Substituted benzothiophene or benzofuran derivatives as a novel class of bone morphogenetic protein-2 up-Regulators: Synthesis, structure-activity relationships, and preventive bone loss efficacies in senescence accelerated mice (SAMP6) and ovariectomized

Article abstract of DOI:10.1021/jm901685n

In this work, substituted benzothiophene and benzofuran compounds were found to be a new class of potential anabolic agents by enhancing BMP-2 expression. A series of benzothiophene and benzofuran derivatives have been synthesized, and their activities of

Full text of DOI:10.1021/jm901685n

J. Med. Chem. 2010, 53, 1819–1829 1819
DOI: 10.1021/jm901685n
Substituted Benzothiophene or Benzofuran Derivatives as a Novel Class of Bone Morphogenetic
Protein-2 Up-Regulators: Synthesis, Structure-Activity Relationships, and Preventive Bone Loss
Efficacies in Senescence Accelerated Mice (SAMP6) and Ovariectomized Rats
Hui-fang Guo,† Hua-yi Shao,† Zhao-yong Yang,† Si-tu Xue, Xue Li, Zong-ying Liu, Xiao-bo He, Jian-dong Jiang,
Yue-qin Zhang, Shu-yi Si,* and Zhuo-rong Li*
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050,
People’s Republic of China. †These authors made equal contributions to this work.
Received November 14, 2009
In this work, substituted benzothiophene and benzofuran compounds were found to be a new class of
potential anabolic agents by enhancing BMP-2 expression. A series of benzothiophene and benzofuran
derivatives have been synthesized, and their activities of up-regulating BMP-2 and bone loss prevention
efficacies in SAMP6 mice and OVX rats have been studied. Benzothiophenes 1, 3, 14, 4a, 7a, 8a, and
benzofuran analogue 2 showed higher BMP-2 up-regulation rates in vitro. Compound 8a was found to
significantly affect the bone formation rate of tested SAMP6 mice. Compound 1 showed an improved
bone quality in SAMP6 mice and also showed activity in OVX rats. We have demonstrated that
substituted benzothiophene and benzofuran derivatives, especially compounds 1 and 8a, enhance BMP-
2 expression in vitro and in vivo and stimulate bone formation and trabecular connectivity restoration
in vivo. The compounds represent potential leads in the development of a new class of anabolic agents.
Introduction
orthotopic bone in a variety of experimental systems, includ-
ing femoral defects in rats, tibial and ulnar defects in rabbits,
femoral defects in sheep, mandibular defects in dogs, spinal
fusionindogs, andporous in-growthin rats.9 Recently, results
indicate the high potency of BMP-2 as an inducer of osteo-
genesis and that BMP-2 may be a novel therapeutic agent for
diseases associated with bone loss and requiring bone repair.10
Transfer of BMP-2 cDNA with a recombinant adenovirus
into an established osteoporotic sheep model enhanced frac-
ture healing.11 Recombinant human bone morphogenetic
protein-2 (rhBMP-2) can accelerate the repair of bone defects
and has three FDA approvals for use in orthopedics.12
An alternative approach to exploit the beneficial actions of
BMP-2 on bone formation is to discover agents that activate
BMP-2 gene expression in bone cells. Mundy et al.13 reported
an assay to search for small molecules that activate the
promoter of the BMP-2 gene. In this report, it was demon-
strated that the effects of statins to enhance new bone forma-
tion in vitro and in rodents were associated with an increase in
the expression of the BMP-2 gene in bone cells. Following this
report, many knownbioactive compoundshave beenreported
to increase the gene expression and/or protein expression of
BMP-2. Estrogen replacement therapy is one of the most
common and effective strategies used to prevent osteoporosis
inpostmenopausalwomen. Estrogensarebelieved toworkex-
clusively by inhibiting bone resorption. The results of Zhou10
demonstrated that estrogens increase mouse BMP-2 mRNA,
suggesting that estrogens may also enhance bone formation.
Resveratrol, a naturally occurring compound possessing estro-
genic activity, is thought to have considerable potential as a
therapy against osteoporosis. The results of Chang14 indicate
that piceatannol stimulates osteoblast differentiation at vari-
ous stages (from maturation to terminally differentiated osteo-
blasts), and the induction of differentiation by piceatannol
Osteoporosis is a worldwide health problem, observed most
frequently in postmenopausal women and in aging men. The
number of people at risk is growing as the population of
elderly increases. Agents for the treatment of osteoporosis are
classified as either antiresorptive or anabolic. Antiresorptive
agents, a basic treatment for osteoporosis in the clinic, prevent
bone loss by inhibiting the activity of osteoclasts and, there-
fore, reducing bone resorption. Anabolic agents act by stimu-
lating the formation of new bone and thus provide an
additional option for osteoporosis patients and represent a
major advance in the treatment of osteoporosis.1-6 Anabolic
agents are capable of increasing bone mass to a greater degree
than antiresorptive agents and also have the capacity to
improve bone quality and increase bone strength.1 The re-
combinant human PTH (teriparatide) is the only FDA-
approved anabolic agent, which is currently available in the
U.S. and Europe in the forms of PTH (1-34) and PTH (1-84),
respectively. Because of the paucity of available anabolic agents
for osteoporosis treatment, there is an urgent need to develop
small molecular compounds to treat this disease that are
nontoxic, cost-effective, and easy to administer.7
Bone morphogenetic protein-2 (BMP-2) is expressed by
normal osteoblasts and is a crucial regulator of osteogenic
differentiation that has been shown to stimulate osteoblast
differentiation and osteogenic nodule formation in vitro, as
well as bone formation in vivo. BMP-2 plays an important
role in osteoblast differentiation and bone generation and
regeneration.3,8 Human BMP-2 induces structurally sound
*To whom correspondence should be addressed. For S-y.S.: phone,
86-10-63180604; fax, 86-10-63017302; e-mail, sisyimb@hotmail.com.
For Z.-r.L.: phone, 86-10-63027185; fax, 86-10-63017302; e-mail, l-z-r@
263.net.
r
2010 American Chemical Society
Published on Web 01/20/2010
pubs.acs.org/jmc

1820 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Guo et al.
Table 1. Structures and the Up-Regulated Activities of BMP-2 Expression (UpR) of the Tested Benzothiophene and Benzofuran Analoguesa in Vitro
compd
X
R1
R2
R3
R4
R5
R6
UpR (%)
1
S
COMe
COMe
COMe
COMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
Cl
H
H
H
H
H
H
H
H
H
35.6
33.5
41.4
37.1
15.4
2.0
2
O
S
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
F
3
COOMe
OMe
H
14
1c
1d
1e
2c
2d
3c
3d
3e
3f
S
OMe
H
S
2E-3-phenylpropenone
2E-3-(2-chloro-phenyl)propenone
2E-3-(4-methoxyphenyl)propenone
2E-3-phenyl-propenone
2E-3-(2-chloro-phenyl)propenone
2E-3-phenylpropenone
2E-3-(2-chlorophenyl)propenone
2E-3-(4-methoxyphenyl)propenone
2E-3-(3,4-dimethoxyphenyl)propenone
2E-3-(2,3,4-trimethoxyphenyl)propenone
COOC2H5
H
S
H
H
S
H
H
27.3
24.4
1.8
O
O
S
H
H
H
H
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOC2H5
COOMe
H
H
12.6
24.0
1.7
S
H
S
H
S
H
-9.6
-21.5
49.8
8.2
3g
4a
5a
6a
7a
8a
9a
10a
11a
12a
5b
6b
7b
8b
9b
5c
8c
Lovb
S
H
S
H
S
COOC2H5
COOC2H5
H
S
H
OMe
H
OMe
-0.6
49.9
64.2
7.1
S
COOC2H5
COOC2H5
S
-OCH2O-
O
O
O
O
S
COOC2H5
COOC2H5
H
H
Cl
H
H
-1.6
-18.1
12.4
19.7
18.5
13.5
0.6
COOC2H5
COOC2H5
H
H
H
OMe
H
H
H
H
H
H
H
COCH2SOMe
COCH2SOMe
H
H
S
H
OMe
H
OMe
S
COCH2SOMe
COCH2SOMe
S
-OCH2O-
-OCH2O-
O
S
COCH2SOMe
COCH2SO2Me
COCH2SO2Me
H
H
H
H
3.9
5.1
S
-9.3
20.9
a Compound concentrations were 4.0 μM. b Concentration of Lov was 0.4 μM.
was associated with an increase in BMP-2 production. Flavo-
noids15-17 and coumarin derivatives,18-20 present in many medi-
cinal plants, have a direct stimulatory effect on the prolifera-
tion and differentiation of cultured human osteoblast cells
in vitro. This proliferative and differentiation effect is mediated
by increasing production of BMP-2 in the human osteoblasts.
