Synthesis and evaluation of benzo[b]thiophene derivatives as inhibitors of alkaline phosphatases

Article abstract of DOI:10.1016/j.bmc.2009.08.048

Presence of basic calcium phosphate in knee joints of osteoarthritis patients could be prevented by inhibiting tissue non-specific alkaline phosphatase (TNAP) activity. Levamisole or the L stereoisomer of tetramisole (a known TNAP inhibitor) has been used

Full text of DOI:10.1016/j.bmc.2009.08.048

Bioorganic & Medicinal Chemistry 17 (2009) 7290–7300
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of benzo[b]thiophene derivatives as inhibitors
of alkaline phosphatases
a,b,c,d,e
Lina Li a,b,c,d,e,f, Lei Chang a,b,c,d,e, Stéphane Pellet-Rostaing a,b,c,d,e, , François Liger
,
*
a,b,c,d,e,
f
Marc Lemaire a,b,c,d,e, , René Buchet
, Yuqing Wu
*
*
a Université de Lyon, Villeurbanne F-69622, France
b Université Lyon 1, Villeurbanne F-69622, France
c INSA de Lyon, Villeurbanne F-69621, France
d CPE Lyon, Villeurbanne F-69616, France
e CNRS UMR 5246 ICBMS, Villeurbanne F-69622, France
f State Key Laboratory for Supramolecular Structure and Materials, Jilin University, Changchun 130012, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Presence of basic calcium phosphate in knee joints of osteoarthritis patients could be prevented by inhib-
iting tissue non-specific alkaline phosphatase (TNAP) activity. Levamisole or the L stereoisomer of
tetramisole (a known TNAP inhibitor) has been used as a treatment for curing rheumatoid arthritis but
its therapeutical use is limited due to side effects. We report the synthesis and the TNAP inhibition prop-
erty of benzo[b]thiophene derivatives, among which benzothiopheno-tetramisole and benzothiopheno-
2,3-dehydrotetramisole, which could be involved in a drug therapy for osteoarthritis. Two water soluble
racemic benzothiopheno-tetramisole and -2,3-dehydrotetramisole with apparent inhibition constants
Received 13 March 2009
Revised 17 August 2009
Accepted 21 August 2009
Available online 3 September 2009
Keywords:
Tetramisole
Benzo[b]thiophene
TNAP
Ki = 85
6
l
M and 135
3
l
M (n = 3) comparable to that of enantiomeric levamisole 93
4 lM were
found. Several novel derivatives showed more pronounced inhibition properties towards intestinal alka-
line phosphatase than TNAP.
Inhibitor
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
first functional genes expressed in the process of calcification.
The crucial role of TNAP in the mineralization process is evidenced
Calcium-containing crystals are present in synovial fluid ex-
tracted from the knee joints of up to 70% of osteoarthritis patients,
indicating that pathological calcification occurs in the majority of
osteoarthritis.1–4 Calcified diseases associated with osteoarthritis
are correlated with the presence of calcium pyrophosphate dihy-
drate (CPPD) crystals (25–55% of the time) and/or of the occurence
of basic calcium phosphate (BCP) crystals (35–70% of the time)
consisting of carbonate-substituted hydroxyapatite (HA) and octa-
calcium phosphate.5–8 The origin of CPPD crystals is associated
with the increase in inorganic pyrophosphate (PPi) concentration.
Upregulation of nucleotide pyrophosphatase phosphodiesterase 1
(NPP1) and of ankylosis protein (ANK, a PPi transporter) expres-
sions in articular cartilage can contribute to an extracellular PPi
excess,9 leading possibly to CPPD deposition. Consistent with this
mechanism, some mutations affecting ank gene, upregulating
ANK activity, cause chondrocalcinosis in humans.10,11 In addition,
matrix vesicles (MVS) of affected cartilage in osteoarthritis in-
crease tissue non-specific alkaline phosphatase (TNAP) activity as
much as 30-fold and induce HA deposition.12 TNAP is among the
in the case of hypophosphatasia patients, whose disease results
from mutations in the gene coding TNAP leading to a decreased
or absent TNAP activity.13,14 Therefore, the formation of BCP crys-
tals could be prevented by inhibiting TNAP activity. Levamisole
(Fig. 1) or the L stereoisomer of tetramisole (a known TNAP
inhibitor15) have been used as a treatment for curing rheumatoid
arthritis.16–18 However, skin rashes and agranulocytosis reported
as side effects for Levamisole19,20 have limited its therapeutical
use. Although numerous levamisole analogs (6-aryl,heteroarylimi-
dazo[2,1-b]thiazole) have been described in the literature as
anthelmintics,21–33 few of them have been tested as inhibitors of
alkaline phosphatase22,30 and, as far as we know, none of these
contained heterocyclic moieties (thiophen, pyridine, benzofu-
ran. . .). More recently, Sidique et al. described the screening
test of a library of pyrazolo derivatives as potent and selective
H
N
N
S
Levamisole (L-tetramisole)
* Corresponding authors. Tel.: +33 0 72 44 85 07; fax: +33 0 4 72 43 14 08.
E-mail address: pellet@univ-lyon1.fr (S. Pellet-Rostaing).
Figure 1. Structure of levamisole.
