Synthesis and evaluation of 1-(benzo[b]thiophen-2-yl)ethanone analogues as novel anti-osteoporosis agents acting on BMP-2 promotor

Article abstract of DOI:10.1016/j.bmcl.2009.05.118

A novel series of 1-(benzo[b]thiophen-2-yl)ethanone analogues were prepared and evaluated for enhancing BMP-2 expression. Compounds 1-5, 7, 8, 12, 13 and 16, with upregulation rate values of 35.6%, 27.9%, 39.8%, 32.0%, 37.1%, 30.2%, 28.0%, 33.5%, 22.8% an

Full text of DOI:10.1016/j.bmcl.2009.05.118

Bioorganic & Medicinal Chemistry Letters 19 (2009) 4167–4170
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and evaluation of 1-(benzo[b]thiophen-2-yl)ethanone analogues
as novel anti-osteoporosis agents acting on BMP-2 promotor
Zong-ying Liu , Xiao-bo He , Zhao-yong Yang, Hua-yi Shao, Xue Li, Hui-fang Guo, Yue-qin Zhang,
*
*
Shu-yi Si , Zhuo-rong Li
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050, People’s Republic of China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 1-(benzo[b]thiophen-2-yl)ethanone analogues were prepared and evaluated for enhanc-
ing BMP-2 expression. Compounds 1–5, 7, 8, 12, 13 and 16, with upregulation rate values of 35.6%, 27.9%,
39.8%, 32.0%, 37.1%, 30.2%, 28.0%, 33.5%, 22.8% and 27.3% in vitro, respectively, at a concentration of 4 lM,
exhibited potent effect for enhancing BMP-2 expression. We also found that compounds 1 and 12 pro-
duced a dose-dependent increase on bone histology and histomorphometry, and effectively reduced bone
defects induced by ovariectomy in an ovariectomized rat model (OVX).
Received 23 January 2009
Revised 14 May 2009
Accepted 29 May 2009
Available online 2 June 2009
Keywords:
Synthesis
Ó 2009 Elsevier Ltd. All rights reserved.
BMP-2 promotor
Anti-osteoporosis
Osteoporosis is a reduction in skeletal mass due to an imbalance
between bone formation modulated by osteoblasts and bone
resorption modulated by osteoclasts.1 There are no safe and effec-
tive methods to help porous bone to recover. The mainstay of ther-
apy for osteoporosis is bone resorption. The effects of currently
available drugs in increasing or recovering bone mass are relatively
small, generally less than 10% over 3 years. Therefore, there is a
need for developing new bone-building (anabolic) agents. Anabolic
agents directly stimulate bone formation and correct the imbal-
ance of bone formation and resorption in established osteoporo-
sis.2 Not only are anabolic agents able to increase bone mass, but
they also have the capacity to improve bone quality and increase
bone strength. The only anabolic agent currently available is
recombinant human parathyroid hormone PTH.
Bone morphogenetic protein (BMP) plays important roles in
osteoblastic differentiation and bone formation.2a,3 Among the
BMP family members, BMP-2 has been extensively studied. BMP-
2 is expressed by normal osteoblasts and has been shown to
stimulate osteoblast differentiation and bone formation during
embryonic skeletal development and postnatal bone remodeling
in vitro, as well as bone formation in vivo.2a,4 Application of
rhBMP-2 (recombinant human bone morphogenetic protein-2) to
an open tibial fracture has been reported, however, this product
is very expensive.5
Significant progress has been made in osteoporosis therapy, but
drugs used in the clinic lack specificity, and generally have severe
side effects. Owing to the inherent difficulties associated with
these current anabolic treatments (dose route, narrow therapeutic
index, bone quality), there remains considerable interest in small
molecules which produce new bone growth as potential treat-
ments for osteoporosis. Mundy et al. reported that statins en-
hanced new bone formation in vitro and in rodents. Such effects
were associated with increased expression of the BMP-2 gene in
bone cells.6 Within a program of identifying agents with strong tis-
sue specificity in improving metabolic balance of osteoporosis, and
with the help of an anti-osteoporosis screening model targeting
the promoter of the BMP-2 gene, we have evaluated thousands of
compounds and observed that 1-(benzo[b]thiophen-2-yl)ethanone
(1) has potent activity in enhancing BMP-2 expression and has the
potential to be developed in the future as a clinical drug. To devel-
op a more thorough understanding of the SAR, we wish to explore
substituent effects on the phenyl ring, and both the C-2 and C-3
positions of compound 1. We now wish to report our findings for
these compounds which enhance BMP-2 expression in vitro. Com-
pounds 1 and 12 have also shown bone anabolic effects in vivo in
the ovariectomized rat model.
