Synthesis of benzofuran derivatives as selective inhibitors of tissue-nonspecific alkaline phosphatase: Effects on cell toxicity and osteoblast-induced mineralization

Article abstract of DOI:10.1016/j.bmcl.2016.01.061

Tissue-nonspecific alkaline phosphatase (TNAP) by hydrolyzing pyrophosphate, an inhibitor of apatite formation, promotes extracellular matrix calcification during bone formation and growth, as well as during ectopic calcification under pathological conditions. TNAP is a target for the treatment of soft tissue pathological ossification. We synthesized a series of benzofuran derivatives. Among these, SMA14, displayed TNAP activity better than levamisole. SMA14 was found to be not toxic at doses of up to 40 μM in osteoblast-like Saos-2 cells and primary osteoblasts. As probed by Alizarin Red staining, this compound inhibited mineral formation in murine primary osteoblast and in osteoblast-like Saos-2 cells.

Full text of DOI:10.1016/j.bmcl.2016.01.061

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of benzofuran derivatives as selective inhibitors of tissue-
nonspecific alkaline phosphatase: effects on cell toxicity and
osteoblast-induced mineralization
Stéphanie Marquès a, René Buchet b, Florence Popowycz a,c, Marc Lemaire a, Saïda Mebarek b,
⇑
a Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, UMR-CNRS 5246, Equipe Catalyse Synthèse Environnement, Université de Lyon, Université Claude Bernard–Lyon
1, Bâtiment Curien, 43 Bd du 11 Novembre 1918, F-69622 Villeurbanne, France
b Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, UMR-CNRS 5246, Equipe Organisation et Dynamique des Membranes Biologiques, Université de Lyon, Université
Claude Bernard–Lyon 1, Bâtiment Raulin, 43 Bd du 11 Novembre 1918, F-69622 Villeurbanne, France
c Equipe Chimie Organique et Bioorganique, Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, Institut National des Sciences Appliquées (INSA Lyon), Bâtiment
Jules Verne, 20 Avenue Albert Einstein, F-69621 Villeurbanne Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Tissue-nonspecific alkaline phosphatase (TNAP) by hydrolyzing pyrophosphate, an inhibitor of apatite
formation, promotes extracellular matrix calcification during bone formation and growth, as well as dur-
ing ectopic calcification under pathological conditions. TNAP is a target for the treatment of soft tissue
pathological ossification. We synthesized a series of benzofuran derivatives. Among these, SMA14, dis-
Received 30 November 2015
Revised 20 January 2016
Accepted 21 January 2016
Available online xxxx
played TNAP activity better than levamisole. SMA14 was found to be not toxic at doses of up to 40 lM
in osteoblast-like Saos-2 cells and primary osteoblasts. As probed by Alizarin Red staining, this compound
inhibited mineral formation in murine primary osteoblast and in osteoblast-like Saos-2 cells.
Ó 2016 Elsevier Ltd. All rights reserved.
Keywords:
Alkaline phosphatase
Mineralization
Benzofuran derivatives
Calcification disease
Inhibitors
2-aminothiazole (1.1 eq)
iPrOH, reflux, 1 h
Br2 (1 eq)
In humans, three of the four alkaline phosphatase isozymes are
tissue-specific, the intestinal (IAP), placental (PLAP), and germ cell
(GCALP), whereas the fourth is tissue-nonspecific (TNAP) and is
expressed in bone, liver, kidney and the central nervous sys-
tem.1–3 TNAP hydrolyzes extracellular inorganic pyrophosphate
(PPi), a potent mineralization inhibitor, to enable the physiological
deposition of hydroxyapatite (HA).4 Mineralized cartilage, bone,
and teeth are the only tissues in which deposition of calcium phos-
phate in the form of hydroxyapatite is intended. Ectopic calcifica-
tion in soft tissues, under pathological conditions, is associated
with increased levels of TNAP activity. Therefore TNAP is a thera-
peutic target for the treatment of soft tissue ossification abnormal-
ities including ankylosis, osteoarthritis, and arterial calcification.5
Presence of TNAP enriched matrix vesicle in human atherosclerotic
lesions was evidenced in vascular calcification.6 Increase of TNAP
activity in arteries was sufficient to remove PPi secreted by smooth
muscle cells and promote vascular calcification.7
Br
CH2Cl2, 1 h, rt
O
O
O
O
76%
98%
2
1
HN
N
HN
N
SOCl2 (35 eq)
CH2Cl2
S
NaBH4 (2.2 eq)
MeOH, rt, 14 h
S
rt, 1 h
74%
O
O
O
OH
SMA2
99%
SMA7
H2N
N
S
N
O
SMA14
Scheme 1. Synthetic pathway.
Several inhibitors were found to be more potent than levami-
a series of inhibitors easily synthesizable and more available for
exploring the role of TNAP in cells and in tissues. We replaced
benzo[b]thiophene13 scaffold by bioisosteric benzo[b]furan skele-
ton. Chemistry could be easily synthesized and adaptable in the
already developed conditions.
sole.8–13 Some of them were patented. In this Letter, we provided
⇑
Corresponding author. Tel.: +33 42 746 5721.
E-mail address: saida.youjil@univ-lyon1.fr (S. Mebarek).
http://dx.doi.org/10.1016/j.bmcl.2016.01.061
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Marquès, S.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.01.061

