Inhibitors of tissue-nonspecific alkaline phosphatase: Design, synthesis, kinetics, biomineralization and cellular tests

Article abstract of DOI:10.1016/j.bmc.2013.09.053

Chronic kidney disease (CKD) is associated with numerous metabolic and endocrine disturbances, including abnormalities of calcium and phosphate metabolism and an inflammatory syndrome. The latter occurs early in the course of CKD and contributes to the development and progression of vascular calcification. A few therapeutic strategies are today contemplated to target vascular calcification in patients with CKD: vitamin K2, calcimimetics and phosphate binders. However, none has provided complete prevention of vascular calcification and there is an urgent need for alternate efficient treatments. Recent findings indicate that tissue-nonspecific alkaline phosphatase (TNAP) may represent a very promising drug target due to its participation in mineralization by vascular smooth muscle cells. We report the synthesis of four levamisole derivatives having better inhibition property on TNAP than levamisole. Their IC₅₀, K_i and water solubility have been determined. We found that the four inhibitors bind to TNAP in an uncompetitive manner and are selective to TNAP. Indeed, they do not inhibit intestinal and placental alkaline phosphatases. Survival MTT tests on human MG-63 and Saos-2 osteoblast-like cells have been performed in the presence of inhibitors. All the inhibitors are not toxic at concentrations that block TNAP activity. Moreover, they are able to significantly reduce mineralization in MG63 and Saos-2 osteoblast-like cells, indicating that they are promising molecules to prevent vascular calcification.

Full text of DOI:10.1016/j.bmc.2013.09.053

Bioorganic & Medicinal Chemistry 21 (2013) 7981–7987
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibitors of tissue-nonspecific alkaline phosphatase: Design,
synthesis, kinetics, biomineralization and cellular tests
a,b,
, Lei Chang a, , Stéphanie Marquès , Stéphane Pellet-Rostaing , Le Duy Do b,d,
a
a,c
Julien Debray
b
b,
⇑
b
e,
⇑
a,
⇑
Saida Mebarek , René Buchet , David Magne , Florence Popowycz , Marc Lemaire
a
Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, UMR-CNRS 5246, Equipe Catalyse Synthèse Environnement, Université de Lyon, Université Lyon 1, F-69622
Villeurbanne, France
Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, UMR-CNRS 5246, Equipe Organisation et Dynamique des Membranes Biologiques, Université de Lyon, Université
b
Lyon 1, F-69622 Villeurbanne, France
c
Institut de Chimie Séparative de Marcoule, UMR-CNRS 5257, site de Marcoule, Université Montpellier 2, F-30207 Bagnols sur Cèze, France
Department of Biochemistry, Nencki Institute of Experimental Biology, Polish Academy of Sciences, 02-093 Warsaw, Poland
Institut de Chimie et Biochimie Moléculaires et Supramoléculaires, UMR-CNRS 5246, Equipe Chimie Organique et Bioorganique, INSA Lyon, F-69621 Villeurbanne, France
d
e
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 31 July 2013
Revised 16 September 2013
Accepted 20 September 2013
Available online 17 October 2013
Chronic kidney disease (CKD) is associated with numerous metabolic and endocrine disturbances, includ-
ing abnormalities of calcium and phosphate metabolism and an inflammatory syndrome. The latter
occurs early in the course of CKD and contributes to the development and progression of vascular calci-
fication. A few therapeutic strategies are today contemplated to target vascular calcification in patients
with CKD: vitamin K2, calcimimetics and phosphate binders. However, none has provided complete pre-
vention of vascular calcification and there is an urgent need for alternate efficient treatments. Recent
findings indicate that tissue-nonspecific alkaline phosphatase (TNAP) may represent a very promising
drug target due to its participation in mineralization by vascular smooth muscle cells. We report the syn-
thesis of four levamisole derivatives having better inhibition property on TNAP than levamisole. Their
Keywords:
Alkaline phosphatase
Benzo[b]thiophen
Enzyme
Inhibitor
i
IC50, K and water solubility have been determined. We found that the four inhibitors bind to TNAP in
Levamisole
Vascular calcification
an uncompetitive manner and are selective to TNAP. Indeed, they do not inhibit intestinal and placental
alkaline phosphatases. Survival MTT tests on human MG-63 and Saos-2 osteoblast-like cells have been
performed in the presence of inhibitors. All the inhibitors are not toxic at concentrations that block TNAP
activity. Moreover, they are able to significantly reduce mineralization in MG63 and Saos-2 osteoblast-
like cells, indicating that they are promising molecules to prevent vascular calcification.
Ó 2013 Elsevier Ltd. All rights reserved.
1
. Introduction
End-stage of renal disease [ESRD or stage-5 chronic kidney dis-
provided complete prevention of vascular calcification and there
is an urgent need for alternate efficient treatments. A few years
ago, it was still commonly thought that vascular calcification in
ESRD was solely due to an increased Ca  Pi product resulting from
hyperphosphatemia. However, several reports have challenged this
point of view. For instance, medial calcification also occurs in aging
and diabetes, when calcium and phosphate concentrations are in
the normal range. Moreover, the mere increase in Ca  Pi product
ease (CKD)] is associated with numerous metabolic and endocrine
disturbances, including abnormalities of calcium and phosphate
metabolism and an inflammatory syndrome. CKD contributes to
the development and progression of vascular calcification. The
prevalence of CKD has reached epidemic proportions with
1
8
1
0–13% of the population in Japan or Canada for instance.
in vivo is not sufficient to induce any calcification in mice. Medial
The coronary calcium score is recognized as a strong predictor
calcifications much more likely result from the tissue-nonspecific
alkaline phosphatase (TNAP) induced removal of inorganic pyro-
phosphate (PPi), which is a potent mineralization inhibitor. PPi is
normally produced in the vasculature and other tissues to inhibit
hydroxyapatite (HA) crystal formation. In bone and growth plate
cartilage, PPi is physiologically removed by TNAP, an ectoenzyme
expressed by osteoblasts and growth plate chondrocytes to
promote mineralization. In humans, TNAP functional deficiency
leads to severe hypomineralization and death in utero.9 On the
opposite, PPi deficiency due to mutations in the gene encoding the
2
of incident coronary heart disease in the general population and
3
–6
even more in patients with ESRD
who develop exacerbated vas-
7
cular calcification. A few therapeutic strategies are today contem-
plated to target vascular calcification in patients with CKD: vitamin
K2, calcimimetics and phosphate binders. However, none has
1
⇑
Corresponding authors. Tel.: +31 4 72 43 13 20; fax: 31 4 72 43 15 43.