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.08.048

L. Li et al. / Bioorg. Med. Chem. 17 (2009) 7290–7300
7291
COOMe
Ar
R1
NHBz
Ar
R2
or
Ar
S
S
1) C-H
S
51-56
7-50
COOMe
activation
57-59
C-H
activation
2) Erlenmeyer
NHBz
Ar
S
and
60-63
R1
R1
O
1) SNAr
R2
S
1) C-H
R3
X
activation
2) C-H
activation
S
X
R1, R2
2) Friedel-Crafts
=
O
Ar2
H, Br, CHO, CN,
OCH3, OCH2CF3
N
1-6
Ar1
R3 = H, Ar
X = O, nil
S
R3
S
112-124
78-111
X
Scheme 1. General synthesis of benzo[b]thiophene derivatives35–40 7–63 and 78–124.
inhibitors of TNAP34 demonstrating the great interest in the re-
search of new active heterocycles. Recently we developed several
strategies to synthesize benzothiophene derivatives,35–40 which
we could derivative easily, and having a large library, we firstly
checked their inhibitory properties to several alkaline phospha-
tases. Some of them had significant inhibitory properties. Here,
we present the synthesis and the BIAP (bovine intestinal alkaline
phosphatase)/TNAP (porcine kidney tissue non-specific alkaline
phosphatase and chicken femur tissue non-specific alkaline phos-
phatase) inhibition properties of a library of benzothiophene deriv-
atives among which the 6-benzothiopheno-imidazo[2,1-b]thiazole
derivatives (benzothiopheno-tetramisole and benzothiopheno-2,3-
dehydrotetramisole), which could be implemented in a drug ther-
apy for osteoarthitis.
them were synthesized as previously reported in the literature.35–40
Thus, 2- and 3-aryl-Benzo[b]thiophenes 7–56 (Tables 1 and 2)
were obtained from the corresponding benzo[b]thiophene and
2- or 3-cyano, methoxy, carbaldehyde, (2,2,2-trifluoroethoxy)-
benzo[b]thiophene by a one-step palladium coupling with various
aryl bromides as described by us in previous works35,39,40(Scheme
1). Compounds 57–63 were obtained as described40 from the corre-
sponding 2-arylbenzo[b]thiophenes-3-carbaldehydes involving an
Erlenmeyer condensation with hippuric acid, followed by an elec-
trophilic opening of azalactone intermediates and a hydrogenation
of the alkenes. 2-Aryl-3-amino or phenoxybenzo[b]thiophenes 78–
11136 were obtained from the starting 3-bromo-benzo[b]thiophene
1-oxide 77 (Table 3) by using an aromatic nucleophilic substitution
reaction affording the 3-amino and 3-phenoxybenzo[b]thiophenes
1-oxide followed by a palladium coupling involving the corre-
sponding aryl-bromides. Aroyl-Benzo[b]thiophenes 112–124
were synthesized by a direct acylation of benzo[b]thiophene or
2-aryl-benzo[b]thiophene.37
2. Results and discussion
2.1. Chemistry
Finally, 2-aryl-3-methylamino-benzo[b]thiophene type-inter-
mediates 72–76 (Table 2) were obtained by the reduction of the
corresponding 2-aryl-3-cyano-benzo[b]thiophene derivatives with
The synthetic chemistry used for the preparation of the benzo-
thiophene derivatives 7–124 is shown in Schemes 1–3. Some of
CF2H
OH
N
S
COOH
X
64: X = H (76%)
68: X = N (62%)
S
X
S
67: X = H (95%)
71: X = N (95%)
NaClO2
NH2SO3H
dioxane
65 (68%)
DAST
r.t.
NH2OH, HCl
py, EtOH
CH2OH
CHO
COOMe
NaBH4
MeOH
NaCN, MnO2
MeOH
S
X
S
X
S
N
66: X = H (78%)
70: X = N (100%)
21: X = H
24: X = N
69 (93%)
Scheme 2. Synthesis of compounds 65–71.

7292
L. Li et al. / Bioorg. Med. Chem. 17 (2009) 7290–7300
S
S
S
HN
H3COCN
O
S
H3COCN
HO
N
N
N
N
O
N
d, e
43%
a
c
b
HCl
65%
66%
S
S
S
S
128
127
126
129.HCl
Br
O
S
S
S
HN
H3COCN
O
S
H3COCN
HO
S
125
N
N
N
N
O
N
c
b
d,e
HCl
76%
f
40%
22%
S
S
S
S
131
132
130
133.HCl
Scheme 3. Reagents and conditions: (a) 2-aminothiazole, 2-propanol, reflux, (78%); (b) Ac2O, pyridine, chloroform, reflux; (c) NaBH4, MeOH, rt; (d) SOCl2, Ac2O, reflux; (e)
HCl, MeOH (f) 2-aminothiazoline, CH3CN, reflux, (89%).
BH3–THF.38 New 2-aryl-benzothiophenes 65–71 were synthesized
from the 2-phenyl-benzo[b]thiophene-3-carbaldehyde 21 and the
2-(30-pyridine)-benzo[b]thiophene-3-carbaldehyde 24 (Scheme
2). Fluorination with DAST and amination with hydroxylamine
afforded benzo[b]thiophenes 64, 67, 68 and 71. Reduction of 21
and 24 with sodium borohydride gave the corresponding hydroxy-
methyl derivatives 6641 and 70. Although it was already synthe-
sized from thianaphthen-2-one,42 the carboxylic acid 65 was
easily obtained by oxidation of 21 with sodium chlorite and sulfa-
mic acid. Finally, the compound 24 was oxidized43 to a methyl es-
ter 69 with manganese dioxide in the presence of sodium cyanide.
Benzo[b]thiopheno-2,3-dehydrotetramisole 129 and -tetrami-
sole 133 were obtained in five steps from the known 3-(2-bromo-
acetyl)benzo[b]thiophene 125 previously synthesised from
benzo[b]thiophene as reported in the literature44 (Scheme 3). Fol-
lowing the synthetic methodology already described in the litera-
ture,21,22 129 was prepared in an overall yield of 14% by
condensation of 125 with the 2-aminothiazole followed by the
acylation of 126, sodium borohydride reduction of 126 and ring
closure of 128 with thionyl chloride and acetic anhydride. Follow-
ing the similar strategy, the condensation of 125 with the 2-amino-
thiazoline gave the intermediate 130 which was converted into the
levamisole derivative 133.