The BMP pathway is a promising new area to target
therapeutic agents for the treatment of low bone mass. In
our research, a new class of small molecules, substituted
benzothiophene or benzofuran derivatives, were found to be
potential anabolic agents targeting BMP-2. We have pre-
viously established a novel cell-based high-throughput screen
assay for the identification of BMP-2 up-regulators.21 The
assay used murine calvarial MC3T3-E1 cells that were stably
transfected with pGL4-BMP2, a firefly luciferase reporter
gene fused downstream of the 50-flanking promoter region
of the mouse BMP-2 gene. We tested more than 13000
samples from a synthetic and fermentation products collec-
tion and identified that 1-(benzo[b]thiophen-2-yl)thanone (1)
and 1-(benzo[b]furan-2-yl)thanone (2) specifically enhanced
the expression of BMP-2 in cultured cells, which increased
promoter activity in a dose-dependent manner and stimulated
the expression of BMP-2 at both the RNA and protein levels.
In this work, to identify whether the substituted benzo-
thiophene or benzofuran derivatives have the potency to be
developed as new clinical anabolic agents by increasing BMP-
2 expression, we synthesized a novel series of benzo[b]thio-
phen and benzo[b]furan derivatives and evaluated their ability
to enhance BMP-2 expression in vitro. Moreover, we tested
the activity of the compounds in preventing bone loss efficacy
in SAMP6 mice and in OVX rats. The in vitro structure-
activity relationships of the compounds were also investi-
gated.
Chemistry
Our previous study revealed that the C-3 methyl group in
the benzothiophene or benzofuran moiety did not enhance
BMP-2 expression.22 In the present study, we retained the
hydrogen atom at the C-3 position and focused an SAR
analysis on the variations of the C-2 groups as well as
substituents at the 4, 5, 6, and 7 positions of the phenyl ring.
The effect of changing the sulfur atom to an oxygen atom in
the five-membered heterocycles was also investigated. On the
basis of this strategy, 20 substituted benzothiophene analo-
gues and 7 substituted benzofuran analogues were designed
and synthesized (Table 1). The synthetic routes are described
in Scheme 1. Compound 14 was synthesized using the method
in ref 22.
Scheme 1 includes three synthetic routes. Desired com-
pounds (1c-e, 2c,d, 3c-g) with different cinnamoyl groups

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1821
Scheme 1. Synthesis of Substituted Benzothiophene and Benzofuran Derivativesa
a Reagents and conditions: (a) C2H5ONa, C2H5OH, room temperature; (b) HSCH2COOC2H5, K2CO3, DMF, 60-100 °C; (c) BrCH2COOC2H5,
K2CO3, DMF, 60-100 °C; (d) DMSO, NaH, THF, 70-75 °C; (e) 30% H2O2, acetic acid, 0 °C; (f) HSCH2CH3, K2CO3, DMF, room temperature;
(g) ClCH2COCH3, CaO, DMF, 90 °C.
at the C-2 position and diverse substituents on the phenyl ring
were synthesized according to route A, using 1, 2, and 3
(compounds 1 and 2 are commercially available; 3 was
synthesized by route C) as the starting materials. Compounds
1-3 reacted with substituted benzaldehyde in the presence of
sodium ethoxide to afford the desired products (1c-e, 2c,d,
3c-g). Compounds 2c and 2d were derivatives of 2, while
compounds 1c-e and 3c-g were derivatives of 1.
ethyl mercaptide in dimethylformamide (DMF) at room
temperature in the presence of anhydrous potassium carbo-
nate to yield intermediate 13. Compound 13 was then reac-
ted with chloroacetone in DMF at 90 °C in which the reaction
was catalyzed using calcium oxide to yield the desired com-
pound 3.
Results and Discussion
In route B, compounds 4-12 were commercial available
starting materials. Desired products 4a-8a were obtained
through the reaction of 4-8 with ethyl thioglycolate, respec-
tively, in the presence of anhydrous potassium carbonate at
60-100 °C.23 Compounds 9a-12a were obtained using a
similar method employed to produce 4a-8a, with the starting
materials 9-12 and ethyl bromoacetate.24,25 Compounds
5b-9b were obtained with 5a-9a and methylsulfinyl carba-
nion (CH3SOCH2-), which was prepared from dimethyl
sulfoxide and sodium hydride (NaH).26 The reaction should
be conducted with rigorous exclusion of oxygen, carbon
dioxide, and water, since the methylsulfinyl carbanion reacts
rapidly with these substances. Oxidation of 5b and 8b with
30% H2O2 in acetic acid at 0 °C afforded 5c and 8c.
In Vitro Activities of Up-Regulated BMP-2 Expression and
SAR Analysis. All of the 28 synthesized benzothiophene or
benzofuran derivatives were examined for the up-regulation
of the BMP-2 promoter activities in vitro at 4.0 μM. Lovas-
tatin (Lov, 0.4 μM) was the positive control, and the nega-
tive control was 0.1% DMSO. The in vitro assay was
performed following the reported method.21 BMP-2 up-
regulation rates (UpRa) were defined as (RLUtest compound
-
RLUnegative control/RLUnegative control) ꢀ 100, in which RLU is
the relative luminescence units. Structures of the tested
a Abbreviations: UpR, up-regulation rate; TBV%, trabecular bone
volume percentage; TFS%, trabecular formation surface percentage;
TRS%, trabecular resorption surface percentage; OSW, average osteoid
width; MAR, mineral apposition rate; mAR, osteo cortex mineraliza-
tion rate; ROI, regions of interest.
Compound 3 was synthesized by route C using commer-
cially available 4 as the starting material, which reacted with

1822 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Guo et al.
Figure 1. ROIs in which BMD values were measured (A). The BMD data of whole body (total, B), spine (C), and left hind leg (D) of each group
at 0, 1, 2, and 3 months after treatment were collected.
compounds and their up-regulation activities are shown in
Table 1. Six compounds (8a, 7a, 5a, 3, 2, and 1) showed
higher UpR (over 30%) than Lov (UpR was about 21%), of
which the activities of compounds 8a, 7a, 5a, and 3 (UpR
over 40%) were higher than that of compound 1 (UpR was
35.6%).
and 1, the 5,6-dimethoxy group (7a, 49.9%), the 5,6-methyl-
enedioxy group (8a, 64.2%), and the 5-methyl carbonate
group (4a, 49.8%) on the benzo[b]thiophene phenyl ring
significantly improved the activities. The UpR of 8a was
almost 2-fold higher than the rate observed for compound 1.
Compared with 9a and 2, the 5,7-dichloro group (10a,
-1.6%), the 7-fluoro group (11a, -18.1%), and the 7-
methoxy group (12a, 12.4%) on the benzo[b]furan phenyl
ring led to reductions in activities.
Modification of the C-2 group within the benzo[b]thio-
phene/furan framework, by the introduction of methylsulfi-
nyl and methylsulfonyl groups at the β position of the C-2
carbonyl group, decreased the activities when compared with
1 and 2. Decreasing activities were observed for 5c (5.11%)
versus 5b, 7b (13.5%) versus 7a, 8b (0.6%) versus 8a, 8c
(-9.3%) versus 8b, and 9b (3.9%) versus 9a. However, there
was still some augment of the activities when compared to
their respective analogues: 5b (19.7%) versus 5a, 6b (18.5%)
versus 6a.
The SAR analysis showed that the structure of 8a gave
optimal up-regulation of BMP-2 promoter activity in vitro.