E-mail address: rbuchet@univ-lyon1.fr (R. Buchet).
Both Julien Debray and Lei Chang contributed equally.
0
968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.09.053

7
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J. Debray et al. / Bioorg. Med. Chem. 21 (2013) 7981–7987
PPi-producing enzyme NPP1 leads to severe, fatal arterial calcifica-
from benzo[b]thiophene as reported in the literature
(Scheme 1).
1
0
27,28
tion. Plasma PPi levels are negatively associated with vascular
1
1
calcification in ESRD which highlights the central role of TNAP.
TNAP activity is significantly higher in vascular smooth muscle
cells (VSMCs) from aortas of uremic rats as compared with control
352 was prepared in an overall yield of 22% by condensation of 1
with the 2-aminothiazole followed by the acetylation of 2, sodium
borohydride reduction of 4 and ring closure of 6 with thionyl chlo-
ride. A similar sequence of reactions was employed for the conden-
sation of the bromomethyl aryl ketone 1 with 2-aminothiazoline.
Following the similar synthetic sequence developed for 352, the
intermediate 3 was converted into levamisole derivative 354. To in-
crease the hydrophilic character of the 352 benchmark compound,
1
2
ones. In this context, administration of PPi and/or inhibition of
TNAP appear as particularly appealing strategies. PPi administra-
tion in uremic rodents strongly decreases vascular calcifica-
1
1,13
tion.
However, PPi is
a
short-lived molecule in the
circulation, with a half-life of about 30 min. It is therefore uncer-
tain that PPi may be a suitable long-term treatment option in
amino group was introduced from the first step of the synthetic se-
1
4
29
humans. Therefore, we have privileged the strategy of TNAP inhi-
quence starting from 2-acetyl-3-aminobenzo[b]thiophene.
To
1
5–18
bition.
The most potent commercial TNAP inhibitor known so
complete the series of compounds 352-NO and 352-NH were pre-
2
2
far, levamisole, cannot be used in vivo, since 1 mM is necessary to
block calcification in cultures of aortas from uremic rats, a level
which cannot be achieved in vivo.12 To our knowledge, only one
laboratory in the United States developed new TNAP inhibitors
having higher potency than levamisole and outstanding selectiv-
pared (Fig. 1) (Synthetic scheme and analytical data are available in
the Supplementary data).
2.2. Inhibition and solubility properties of benzothiophenyl
derivatives as compared to those of levamisole
1
9–24
ity.
Recently, we patented a family of compounds showing a
1
8
several-fold higher potency than levamisole to inhibit TNAP.
The inhibition property of each compound was determined by
measuring TNAP activity at alkaline pH (optimum conditions)
and at physiological pH, IC50 values determined at both pH
(Table 1) indicated that all four synthetized inhibitors (352, 354,
Their chemical structures1
9–24
are completely different from that
of our molecules. In this article, we report the synthesis of four lev-
amisole derivatives having better TNAP inhibition properties as
compared with levamisole. Their IC50, K
i
and water solubility have
2 2
352-NO and 352-NH ) have better inhibition properties than lev-
been determined. We found that the four inhibitors bind selec-
tively to TNAP in an uncompetitive manner. They do not inhibit
intestinal alkaline phosphatase (IAP) or placental alkaline phos-
amisole. Indeed their IC50 values either at pH 10.4 or at pH 7.8 were
significantly lower as compared with those of levamisole. Standard
internal reference levamisole has been also included consistently
all along the biological assays. Each compound behaves in an
uncompetitive manner and is selective to TNAP. At 400 mM—that
is, thirteen to hundred times more to inhibit TNAP- all compounds
did not inhibit IAP or PLAP indicating specificity of the inhibition
(Table 1).
phatase (PLAP). All inhibitors are not toxic at 10 lM concentration
and are able to reduce the ascorbic acid (AA) and b-glycerophos-
phate (b-GP) induced mineralization in MG63 and in Saos-2 cells
indicating that the development of TNAP inhibitors may be a
promising strategy to prevent vascular calcification.
Assuming that each inhibitor acts in an uncompetitive manner
having Michaelis–Menten kinetics, K
52-NO —the best inhibitor-as compared with that of levamisole
which was 30.5 ± 0.5 M while other have K ranging from
.9 ± 0.2 (352), 5.0 ± 0.2 (354) to 10.3 ± 0.1
352-NH ), confirming that all synthesized inhibitors can inhibit
TNAP at thirty (352-NO ) to three time (352-NH ) lower concen-
tration than levamisole (Table 1). Solubility property is an impor-
tant factor which can control the accessibility of the inhibitor in
hydrophobic or hydrophilic binding sites. Therefore we synthe-
sized a series of inhibitors having different water solubility. The
i
amounted to 1.3 ± 0.1 lM for
2
2
. Results
3
2
l
i
.1. Organic synthesis
3
(
l
M
l
M
lM
In 1977, a series of meta- and para-substituted phenyl deriva-
2
tives of (±)-2,3-dehydrotetramisole and (±)-tetramisole was syn-
thesized.
described above, benzo[b]thiopheno-2,3-dehydrotetramisole 352
and (±)-tetramisole 354 were obtained in five steps from the known
2
2
2
5,26
Following the synthetic methodology already
3
-(2-bromoacetyl)benzo[b]thiophene 1 previously synthesized
1
2
.BuLi (1.5eq), DMA (1.05 eq)
Et O, -78°C to reflux, 3h
2
HN
2
-aminothiazole or
N
.
-
2-aminothiazoline (2 eq)
S
Br (1.2 eq), AcOH
3
N
Br
i
5
0°C, 30 min then rt,1h
PrOH, reflux,1 h
S
S
O
6
5%
S
O
1
2
3
CH=CH (95%)
CH -CH (86%)
2
2
Ac 2O (2 eq), pyridine (8 eq)
CHCl3, reflux, 14 h
AcN
Ac N
N
HCl
NaBH4 (2.4 eq), MeOH
rt, 14 h
S
N
1
. SOCl2, CH2Cl2, 0°-rt, 2h
S
N
S
(
±)
2. HCl. Et O, acetone
2
N
S
O
3
S
S
O H
3
3
52 CH=CH (61%)
54 CH -CH2 ( 52%)
4
5
CH=CH (63%)
CH -CH (96%)
6
7
CH=CH ( 96%)
2
CH -CH (95%)
2
2
2
2
Scheme 1. Reaction scheme.