The synthesis of substituted 1-(benzo[b]thiophen-2-yl)etha-
none analogues 1–11 is shown in Scheme 1. The key intermediates
2-(ethylthio)benzaldehyde analogues 1b–11b were obtained in
good yield from commercially available substituted 2-chlorobenz-
aldehyde and ethanethiol sodium salt, using tetra-n-butylammo-
* Corresponding authors. Tel.: +86 10 63180604; fax: +86 10 63017302 (S.S.);
tel.: +86 10 63027185; fax: +86 10 63017302 (Z.L.).
nium bromide as
a phase transfer catalyst. The ethanethiol
sodium salt was made from ethanethiol and a sodium hydroxide
solution. Reaction of the 2-(ethylthio)benzaldehyde analogues
E-mail addresses: sisyimb@hotmail.com (S.S.), l-z-r@263.net (Z.L.).
These authors made equal contribution to this work.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.118

4168
Z. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4167–4170
O
S
R1
pounds (2–5, 7) showed potent activities, with upregulation rate
O
values of 27.9%, 39.8%, 32.0%, 37.1%, 30.2%. The effect of the 5,6-
(OMe)2-substituted compound 5 is similar to that found for com-
pound 1. The 4-Cl-5-Me-substituted compound 3 showed a little
bit higher activity than compound 1, and its isomer 4-Cl-7-Me-
substituted compound 4 showed a little bit lower activity com-
pared with compound 1. The 7-CF3-substituted compound 7 is
more potent than its 5-CF3 isomer compound 6. These results show
that electron donating groups cause higher upregulation rate val-
ues than electron-withdrawing groups in this system.
By comparing the results for 3 and 4, one can see that C-5 posi-
tion is more favorable for electron donating group attachment than
the C-7 position. However, C-7 position is more favorable for elec-
tron-withdrawing groups than the C-5 position based on the
results for 6 and 7.
R1
R2
R1
R2
b
R2
R
a
S
Cl
O
1b R1 = H, R2 = H
R1 = H, R2 = H
1a
1 = H, R2 = H
1
2
3
4
5
6
7
8
9
2a R1 = H, R2 = 6-Cl
R1 = H, R2 = 4-Cl
2b
R
1 = H, R2 = 6-Cl
3a R1 = H, R2 = 5-Me-6-Cl
3b R1 = H, R2 = 5-Me-6-Cl
4b R1 = H, R2 = 3-Me-6-Cl
5b R1 = H, R2 = 4,5-(OMe)2
6b R1 = H, R2 = 5-CF3
7b R1 = H, R2 = 3-CF3
8b R1 = Me, R2 = H
R1 = H, R2 = 4-Cl-5-Me
R1 = H, R2 = 4-Cl-7-Me
4a
R
1 = H, R2 = 3-Me-6-Cl
5a R1 = H, R2 = 4,5-(OMe)2
6a R1 = H, R2 = 5-CF3
7a R1 = H, R2 = 3-CF3
8a R1 = Me, R2 = H
R
1 = H, R2 = 5,6-(OMe)2
R1 = H, R2 = 5-CF3
R1 = H, R2 = 7-CF3
R1 = Me, R2 = H
9b R1 = Me, R2 = 6-Cl
1 = Me, R2 = 4-Cl
9a
R
1 = Me, R2 = 6-Cl
R
10a R1 = Me, R2 = 5-Me-6-Cl
11a R1 = Me, R2 = 5-CF3
10b R1 = Me, R2 = 5-Me-6-Cl
11b R1 = Me, R2 = 5-CF3
10 R1 = Me, R2 = 4-Cl-5-Me
11 R1 = Me, R2 = 5-CF3
Scheme 1. Reagents and conditions: (a) CH3CH2SH, NaOH, H2O, 1 h, then 1a,
(n-Bu)4NBr, reflux 4 h; (b) ClCH2COCH3, CaO, reflux 3 h.