Although 2-aryl-benzo[b]thiophene-3-carbaldehydes are glob-
ally more efficient than the 3-H, 3-cyano, 3-methoxy and 3-OCH2-
CF3 analogs, 2-phenyl-benzo[b]thiophen-3-oxime 67 (Table 2 and
Fig. 2A) was one of the most promising. 0.1 mM of it inhibited al-
most totally the pNPP hydrolysis by BIAP (Fig. 3A) and 0.4 mM of it
caused a residual TNAP-activity of 68% (Table 2). Figure 3B shows
the inhibition of 67 on the bone TNAP activity of matrix vesicles
(MVs). The inhibition was found to be dependent on the concentra-
tion of DMSO (data not shown), while DMSO (up to 4% v/v) alone
had nearly no effect on BIAP, porcine kidney TNAP or bone TNAP
activities. In this series (Table 2), no significant inhibition was ob-
served with 3-aryl-benzo[b]thiophene derivatives 51–56 towards
BIAP or porcine kidney TNAP and 3-aminomethyl compounds 72,
74 and 76 increased the activity of the BIAP.
Whatever the nature of the phenolic or amino moieties incorpo-
rated in the benzo[b]thiophene-sulfoxide 78–86 (Table 3) and
2-aryl-benzo[b]thiophene 100–110, no significant inhibition was
observed on BIAP and porcine kidney TNAP with compound
concentrations of 0.1 mM and 0.4 mM, respectively. Only the cyano
derivative 81 exhibited a relative residual porcine kidney TNAP-
activity of 67% and the carbaldehyde 90 induced a residual
BIAP-activity of 9%. Finally, in the series of 3-aroyl compounds
112–124 (Table 4), only 2-H derivatives 112 and 113 promoted,
respectively 43% and 40% residual BIAP activities. No significant
inhibition was observed on porcine kidney TNAP with compounds
112–124.
2.2. Evaluation of BIAP and TNAP inhibition
The library of benzothiophene compounds 1–124 (0.1 mM in
bovine intestinal alkaline phosphatase (BIAP) reactive medium
containing p-nitrophenyl phosphate (pNPP) as substrate with final
dimethyl sulfoxide (DMSO) 1% (v/v) and 0.4 mM in porcine kidney
TNAP reactive medium with final DMSO 4% (v/v), respectively) was
tested for the potential inhibition activity of the bovine intestinal
alkaline phosphatase BIAP and the porcine kidney TNAP, respec-
tively (Tables 1–4). As observed in Figure 2A and B, most of benzo-
thiophene derivatives were ineffective or tend to inhibit towards
BIAP and were totally ineffective towards TNAP. Among the 2-aryl-
benzo[b]thiophenes 7–50 (Table 1 and Fig. 2A), 14 and 23 pre-
sented the more interesting effect with 95% and 91% inhibition of
BIAP (corresponding, respectively to a residual activity of 5% and
9% as stated in Table 1), no inhibition was observed with the 3-H
derivatives 43–50 and only the 2-(3’-pyridine)-benzo[b]thiophene
35 was the best porcine kidney TNAP inhibitor with a residual
activity of 46% (Table 1 and Fig. 2B) whereas all of the other 2-(2’
or 3’-pyridine)-benzo[b]thiophene are totally inefficient towards
BIAP and porcine kidney TNAP.
As well as for the most promising compounds 35 and 81 (inhib-
iting specifically porcine kidney TNAP) or 4 and 67 (inhibiting both
BIAP and porcine kidney TNAP), the solubilities of benzothiophene
derivatives 1–124 were not high enough in aqueous buffer and
DMSO (up to 4% v/v) was generally added to solubilize them. Addi-
tion of 4% v/v DMSO into aqueous mineralization medium induced
spontaneously hydroxyapatite formation45 as in the case of matrix
vesicles which are released from hypertrophied chondrocytes dur-
ing physiological endochondral ossification46–48 or from osteoar-
thritic articular chondrocytes.49–52
DMSO is very often used as solvent for water- insoluble drugs and
in several human therapeutic situations.53 Although DMSO has
some beneficial effects,53 several reports indicate that care must
be taken in the experiments with DMSO54,55 or using DMSO as a drug
vehicle.56 DMSO induced hydroxyapatite formation in synthetic car-
tilage lymph.45 Although we found that several water-insoluble
benzothiophene molecules could inhibit TNAP, their effects were
dependent on the DMSO concentration. Whether, DMSO could take
the place of the inhibitor or alter the accessibility of the active site is

L. Li et al. / Bioorg. Med. Chem. 17 (2009) 7290–7300
7293
Table 1
Inhibition effects of benzo[b]thiophene derivatives 1–3 and 7–50 on BIAP and on porcine kidney TNAP activities at pH 10.4 and at 37 °C
General structures
Compound
number
BIAP + 0.1 mM inhibitor
with 1% DMSO
TNAP + 0.4 mM inhibitor
with 4% DMSO
Relative activity (%)
Relative activity (%)
CN: 1
CHO: 2
OMe: 3
92
120
90
92
85
93
R
S
Ph: 7
76
94
96
18
106
94
90
5
95
115
69
97
88
82
N.T.
N.T.
97
4-OMe–Ph: 8
4-CF3–Ph: 9
2-Napht: 10
3-py: 11
99
CN
N.T.
N.T.
N.T.
85
N.T.
N.T.
N.T.
N.T.
91
2-py: 12
Ar
S
2-CN–Ph: 13
2-Me–Ph: 14
3-Quinoline: 15
3-Cl–Ph: 16
4-Cl–Ph: 17
3-OMe–Ph: 18
3,4,5-OMe–Ph: 19
4-N(Me)2–Ph: 20
83
Ph: 21
41
37
9
119
110
110
92
116
30
77
99
N.T.
93
79
N.T.
105
78
80
95
4-OMe–Ph: 22
4-Cl–Ph: 23
3-Py: 24
2-CN–Ph: 25
3-Quinoline: 26
4-CN–Ph: 27
2-NO2–Ph: 28
4-CF3–Ph: 29
3-Cl–Ph: 30
2-Cl–Ph: 31
CHO
Ar
S
62
63
97
Ph: 32
80
57
78
112
102
73
87
79
97
44
N.T.