J. Debray et al. / Bioorg. Med. Chem. 21 (2013) 7981–7987
7983
N
S
N
S
H2N
N
(±)
S
(
±)
(±)
O 2N
N
±)
S
(
N
N
N
S
S
N
S
S
3
52 HCl
354 HCl
352-NO2 HCl
352-NH2 HCl
Figure 1. Molecules synthesized.
upper limit of concentrations to solubilize them in water were
calcification44 This has convinced us15–18 and others19–24,43 to
search for TNAP inhibitors to be used as a drug therapy to target
vascular calcification induced by VSMCs. So far, their developments
are still in infancy and no in vivo data have been reported yet.
Therefore, there is a need to develop specific TNAP inhibitors that
are more efficient than levamisole. Based on the fact that a series
0
3
.45 ± 0.09 mM for 354, 1.3 ± 0.4 mM for 352, 1.77 ± 0.2 mM for
52-NH and 6.9 ± 1 mM for 352-NO
2
2
.
2
.3. Effects of inhibitors on the viability of Saos-2 and MG63
cells
1
5
12
of benzothiophenes as well as levamisole are both able to inhi-
bit TNAP, levamisole coupled with benzothiophenes were synthe-
sized and tested for their inhibition properties. We found four
hits which inhibited TNAP selectively and at lower concentration
than levamisole, validating the strategy of organic synthesis and
search of TNAP inhibitors. In addition, the four inhibitors, like lev-
amisole, did not inhibit IAP or PLAP. Since the water solubility of
the compound 352, which was 1.3 mM and that of compound 354
amounting to 0.45 mM were much lower than that of levamisole
which was 200 mM, we reasoned that we may gain better inhibi-
tion if we could increase their solubility. That was the case for the
Human osteosarcoma Saos-2 cells produce a collagenous extra-
3
0
cellular matrix
and spontaneously release mineralization-
competent matrix vesicles (MVs).3
1,32
Extracellular MVs can not
only initiate mineral formation under normal skeletal calcification
but also during vascular calcification.3
3–37
VSMCs undergoing
pathologic mineralization have been shown to mimic osteoblasts
and/or growth plate chondrocytes, particularly in their ability to
produce MVs enriched in TNAP.38 Therefore, we selected osteo-
blast-like Saos-2 cell cultures as a convenient model of osteoblast
mineralization and vascular calcification induced by VSMCs to ana-
lyze the effects of inhibitors on the cellular viability and minerali-
zation. In addition to Saos-2 cells, we used MG63 cells that upon
352-NO
2
compound having a water solubility of 6.9 mM which is
com-
a better inhibitor than compound 352. However the 352-NH
2
AA and b-GP treatment undergo osteoblast differentiation, as
pound with a water solubility amounting to 1.77 mM was a weaker
inhibitor than compound 352. Furthermore, water solubility of the
inhibitor could be an important issue considering the fact that TNAP
is an ectoenzyme facing the extracellular medium. All the inhibitors
particularly illustrated by enhanced TNAP activity.3
9,40
Both cell
lines were used to monitor cell survival and mineralization. Incu-
bation of up to 10 M of levamisole or compounds 352 and 354
l
did not affect MG63 cell surviving (Fig. 2).
at the concentration of 10
lM are not cytotoxic. Although the best
This was further confirmed by using Saos-2 cells tested with all
inhibitor 352-NO can inhibit TNAP at least at ten time less concen-
2
inhibitors including 352-NO
2
and 352-NH
2
(Fig. 3) indicating that
trated than levamisole (Table 1), it reduced the AA and b-GP in-
exposure of levamisole (used as a control) and each of all synthe-
sized inhibitors are not toxic to these cells.
duced mineralization in Saos-2 cells four times less from around
100% (control) to around 25% (352-NO ) as compared with levam-
2
isole that was three times less from around 100% (control) to 33%
(Levamisole) (Fig. 5). Compound 352 was slightly better than lev-
amisole to inhibit the AA and b-GP induced mineralization in
MG63 cells (Fig. 4) while both 352 and levamisole acted in a similar
manner in Saos2-cells (Fig. 5). In contrast 354 inhibited less well
than levamisole the AA- and b-GP-induced mineralization in
MG63 (Fig. 4) and Saos-2 cells (Fig. 5). The extent of TNAP inhibition
2
cells
.4. Effects of inhibitors on mineralization in Saos-2 and MG63
Ascorbic acid (AA) and b-glycerophosphate (b-GP) are two fac-
tors commonly used to stimulate osteoblast differentiation and
mineralization of MG63 cells39 and Saos-2 cells. As expected,
the mineral deposition-revealed by Alizarin RED-S (AR-S) staining-
41
i
(IC50 or K values), as determined from purified porcine TNAP may
was enhanced by the concomitant addition of 50
.5 mM b-GP in Saos-2 cells as well as in MG63 cells (data not
shown). Incubation of 10 M of levamisole, of 10 M of 352 or of
M 354 decreased the AA- and b-GP induced mineralization
l
g/ml AA and
not perfectly coincide with the extent of mineralization as deter-
mined from mineralization assay on MG63 cells and on human
osteoblast-like Saos-2 cells. The mineralization assay on the whole
cell is a much more stringent test than TNAP inhibition obtained
from purified enzyme since TNAP inhibitor need to be fully accessi-
ble and to bind selectively to TNAP within the whole cell to block
mineralization. Porcine TNAP enzyme provided a direct proof that
the inhibitor can inhibit mammalian TNAP, while measuring min-
eral formation in human Saos-2 cells indicates the efficacy of our
molecules to inhibit human TNAP on the whole cells. Not only the
TNAP inhibition property but water solubility of the inhibitor and
the TNAP accessibility to inhibitors within the cells have to take
into account to reduce efficiently the AA and b-GP induced miner-
alization in MG63 and Saos-2 cells. Taken together these findings
indicate that TNAP inhibitors blocking mineralization as a proof of
principle has been verified, as reported on other inhibitors.2
7
l
l
10 l
in osteoblast-like MG63 cells (Fig. 4).