Next we evaluated the effects of different heterocycles on
enhancing BMP-2 expression. When the sulfur atom in compound
1 was replaced by oxygen to yield 12, the upregulation rate value is
similar with that found for compound 1. When the sulfur atom in
compound 1 was replaced by nitrogen, and the position of the acet-
yl group was changed from C-2 to C-3 to yield 13, the upregulation
rate value decreased to 22.8%. Furthermore, when the position of
the acetyl group of compound 1 was changed from C-2 to C-3 to
yield 14, the upregulation rate value decreased to 11.9%. The re-
sults demonstrated that the 2-acetyl benzoheterocycle is much
better than the 3-acetyl benzoheterocycle.
We also evaluated analogues of compound 1 with different sub-
stituents at C-2 position on enhancing BMP-2 expression. Replace-
ment of the 2-acetyl group in compound 1 with a carboxyl group,
to yield 15, resulted in decreased activity. Replacing the 2-acetyl
group of compound 1 with methyl formate, formamide and form-
hydrazide (yielding 16–18) resulted in decreased activities com-
pared with compound 1. These results demonstrate that the
2-acetyl group is very important for the potent effect of compound 1.
In an effort to further understand the substituent effects, com-
pounds 8–11 were synthesised. All those compounds have a methyl
with chloroacetone in the presence of calcium oxide as a solid base
furnished the corresponding target compounds 1-(benzo[b]thio-
phen-2-yl)ethanone analogues 1–11 using well established litera-
ture methodology.7
The activities of 18 compounds (compounds 12–18 were
bought from Acros Organics) in enhancing BMP-2 expression were
evaluated using lovastatin as a positive control employing recom-
binant plasmid PMB and rat skull cells MC3T3-E1. The results are
summarized in Table 1.6,8 Many compounds exhibited strong activ-
ities in enhancing BMP-2 expression with upregulation rate >21%
(1–5, 7, 8, 12, 13, 16) or >10% to <21% (6, 9, 11, 14, 15, 17). The lead
compound 1-(benzo[b]thiophen-2-yl)ethanone (1), at a concentra-
tion 10-fold higher than that of the positive control, lovastatin,
significantly enhanced BMP-2 expression as reflected by the upreg-
ulation rate value of 35.6%, almost twofold higher than that of
lovastatin.
We first evaluated the effects of substituents and their location
on the phenyl ring of compound 1 on enhancing BMP-2 expression.
4-Cl, 4-Cl-5-Me, 4-Cl-7-Me, 5,6-(OMe)2, 7-CF3 substituted com-
Table 1
a
BMP-2 expression enhancing activities of 1-(benzo[b]thiophen-2-yl)ethanone analogues in vitro
R3
R4
R5
R2
R1
4
5
3
2
6
7
X
R6
Compound
X
R1
R2
R3
R4
R5
R6
Upregulation rate (%)
1
2
3
4
5
6
7
8
S
S
S
S
S
S
S
S
S
S
S
O
NH
S
S
S
COMe
COMe
COMe
COMe
COMe
COMe
COMe
COMe
COMe
COMe
COMe
COMe
H
H
H
H
H
H
H
H
Me
Me
Me
Me
H
COMe
COMe
H
H
Cl
Cl
Cl
H
H
H
H
Cl
Cl
H
H
H
H
H
H
H
H
H
H
H
H
H
H
OMe
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
CF3
H
H
H
H
H
H
H
H
H
H
H
35.6
27.9
39.8
32.0
37.1
18.5
30.2
28.0
16.5
6.5
16.7
33.5
22.8
11.9
10.8
27.3
11.2
7.8
Me
H
OMe
CF3
H
H
H
Me
CF3
H
H
H
H
H
H
H
9
10
11
12c
13c
14c
H
15c
CO2H
CO2Me
CONH2
CONHNH2
16c
H
H
H
17c
S
S
18c
H
Lovastatinb
20.9
a
Compound concentration for 1–18 is 4 lM.