The activities of 1e and 5b were the best among the analogues
with C-2 cinnamoyl groups and C-2 methylsulfinyl/methyl-
sulfonyl groups, respectively. Taken together, we selected 8a,
1e, and 5b of each C-2 group category to evaluate their
in vivo activities in SAMP6 mice. Compound 1, initially
identified to be active for enhancing expression of BMP-2
in our work, was chosen for in vivo efficacy evaluation in
SAMP6 mice and also in OVX rats.
Effects of the Tested Compounds on Bone Mineral Density
in SAMP6 Mice. In vitro efficacies of compounds 1, 1e, 5b,
and 8a were evaluated in SAMP6 mice, using Lov (purchased
from Tianzun Chemicals, Nanjing, China) as the positive
control. Male and female SAMP6 mice (5-months old) were
administered Lov, 1, 1e, 5b, and 8a orally at doses of 10, 30,
30, 30, and 30 (mg/kg)/day, respectively, for 3 months. The
mice of the untreated group (MC) were given the vehicle
(0.5% methylcellulose) by gavage.
The SAR study was initially focused on the 1-(benzo-
[b]thiophen/furan-2-yl)thanone analogues (compounds 1, 3,
and 14). Previous studies have indicated that electron donat-
ing groups on the phenyl ring cause a higher UpR than
electron-withdrawing groups when R1 was acetyl. The UpR
of compound 3 was higher than the lead compounds 1 and 14
with two methoxy groups on the phenyl ring. This implied
that the phenyl ring substituted by a methyl carboxylate may
be beneficial to the activity of enhancing BMP-2 expression.
All the UpRs of the generated analogues 1c-e, 2c,d, and
3c-g were significantly reduced when the acetyl group at the
C-2 position in compounds 1, 2, and 3 was replaced with
cinnamoyl groups. Compared with 1c (UpR = 15.4%), the
UpR of 1e (27.3%) was much higher; however, the activity of
1d (UpR = 2.0%) decreased. The electron donating group
was better than the electron-withdrawing group in this
system. The UpR of compound 2d was 1.8%, much less than
that of 2c (24.4%), indicating that the 20-chloride on the
phenyl ring of the cinnamoyl group was not favorable for up-
regulating the activity of BMP-2. By comparing 3d (24.0%),
3e (1.7%), 3f (-9.6%), and 3g (-21.5%) with 3c (12.6%), we
found that the introduction of an ethyl carbonate group at
the C-5 position of the phenyl ring was favorable to the
activity while the electron-donating methoxy group in the
phenyl ring of the cinnamoyl group decreased the activity. As
such, the more methoxy groups there are, the lower is the
activity.
The SAR was further explored when the acetyl group of 1
and 2 was replaced by an ethylcarboxylate group. The UpRs
of 5a (8.2%) and 9a (7.1%) were much lower than that of 1
and 2. Replacing the C-4 hydrogen atom with chlorine (6a,
-0.6%) decreased the activity. However, compared to 5a

Article
The effects of the tested compounds on bone mineral
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1823
Insignificant variation of the left hind leg BMD values was
observed among the six groups; however, there was a slight
enhancement in the 8a group.
density (BMD) of the SAMP6 mice were initially observed.
BMD measurements of SAMP6 mice were performed by
dual-energy X-ray absorptiometry (DXA) (OTEOCORE 3,
MEDILINK, France) at 0, 1, 2, and 3 months after treat-
ment. The whole body (total), spine, and left hind leg BMD
values were analyzed using the small animals software
package of OTEOCORE 3 as the regions of interest (ROIs,
Figure 1A).
Effects of the Tested Compounds on Bone Histomorpho-
metry in SAMP6 Mice. Following treatment for 3 months,
the right femora of the tested SAMP6 mice were harvested,
and the undecalcified bone sections were obtained (Figure 2).
The trabecula of all the treated groups was denser than that
of the MC group. Bone histomorphometry was analyzed
using a Leica Qwin image analysis system (Table 3). Trabe-
cular bone volume percentage (TBV%) of each treated
group increased compared to that of the untreated MC
group. The Lov, 1, 1e and 8a groups increased more sub-
stantially suggesting an enhancement of the mice bone
masses of these groups. Trabecular formation surface per-
centage (TFS%) of each treated group was also enhanced
and the Lov, 1, 1e groups had a more obvious rise, indicating
an improvement of osteoblast activity in these groups.
Trabecular resorption surface percentage (TRS%) of all
groups did not significantly vary, which indicated that there
were no noticeable differences of the osteoclast activities.
Average osteoid width (OSW) of the Lov group mice was
much larger than that of other groups, indicating that only
Lov among the 6 compounds can increase the osteoblast
activity of the osteoid in SAMP6 mice. Compared with the
MC group, the bone formation related histomorphometries
(TBV% and TFS%) of all treated groups increased; how-
ever, bone resorption related histomorphometry (TRS%)
were clearly not affected.
The total BMD of all tested animals except the Lov group
decreased during the first month (Figure 1B). Then the total
BMD of all groups increased in the following 2 months,
while there was a reduction in group 1 during the third
month. Total BMD of groups Lov, 5b, and 8a showed
persistent and greater enhancement than the MC group
following the first month of treatment. The spine BMD
values of all groups were similar (Figure 1C), while the Lov
and 8a groups spine BMD values increased more than the
MC group in the last 2 months. Spine BMD values of group 1
were obviously enhanced during the first 2 months but
decreased in the third month, which was also observed in
the total BMD and left hind leg BMD values (Figure 1D).
BMD values of the left hind leg of all groups decreased
during the 3 months, while the Lov, 1, and 8a groups showed
a slower rate of decrease when compared with the MC group.
After 3 months of treatment, the total BMD of the Lov
group was enhanced compared with the MC group (Table 2).
The total BMD of the 1e group was almost the same as the
Lov group, whereas the 5b and 8a groups were higher than
those of the MC and Lov groups. The spine BMD of all
tested compound groups was enhanced compared with
the MC group after 3 months of treatment, and the spine
BMD of the Lov group was observed to increase the most.
Effects of the Tested Compounds on BMP-2 Expression in
SAMP6 Mice. The compounds were selected for in vivo
efficacy evaluation mainly according to the UpR value of
BMP-2 promoter activity. After 3 months of treatment, to
Table 3. Bone Histomorphometry of the SAMP6 Mice after 3 Months
of Treatment with the Tested Compounds
Table 2. BMD Values of the SAMP6 Mice after 3 Months of Treatment
BMD (g/cm2)
group
TBV% (%)
TFS% (%)
TRS% (%)
OSW (μm)
group
total
spine
left hind leg
Lov
MC
1
7.44 ( 2.05a
3.88 ( 0.58
4.95 ( 0.80a
8.94 ( 3.68a
5.95 ( 1.93
7.10 ( 2.15a
6.22 ( 0.97a
4.46 ( 0.47
5.89 ( 0.61b
6.54 ( 0.81b
5.13 ( 0.82
5.55 ( 0.93
6.64 ( 1.64
7.47 ( 1.12
7.09 ( 1.31
6.29 ( 1.48
6.45 ( 0.60
6.45 ( 0.82
6.59 ( 0.29a
5.93 ( 0.42
5.55 ( 0.82
5.50 ( 0.82
5.85 ( 0.64
5.76 ( 0.58
Lov
MC
1
0.070 ( 0.003
0.067 ( 0.002
0.068 ( 0.002
0.070 ( 0.006
0.072 ( 0.005
0.072 ( 0.003a
0.095 ( 0.007a
0.087 ( 0.007
0.086 ( 0.004
0.089 ( 0.013
0.090 ( 0.013
0.091 ( 0.008
0.046 ( 0.003
0.046 ( 0.002
0.050 ( 0.001
0.050 ( 0.007
0.050 ( 0.004
0.053 ( 0.008
1e
5b
1e
5b
8a
8a
a p < 0.05 (n = 5) compared to that of the MC group. b p < 0.005
(n = 5) compared to that of the MC group.
a p < 0.05 (n = 5) compared to that of the MC group.
Figure 2. Bone histology of the right femora of the SAMP6 mice after 3 months of treatment with the tested compounds. The dark-blue bands
in the pictures (1 ꢀ 200) represent the bone trabecular.