Similarly, in Saos-2 cells, treatment with 10 lM of levamisole or
of 10
lM of each inhibitor significantly reduced mineralization
(Fig. 5). Although inhibition of TNAP by levamisole and the synthe-
sized inhibitors (Table 1) were different (order of magnitude
change was around ten) the effects on mineralization induced by
MG63 and Saos-2 cells were less variable (order of magnitude of
change was around two) (Figs. 4 and 5).
3
. Conclusion
1,22
TNAP is a key enzyme in the control of mineralization. Its lack of
function in hypophosphatasia leads to impaired mineralization,
while its overexpression or increased activity induces ectopic
4. Experimental section
2
,8,11
calcification.
Importantly, recombinant human TNAP as an
enzyme-replacement therapy can reverse life-threatening hypo-
4.1. Reagents and spectrometry
4
2
phosphatasia
while TNAP inhibitors can suppress VSMCs
4
3
calcification. Due to its key function in mineralization, TNAP is
an obvious target to prevent ectopic calcification including vascular
The commercially available reagents and solvents were used
without further purification. The reactions were monitored by

7
984
J. Debray et al. / Bioorg. Med. Chem. 21 (2013) 7981–7987
Table 1
Inhibition constants IC50 on TNAP, PLAP, and IAP at the indicated pH and water solubility of inhibitors
Structure
TNAP
M) pH
PLAP
IC50
10.4
IAP
IC50
10.4
Solubility
mM)
(
IC50
.8
(lM) pH
IC50
(l
K
7.8
i
(
lM) pH
IC50
7.8
(lM) pH
(
lM) pH
IC50
7.8
(lM) pH
(lM) pH
7
10.4
H
N
S
200a
N
23.2 ± 0.9
62.9 ± 1.6
30.5 ± 0.5
>400
>400
>400
>400
Levamisole
N
S
(
±)
N
S
4.4 ± 0.2
5.5 ± 0.3
13.1 ± 0.1
16.4 ± 0.7
3.9 ± 0.2
5.0 ± 0.2
>400
>400
>400
>400
>400
>400
>400
>400
1.3 ± 0.4
3
5 2 H Cl
N
S
(
±)
N
S
0.45 ± 0.09
3
5 4 HC l
O2N
H2N
N
S
(
±)
N
2.11 ± 0.03
18.3 ± 0.6
3.31 ± 0.02
23.2 ± 2.2
1.3 ± 0.1
>400
>400
>400
>400
>400
>400
>400
>400
6.9 ± 1
S
3
52-NO2 HCl
N
±)
S
(
N
S
10.3 ± 0.1
1.77 ± 0.2
3
52-NH2 HCl
a
Water solubility of levamisole was determined by Sigma.
TLC plates (detection by UV light). The NMR spectra of all the
compounds were recorded on Bruker ALS 300 and DRX 300 spec-
(Et
2
O); 1H NMR (300 MHz, CDCl
3
) d (ppm) 8.07 (s, 1H, H
3
), 7.94–
);
7.88 (m, 2H, Harom), 7.53–7.41 (m, 2H, Harom), 4.46 (s, 2H, CH
2
1
3
trometers in CDCl
3
, DMSO-d
6
, or CD
3
OD-d
4
. Chemical shifts were
C NMR and MS were conform to the literature values (25).
assigned relative to the reference solvent in ppm. Electrospray ion-
ization (ESI) mass spectrometry (MS) experiments were performed
on a Thermo Finnigan LCQ Advantage mass spectrometer. High-
resolution mass spectra (HRMS) were recorded on a Finnigan
Mat 95 Â L mass spectrometer using electrospray. Purifications
were carried out by flash chromatography on silica gel (230–
4
.2.2. 1-(Benzo[b]thiophen-2-yl)-2-(2-iminothiazol-3(2H)-
yl)ethanone (2)
-Aminothiazole (204 mg, 2.04 mmol, 2 equiv) was added to a
solution of 1 (260 mg, 1.02 mmol, 1 equiv) in PrOH (3 ml). The
2
i
resulting mixture was heated at reflux for 1 h. The precipitate
4
00 mesh; 40–63
lm). Elemental analyses were performed by Ser-
was filtered and washed with an aqueous solution of Na
10%), then with Et O to provide 2 (266 mg, 95%) as a yellow crys-
talline solid. Mp = 220–223 °C (Et
) d 8.56 (s, 1H), 8.13 (dd, 2H, J = 1.5, 7.7 Hz); 7.62–7.50 (m,
H); 7.44 (d, 1H, J = 4.5 Hz), 7.09 (d, 1H, J = 4.5 Hz); 5.95 (s, 2H);
2 3
CO
vice Central d’Analyses (UMR 5280).
(
2
1
2
O). H NMR (300 MHz, DMSO-
4
4
1
.2. Organic synthesis
d
6
2
.2.1. 1-(Benzo[b]thiophen-2-yl)-2-bromoethanone (1)
N-Butyl lithium (2.5 M in hexane) (4.5 ml, 11.2 mmol,
.5 equiv) was added dropwise to a solution of benzo[b]thiophene
1
H NMR (300 MHz, CDCl
.88 (d, 1H, J = 7.4 Hz); 7.49 (dt, 1H, J = 1.5, 7.4), 7.42 (dt, 1H,
J = 1.3, 7.4), 6.50 (d, 1H, J = 5.1 Hz), 5.85 (d, 1H, J = 5.1 Hz), 5.17
3
) d 8.19 (s, 1H); 7.92 (d, 1H, J = 7.3 Hz);
7
(
1 g, 7.45 mmol, 1 equiv) in anhydrous Et
mixture was stirred at À78 °C for 30 min then refluxed for 1 h. A
solution of DMA (7.8 mmol, 0.7 ml, 1.05 equiv) in anhydrous Et
5 ml) was added dropwise to the mixture. The mixture was re-
fluxed for 2 h. After addition of aqueous HCl 1 N (20 ml), the aque-
ous layer was extracted with Et
O (3 Â 10 ml). The combined
organic layers were dried over anhydrous MgSO , filtered and con-
2
O (5 ml) at À78 °C. The
1
3
(
1
(
s, 2H); C NMR (75 MHz, DMSO-d
41.4 (C ); 139.2 (C ); 138.9 (C ); 132.1 (CH); 130.6 (CH); 128.3
CH); 126.6 (CH); 125.7; (CH) 123.3 (CH); 107.5 (CH); 54.6 (CH );
MS (ESI) m/z = 259 (C13 S); HRMS-ESI calculated for C13
: 275.0307; found: 275.0308.