2
S. Marquès et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 1
Inhibition constants of inhibitors of TNAP, PLAP and IAP
Structure
TNAP
IC50 (pH 10.4)
M)
PLAP
IAP
IC50 (pH 10.4)
M)
IC50 (pH 7.8)
M)
Ki (pH 7.8)
M)
IC50 (pH 7.8)
M)
IC50 (pH 10.4)
M)
IC50 (pH 7.8)
M)
(
l
(l
(
l
(
l
(l
(
l
(l
H
N
41
1
256 98
>400
47
nd
2
>400
>400
>400
>400
>400
>400
>400
>400
N
S
N
levamisole
NH
S
O
>400
>400
O
sma 2
NH
O
N
S
>400
nd
32
>400
>400
>400
>400
>400
>400
>400
>400
OH
sma 7
O
N
28
2
124 31
1
N
S
sma 14
The synthetic chemistry used for the preparation of the benzo-
furan derivatives is shown in Scheme 1. Bromination of 2-acetyl-
benzo[b]furan in dichloromethane at room temperature provided
compound 2 with 76% yield. Subsequent condensation with 2-
aminothiazole was performed almost quantitatively. Ketone
SMA2 was reduced classically in the presence of a slight excess
of sodium borohydride at room temperature. Upon treatment with
thionyl chloride, alcohol SMA7 furnished cyclized derivative
SMA14. The overall yield of the synthesis was estimated to 55%
for 4 steps (Scheme 1).
The inhibition property of each compound was determined by
measuring TNAP activity at alkaline pH (optimum conditions)
and at physiological pH. Analog of levamisole having a benzofuran
ring was synthesized. This analog and the main synthetic interme-
diates were evaluated. SMA14 had better inhibition properties
than levamisole (Table 1). Its IC50 values either at pH 10.4 or at
pH 7.8 were significantly lower as compared with those of levami-
sole. SMA-14 behaved in an uncompetitive manner. At 400 M,
SMA-14 inhibited neither IAP nor PLAP indicating specificity of
the inhibition (Table 1).
None of the synthetic intermediates to access SMA14 was active
on any alkaline phosphatase, while SMA2, SMA7 and other dis-
l
played IC50 over 400 lM.
The cytotoxicity of SMA14 was tested on human osteoblast like
SAOS-2 and MG63, on mouse vascular smooth muscle cell line
MOVAS-1 and on mouse primary osteoblast cells, using MTT
assay.14 The cells were incubated without or with inhibitors for
3 days. SMA14 and levamisole (used as control) were not toxic in
the concentration range from 10 to 40
(Fig. 1).
lM on different type of cells
We selected osteoblast-like Saos-2 and primary osteoblast cells
to analyze the effects of inhibitors on the mineralization. Ascorbic
Figure 1. Effect of inhibitors on the viability of (A) mouse primary osteoblast, (B) MG63, (C) SAOS-2, (D) MOVAS measured using MTT assay. Results are expressed as
percentage of control containing DMSO 0.1% (mL:mL) without inhibitor. The experiments were repeated in triplicate for each inhibitor (n = 9).
Please cite this article in press as: Marquès, S.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.01.061

S. Marquès et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Figure 2. Effect of SMA14 inhibitor on osteoblast-mediated mineralization, induced by (A) Saos-2 cells and (B) primary osteoblasts as monitored by AR-S staining at the
indicated concentrations. Results are expressed as percentage of control containing DMSO 0.1% (mL:mL) without inhibitor. The experiments were repeated in triplicate for
each inhibitor (n = 9).
acid (AA) and b-glycerophosphate (b-GP) served to stimulate
osteoblast differentiation and mineralization of Saos-2 cells and
primary osteoblast cells.15,16
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from 20 to 40
lM while levamisole decreased it at 30–40
lM to
around 40% of control (Fig. 2A). SMA14 at 10
lM already inhibited
mineralization by 20% in primary cells (Fig. 2B) while levamisole
did not. Although the effects on mineralization are nearly similar,
the solubility property of these two compounds is different.
Levamisole is more soluble in water than SMA14 which may affect
tissue selectivity.
In conclusion, we developed a TNAP inhibitor which could be
easily synthesized with good inhibition of mineralization induced
by osteoblasts and which could be implemented in a drug therapy
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Acknowledgments
The work was supported by CNRS and a Grant of Lyon Science
Transfert, France. We acknowledge Dr. Julien Debray for
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Supplementary data
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Supplementary data (experimental methods) associated with
this article can be found, in the online version, at http://dx.doi.
org/10.1016/j.bmcl.2016.01.061.
Please cite this article in press as: Marquès, S.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.01.061