1824 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Guo et al.
Figure 3. Immunohistochemical analysis of BMP-2 expression in the femora of SAMP6 mice. Brownish-red dots in the pictures (1 ꢀ 400) were
cells that expressed BMP-2. Five visual fields in the medullary cavity under the epiphyseal plate were uptaken, and the percentage of the area of
BMP-2 expression was calculated for each visual field. The percentage of the area of BMP-2 expression was the ratio of the area of cells
showing a positive reaction (brownish red dots) to the total bone tissue area in a visual field. (A) Shown is the average percentage of area of
BMP-2 expression: (/) p<0.05 (n=5) compared to that of the untreated group (MC); (//) p<0.005 (n=5) compared to that of untreated
group (MC).
Figure 4. Efficacy of compound 1 preventing bone loss in OVX rats. Dosages of compound 1 are 7.5 (mg/kg)/day (1L), 15 (mg/kg)/day (1M),
and 30 (mg/kg)/day (1H). MAR is mineral apposition rate, and mAR is osteo cortex mineralization rate: (/) p<0.05 (n=7) compared to the
SHAM group; (#) p < 0.05 (n = 7) compared to the OVX group. Dark-blue bands in the pictures illustrate trabecular of left proximal tibia
(1 ꢀ 50).
determine whether the compounds increased the BMP-2
expression in vivo, the left femora of all SAMP6 mice were
harvested to obtain frozen bone section lengthways using a
freezing microtome. Immunohistochemistry was performed
to observe the expression levels of BMP-2 using a Leica Qwin
image analysis system. The positive reaction cells of the
osteoblast and marrow stroma stained as brownish red dots
(Figure 3). No cartilage cells were found to express BMP-2 in
all groups. Moreover, cartilage cells were seldom observed
which may be due to the senescence of this senile osteoporo-
tic model. The percentage of area of BMP-2 expression of all
treated groups had increased compared with that of the MC
group (Figure 3A). The observed increases of the Lov, 1, 5b,
and 8a groups were more significant, and the BMP-2 expres-
sion areas of the 5b and 8a groups were higher than the area
of the Lov group.
Compounds 1, 1e, 5b, and 8a up-regulated the BMP-2
promoter activity in vitro and also increased BMP-2 expres-
sion in vivo, which implied these compounds improved bone
quality possibly through the BMP-2 pathway.
Bone Loss Preventing Efficacy in OVX Rats. The in vivo
bone loss preventing efficacy of compound 1 was also evalu-
ated in OVX rats (OVX) model. Six groups were assigned,
including sham-operated (SHAM), OVX with vehicle (OVX),
OVX with raloxifene (Ralo, 5 (mg/kg)/day, orally for 3 months),
and OVX with compound 1 (1L, 7.5 (mg/kg)/day; 1M, 15
(mg/kg)/day; 1H, 30 (mg/kg)/day; orally for 3 months, respec-
tively).
Following treatment for 3 months, the right left proximal
tibia of all rats were harvested and the undecalcified bone
sections were obtained (Figure 4). In the 1H group receiving
a dose of 30 (mg/kg)/day and the 1M group receiving the

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1825
Figure 5. Pathological sections (1 ꢀ 200) of the hearts, livers, spleens, lungs, and kidneys after 3 months of treatment. (/) SAMP6 mice were
administered tested compounds with a dose of 30 (mg/kg)/day orally. (//) Rats were administered compound 1 as 150 (mg/kg)/day orally.
dose of 15 (mg/kg)/day, the number, thickness, area, and
connectivity of trabecula had clearly increased when com-
pared to the OVX group. Ovariectomy (see OVX group) is
associated with a lower trabecular bone volume and a higher
trabecula resorption surface percentage compared with
SHAM (Figure 4). Compound 1 improved the trabecula in
a dose-dependent manner. According to the bone histomor-
phometry analyzed using a Leica Qwin image analysis
system, all doses of compound 1 increased TBV% and
decreased the trabecular resorption surface percentage
(TRS%) when compared to OVX. TBV% of 1H was con-
sistent with that of the Ralo group.
Safety Evaluation of Tested Compounds. Following the
treatment of 1, 1e, 5b, and 8a for 3 months, the hearts, livers,
spleens, lungs, and kidneys of SAMP6 mice were harvested
and a histological examination was performed (Figure 5).
The histomorphology of the organs of the treated and
untreated animals was similar, and no pathological changes
were found. Organ indexes (organ weight/body weight) of
the six groups were similar to each other. Body weights of all
mice increased slowly during the 3 months, and the 24 h
consumption of water (g/g body weight) and food (g/g body
weight) of the treated groups was similar to the untreated
group.
Preliminary acute toxicity in the Kunming mice was
performed. Compounds 1e, 5b, and 8a were given orally at
doses of 500, 700, and 500 (mg/kg)/day for 3 days, and the
animals were closely monitored during the 3 days and the
following 14 days. No animal died during the 17 days,
indicating that the LD50 values of 1e, 5b, and 8a were larger
than 500, 700, and 500 mg/kg orally. Rats were given
compound 1 orally at a dose of 150 (mg/kg)/day for 3
months; no pathological changes were seen of the main
organs (Figure 5). The body weights of the animals increased
slowly. The animals were healthy in behavior and had shiny
fur. The LD50 of 1 on the Kunming mice was over 5 g/kg
(orally) and 1.58 g/kg (intraperitoneally). All these data
indicated that compounds 1, 1e, 5b, and 8a were considerably
safe in vivo.
Conclusion
The BMP pathway is a promising new target to design
therapeutic agents for the treatment of low bone mass. Sub-
stituted benzothiophene and benzofuran derivatives were
found to be a new class of small molecules that act as potential
anabolic agents targeting BMP-2. Twenty-seven substituted
benzothiophene and benzofuran derivatives have been
synthesized as a novel class of BMP-2 up-regulators and also
as a new class of potent antiosteoporosis agents. The synthe-
sis, structure-activity relationships, and bone loss prevention
efficacies of these compounds in different animal models have
been presented. At concentrations 10-fold higher than that of
Lov, compounds 1, 2, 3, 14, 4a, 7a, and 8a were found to
exhibitmorepotent activitiesonenhancingBMP-2 expression
in vitro, whereas compounds 1e, 2e, 3d, 5b, and 6b were found
to exhibit similar BMP-2 up-regulating rates as Lov. The SAR
information revealed that (i) the phenyl ring of benzothio-
phene substituted by methyl carboxylate may be good for
activity, (ii) the C-2 acetyl group of benzothiophene or
benzofuran replaced by a cinnamoyl group decreased the
activity in vitro, (iii) the C-2 ethylcarboxylate group made
C-5, C-6 methoxyl or methylenedioxy groups more favorable
to the activities, and (iv) the introduction of methylsulfinyl
and methylsulfonyl groups at the β-position of the C-2
carbonyl group decreased the activities compared with com-
pounds 1 and 2.
Compounds 1, 1e, 5b, and 8a, based on the structures and
in vitro activities, were chosen to test in vivo efficacy in
SAMP6 mice. The four tested compounds significantly
enhanced mouse BMP-2 expression in vivo and improved
mouse bone quality. Compared with the MC group, the
bone formation related histomorphometries (TBV% and
TFS%) of all treated groups were increased; however, bone
resorption related histomorphometry (TRS%) was not
affected. This observation indicated that these types of
compounds improve bone quality possibly by stimulating
the formation of new bone growth through the BMP-2
pathway.

1826 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Guo et al.
Groups treated with compounds 1 and 8a showed higher
UpRs in vitro and significantly enhanced mouse BMP-2
expression in vivo when compared with the values observed
in the untreated group. Compound 8a enhanced the total
BMD and bone mass of SAMP6 mice at a dose of 30 (mg/kg)/
day orally for 3 months. In the SAMP6 mice model, com-
pound 8a showed much stronger effects on the bone forma-
tion rate of the tested mice. Compound 1, first found to be a
BMP-2 up-regulating agent, improved bone quality in
SAMP6 mice and also showed evident activity in OVX rats.