96
86
90
2-CN–Ph: 33
4-OMe–Ph: 34
3-Py: 35
2-Napht: 36
2-Me–Ph: 37
3-Quinoline: 38
2-Py: 39
OMe
Ar
S
64
99
CH2CF3
2-CN–Ph: 40
3-Py: 41
4-OMe–Ph: 42
70
122
107
87
84
94
Ar
S
2-Py: 43
2-NO2–Ph: 44
2-NH2–Ph: 45
2-NO2-4-Me–Ph: 46
2-NO2-4-Cl–Ph: 47
2-NH2-4-Me–Ph: 48
3-Cl–Ph: 49
119
121
93
127
107
107
95
94
86
70
97
93
93
96
96
Ar
S
2-NO2-4-OMe–Ph: 50
99
not yet clear. Such behavior has been previously reported in the case
of diethanolamine affecting the inhibition of a 2,3,4-trichlorophenyl
derivative of pyrazole on TNAP activity.34 Since DMSO is a promoter
of mineralization, water-soluble benzothiophene derivatives have
to be synthesized. We reasoned that water-soluble tetramisole
could be derivatized with benzothiophene compounds to increase
the solubility of benzothiophene moiety in water and to increase
the inhibition effect having two active sites in one molecule. There-
fore, we designed and synthesized water-soluble benzothiophene
compounds.
chlorhydrate (Fig. 4), 129ꢀHCl (Fig. 5) and 133ꢀHCl (Fig. 6) inhibited
the porcine kidney TNAP. Both samples as levamisole inhibited, in
an uncompetitive manner, porcine kidney TNAP activity, being
consistent with the fact that the location of the binding site of
the inhibitor is controlled by the tetramisole moiety. Their plots
ꢁ1
of
v
versus inhibitor concentration (Figs. 4B, 5B and 6B) allowed
max
us to determine their apparent inhibition constants Ki (pH 7.8;
37 °C and without DMSO). Small differences in their apparent
inhibitor constants (Ki) were observed. A better stabilization of
the interactions between inhibitor and porcine kidney TNAP was
As levamisole, 129 and 133 hydrochloride salt did not inhibit
intestinal alkaline phosphatase (Table 5). However, levamisole
evidenced for 129ꢀHCl (Ki = 85
l
M) as compared with 133ꢀHCl
(Ki = 135 M) (Table 6).
l

7294
L. Li et al. / Bioorg. Med. Chem. 17 (2009) 7290–7300
Table 2
tion effects than levamisole, their side effects could be entirely
different than those of levamisole. The strategy to develop drug-
like-TNAP soluble inhibitors for therapeutic use for treating patho-
logical soft tissue mineralization disorders34,57,58 looks promising.
Inhibition effects of benzo[b]thiophene derivatives 4–6 and -51–76 on BIAP and on
porcine kidney TNAP activities at pH 10.4 and at 37 °C
General
structures
Compound
number
BIAP + 0.1 mM TNAP + 0.4 mM
inhibitor
with 1%
DMSO
inhibitor
with 4% DMSO
3. Conclusion
Activity (%)
Activity (%)
Some benzothiophene derivatives showed more pronounced
inhibition properties towards BIAP than porcine kidney TNAP. Such
compounds may have a clinical application, since the intestinal type
of alkaline phosphatase increased in the urine of patients with renal
disease.60 On the other hand, 6-benzothiopheno-imidazo[2,1-b]thi-
azole derivatives (benzothiopheno-tetramisole and benzothiophe-
no-2,3-dehydrotetramisole), proved to be efficient in the porcine
kidney TNAP and in chicken femur TNAP inhibitions, which could
be implemented in a drug therapy for osteoarthritis. Two water-sol-
uble racemic benzothiopheno-tetramisole and -2,3-dehydrotetra-
misole (129ꢀHCl and 133ꢀHCl) with apparent inhibition constants
I: 4
OCH2CF3: 5
CN: 6
41
116
103
54
100
105
R
R
S
R = H,
Ar =4-OMe–Ph: 51 100
R = CN,
Ar = 4-CF3–Ph: 52 115
Ar = 3-Py: 53
R = OCH2CF3,
Ar = 2-CN–Ph: 54
Ar = 3-Py: 55
R = 2-NO2–Ph
N.T.
Ar
N.T.
N.T.
120
S
101
102
87
97
Ki = 85
6
lM and 135
3
l
M (n = 3) comparable to that of enantio-
Ar = 2-NO2–Ph: 56 108
88
meric levamisole 93
cine kidney TNAP), indicating some potential to synthesize and
optimize enantiomeric benzothiopheno-tetramisole.
4
l
M were found (as determined with por-
COOMe
NBz
4-CN–Ph: 57
4-CF3–Ph: 58
4-OMe–Ph: 59
108
35
77
N.T.
87
91
Ar
4. Experimental section
4.1. Materials
S
S
COOMe
NBz
4-CN–Ph: 60
4-CF3–Ph: 61
4-OMe–Ph: 62
H: 63
115
63
91
N.T.
N.T.
86
Reactants and solvents were supplied by Aldrich, Acros, Lancas-
ter, Alfa Aeser and Fluka and purchased at the highest commercial
quality and used without further purification. Porcine kidney tis-
sue non-specific alkaline phosphatase (TNAP), bovine intestinal
alkaline phosphatase (BIAP) and levamisole hydrochloride were
purchased from Sigma and used without further purification.
p-Nitrophenylphosphate was obtained from Fluka.
116
74
Ar
R
CF2: 64
94
113
103
1
90
78
87
68
COOH: 65
CH2OH: 66
C@N–OH: 67
S
All reactions were carried out under an argon atmosphere with
dry solvents under anhydrous conditions, unless otherwise stated.
NMR spectra were recorded on a Bruker DPX-300 (1H: 300 MHz;
13C: 300 MHz) instrument using CDCl3 and DMSO as solvents.