Compound concentration for lovastatin is 0.4
Commercially available.
b
c
lM.

Z. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4167–4170
4169
Figure 1. Histology of left proximal tibia, illustrating trabecula (dark blue). (1) Sham operated group. (2) OVX group. (3) Raloxifene (5 mg/kg/day, positive control) treated
group. (4) 12L (compound 12 10 mg/kg/day) treated group. (5) 12M (compound 12 20 mg/kg/day) treated group. (6) 12H (compound 12 40 mg/kg/day) treated group. (7) 1L
(compound 1 7.5 mg/kg/day) treated group. (8) 1M (compound 1 15 mg/kg/day) treated group. (9) 1H (compound 1 30 mg/kg/day) treated group.
group at the C-3 position. However, the activities of 8–11 decreased
when compared their respective analogs, 8 versus 1, 9 versus 2, 10
versus 3, and 11 versus 6. This finding revealed that the C-3 methyl
group is not favored for enhancing BMP-2 expression.
area and connectivity of the trabecula have significantly increased
compared with the control. In the OVX groups receiving 1 (7.5 mg/
kg/day, 15 mg/kg/day, 30 mg/kg/day) and 12 (10 mg/kg/day,
20 mg/kg/day and 40 mg/kg/day), the number, area, thickness
and connectivity of trabecula increased compared with the OVX
group in a dose-dependent manner.
Table 2 summarizes the effects of the OVX group, the groups
after treatment with raloxifene, compound 1 and compound 12
on bone histomorphometry of rats evaluated in terms of trabecular
bone volume percentage (TBV%), trabecula resorption surface per-
centage (TRS%), trabecula formation surface percentage (TFS%),
Compounds 1 and 12 were chosen for in vivo efficacy evaluation
using the ovariectomized Sprague–Dawley rat model (OVX).9 Test
rats were assigned groups: sham-operated (Sham), OVX with vehi-
cle (OVX, control); OVX with raloxifene (5 mg/kg/day, orally, for
90 days, positive control); OVX with compound 1 (orally for
90 days); and OVX with compound 12 (orally for 90 days).
Bone histology under light microscopy is shown in Figure 1. The
trabecula is dense and compact in the sham-operated group, but
for the OVX group with vehicle, it is turbulent and results in tra-
becula sparse rarefaction. In the OVX groups receiving raloxifene
and compound 1 (30 mg/kg/day), the number, the thickness, the
mineral apposition rate (MAR,
lM/d), osteo cortex mineralization
rate (mAR, M/d) and average osteoid width (OSW,
l
lM).
Clearly, all parameters in the femora of OVX are statistically sig-
nificant compared with the sham group, p < 0.05 on TFS% and mAR,
Table 2
Effects of OVX, oral treatment with raloxifene, compound 1 and compound 12 on bone histomorphometry
Group
TBV (%)
TRS (%)
TFS (%)
MAR(l
M/d)
mAR (
lM/d)
OSW (lM)
SHAM
OVX
Ralo
12L
12M
12H
1L
27.38 1.67
10.34 1.62b
22.31 1.93d
12.39 1.50c
13.13 1.39d
17.50 1.62d
14.41 1.50d
19.39 1.95d
21.02 1.85d
3.49 1.19
8.18 1.75b
3.34 1.03d,e
7.84 1.85
7.15 1.93
5.30 0.79d
7.81 1.67
5.16 1.34d
4.74 1.62d,e
3.97 1.33
6.16 1.76a
6.52 1.76
7.26 1.80
6.75 1.90
6.74 0.93
7.40 1.60
6.64 1.95
6.39 1.85
1.61 0.24
2.28 0.36b
2.14 0.49
2.31 0.49
2.34 0.33
2.20 0.18
2.32 0.32
2.19 0.29
2.18 0.51
2.42 0.36
3.22 0.68a
3.15 0.84
3.28 0.70
3.51 0.68
3.30 0.21
3.42 0.50
3.14 0.61
3.22 0.33
8.65 0.99
11.33 1.91b
11.08 1.59
11.25 1.38
11.62 1.60
10.99 0.66
11.44 1.52
10.86 1.09
10.76 1.49
1M
1H
12L, 12M and 12H: separately treated with compound 12 10 mg/kg/day, 20 mg/kg/day and 40 mg/kg/day for 90 days; 1L, 1M and 1H: separately treated with compound 1
7.5 mg/kg/day, 15 mg/kg/day and 30 mg/kg/day for 90 days.