In addition, histological examination of the main organs of
the SAMP6 mice and rats proved that compounds 1 and 8a
were considerably safe in vivo.
Most of the antiosteoporosis agents currently used in the
clinic can effectively prevent further bone loss and stabilize
bonemass butdonot have substantialinfluence onfacilitating
new bone formation. A safe, efficacious drug that stimulates
the formation of bone and restoration of trabecular connec-
tivity would represent a major therapeutic advance in the
osteoporosis field. In this work, it was demonstrated that
substituted benzothiophene and benzofuran derivatives, espe-
cially compounds 1 and 8a, enhance BMP-2 expression in
vitro and in vivo and stimulate bone formation and trabecular
connectivity restoration in vivo. The compounds represent
potential leads in the development of a new class of anabolic
agents.
4-methoxybenzaldehyde (e) using a procedure similar to that
for compound 1c. Yield, 70%; yellow solid; mp 116-119 °C. 1H
NMR (CDCl3) δ: 3.87 (s, 3H), 6.96 (d, J = 8.4 Hz, 2H),
7.40-7.49 (m, 3H), 7.64 (d, J = 8.8 Hz, 2H), 7.87 (d, J = 15.2
Hz, 1H), 7.92 (d, J = 8.4 Hz, 2H), 8.09 (s, 1H). HRMS (EIþ)
calcd for C18H14O2S [M]þ 294.0715; found 294.0711.
(E)-1-(Benzofuran-2-yl)-3-phenylprop-2-en-1-one (2c). The
title compound was obtained from 2 and c using a procedure
similar to that for compound 1c. Yield, 69%; yellow solid; mp
98-101 °C. 1H NMR (CDCl3) δ: 7.24-7.28 (m, 5H), 7.37-7.40
(m, 2H), 7.43 (d, J=7.6 Hz, 1H), 7.47-7.48 (m, 1H), 7.51-7.52
(m, 1H), 7.56 (d, J=7.6 Hz, 1H), 7.59 (s, 1H), 7.62-7.63 (m, 1H),
7.67 (d, J=8.0 Hz, 1H), 7.74 (dd, J=7.6 Hz, 2.8 Hz, 1H), 7.89 (d,
J=15.6 Hz, 1H), 8.27 (d, J=15.6 Hz, 1H). HRMS (ESIþ) calcd
for C17H13O2 [M þ H]þ 249.09155; found 249.09089.
(E)-1-(Benzofuran-2-yl)-3-(2-chlorophenyl)prop-2-en-1-one (2d).
The title compound was obtained from 2 and d using a proce-
dure similar to that for compound 1c. Yield, 61%; yellow solid;
mp 108-112 °C. 1H NMR (CDCl3) δ: 7.33 (d, J=7.6 Hz, 1H),
7.36-7.38 (m, 2H), 7.46-7.49 (m, 1H), 7.51 (d, J=7.6 Hz, 1H),
7.58 (d, J=15.6 Hz, 1H), 7.63 (d, J=8.8 Hz, 1H), 7.67 (s, 1H),
7.74 (d, J=7.6 Hz, 1H), 7.83 (dd, J=7.6 Hz, 2.8 Hz, 1H), 8.35
(d, J=15.6 Hz, 1H). HRMS (EIþ) calcd for C17H11ClO2 [M]þ
283.0526; found 283.0545.
Ethyl 2-Cinnamoylbenzo[b]thiophene-5-carboxylate (3c). The
title compound was obtained from 3 and c using a procedure
similar to that for compound 1c. Yield, 62%; yellow solid; mp
127-128 °C. 1H NMR (CDCl3) δ: 1.44 (t, J=7.2 Hz, 3H), 4.44
(q, J = 7.2 Hz, 2H), 7.54 (s, 3H), 7.59 (d, J = 15.2 Hz, 1H),
7.68-7.69 (m, 2H), 7.91 (d, J=15.2 Hz, 1H), 7.94 (d, J=8.0 Hz,
1H), 8.10 (d, J=8.0 Hz, 1H), 8.15 (s, 1H), 8.64 (s, 1H). HRMS
(EIþ) calcd for C20H16O3S [M]þ 336.0820; found 336.0796.
Ethyl (E)-2-(3-(2-Chlorophenyl)acryloyl)benzo[b]thiophene-5-
carboxylate (3d). The title compound was obtained from 3 and d
using a procedure similar to that for compound 1c. Yield, 62%;
yellow solid; mp 91-93 °C. 1H NMR (CDCl3) δ: 1.44 (t, J=7.2
Hz, 3H), 4.44 (q, J = 7.2 Hz, 2H), 7.35-7.37 (m, 2H), 7.47 (d,
J=8.8 Hz, 1H), 7.50 (d, J=15.6 Hz, 1H), 7.79 (dd, J=8.0 Hz,
2.4 Hz, 1H), 7.95 (d, J=8.4 Hz, 1H), 8.13 (d, J=8.4 Hz, 1H),
8.17 (s, 1H), 8.30 (d, J=15.6 Hz, 1H), 8.65 (s, 1H). HRMS (EIþ)
calcd for C20H15O3SCl [M]þ 370.0430; found 370.0418.
Ethyl (E)-2-(3-(4-Methoxylphenyl)acryloyl)benzo[b]thiophene-
5-carboxylate (3e). The title compound was obtained from 3 and
e using a procedure similar to that for compound 1c. Yield, 34%;
flavogreen solid; mp 146-148 °C. 1H NMR (CDCl3) δ: 1.45 (t,
J =7.2 Hz, 3H), 3.88 (s, 3H), 4.44 (q, J=7.2 Hz, 2H), 6.97 (d,
J=8.4 Hz, 2H), 7.42 (d, J=15.2 Hz, 1H), 7.65 (d, J=8.4 Hz,
2H), 7.89 (d, J=15.2 Hz, 1H), 7.94 (d, J=8.4 Hz, 1H), 8.12 (d,
J=8.4 Hz, 1H), 8.15 (s, 1H), 8.64 (s, 1H). HRMS (EIþ) calcd for
C21H18O4S [M]þ 366.0926; found 366.0916.
Experimental Section
Chemistry. Melting points (mp) were determined with an
X6 microscope melting point apparatus and were uncorrected.
1H NMR spectra were recorded on a Varian Mercury-400
spectrometer. HRMS spectra were recorded on an AccuTOF
CS mass spectrometer. The area normalization purities of tested
compounds were >95% as determined by a high-pressure
liquid chromatography (HPLC). HPLC was performed using
an Agilent 1200 series, diode array detector. The eluting process
was performed on a Dikma PlatisilTM ODS 5 μm, 250 mm ꢀ 4.6 mm
column using a mobile phase of water-methanol (20:80 v/v for
compounds 1, 2, 3, 4a, 5a, 6a, 7a, 8a, 9a, 10a, 11a, 5b, 6b, 7b, 8b,
9b, 5c, and 8c; 10:90 v/v for compounds 1c, 1d, 1e, 2c, 2d, 3c, 3d,
3e, 3f, and 3g), an injection volume of 1 μL controlled by an
autosampler, and a flow rate of 1.0 mL/min.
(E)-1-(Benzo[b]thiophen-2-yl)-3-phenylprop-2-en-1-one (1c).
1-(Benzo[b]thiophen-2-yl)ethanone (1, 500 mg, 2.8 mmol), so-
dium ethylate (191 mg, 2.8 mmol), and benzaldehyde (c, 361 mg,
3.4 mmol) were added to 25 mL of ethanol. The reaction mixture
was stirred at room temperature for 1.5 h and then evaporated
under vacuum. The residue was dissolved in chloroform and
washed with water. The organic layer was dried over anhydrous
magnesium sulfate and then filtered to remove the precipitate
and concentrated in vacuo. The residue was purified by flash
chromatography over silica gel, affording the title compound
(349 mg, 60%). Yellow solid; mp 119-122 °C. 1H NMR
(CDCl3) δ: 7.41-7.51 (m, 5H), 7.55 (d, J=15.6 Hz, 1H), 7.68-
7.70 (m, 2H), 7.89-7.94 (m, 3H), 8.12 (s, 1H). HRMS (EIþ)
calcd for C17H12OS [M]þ 264.0609; found 264.0595.
