stained for tartrate-resistant acid phosphate (TRAP) (Sigma) activity
according to the recommendation of the manufacturer. TRAP+
multinucleated cells with more than three nuclei were counted as
osteoclasts.
Cytotoxicity Assay of Compound 20 on Precursor Osteo-
clasts. The proliferation effect of 20 was determined by the MTS
method as described previously20 with a VersaMax microplate reader
(Molecular Devices).
Compound 20 Inhibits the Expression of Osteoclast Mark
Gene TRAP and Cathepsin K. For the real-time RT-PCR analysis,
total RNA was extracted from cells with TRIzol (Invitrogen).
Cathepsin K and TRAP transcripts were quantified on Mx 3005P
(Stratagene) using SYBR green dye and normalized with β-actin. The
following primer sets were used: mouse TRAP, (forward) 5′-
GCTGGAAACCATGATCACCT-3′, (reverse) 5′-GAGTTGCCACA-
CAGCATCAC-3′; mouse cathepsin K, (forward) 5′-CTTCCAA-
TACGTGCAGCAGA-3′, (reverse) 5′-TCTTCAGGGCTTTCTC-
GTTC-3′.
In Vivo Experiments and Bone Histomorphometry. Eight-
week-old C57/BL6 mice were purchased from SIBS (Shanghai,
China). Five days after ovariectomy, mice were divided into three
groups (n = 8): sham-operated mice (Sham) and ovariectomized mice
treated with vehicle (OVX) or with 20 (10 mg/kg) every 2 days (OVX
+ 20) for 90 days. Sham and OVX groups were intraperitoneally (ip)
injected with 50 μL of DMSO. The OVX + 20 group was
intraperitoneally injected with 20 that was dissolved in 50 μL of
DMSO. We also found that the weight of all the tested mice of the
three groups has no big difference after 90 days. After then, all mice
were euthanized with excess amounts of anesthetic. Von Kossa staining
was performed on the sections of lumbar vertebrae (L3) from sham-
operated mice, OVX mice, and OVX + 20 mice as described
previously.20 Bone value/total value (BV/TV), trabecular number
(Tb.N), and trabecular space (Tb.Sp) were analyzed by histomorpho-
metric analysis using the OsteoMeasure analysis system (Osteometrics,
Atlanta, GA, U.S.) according to standard criteria. For in vivo osteoclast
activity, TRAP assay was performed. Tibia bones were fixed in 10%
paraformaldehyde (PFA), and decalcification was achieved by 10%
EDTA immersion for 2 weeks. The samples were embedded in
paraffin and sectioned for TRAP staining (Sigma) as described
previously.20 Mouse maintenance, use, and treatment were in
accordance with accepted standards of the Ethics Committee of
ECNU.
Compound 34. To a solution of 32 (230 mg, 0.44 mmol) in dry
DMF (15 mL) was added lithium iodide (590 mg, 4.4 mmol) at 23 °C.
The reaction mixture was heated under reflux for 8 h under N2. After
cooling, the mixture was poured into H2O (20 mL) and extracted with
AcOEt (30 mL × 3). The organic layer was washed with brine, dried
over anhydrous Na2SO4, and concentrated. The residue was purified
by silica gel chromatography (petroleum ether/AcOEt, 4/1 v/v) to
1
give 34 (115 mg, 52%) as a white solid, mp 256−258 °C. H NMR
(CDCl3, 500 Hz) δ: 8.21 (s, 1H), 4.76 (s, 1H), 4.62 (s, 1H), 3.08−
3.03 (m, 1H), 2.73−2.72 (m, 1H), 2.67 (s, 3H), 2.33−2.31 (m, 2H),
2.21−2.18 (m, 1H), 2.01−1.99 (m, 2H), 1.80−1.77 (m, 1H), 1.71 (s,
3H), 1.68−1.28 (m, 18H), 1.22 (s, 3H), 1.02 (s, 3H), 1.01 (s, 3H),
0.75 (s, 3H). 13C NMR (CDCl3, 125 MHz) δ: 181.1, 172.9, 165.3,
157.2, 150.5, 124.1, 109.7, 56.3, 53.1, 49.2, 48.8, 46.9, 42.5, 42.0, 40.6,
39.3, 38.4, 37.0, 36.2, 33.3, 32.1, 30.8, 30.6, 29.7, 29.6, 25.5, 25.3, 23.3,
21.4, 19.9, 19.3, 15.6, 15.5, 14.6. ESI-HRMS (m/z) [M + H]+ calcd for
C33H49N2O2, 505.3794, found 505.3838.