6 q q
) d 185.5 (C ); 169.1 (C );
q
q
q
2
O
2
(
H
10
N
2
O
2
H
11-
+
2 2
N OS
2
4
centrated in vacuum to give a crude product which was purified by
flash chromatography on silica gel (cyclohexane/EtOAc: 90:10) to
provide intermediate 2-acetylbenzo[b]thiophene (1.03 g, 78%) as
a crystalline yellow solid. Trimethylphenylammonium tribromide
4.2.3. 1-Benzo[b]thiophen-2-yl-2-(2-imino-thiazolidin-3-yl)-
ethanone (3)
2-Aminothiazoline (2.04 g, 20 mmol, 2 equiv) was added to a
i
solution of 1 (2.5 g, 10 mmol, 1 equiv) in PrOH (25 ml). The result-
(
677 mg, 1.8 mmol, 1.2 equiv) in small portions was added into
ing mixture was heated at reflux for 2 h. The precipitate was fil-
the solution of 2-acetylbenzo[b]thiophene (270 mg, 1.5 mmol,
tered and washed with a saturated aqueous solution of NaHCO
water and finally Et O to provide 3 (2.38 g, 86%) as a yellow crys-
talline solid. H NMR (300 MHz, CDCl ) d 8.15 (s, 1H); 7.91 (dd,
3
,
1
3
equiv) in acetic acid (3 ml). The mixture was stirred at 50 °C for
0 min and then at room temperature for 1 h. The mixture was
2
1
3
evaporated in vacuum. The solid was dissolved in CH
washed with a saturated sodium bicarbonate solution (3 Â 5 ml)
and dried over anhydrous MgSO . The solvent was removed under
reduced pressure and the product was purified by flash chromatog-
raphy on silica gel (cyclohexane/EtOAc: 95:5) to provide 1
2
Cl
2
(20 ml),
J = 1.2; 7.7 Hz, 1H); 7.88 (dd, J = 1.7; 7.0 Hz, 1H); 7.52–7.40 (m,
2H); 5.12 (s, 2H); 3.78 (t, J = 7.8 Hz, 2H); 3.21 (t, J = 7.8 Hz, 2H);
1
3
C NMR (75 MHz, CDCl
140.7 (C ); 139.4 (C ); 130.3 (CH); 128.1 (CH); 126.3 (CH); 125.4
(CH); 123.1 (CH); 53.1 (CH ); 50.0 (CH ); 27.3 (CH ); HRMS-ESI cal-
: 277.0464; found: 277.0459.
3 q q q
) d 187.7 (C ), 171.6 (C ); 142.6 (C );
4
q
q
2
2
2
+
(
300 mg, 84%) as a crystalline yellow solid. Mp = 114–116 °C
13 2 2
culated for C13H N OS

J. Debray et al. / Bioorg. Med. Chem. 21 (2013) 7981–7987
7985
Figure 2. Effect of inhibitors (concentration: 10
l
M) on the viability of MG63 cells
measured using the MTT assay. Results are expressed as percentage of control
Figure 5. Saos-2 cells were incubated for 7 days in presence of 50
l
g/ml AA and
(
(
without inhibitor) and as means ± SD. One pool of sample was repeated three times
n = 3).
7.5 mM b-GP without (control) or with 10 lM inhibitors stained with AR-S to detect
calcium nodules. AR-S was solubilized by cetylpyridinium chloride and quantified
at 562 nm. Results are presented as percentage of control and are mean ± SD with
n = 9–12.
during 30 min. Filtration and drying provided 4 (500 mg, 63%) as
1
a
(
white crystalline solid. Mp = 216–218 °C (Et
300 MHz, DMSO-d
J = 1.3, 7.1 Hz), 7.52 (dt, 1H, J = 1.1, 7.1 Hz), 7.48 (d, 1H,
2
O).
H NMR
6
) d 8.66 (s, 1H), 8.10 (m, 2H), 7.59 (dt, 1H,
J = 4.7 Hz), 6.99 (d, 1H, J = 4.7 Hz), 5.86 (s, 2H), 2.02 (s, 3H); 13
NMR (75 MHz, DMSO-d ) d 187.1 (C ), 178.4 (C ), 166.8 (C
41.5 (C ), 139.8 (C ), 139.0 (C
CH), 126.6 (CH), 125.6 (CH), 123.3 (CH), 108.0 (CH), 53.8 (CH
C
),
6
q
q
q
1
(
2
q
q
q
), 131.7 (CH), 128.2 (CH), 127.6
),
3 13 2 2 2
). HRMS-ESI calculated for C15H N O S : 317.0413;
2
+
6.7 (CH
found: 317.0414.
4
2
.2.5. N-[3-(2-Benzo[b]thiophen-2-yl-2-oxo-ethyl)-thiazolidin-
-ylidene]-acetamide (5)
To a solution of compound 3 (400 mg, 1.45 mmol, 1 equiv) and
Figure 3. Effect of inhibitors on the viability of Saos-2 cells measured using the
MTT assay. Results are expressed as means ± SD. Four independent pools of sample
were repeated each three times (n = 12).
pyridine (934 ll, 20 mmol, 8 equiv) in chloroform (20 ml), was
added acetic anhydride (273 ml, 2.9 mmol, 2 equiv). The mixture
was refluxed for 14 h. After removal of the solvent, the oily residue
was taken into water (20 ml) and stirred at room temperature dur-
ing 30 min. Filtration, washing with water (20 ml) and ether
(
10 ml) provided 5 (438 mg, 96%). Mp = 177–179 °C (Et
NMR (300 MHz, DMSO-d ) d 8.55 (s, 1H); 8.09 (dd, J = 7.5; 8.0 Hz,
H); 7.58 (dt, J = 1.3, 7.5, 1H); 7.54 (dt, J = 1.1, 8.0 Hz, 1H); 5.26
s, 2H); 3.76 (t, J = 7.7 Hz, 2H); 3.22 (t, J = 7.7 Hz, 2H); 1.96 (s,
O). 1
H
2
6
1
(
3
(
1
3
H); C NMR (75 MHz, CDCl
); 142.9 (C ); 141.0 (C ); 139.3 (C
26.6 (CH); 125.7 (CH); 123.4 (CH); 53.4 (CH
); 27.6 (CH ); HRMS-ESI calculated for
19.0569; found: 319.0573.