Ethyl (E)-2-(3-(3,4-Dimethoxylphenyl)acryloyl)benzo[b]thio-
phene-5-carboxylate (3f). The title compound was obtained
from 3 and 3,4-dimethoxybenzaldehyde (f) using a procedure
1
similar to that for compound 1c. Yield, 10%; yellow oil. H
NMR (CDCl3) δ: 1.44 (t, J=7.2 Hz, 3H), 3.94 (s, 3H), 3.98 (s,
3H), 4.45 (q, J=7.2 Hz, 2H), 6.93 (d, J=8.4 Hz, 1H), 7.20 (s,
1H), 7.29 (d, J=8.4 Hz, 1H), 7.40 (d, J=15.2 Hz, 1H), 7.88 (d,
J=15.2 Hz, 1H), 7.95 (d, J=8.8 Hz, 1H), 8.12 (d, J=8.8 Hz,
1H), 8.17 (s, 1H), 8.65 (s, 1H). HRMS (EIþ) calcd for C22H20-
O5S [M]þ 396.1031; found 396.1032.
Ethyl (E)-2-(3-(2,3,4-Trimethoxylphenyl)acryloyl)benzo[b]thio-
phene-5-carboxylate (3g). The title compound was obtained
from 3 and 3,4,5-trimethoxybenzaldehyde (g) using a procedure
similar to that for compound 1c. Yield, 30%; yellow solid; mp
113-115 °C. 1H NMR (CDCl3) δ: 1.44 (t, J=7.2 Hz, 3H), 3.91
(s, 3H), 3.94 (s, 3H), 3.99 (s, 3H), 4.44 (q, J=7.2 Hz, 2H), 6.75
(d, J=8.8 Hz, 1H), 7.42 (d, J=8.8 Hz, 1H), 7.59 (d, J=15.6 Hz,
1H), 7.94 (d, J = 8.4 Hz, 1H), 8.08 (d, J = 15.6 Hz, 1H),
8.11-8.14 (m, 2H), 8.64 (s, 1H). HRMS (EIþ) calcd for
C23H22O6S [M]þ 426.1137; found 426.1147.
(E)-1-(Benzo[b]thiophen-2-yl)-3-(2-chlorophenyl)prop-2-en-1-one
(1d). The title compound was obtained from 1 and 2-chloro-
benzaldehyde (d) using a procedure similar to that for com-
pound 1c. Yield, 67%; yellow solid; mp 105-108 °C. 1H NMR
(CDCl3) δ: 7.34-7.38 (m, 2H), 7.43-7.51 (m, 3H), 7.52 (d, J=
15.6 Hz, 1H), 7.78-7.81 (m, 1H), 7.91 (d, J=8.4 Hz, 1H), 7.93
(d, J = 8.0 Hz, 1H), 8.12 (s, 1H), 8.28 (d, J = 15.6 Hz, 1H).
HRMS (FABþ) calcd for C17H11ClOS [M þ H]þ 299.0297;
found 299.0313.
(E)-1-(Benzo[b]thiophen-2-yl)-3-(4-methoxylphenyl)prop-2-en-
1-one (1e). The title compound was obtained from 1 and

Article
Ethyl 5-Methylbenzo[b]thiophene-2,5-dicarboxylate (4a). To a
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1827
(CDCl3) δ: 1.42 (t, J = 7.2 Hz, 3H), 4.02 (s, 3H), 4.44 (q, J =
7.2 Hz, 2H), 6.92 (d, J=7.6 Hz, 1H), 7.21 (d, J=7.6 Hz, 1H),
7.22 (dd, J = 7.6 Hz, 3.2 Hz, 1H), 7.52 (s, 1H). HRMS (EIþ)
calcd for C12H12O4 [M]þ 220.0736; found 220.0733.
mixture of 4 (2.0 g, 9.6 mmol) and anhydrous potassium
carbonate (1.6 g, 11.6 mmol) in DMF at 0 °C, ethyl thioglycolate
(1.4 g, 11.7 mmol) was added dropwise.23 The system was stirred
at 0 °C for 30 min and then at 60 °C for 12 h. The solution was
filtered to remove the precipitate, and the filtrate was concen-
trated under vacuum. The residue was dissolved in chloroform
and washed with water. The organic layer was dried over
anhydrous magnesium sulfate and then filtered to remove the
precipitate and concentrated in vacuo. The residue was purified
by flash chromatography over silica gel, affording the title
compound (2.5 g, 97%) as a white solid; mp 78-79 °C. 1H
NMR (CDCl3) δ: 1.43 (t, J=7.2 Hz, 3H), 3.97 (s, 3H), 4.43 (q,
J = 7.2 Hz, 2H), 7.91 (d, J = 8.4 Hz, 1H), 8.10 (d, J = 8.4 Hz,
1H), 8.12 (s, 1H), 8.58 (s, 1H). HRMS (EIþ) calcd for C13H12-
O4S [M]þ 264.0456; found 264.0452.
1-(Benzo[b]thiophen-2-yl)-2-(methylsulfinyl)ethanone (5b).
Sodium hydride (60%, 280 mg, 7.0 mmol) was washed three
times with petroleum ether, dried under vacuum, and then mixed
with dry dimethyl sulfoxide (8 mL). The system was protected
with nitrogen and heated to 70-75 °C.26 The reaction system
was cooled to 0 °C when effervescence stopped. 5a (200 mg,
1.0 mmol) in 8 mL of dry tetrahydrofuran was added slowly. The
reaction system was then stirred for 30 min at room temperature
and quenched with 60 mL of ice cold water. The pH of the
solution was adjusted to 3-4 with 2 N HCl. The mixture was
extracted with chloroform (80 mL ꢀ 2). The combined organic
layer was washed with water (160 mL ꢀ 3). The organic layer
was dried over anhydrous magnesium sulfate and then filtered
to remove the precipitate and concentrated in vacuo. The
residue was purified by flash chromatography over silica gel,
affording the title compound (202 mg, 87%). White solid; mp
120-123 °C. 1H NMR (CDCl3) δ: 2.79 (s, 3H), AB system (4.33,
4.41, J = 13.2 Hz, 2H), 7.43 (t, J = 7.0 Hz, 1H), 7.51 (t, J =
7.0 Hz, 1H), 7.88 (d, J=7.0 Hz, 1H), 7.94 (d, J=7.0 Hz, 1H),
8.09 (s, 1H). HRMS (EIþ) calcd for C11H10O2S2 [M]þ 238.0122;
found 238.0102.
1-(4-Chlorobenzo[b]thiophen-2-yl)-2-(methylsulfinyl)ethanone
(6b). The title compound was obtained from 6a and dimethyl
sulfoxide using a procedure similar to that for compound 5b.
Yield, 59%; yellow solid; mp 156-159 °C. 1H NMR (CDCl3) δ:
2.83 (s, 3H), AB system (4.38, 4.45, J=13.5 Hz, 2H), 7.46 (d, J=
4.5 Hz, 2H), 7.79 (t, J=4.5 Hz, 1H), 8.24 (s, 1H). HRMS (EIþ)
calcd for C11H9O2S2Cl [M]þ 271.9733; found 271.9733.
1-(5, 6-Dimethoxylbenzo[b]thiophen-2-yl)-2-(methylsulfinyl)-
ethanone (7b). The title compound was obtained from 7a and
dimethyl sulfoxide using a procedure similar to that for compound
5b. Yield, 96%; yellow solid; mp 161-164 °C. 1H NMR (CDCl3)
δ: 2.78 (s, 3H), 3.96 (s, 3H), 3.99 (s, 3H), AB system (4.28, 4.35, J=
13.6 Hz, 2H), 7.25 (s, 1H), 7.28 (s, 1H), 7.96 (s, 1H). HRMS (EIþ)
calcd for C13H14O4S2 [M]þ 298.0334; found 298.0306.
Ethyl Benzo[b]thiophene-2-carboxylate (5a). The title com-
pound was obtained from 2-nitrobenzaldehyde (5) and ethyl
thioglycolate using a procedure similar to that for compound 4a.