The chemical shifts (d ppm) and coupling constants (Hz) are re-
ported in the standard fashion. The following abbreviations are
used to explain the multiplicities: s = singlet, d = doublet, t = trip-
let, q = quartet, m = multiplet, br = broad. Electrospray ionization
(ESI) mass spectrometry (MS) experiments were performed on a
thermo Finnigan LCQ Advantage mass. High-resolution mass spec-
tra (HRMS) were recorded on a Finnigan Mat 95xL mass spectrom-
eter using CI. Analytical thin-layer chromatography was effected
on silica gel Merck 60 D254 (0.25 mm). Flash chromatographies
R
CF2: 68
103
117
104
103
79
98
106
71
COOMe: 69
CH2OH: 70
C@N–OH: 71
S
S
N
NH2
Ph: 72
148
105
142
76
111
94
3-Py: 73
2,3,4-(OMe)3-Ph:
74
2-NH2-4-OMe–Ph: 103
75
Ar
103
84
3-N(CH3)2: 76
134
were performed on Merck Si 60 silica gel (40–63
num oxide 90 active neutral (63–200 m).
lm) Merck alumi-
2.3. Comparison of inhibition effects of levamisole and
benzothiopheno-tetramisole derivatives
l
4.2. Chemistry. General procedure of 2-aryl-3(difluoromethyl)-
benzo[b]thiophenes 64 and 68
Although porcine kidney TNAP activity is usually measured at
alkaline pH as to screen putative inhibitors (Table 5), the inhibition
property of samples and kinetic parameters were determined at pH
7.8 and at 37 °C to match physiological conditions.57 The apparent Ki
DAST (3.08 mL, 25.2 mmol) was added dropwise to the solid 2-
aryl-benzo[b]thiophene-3-carboxaldehyde (0.84 mmol) 21 or 24
and the resulting red solution was stirred at room temperature
for 16 h. under argon. The mixture was then poured dropwise into
ice-cold water (50 mL) and extracted with CH2Cl2 (2 ꢂ 50 mL). The
organic phase was dried over MgSO4, filtered and concentrated.
The residue was purified by flash column chromatography (SiO2)
to obtain the pure desired compound.
of porcine kidney TNAP for levamisole was 93
lM at pH 7.8 and at
37 °C (Table 6). The Ki of human TNAP for levamisole amounted to
16 lM or to 136 lM for that of chicken TNAP measured at pH
9.8.57 The difference in Ki of human TNAP and that of chicken TNAP
was attributed to the presence of His-434 residue.58 The source of
alkaline phosphatase (tissues/organs) could modify the inhibition
effects of inhibitors.59 The Ki of racemic 129zꢀHCl (Ki = 85
lM) and
lM) were slightly distinct from
that of racemic 133ꢀHCl (Ki = 135
4.2.1. 2-Phenyl-3-(difluoromethyl)-benzo[b]thiophene 64
Eluent: cyclohexane. Yield: 75%. 1H NMR (300 MHz, CDCl3) d
8.16 (dd, J = 0.9 Hz, 6.8 Hz, 1H), 7.85 (dd, J = 0.9 Hz, 6.8 Hz, 1H),
that of enantiomeric levamisole (Ki = 93 lM) indicating that there
is some potential to synthesize and optimize enantiomeric levam-
isole derivatives. Although 129ꢀHCl and 133ꢀHCl had similar inhibi-

L. Li et al. / Bioorg. Med. Chem. 17 (2009) 7290–7300
7295
Table 3
Inhibition effects of benzo[b]thiophene derivatives 77–111 on BIAP and on porcine kidney TNAP activities at pH 10.4 and at 37 °C
General structures
Compound number
BIAP + 0.1 mM inhibitor
with 1% DMSO
TNAP + 0.4 mM inhibitor
with 4% DMSO
Relative activity (%)
Relative activity (%)
Br
77
105
100
S
O
Ph: 78
102
107
122
106
109
127
104
100
103
67
79
101
Ar
O
4-OMe–Ph: 79
4-Cl–Ph: 80
4-CN–Ph: 81
4-F–Ph: 82
S
3,4,5-(OMe)3-Ph: 83
O
X
CH2: 84
N–CH3: 85
O: 86
110
102
97
101
95
111
N
S
Ph: 87
4-OMe–Ph: 88
4-F–Ph: 89
4-CHO–Ph: 90
4-Cl–Ph: 91
t-Bu–Ph: 92
4-CH2NH2–Ph: 93
4-CN–Ph: 94
3,4,5-(OMe)3-Ph: 95
104
96
104
9
107
113
105
87
89
82
78
N.T.
94
100
83
85
O
Ar
O
S
129
101
X
N
N-(2-OH)-Ph: 96
N–CH3: 97
O: 98
49
107
108
92
109
97
99
CH2: 99
80
S
4-F–Ph: 100
Ph: 101
2,3,4-(OMe)3-Ph: 102
4-Cl–Ph: 103
4-OMe–Ph: 104
4-CN–Ph: 105
66
110
121
101
106
82
85
91
106
79
98
97
Ar
O
S
NC
O
4-OMe-Ph: 106
Ph: 107
105
104
94
91
Ar
S
X
N
X = CH2; R=2-CN: 108
X = CH2; R = 4-OMe: 109
X = CH2; R = H: 110
99
106
99
100
91
81
R
X = O; R = 2-CN: 111
107
93
S
7.53–7.38 (m, 7H), 6.77 (t, J = 54 Hz, 1H, CF2H). 13C NMR (75 MHz,
CDCl3) d 147.1, 139.1, 136.9, 132.1, 129.9, 129.5, 129.1, 125.2,
124.9 (t, J = 24.5 Hz), 124.0, 122.2, 112.7 (t, J = 232 Hz). 19F NMR
(CDCl3): ꢁ108.9. MS (EI+): m/z 260([M+H]+).
CDCl3) d 150.4, 149.8, 142.5 (t, J = 9.82 Hz), 139.1, 136.9, 136.6,
128.3, 125.9 (t, J = 25.1 Hz), 125.6, 125.4, 123.8 (t, J = 2.2 Hz),
123.5, 122.1, 112.0 (t, J = 234 Hz). 19F NMR (CDCl3): ꢁ109.2. MS
(EI+): m/z 261([M+H]+).