3
) d 188.1 (C
); 130.5 (CH); 128.3 (CH);
); 50.3 (CH ); 27.7
q q
), 182.6 (C ); 171.9
C
q
q
q
q
1
2
2
+
(
CH
3
2
15 15 2 2 2
C H N O S :
3
4
.2.6. N-[3-(2-Benzo[b]thiophen-3-yl-2-hydroxy-ethyl)-3H-
thiazol-2-ylidene]-acetamide (6)
Figure 4. MG63 cells were incubated for 7 days in presence of 50
lg/ml AA and
7
.5 mM b-GP without (control) or with 10 M inhibitors stained with AR-S to detect
l
Sodium borohydride (26 mg, 0.68 mmol, 1.8 equiv) was added
portionwise to a solution of ketone 4 (120 mg, 0.38 mmol, 1 equiv)
in MeOH (5 ml). The mixture was stirred at rt for 12 h. The second
calcium nodules. AR-S was solubilized by cetylpyridinium chloride and quantified
at 562 nm. Results are presented as percentage of control and are mean ± SD with
n = 3.
4
part of NaBH (9 mg, 0.23 mmol, 0.6 equiv) was added. The mix-
ture was maintained at rt for an additional time of 2 h, concen-
trated under reduced pressure and the solid obtained was
4
2
.2.4. N-[3-(2-Benzo[b]thiophen-3-yl-2-oxo-ethyl)-3H-thiazol-
-ylidene]-acetamide (4)
To a solution of compound 2 (690 mg, 2.5 mmol, 1 equiv) and
suspended in water. The mixture was extracted with CH
2 2
Cl
(3 Â 20 ml). The organic layer was dried on anhydrous MgSO
4
, fil-
tered and concentrated under reduced pressure to provide 6
pyridine (1.6 ml, 20 mmol, 8 equiv) in chloroform (25 ml), was
added acetic anhydride (472 ml, 5 mmol, 2 equiv). The mixture
was refluxed for 14 h. After removal of the solvent, the oily residue
was taken into water (50 ml) and stirred at room temperature
(115 mg, 96%) as a white crystalline solid. Mp = 174–176 °C
1
(Et
2
O). H NMR (300 MHz, CDCl
3
) d 7.84 (d, 1H, J = 7.4 Hz), 7.74
(dd, 1H, J = 1.7, 7.7 Hz), 7.31–7.41 (m, 2H), 6.83 (d, 1H, J = 4.6 Hz),
6.55 (d, 1H, J = 4.6 Hz), 5.55 (d, 1H, J = 1.5, 6.3 Hz), 4.78 (dd, 1H,

7
986
J. Debray et al. / Bioorg. Med. Chem. 21 (2013) 7981–7987
J = 1.5, 14.1 Hz), 4.44 (dd, 1H, J = 6.3, 14.1 Hz), 2.37 (s, 3H); 13C
NMR (75 MHz, CDCl ) d 179.2 (C ), 168.3 (C ), 145.7 (C ), 139.6
), 139.4 (C ), 127.5 (CH), 124.6 (CH), 124.4 (CH), 123.7 (CH),
22.6 (CH), 120.7 (CH), 109.2 (CH), 70.5 (CH), 57.4 (CH ), 26.5
: 319.0569; found:
lid. After washing with acetone, the hydrochloride salt of 354 was
obtained. Mp = 230 °C (Et O, dec). H NMR (300 MHz, MeOD) d
2
7.81 (dd, J = 1.4; 7.2 Hz, 1H); 7.72 (dd, J = 1.3; 6.7 Hz, 1H); 7.34–
1
3
q
q
q
(
1
(
C
q
q
2
7.27 (m, 2H); 7.25 (s, 1H); 5.71 (t, J = 8.7 Hz, 1H); 3.79 (t, J = 8.7,
+
1
CH
3
). HRMS-ESI calculated for C15
H
15
N
2
O
2
S
2
1H); 3.76–3.62 (m, 2H); 3.48–3.41 (m, 1H); 3.30–3.22 (m, 2H); H
3
19.0570.
3
NMR (300 MHz, CDCl ) d 7.79 (dd, J = 1.4; 7.2 Hz, 1H); 7.69 (dd,
J = 1.3; 6.7 Hz, 1H); 7.29 (m, 2H); 7.18 (s, 1H); 5.71 (dd, J = 2.5;
2.5 Hz, 1H); 4.28 (t, pseudot, 1H); 4.01 (t, J = 7.3 Hz, 2H); 3.86 (t,
pseudot, 1H); 3.83 (t, J = 7.3 Hz, 2H); 3.30 (ddd, J = 4.9; 6.5; 8.6 Hz,
4
.2.7. N-[3-(2-Benzo[b]thiophen-2-yl-2-hydroxy-ethyl)-
thiazolidin-2-ylidene]-acetamide (7)
1
3
Sodium borohydride (45 mg, 1.15 mmol, 2 equiv) was added
portionwise to a solution of ketone 5 (183 mg, 0.57 mmol, 1 equiv)
in MeOH (3 ml). The mixture was stirred at rt for 12 h. The second
1H); 3.18 (t, J = 7.3 Hz, 1H); 3.16–3.07 (m, 1H); C NMR (75 MHz,
DMSO-d ) d 176.2; 142.1; 139.0; 138.8; 125.1; 124.9; 124.1;
123.2; 122.8; 62.9; 54.8; 47.7, 36.9; HRMS-ESI calculated for
6
+
part of NaBH
4
(11 mg, 0.34 mmol, 0.6 equiv) was added. The mix-
C H
13 13
N
S
2 2
: 261.0515; found: 261.0516. Anal. Calcd for C13
H
13-
ture was maintained at rt for 2 h, concentrated under reduced
pressure and the solid obtained was suspended in water. The mix-
ClN S : C, 52.60; H, 4.41. Found: C, 52.45; H, 4.12.
2 2
ture was extracted with CH
dried on anhydrous MgSO
duced pressure to provide 7 as a white solid (175 mg, 95%).