1
Yield, 94%; yellow solid; mp 36-38 °C. H NMR (CDCl3) δ:
1.42 (t, J=7.0 Hz, 3H), 4.42 (q, J=7.0 Hz, 2H), 7.39-7.47 (m,
2H), 7.86-7.89 (m, 2H), 8.06 (s, 1H). HRMS (EIþ) calcd for
C11H10O2S [M]þ 206.0397; found 206.0397.
Ethyl 4-Chlorobenzo[b]thiophene-2-carboxylate (6a). The title
compound was obtained from 2-chloro-6-fluorobenzaldehyde
(6) and ethyl thioglycolate using a procedure similar to that for
compound 4a. Yield, 12%; yellow solid; mp 55-57 °C. 1H NMR
(CDCl3) δ: 1.43 (t, J = 7.0 Hz, 3H), 4.43 (q, J = 7.0 Hz, 2H),
7.35-7.41 (m, 2H), 7.73-7.75 (m, 1H). HRMS (EIþ) calcd for
C11H9O2SCl [M]þ 240.0012; found 240.0014.
Ethyl 5,6-Dimethoxylbenzo[b]thiophene-2-carboxylate (7a).
The title compound was obtained from 6-nitroveratraldehyde
(7) and ethyl thioglycolate using a procedure similar to that for
compound 4a. Yield, 63%; white solid; mp 82-84 °C. 1H NMR
(CDCl3) δ: 1.405 (t, J = 7.2 Hz, 3H), 3.95 (s, 3H), 3.98 (s, 3H),
4.38 (q, J=7.2 Hz, 2H), 7.24 (s, 1H), 7.25 (s, 1H), 7.93 (s, 1H).
HRMS (EIþ) calcd for C13H14O4S [M]þ 266.0631; found
266.0613.
Ethyl 5,6-Dimethylenedioxybenzo[b]thiophene-2-carboxylate
(8a). The title compound was obtained from 6-nitropiperonal
(8) and ethyl thioglycolate using a procedure similar to that for
1-(5, 6-Methylenedioxybenzo[b]thiophen-2-yl)-2-(methylsulfinyl)-
ethanone (8b). The title compound was obtained from 8a and
dimethyl sulfoxide using a procedure similar to that for com-
pound 5b. Yield, 90%; yellow solid; mp 182-184 °C. 1H NMR
(CDCl3) δ: 2.77 (s, 3H), AB system (4.26, 4.33, J=13.2 Hz, 2H),
6.08 (s, 1H), 7.22 (s, 1H), 7.24 (s, 1H), 7.91 (s, 1H). HRMS (EIþ)
calcd for C12H10O4S2 [M]þ 282.0021; found 282.0002.
1
compound 4a. Yield, 69%; white solid; mp 110-112 °C. H
NMR (CDCl3) δ: 1.39 (t, J = 7.2 Hz, 3H), 4.37 (q, J = 7.2 Hz,
2H), 6.04 (s, 1H), 7.19 (s, 1H), 7.20 (s, 1H), 7.89 (s, 1H). HRMS
(EIþ) calcd for C12H10O4S [M]þ 250.0300; found 250.0305.
Ethyl Benzofuran-2-carboxylate (9a). 9a24,25 was obtained
from salicylaldehyde (9) and ethyl bromoacetate using a proce-
dure similar to that for compound 4a. Yield, 56%; yellow oil. 1H
NMR (CDCl3) δ: 1.35 (t, J = 7.2 Hz, 3H), 4.37 (q, J = 7.2 Hz,
2H), 7.22 (t, J = 7.6 Hz, 1H), 7.36 (t, J = 7.6 Hz, 1H), 7.45 (s,
1H), 7.51 (d, J=8.0 Hz, 1H), 7.60 (d, J=8.0 Hz, 1H). HRMS
(EIþ) calcd for C11H10O3 [M]þ 190.0630; found 190.0616.
Ethyl 5,7-Dichlorobenzofuran-2-carboxylate (10a). The title
compound was obtained from 3,5-dichlorosalicylaldehyde (10)
and ethyl bromoacetate using a procedure similar to that for
compound 4a. Yield, 65%; white solid; mp 78-79 °C. 1H NMR
(CDCl3) δ: 1.44 (t, J=7.2 Hz, 3H), 4.46 (q, J=7.2 Hz, 2H), 7.46
(d, J=1.6 Hz, 1H), 7.48 (s, 1H), 7.57 (d, J=1.6 Hz, 1H). HRMS
(EIþ) calcd for C11H8O3Cl2 [M]þ 257.9850; found 257.9840.
Ethyl 7-Fluorobenzofuran-2-carboxylate (11a). The title com-
pound was obtained from 3-fluoro-2-hydroxybenzaldehyde (11)
and ethyl bromoacetate using a procedure similar to that for
compound 4a. Yield, 47%; yellow oil. 1H NMR (CDCl3) δ: 1.43
(t, J=7.2 Hz, 3H), 4.45 (q, J=7.2 Hz, 2H), 7.14-7.25 (m, 2H),
7.43 (d, J=8.0 Hz, 1H), 7.54 (d, J=2.8 Hz, 1H). HRMS (EIþ)
calcd for C11H9O3F [M]þ 208.0536; found 208.0531.
1-(Benzofuran-2-yl)-2-(methylsulfinyl)ethanone (9b). The title
compound was obtained from 9a and dimethyl sulfoxide using a
procedure similar to that for compound 5b. Yield, 30%; yellow
1
solid; mp 124-127 °C. H NMR (CDCl3) δ: 2.85 (s, 3H), AB
system (4.30, 4.39, J = 13.2 Hz, 2H), 7.35 (t, J = 7.2 Hz, 1H),
7.54 (t, J=7.2 Hz, 1H), 7.61 (d, J=8.0 Hz,1H), 7.68 (s, 1H), 7.75
(d, J = 8.0 Hz, 1H). HRMS (EIþ) calcd for C11H10O3S [M]þ
222.0351; found 222.0350.
1-(Benzo[b]thiophen-2-yl)-2-(methylsulfonyl)ethanone (5c). 5b
(100 mg, 0.42 mmol) was dissolved in 4 mL of acetic acid. Then
2 mL of 30% H2O2 was added to the solution at 0 °C. The reaction
system was stirred at room temperature for 5 h. The mixture was
extracted with chloroform (4 mL ꢀ 3). The combined organic
layer was washed with water (15 mL ꢀ 3). The organic layer was
dried over anhydrous magnesium sulfate and then filtered to
remove the precipitate and concentrated in vacuo. The residue
was purified by flash chromatography over silica gel, affording
the title compound (48 mg, 45%). White solid; mp 158-160 °C.
1H NMR (CDCl3) δ: 3.17 (s, 3H), 4.60 (s,2H), 7.45 (t, J=7.0 Hz,
1H), 7.53 (t, J=7.0 Hz, 1H), 7.89 (d, J=8.0 Hz, 1H), 7.96 (d, J=
8.0 Hz, 1H), 8.11 (s, 1H). HRMS (EIþ) calcd for C11H10O3S2
[M]þ 254.0071; found 254.0074.
Ethyl 7-Methoxylbenzofuran-2-carboxylate (12a). The title
compound was obtained from 3-methoxysalicylaldehyde (12)
and ethyl thioglycolate using a procedure similar to that for
compound 4a. Yield, 58%; white solid; mp 82-84 °C. 1H NMR
1- (5,6-Methylenedioxybenzo[b]thiophen-2-yl)-2-(methylsulfonyl)-
ethanone (8c). The title compound was obtained from 8b and

1828 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4
Guo et al.
30% H2O2 using a procedure similar to that for compound 5c.
Yield, 80%; yellow solid; mp 203-206 °C. 1H NMR (CDCl3) δ:
3.15 (s, 3H), 4.53 (s, 2H), 6.09 (s, 2H), 7.22 (s, 1H), 7.24 (s, 1H),
7.94 (s, 1H). HRMS (EIþ) calcd for C12H10O5S2 [M]þ 297.9970;
found 297.9967.
(Ralo, 5 (mg/kg)/day, orally for 3 months), and OVX with
compound 1 (7.5, 15, and 30 (mg/kg)/day, orally for 3 months,
respectively).