4.2.2. 2-(30-Pyridine)-3-(difluoromethyl)-benzo[b]thiophene 68
Eluent: cyclohexane/AcOEt (8:2). Yield: 62%. 1H NMR (300 MHz,
CDCl3) d 8.77 (d, br, 2H), 8.16 (dd, J = 1.1 Hz, 6.6 Hz, 1H), 7.81 (dd,
J = 1.0 Hz, 6.6 Hz, 1H), 7.75 (ddd, J = 1.7 Hz, 1.7 Hz, 7.9 Hz 1H),
7.47–7.34 (m, 3H), 6.73 (t, J = 53.7 Hz, 1H). 13C NMR (75 MHz,
4.3. General procedure of 2-aryl-3(hydroxymethyl)-
benzo[b]thiophenes 66 and 70
NaBH4 (0.6 mmol) was added to a solution of 21 or 24
(0.4 mmol) in MeOH (2 mL) at 0 °C under argon. After stirring for

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L. Li et al. / Bioorg. Med. Chem. 17 (2009) 7290–7300
Table 4
Inhibition effects of benzo[b]thiophene derivatives 112–124 on BIAP and on porcine kidney TNAP activities at pH 10.4 and at 37 °C
General structures
Compound number
BIAP + 0.1 mM inhibitor with 1% DMSO
Relative activity (%)
TNAP + 0.4 mM inhibitor with 4% DMSO
Relative activity (%)
O
O
Ar
Ph: 112
4-OMe-Ph: 113
43
40
80
96
S
S
R1 = H, R2 = 2-CN: 114
91
70
92
98
107
92
108
98
96
101
R1
R2
R1 = 4-OMe, R2 = 4-OMe: 115
R1 = 2-OMe, R2 = 2-NO2: 116
R1 = 3-OMe,R2 = 2-NO2: 117
R
1 = 4-OMe,R2 = 2-NO2: 118
R
O
H: 119
3-OMe: 120
4-OMe: 121
2-OMe: 122
3, 5-(OMe)2-4-OH: 123
3,4,5-(OMe)3: 124
114
116
95
117
125
124
97
100
100
111
95
OMe
S
O2N
92
2 h at 0 °C, the reaction mixture was quenched with acetone,
poured into NH4Cl 10% (10 mL) and extracted with CH2Cl2
(2 ꢂ 10 mL). The organic layers were collected, dried over MgSO4,
filtered and concentrated. The residue was purified by flash col-
umn chromatography (SiO2) to obtain the pure desired compound.
(d, J = 7.4 Hz, 1H), 8.18 (s, 1H), 8.06 (d, J = 7.7 Hz, 1H), 7.99 (d,
J = 7.7 Hz, 1H), 7.60–7.45 (m, 3H). 13C NMR (75 MHz, DMSO-d6) d
150.0, 149.7, 143.5, 141.0, 138.3, 137.3, 136.9, 128.9, 125.6,
125.5, 125.4, 125.3, 123.9, 122.5. MS (EI+): m/z 254([M+H]+).
4.4.3. 2-Phenyl-benzo[b]thiophene-3-carboxylic acid 65
4.3.1. 2-Phenyl-3-(hydroxymethyl)-benzo[b]thiophene 6641
Eluent: cyclohexane/AcOEt (9:1). Yield: 78%. 1H NMR (300 MHz,
CDCl3) d 7.96 (d, J = 7 Hz, 1H), 7.86 (d, J = 7 Hz, 1H), 7.61 (dd,
J = 1.7 Hz, 6.6 Hz, 2H), 7.51–7.35 (m, 5H), 4.91 (s, 2H). 13C NMR
(75 MHz, CDCl3) d 143.1, 104.1, 139.2, 133.8, 130.4, 129.8, 128.9,
128.6, 124.7, 122.4, 122.3, 56.97. MS (EI+): m/z 240([M+H]+).
Compound 21 (0.09 g, 0.377 mmol) was dissolved in 5.4 mL of
dioxane/H2O (7:3). NaClO2 (0.045 g, 0.5 mmol) and NH2SO3H
(0.209 g, 2.15 mmol) was added and the mixture was stirred for
2 h at room temperature. After addition of 10% NaHCO3 and extrac-
tion with AcOEt, the organic layer was washed with 2 N HCl. The
organic layer was separated, dried over MgSO4, filtered and
evaporated. Purification by flash chromatography (SiO2, cyclohex-
ane/AcOEt 8:2) produced pure 65 in 68% yield (0.065 g). 1H
NMR (300 MHz, CDCl3) d 11.38 (s, 1H), 8.51 (d, J = 8.1 Hz, 1H),
4.3.2. 2-(3’-Pyridine)-3-(hydroxymethyl)-benzo[b]thiophene 70
Eluent: cyclohexane/AcOEt (7:3). Yield: 100%. 1H NMR
(300 MHz, DMSO-d6) d 8.86 (d, J = 2.2 Hz, 1H), 8.66 (dd, J = 1.5 Hz,
4.7 Hz, 1H), 8.08–8.00 (m, 3H), 7.58 (ddd, J = 0.8 Hz, 4.7 Hz,
4.7 Hz, 1H), 7.46 (m, 2H). 13C NMR (75 MHz, DMSO-d6) d 149.4,
149.3, 140.0, 138.4, 136.8, 136.7, 133.4, 129.6, 125.1, 124.7,
123.9, 123.3, 122.3, 54.85. MS (EI+): m/z 241([M+H]+).
160
72
74
140
76
120
100
80
4.4. General procedure of 2-aryl-3-oxime-benzo[b]thiophenes
67 and 71
60
112
21
22
4
113
40
20
0
58
29
Pyridine (1.26 mmol) was added to a solution of 21 or 24 (0.353
mmol) and NH2OHꢀHCl (1.47 mmol) in EtOH (1 mL). The mixture
was refluxed for 1.5 h. After cooling to room temperature, CH2Cl2
(15 mL) was added and the solution was poured into cold water
(30 mL). After addition of CH2Cl2 (15 mL), the organic phasewas sep-
arated, dried over MgSO4 and concentrated under vacuum at 20 °C.
Purification by chromatography (SiO2) provided pure products.