Mp = 123–125 °C (Et
O). 1H NMR (300 MHz, MeOD) d 7.94 (dd,
J = 1.4; 7.2 Hz, 1H), 7.8 (dd, J = 1.3; 6.7 Hz, 1H), 7.34–7.33 (m,
2
Cl
2
(3 Â 10 ml). The organic layer was
4.3. Determination of IC50
4
, filtered and concentrated under re-
To determine IC50, activity of porcine kidney TNAP (Sigma), pla-
cental alkaline phosphatase (PLAP) (Sigma) and intestinal alkaline
phosphatase (IAP) (Sigma) was measured in buffer 1 containing
2
3
3
H), 6.27 (d, 1H, J = 4.9 Hz), 5.26 (m, 1H), 3.93–3.74 (m, 3H),
.67–3.58 (m, 1H), 3.09–3.03 (m, 2H); 13C NMR (75 MHz, CDCl
), 174.1 (C ); 147.3 (C ); 139.9 (C ); 139.6 (C ); 124.8
CH); 124.5 (CH); 123.9 (CH); 122.8 (CH); 120.5 (CH); 71.2 (CH);
6.7 (CH ); 52.5 (CH ); 27.9 (CH ); 27.4 (CH ); HRMS-ESI calcu-
lated for C15 : 321.0726; found: 321.0726.
25 mM piperazine, 25 mM glycylglycine, 5 mM MgCl
at pH 10.4. First we mixed 10 L of TNAP (0.52–0.86
buffer (total volume of 690 L). Then 0–60
ing 10 mM inhibitor in DMSO was added to obtain 0–400
inhibitor in medium. Total volume of DMSO was adjusted to
60 L to obtain a solution of 750 L containing 0–400 M inhibitor
2
, 5
l
l
M ZnCl
2
À1
3
)
l
g
l
L
) in
d 181.6 (C
(
5
q
q
q
q
q
l
lL of solution contain-
lM
2
2
3
2
+
H
17
N
2
O
2
S
2
l
l
l
and alkaline phosphatase (either TNAP, PLAP or IAP) in buffer with
4
.2.8. 2-Benzo[b]thiopheno-tetramisole (352)
To a solution of compound 6 (1.08 mmol, 0.3 g) in dichloro-
the final DMSO concentration of 8% v/v. Final alkaline phosphatase
À1
concentration was 6.93–11.4
lg ml . The 750-lL mixture was
methane (5 ml) maintained at 4 °C was added portionwise thionyl
chloride (3 ml) over a period of 30 min. The mixture was stirred at
room temperature for 2 h, and the solvent was removed under vac-
incubated for 10 min at 37 °C without para nitrophenylphosphate
(pNPP). Finally, the 750 L mixture was added in 750 L of pNPP
0.1 mM in the same buffer and stirred during 5 s and incubated
l
l
uum. The residue was refluxed for another 2 h with 10% Na
solution (20 ml) and CH Cl (20 ml). Aqueous phase was extracted
with DCM (3 Â 20 ml). The organic phases were combined and
dried with MgSO . The solvent was removed to leave a solid which
was purified by flash chromatography (EtOAc) affording 352
0.17 g, 61%) as a white solid. Then 352 was directly dissolved in
2
CO
3
at 37 °C, to initiate the reaction. The activity was quantified at
400 nm, using a molar absorption coefficient of 18.5 cm mM
at pH 10.4. Final DMSO concentration was 4% v/v. The activity of
each sample containing the inhibitor was compared with the con-
À1
À1
2
2
4
trol sample (without inhibitor). IC50 and the inhibition constant K
of inhibitors, either porcine kidney TNAP, PLAP or IAP activity were
also measured at pH 7.8 in 0.1 M Tris–HCl buffer with 5 mM MgCl
and 5 mM ZnCl , at 37 °C. The change in absorbance of released
p-nitrophenolate chromophore was monitored at 420 nm, using a
i
(
acetone (2 ml) and 2 N hydrochloric acid (1 ml) in ether was added.
The mixture was stirred overnight and the solvent was removed to
2
2
leave a white solid. After washing with acetone and drying, the
1
À1
À1
hydrochloride salt of 352 was obtained. Mp = 200 °C (Et
NMR (300 MHz, DMSO-d
dd, J = 1.3; 7.0 Hz, 1H); 7.38–7.30 (m, 2H); 7.29 (s, 1H); 6.67 (d,
J = 4.9 Hz, 1H); 5.93 (d, J = 4.9 Hz, 1H); 5.26 (dd, J = 3.9; 3.9 Hz,
2
O).
H
molar absorption coefficient of 9.2 cm mM at pH 7.8. In all
cases, one unit of the alkaline phosphatase activity (U) was defined
as the amount of enzyme hydrolyzing 1 mmol of pNPP per min
under described conditions. All the experiments were repeated
three times in an independent manner.
6
) d 7.92 (dd, J = 1.3; 7.0 Hz, 1H); 7.77
(
1
H); 4.03 (dd, J = 4.1; 13.8 Hz, 1H), 3.89 (dd, J = 7.7; 13.8 Hz, 1H);
1
3
C NMR (75 MHz, DMSO-d
38.9 (C ); 125.5 (CH); 125.1 (CH); 124.9 (CH); 124.1 (CH); 123.1
); HRMS-ESI cal-
: 259.0358; found: 259.0360. Anal. Calcd
: C, 52.96; H, 3.76. Found: C, 52.85; H, 3.88.
6 q q q
) d 170.0 (C ); 142.5 (C ); 139.0 (C );
1
q
4.4. Solubility determination
(
CH); 122.8 (CH); 112.7 (CH); 62.5 (CH), 54.8 (CH
2
+
culated for C13
for C13 11ClN S
H
11
N
S
2 2
Saturated solutions of inhibitors in water were centrifuged.
Then the concentrations of inhibitors were determined by UV spec-
trometry. Three independents measurements were performed.