ASSOCIATED CONTENT
* Supporting Information
■
S
Compound 35. To a solution of 33 (125 mg, 0.24 mmol) in dry
DMF (15 mL) was added lithium iodide (322 mg, 2.4 mmol) and n-
octylamine (310 mg, 2.4 mmol) at 23 °C. The reaction mixture was
heated under reflux for 48 h under N2. After cooling, the reaction
mixture was poured into H2O (20 mL) and extracted with AcOEt (30
mL × 3). The organic layer was washed with brine, dried over
anhydrous Na2SO4, and concentrated. The residue was purified by
silica gel chromatography (DCM/MeOH, 10/1, v/v) to give 35 (79
Further chemical and biological experimental details, including
HPLC data and NMR spectra. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
■
*For M.L.: phone, +86-21-54345016; fax, +86-21-54344922; e-
mail, myliu@bio.ecnu.edu.cn. For W.-W.Q.: phone, +86-21-
62237950; fax, +86-21-62237950; e-mail, wwqiu@chem.ecnu.
edu.cn. For J.T.: phone, 86-21-62232764; fax, +86-21-
62232100; e-mail, jtang@chem.ecnu.edu.cn.
1
mg, 65%) as a yellow solid, mp 265−267 °C. H NMR (DMSO-d6,
500 MHz) δ: 12.07 (s, 1H), 7.84 (s, 1H), 6.17 (brs, 2H), 4.71 (s, 1H),
4.58 (s, 1H), 2.99−2.94 (m, 1H), 2.56−2.50 (m,1H), 2.31−2.27 (m,
1H), 2.14−2.12 (m, 1H), 2.05−1.98 (m, 1H), 1.82−1.81 (m, 2H),
1.69−1.66 (m, 4H), 1.59−1.23 (m, 15H), 1.17 (s, 3H), 1.10 (s, 3H),
0.97 (s, 3H), 0.93 (s, 3H), 0.69 (s, 3H). 13C NMR (DMSO-d6, 125
MHz) δ: 177.2, 171.8, 162.4, 158.5, 150.3, 115.1, 109.6, 55.4, 52.4,
48.5, 48.1, 46.6, 42.0, 40.9, 40.1, 40.0, 39.8, 39.7, 39.5, 39.3, 39.2, 39.0,
38.8, 37.7, 36.3, 35.8, 30.6, 23.1, 19.0, 15.4, 15.2, 14.4. ESI-HRMS (m/
z) [M + H]+ calcd for C32H48N3O2, 506.3747, found 506.3764.
In Vitro Osteoclastogenesis Assay. RAW264.7 cells (kindly
provided by Dr. Bryant G. Darnay, University of Texas MD Anderson
Cancer Center, Houston, TX, U.S.) were seeded at 3 × 103 cells per
well using 96-well plates and cultured for 24 h in αMEM with 10%
Author Contributions
∥These authors contributed equally to this work.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
The National Natural Science Foundation of China (Grant
20802020), Shanghai Science and Technology Council (Grant
■
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E.; Wronski, T. J. The anabolic effects of parathyroid hormone on
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10142200802), Ph.D. Program Foundation of Ministry of
Education of China (Grant 200802691039), 973 Program
(Grant 2012CB910400), and National Natural Science
Foundation of China (Grants 30800653 and 81071437) are
appreciated for their financial support. We also thank “Lab of
Organic Functional Molecules, the Sino-French Institute of
ECNU” for support.
ABBREVIATIONS USED
■
BA, betulinic acid; TRAP, tartrate-resistant acid phosphatase;
ERT, estrogen-replacement therapy; BP, bisphosphonate;
PTH, parathyroid hormone; RANK, receptor activator of NF-
κB; OPG, osteoprotegerin; SWH, Sambucus williamsii Hance;
AKBA, acetyl-11-keto-β-boswellic acid; ACCX, 25-acetylcimi-
genol xylopyranoside; ALP, alkaline phosphatase; IBX, 2-
iodoxybenzoic acid; DMSO, dimethyl sulfoxide; DMF, N,N-
dimethylformamide
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