10
14
90
23
67
compound
160
140
120
100
80
4.4.1. 2-Phenyl-3-oxime-benzo[b]thiophene 67
Eluent: cyclohexane/AcOEt (9:1). Yield: 85%. 1H NMR (300 MHz,
CDCl3) d 8.63 (d, J = 7.7 Hz, 1H), 8.40 (s, 1H), 7.85 (d, J = 7.7 Hz, 1H),
7.55–7.39 (m, 8H). 13C NMR (75 MHz, CDCl3) d 147.8, 147.0, 138.8,
137.5, 133.1, 130.2, 129.1, 128.9, 125.6, 125.5, 125.2, 123.7, 122.0.
MS (EI+): m/z 253([M+H]+).
60
4
35
40
Levamisole
129
133
20
0
Compound
4.4.2. 2-(3’-Pyridine) -3-oxime-benzo[b]thiophene 71
Eluent: cyclohexane/AcOEt (7:3). Yield: 95%. 1H NMR (300 MHz,
DMSO-d6) d 11.49 (s, 1H), 8.75 (s, 1H), 8.70 (d, J = 4.4 Hz, 1H), 8.58
Figure 2. Inhibition effects of benzo[b]thiophene derivatives 1–124 on bovine
intestinal BIAP (A) and on porcine kidney TNAP (B) activities at pH 10.4 and at 37 °C.

L. Li et al. / Bioorg. Med. Chem. 17 (2009) 7290–7300
7297
3.0
2.5
2.0
1.5
1.0
0.5
0.0
0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
activity U/ml
control 0.8533
0.01mM 0.5372
0.05mM 0.2554
0.1mM
0.0019
OH
N
S
0.0
0.2
0.4
0.6
0.8
1.0
0.0
0.2
0
.4
0.6
0.8
1.0
1.2
1.4
Time (min)
Inhibitor Concentration (mM)
Figure 3. Inhibition of pNPP hydrolysis by 0.2
l
g mlꢁ1 bovine intestinal BIAP with 0–0.1 mM benzothiophenyl compound 67 in 1% DMSO (v/v) (A). Inhibition of pNPP
hydrolysis by 10
l
g protein mlꢁ1 matrix vesicles containing bone TNAP with 0–1 mM compound 67 in 1% DMSO (v/v) at pH 10.4 and at 37 °C (B). pNPP concentration was
0.1 mM. Three independent measurements were made.
7.84 (d, J = 8.1 Hz, 1H), 7.60–7.40 (m, 7H). 13C NMR (75 MHz,
CDCl3) d 169.2, 162.4, 155.1, 138.7, 138.2, 133.8, 129.8, 129.3,
128.3, 125.8, 125.2, 121.8. MS (EI+): m/z 254([M+H]+).
tion then filtered and dried. The crude product was purified by
flash chromatography (EtOAc) to give 127 (2.1 g, 78%) as a milky
solid. Data for compound 126: 1H NMR (300 MHz, DMSO-d6) d
9.63 (s, 1H), 9.16 (s, 1H), 8.53 (dd, 1H, J = 1.5 Hz, 6.8 Hz), 8.17
(dd, 1H, J = 0.75 Hz, 7.1 Hz), 7.55–7.5 (m, 2H), 7.44 (d, 1H,
J = 4.5 Hz), 7.1 (d, 1H, J = 4.5 Hz), 5.88 (s, 2H); 1C NMR (75 MHz,
DMSO-d6) d 185.9, 169.5, 162.2, 141.4, 139.5, 136.5, 131.3, 131.1,
126.5, 126.1, 124.7, 107.8, 55.5; MS(ESI): 275[M+H]+; HR ESIMS
4.4.4. 2-(3’-Pyridine)-benzo[b]thiophene-3-carboxylic acid
methyl ester 69
NaCN (0.195 g, 4 mmol) and MnO2 (1.38 g, 15.8 mmol) were
added to a solution of 24 (0.19 g, 0.8 mmol) in MeOH (30 mL).
The mixture was stirred at room temperature overnight. CH2Cl2
(100 mL) was then added and the resulting precipitate filtered on
Celite. The filtrate was concentrated and dissolved in H2O/CH2Cl2.
The organic phase was washed with water, dried over MgSO4
and evaporated. Purification by flash chromatography (SiO2, cyclo-
hexane/AcOEt 9:1) gave pure 69 in 93% yield (0.2 g). 1H NMR
(300 MHz, CDCl3) d 8.73 (s, 1H), 8.62 (d, J = 4.0 Hz, 1H), 8.41 (d,
J = 7.9 Hz, 1H), 7.79 (d, J = 7.9 Hz, 2H), 7.49–7.31 (m, 3H), 3.74 (s,
3H). 13C NMR (75 MHz, CDCl3) d 163.7, 149.7, 147.9, 138.7, 138.2,
136.8, 130.3, 125.7, 125.4, 125.0, 123.8, 122.8, 121.8, 51.7. MS
(EI+): m/z 269 ([M+H]+).
þ
calcd for C13H11OS2N2 = 275.0313; found = 275.03121.
4.4.6. Compound 127
To a mixture of 126 (7.6 mmol, 2.1 g) and 5 ml of pyridine in
50 ml of chloroform was added 2 ml of acetic anhydride. The mix-
ture was refluxed for 1.5 h and the chloroform was removed to leave
oil. Then the residue was washed by ethyl ether and purified by flash
chromatography (EtOAc) to afford 127 (1.6 g, 66%) as a brown solid.
Data for compound 127: 1H NMR (300 MHz, DMSO-d6) d 8.3 (s, 1H),
8.54 (dd, 1H, J = 0.9 Hz, 2.8 Hz), 8.15 (dd, 1H, J = 1.9 Hz, 7.0 Hz), 7.53–
7.50 (m, 2H), 7.48 (d, 1H, J = 4.7 Hz), 7.0 (d, 1H, J = 4.7 Hz), 5.82
(s, 2H), 2.02 (s, 3H); 13C NMR (75 MHz, DMSO-d6) d 187.7, 178.7,
167.2, 141.0, 139.6, 136.4, 131.8, 128.2, 126.4, 126.1, 124.8, 123.4,
108.3, 54.8, 27.0; MS(ESI): 317 [M+H]+; HR ESIMS calcd for
4.4.5. Compound 126
Benzo[b]thiophene 125 (9.7 mmol, 2.48 g) was refluxed for 1 h
with 2-aminothiazole (9.7 mmol, 0.97 g) in 25 ml of 2-propanol.