a
p < 0.05.
b
p < 0.01 compared with sham group.
p < 0.05.
p < 0.01 compared with OVX group.
p > 0.05 compared with sham group.
c
d
e

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Z. Liu et al. / Bioorg. Med. Chem. Lett. 19 (2009) 4167–4170
p < 0.01 on other parameters. Compounds 1 and 12 showed a dose-
dependent increase in all the parameters as compared to OVX.
Ovariectomy is associated with a lower trabecular bone volume
and a higher trabecula resorption surface percentage compared
with sham (p < 0.01). All doses of compounds 1 and 12 showed sta-
tistically significant (p < 0.05) effects on TBV% compared with OVX.
12H and 1M (see Table 2) groups showed higher TRS% (p < 0.01)
than OVX. The raloxifene and 1L (see Table 2) groups showed
strong anti-osteoclast activities compared with OVX (p < 0.01)
and they returned the trabecula resorption surface percentage
back to a level similar to sham (p > 0.05).
Ovariectomy resulted in a statistically significant (p < 0.05)
increase of TFS%, mAR, and showed a very significantly (p < 0.01)
increased MAR and OSW compared with sham. The treatment with
different doses of compound 1, compound 12 and raloxifene had
no significant effect on these parameters.
than lovastatin. We also have found that 1-(benzo[b]thiophen-2-
yl) ethanone (1) and its analogue 12 produced a dose-dependent
increase on bone histology and histomorphometry and effectively
reduced bone defects induced by ovariectomy in a OVX rat model.
Compounds 1 and 12 showed similar activities in vitro, however,
compound 12 showed lower activities in vivo than compound 1.
The LD50 of compound 1 on mice was over 5 g/kg (orally), and
1.58 g/kg (intraperitoneally). These results suggest that com-
pounds of this class may be useful in the treatment of bone degen-
erative disease, including osteoporosis. Further investigation of the
bioavailabilities and activities of compounds 1 and 12 in senes-
cence accelerated mouse and the bioavailabilities and activities
of compounds 3 in OVX are underway.
Acknowledgment
The LD50 of compound 1 for Kuming mice (female, 18–20 g in
weight) was over 5 g/kg (orally), and was 1.58 g/kg (intraperitone-
ally). In order to further understand the toxicity of compound 1, we
treated the sham rats with a dosage of 150 mg/kg/day (orally) for
90 days. Compound 1 at the treatment dosage showed no signifi-
cant effect on general behavior, blood parameters including
glutamine–oxaloacetic transaminase (GOT), glutamic–pyruvic
transaminase (GPT), total bilirubin (TBIL), phosphatases (ALP), total
protein (TP), albumin (Alb), triglycerides (TG), total cholesterol
(CHO), direct high density lipoprotein (D-HDL), direct low density
lipoprotein (D-LDL), glucose (Glu), blood urea nitrogen (B.U.N.),
creatinine (Cre), Ca2+ and bone histomorphometry (TBV, TRS, TFS,
MAR, mAR, OSW) of the tested rats.
The project was supported by the National Natural Science
Foundation of the PR China (30100225 and 30772646).
References and notes
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