BMD Test. The BMD of mice were measured at 0, 4, 8, and
12 weeks after treatment. Mice were anesthetized with pento-
barbital sodium (50 mg/kg, ip) and placed in the prone position.
The body was extended and fixed with adhesive tape. BMD was
measured by dual-energy X-ray absorptiometry (DXA) (OTE-
OCORE 3, MEDILINK, France) according to the manufac-
turer’s instructions. BMD values were analyzed using the small
animals software package of OTEOCORE 3. The whole body
(total BMD), spine, and left hind leg were chosen as the ROIs.
Undecalcified Bone Section and Bone Histomorphometry.
Proximal ends (1/3) of the right femora of mice/rats were
harvested and fixed in 4% paraformaldehyde. Femora were
dehydrated in ascending concentrations of ethanol (80%, 95%,
100%) and cleared with dimethylbenzene. Samples were im-
mersed in polymer fluids I, II, III for 3 days, respectively.
Polymer fluid I contained 100 mL of methyl methacrylate,
35 mL of n-butyl methacrylate, 5 mL of methyl benzoate, and
1.2 mL of polyethylene glycol (400). Polymer fluid II contained
fluid I and 0.4 g of benzoyl peroxide. Polymer fluid III contained
fluid II and 0.8 g of benzoyl peroxide. An amount of 400 μL of
N,N-dimethyl-p-toluidine was added to 140 mL of fluid III at
4 °C, and the mixture was stirred for 10 min. The mixture (7 mL)
and a sample were placed into a bottle, and the air in the bottle
was removed by flushing with nitrogen. The bottle was kept at
-20 °C for 1 week. After cropping, the embedded tissue was
sliced with a microtome (Reichert-Jung 2040, Oberkochen,
Germany) into 5 μm sections. Sections were stained with
toluidine blue and analyzed using a Leica Qwin image analysis
system for TBV% (trabecular bone volume percentage), TFS%
(trabecular formation surface percentage), TRS% (trabecular
resorption surface percentage), and OSW (average osteoid
width).
Methyl 2-Acetylbenzo[b]thiophene-5-carboxylate (3). Methyl
3-formyl-4-nitrobenzoate (4, 1.95 g, 9.3 mmol), ethyl mercap-
tide (0.7 mg, 11.3 mmol), and anhydrous potassium carbonate
(1.55 g, 11.2 mmol) were added to 60 mL of DMF at 0 °C. The
reaction system was stirred at 0 °C for 10 min and then at room
temperature for 1 h. The solution was filtered to remove the
precipitate, and the filtrate was concentrated under vacuum.
The residue was dissolved in chloroform and washed with water.
The organic layer was dried over anhydrous magnesium sulfate
and then filtered to remove the precipitate and concentrated in
vacuo. The residue was purified by flash chromatography over
silica gel, affording methyl 4-(ethylthio)-3-formylbenzoate (13)
as a white solid.
13 (obtained above, 1.0 g, 4.5 mmol), calcium oxide (125 mg,
2.2 mmol), and chloroacetone (825 mg, 8.9 mmol) were added
to 20 mL of DMF. The reaction mixture was stirred at 90 °C for
10 h. The solution was filtered to remove the precipitate, and the
filtrate was concentrated under vacuum. The residue was dis-
solved in chloroform and washed with water. The organic layer
was dried over anhydrous magnesium sulfate and then filtered
to remove the precipitate and concentrated in vacuo. The
residue was purified by flash chromatography over silica gel,
affording the title compound (929 mg, 89%) as a yellow solid;
mp 158-161 °C. 1H NMR (CDCl3) δ: 2.69 (s, 3H), 3.98 (s, 3H),
7.93 (d, J =8.4 Hz, 1H), 8.00 (s, 1H), 8.11 (d, J= 8.4 Hz, 1H),
8.61(s, 1H). HRMS (EIþ) calcd for C12H10O3S [M]þ 234.0351;
found 234.0339.
Biological Methods. In Vitro Assay. Cell Lines. The murine
calvarial MC3T3-E1 cells were obtained from the Cell Culture
Centre, Institute of Basic Medical Science Chinese Academy of
Medical Sciences (Beijing, China). Cells were grown in DMEM
medium and supplemented with 10% heat-inactivated FBS at
37 °C in a humidified 5% CO2 incubator.
Frozen Bone Section and Immunohistochemistry. The proxi-
mal ends (1/3) of the left femora of the mice were harvested and
fixed in 4% neutral paraformaldehyde for 24 h. The femora were
washed with 0.1 M PBS (pH 7.2-7.4) and decalcified with 10%
Reporter Plasmid Construction.21 The plasmid pCAT4.5X
with the 50-flanking promoter region -3365 bp to -1658 bp of
the mouse BMP-2 gene was kindly provided by Professor
Rogers (Biochemistry and Molecular Biology, New Jersey
Medical School, UMDNJ). After sequence verification, the
1709 bp product was cloned into the pGL4.17-basic vector
(Promega) using SacI and XhoI restriction sites. This con-
structed vector, containing the mouse BMP-2 promoter region
and the neomycin resistance gene, was designated as pGL4-
BMP-2.
EDTA 2Na/0.1 M PBS (pH 7.2-7.4) at 4 °C. The liquid was
3
changed every other day for 4 weeks. The bone tissue was
washed with 0.1 M PBS (pH 7.2-7.4) and dehydrated using a
15% sucrose solution/0.1 M PBS (pH 7.2-7.4) for 24 h. Frozen
bone sections (5 μm) were obtained from a freezing microtome.
Immunohistochemistry was performed to observe the expres-
sion of BMP-2. The primary and secondary antibodies were a
rabbit polyclonal to BMP-2 (Abcam) and a mouse IgG/alkaline
phosphatase (PV6001, Zymed), respectively. The expression of
BMP-2 was observed using a Leica Qwin image analysis system.
Toxicity Test. Male and female Kunming mice (18-21 g) and
female rates were purchased from the Institute of Laboratory
Animal Science, Chinese Academy of Medical Sciences and
Peking Union Medical College. Mice were divided into three
groups with six mice each (three males, three females). The mice
of groups 1e, 5b, and 8a were orally given doses of 500, 700, and
500 (mg/kg)/day for 3 days, and the animals were closely
monitored during the 3 days and the following 14 days to
observe the acute toxicity. Rats (n = 7) were orally given com-
pound 1 at a dose of 150 (mg/kg)/day for 3 months.
Animal Experiments. Male and female SAMP6 mice (5-months
old) were obtained from the Peking University Health Science
Center. Animals were nurtured according to the institutional
guidelines of the Chinese Academy of Medical Science and
housed in an air-conditioned room with a 12 h light and 12 h
dark cycle. There were two female mice per cage and one male
mice per cage. The mice were randomly divided into six groups
based on the initial BMD. The untreated group (n=5; 3 males,
2 females) were given the vehicle 0.5% methylcellulose by
gavage. The treated groups (n = 5; 3 males, 2 females) were
given the study compounds suspended in 0.5% methylcellulose
by gavage. The doses of Lov, 1, 1e, 5b, and 8a were 10, 30, 30, 30,
and 30 (mg/kg)/day, respectively, for 3 months. Hearts, livers,
spleens, lungs, and kidneys of SAMP6 mice of all groups were
harvested and weighed, and histological examinations were
performed to detect and observe pathological changes.
Femal rats (205-235 g) were obtained from the Institute of
Laboratory Animal Sciences, Chinese Academy of Medical
Sciences and the Peking Union Medical College. There were
three to four rats per cage. Rats were randomly divided into six
groups (n = 7) based on the body weight, including sham-
operated (SHAM), OVX with vehicle (OVX), OVX with raloxifene
Acknowledgment. This work was supported by the Nati-
onal Natural Science Foundation of China (Grant 30772646)
and the National Major Science and Technology Project of
China (“Innovation and Development of New Drugs”, Grant
2009ZX09102-065).
Supporting Information Available: Purities and HPLC traces
for the tested compounds in this paper. This material is available
free of charge via the Internet at http://pubs.acs.org.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 4 1829
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