H
2 2
4
.2.9. 6-Benzo[b]thiophen-2-yl-2,3,5,6-tetrahydro-imidazo[2,1-
b]thiazole (354)
4.5. Mineralizing Saos-2 and MG63 cell cultures
To a solution of compound 7 (320 mg, 1.15 mmol) in dichloro-
methane (5 ml) maintained at 4 °C was added portionwise thionyl
chloride (3 ml) over a period of 30 min. The mixture was stirred at
room temperature for 2 h, and the solvent was removed under vac-
Human osteosarcoma Saos-2 cells (ATCC HTB-85) were cultured
in McCoy’s 5A (PAA) supplemented with 100 U/ml penicillin,
100 mg/ml streptomycin (both from Sigma) and 15% (v:v) fetal bo-
vine serum (FBS) (Gibco). The osteoblast-like MG-63 cell line was
uum. The residue was refluxed for another 2 h with 10% Na
solution (20 ml) and CH Cl (20 ml). Aqueous phase was extracted
with DCM. The organic phases were combined and dried with
MgSO . The solvent was removed to leave a solid which was purified
2 3
CO
2
2
cultured in DMEM with 10% FBS and 1 mM L-glutamine supple-
mented with 100 U/ml penicillin, 100 mg/ml streptomycin. Miner-
alization was induced by culturing the confluent cells in growth
medium supplemented with 50 mg/ml ascorbic acid (Sigma) and
4
by flash chromatography (EtOAc) affording 354 (156 mg, 52%) as a
white solid. Then 354 was directly dissolved in acetone (2 ml) and
4
1,45
7.5 mM b-GP (Sigma).
and treated in presence of 50 mg/ml ascorbic acid and 7.5 mM
b-GP with or without 10 M of inhibitors. The inhibitors were
Cell cultures were grown to confluence
2
N hydrochloric acid (1 ml) in ether was added. The mixture was
stirred overnight and the solvent was removed to leave a white so-
l

J. Debray et al. / Bioorg. Med. Chem. 21 (2013) 7981–7987
7987
solubilized in DMSO and were diluted so that final DMSO concen-
tration in cellular cultures never exceeded 0.1% (v/v). Control cul-
tures received an equivalent amount of solvent (0.1%, v/v).
5. Schlieper, G.; Krüger, T.; Djuric, Z.; Damjanovic, T.; Markovic, N.; Schurgers, L.
J.; Brandenburg, V. M.; Westenfeld, R.; Dimkovic, S.; Ketteler, M.; Grootendorst,
D. C.; Dekker, F. W.; Floege, J.; Dimkovic, N. Kidney Int. 2008, 74, 1582.
6
7
8
9
.
.
.
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4
.6. Calcium nodule detection
Cell layers were washed with phosphate-buffered saline (PBS)
Whyte, M. P. Ann. N.Y. Acad. Sci. 2010, 1192, 190.
(
PAA) and stained with 0.5% (w/v) Alizarin Red-S (AR-S) (Sigma)
1
0. Rutsch, F.; Vaingankar, S.; Johnson, K.; Goldfine, I.; Maddux, B.; Schauerte, P.;
Kalhoff, H.; Sano, K.; Boisvert, W. A.; Superti-Furga, A.; Terkeltaub, R. Am. J.
Pathol. 2001, 158, 543.
46
in PBS (pH 5.0) for 30 min at room temperature. After washing
four times with PBS to remove free calcium ions, cell cultures were
destained with 3.6% (w/v) cetylpyridinium chloride (Sigma) in PBS
pH 7.0 for 60 min at room temperature (27). AR-S concentration
was determined by measuring the absorbance at 562 nm.
1
1
1
1. O’Neill, W. C.; Lomashvili, K. A.; Malluche, H. H.; Faugere, M. C.; Riser, B. Kidney
Int. 2011, 79, 512.
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4
.7. Cell viability assay
14. Persy, V. P.; McKee, M. D. Kidney Int. 2011, 79, 490.
Viability of cultured cells (either Saos-2 or MG63 cell lines) was
15. Li, L.; Chang, L.; Pellet-Rostaing, S.; Liger, F.; Lemaire, M.; Buchet, R.; Wu, Y.
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measured using the MTT colorimetric assay (Roche Diagnostics,
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Meylan, France) as described previously.47 Briefly, cells were cul-
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4
tured in 96-well culture plates at a cell density of 3 Â 10 cells/
2
M.; Buchet, R. Bioorg. Med. Chem. Lett. 2011, 21, 2297.
cm . At the end of the treatment period in presence of 50 mg/ml
1
8. Buchet, R.; Pellet-Rostaing, S.; Lemaire, M.; Chang, L.; Li, L.; Mebarek, S.;
Popowycz, F. Demande 2012, FR 2966152 A1 15 20120420; PCT Int. Appl. 2012,
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ascorbic acid and 7.5 mM b-GP, in absence (control) or presence
10 lM inhibitors, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium bromide (MTT) labeling reagent (0.5 mg/ml final con-
centration) was added to each well. The cells were further incu-
1
2
9. Sergienko, E.; Su, Y.; Chan, X.; Brown, B.; Hurder, A.; Narisawa, S.; Millán, J. L. J.
Biomol. Screen. 2009, 14, 824.
0. Dahl, R.; Sergienko, E.; Mostofi, Y. S.; Yang, L.; Su, Y.; Simao, A. M.; Narisawa, S.;
Brown, B.; Mangravita-Novo, A.; Vicchiarelli, M.; Smith, L. H.; O’Neill, W. C.;
Millán, J. L.; Cosford, N. D. P. J. Med. Chem. 2009, 52, 6919.
bated for 4 h. 100 ll of solubilization solution were then added
and plates were allowed to stand overnight at 37 °C in a humidified
atmosphere. The absorbance was measured the next day at 550
and 690 nm wavelength using a Tecan Infinite M200 (Salzburg,
Austria) micro-titre plate reader. The cell viability was directly cor-
related to the difference of absorbance measured at 550 and
21. Sidique, S.; Ardecky, R.; Su, Y.; Narisawa, S.; Brown, B.; Millán, J. L.; Sergienko,
E.; Cosford, N. D. Bioorg. Med. Chem. Lett. 2009, 19, 222.
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6
90 nm. Results were normalized relative to their respective con-
25. Raeymaekers, A. H. M.; Allewijn, F. T. N.; Vandenberk, J.; Demoen, P. J. A.; Van
trols (without inhibitors) taken as 100. For each inhibitor in
Saos-2 cells three to four distinct pools of sample were analyzed
in a triplicate manner (n = 9–12) while for each inhibitor in
MG63 cells one pool of sample was analyzed in a triplicate manner
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(
n = 3).
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