The resulting solid was filtered, triturated with a 10% Na2CO3 solu-
þ
C15H13O2S2N2 = 317.0418; found = 317.04179.
4.4.7. Compound 128
To a solution of 127 (3 mmol, 1 g) in 25 ml of methanol main-
tained at 10 °C was added in small portions 5 mmol of NaBH4.
The solution was stirred at room temperature for 2 h, solvent
was removed under vacuum, and the residue was suspended in
water and extracted with DCM. The DCM layer was dried with
MgSO4 and the solvent was removed to leave a solid which was
purified by flash chromatography (EtOAc/MeOH = 95/5) to afford
128 (0.65 g, 65%) as a white solid. Data for compound 128: 1H
NMR (300 MHz, DMSO-d6) d 8.45 (d, 1H, J = 7.4 Hz), 8.00 (d, 1H,
J = 8.5 Hz), 7.64 (s, 1H), 7.47–7.40 (m, 3H), 6.92 (d, 1H, J = 4.7 Hz),
5.95 (d, 1H, J = 5.1 Hz), 5.38 (s, 1H), 4.63 (dd, 1H J = 2.4 Hz,
13.4 Hz), 4.12 (dd, 1H J = 9.0 Hz, 13.4 Hz), 2.20 (s, 3H); 13C NMR
(75 MHz, DMSO-d6) d 178.5, 166.5, 140.5, 138.2, 137.5, 129.1,
124.9, 124.5, 123.5, 123.4, 122.8, 107.6, 66.8, 54.7, 27.0; MS(ESI):
Table 5
Inhibition effects of levamisole derivatives on BIAP and on porcine kidney TNAP
activities at pH 10.4 and at 37 °C
General structures
Compound
number
BIAP + 0.1 mM TNAP + 0.4 mM
inhibitor inhibitor
with 1% DMSO with 4% DMSO
relative
Relative
activity (%)
activity (%)
H
N
LevamisoleꢀHCl
129ꢀHCl
99
99
10
11
N
S
N
S
319 [M+H]+; HR ESIMS calcd for C15H15O2S2N2 = 319.0575;
þ
S
N
found = 319.05759.
4.4.8. Compound 129
133ꢀHCl
100
19
N
S
Compound 128 (0.63 mmol, 0.2 g) was added in small portion
to 2 ml of thionyl chloride at 5 °C over a period of 30 min. The mix-
S
N

7298
L. Li et al. / Bioorg. Med. Chem. 17 (2009) 7290–7300
20
18
16
14
12
10
8
20
18
16
14
12
10
8
H
N
N
S
6
6
4
4
2
2
0
0
0.00
0.02
0.04
0.06
0.08
0.10
-100
0
100
200
300
400
500
1/pNPP (1/µM)
Levamisole (µM)
Figure 4. Inhibition of pNPP hydrolysis of porcine kidney TNAP by levamisole. HCl at pH 7.8 and at 37 °C without DMSO. (A) Linewaever–Burk plot in the presence of
g mlꢁ1 TNAP with 10, 20, 40, 100 and 1000
l
M pNPP and increasing concentration of 0
l
M (j), 122.5
l
M (d), 245
l
M (ꢀ) and 490
lM (N) levamisole. (B) Plot of
v
ꢁ1
6
l
max
versus levamisole concentration that enabled us to determine apparent Ki. The x intercept gives the negative value of Ki.
20
18
16
14
12
10
8
16
14
12
10
8
N
S
S
N
6
6
4
4
2
2
0
0
0.00
0.02
0.04
0.06
0.08
0.10
-100
0
100
200
300
400
500
1/pNPP (1/µM)
129·HCl (µM)
Figure 5. Inhibition of pNPP hydrolysis of porcine kidney TNAP by 129ꢀHCl at pH 7.8 and at 37 °C without DMSO. (A) Linewaever–Burk plot in the presence of 6
l
g mlꢁ1 TNAP
versus 129ꢀHCl
ꢁ1
with 10, 20, 40, 100 and 1000
l
M pNPP and increasing concentration of 0
l
M (j), 122.5
lM (d), 245
lM (ꢀ) and 490
lM (N)
l
M 129ꢀHCl. (B) Plot of
v
max
concentration that enabled us to determine apparent Ki. The x intercept gives the negative value of Ki.
8
7
6
5
4
3
2
1
0
8
7
6
5
4
3
2
1
0
N
S
S
N
0.00
0.02
0.04
0.06
0.08
0.10
-100
0
100
200
300
400
500
133·HCl (µM)
1/pNPP (1/µM)
Figure 6. Inhibition of pNPP hydrolysis of porcine kidney TNAP by 133ꢀHCl at pH 7.8 and at 37 °C without DMSO. (A) Linewaever–Burk plot in the presence of 6
l
g mlꢁ1 TNAP
versus 133ꢀHCl
ꢁ1
with 10, 20, 40, 100 and 1000
l
M pNPP and increasing concentration of 0
lM (j), 100
l
M (d), 200
l
M (ꢀ) and 400
l
M (N) 133ꢀHCl. (B) Plot of
v
max
concentration that enabled us to determine apparent Ki. The x intercept gives the negative value of Ki.
ture was stirred at room temperature for 1 h, Ac2O (10 ml) was
added, and the acetyl chloride which formed was removed under
vacuum. The mixture was refluxed for another 0.5 h and the excess
Ac2O was removed under vacuum. The residue was washed by 10%
Na2CO3 solution and extracted with DCM. The DCM layer was dried
with MgSO4 and the solvent was removed to leave a solid which

L. Li et al. / Bioorg. Med. Chem. 17 (2009) 7290–7300
7299
Table 6
124.8, 124.3, 123.3, 123.2, 122.9, 67.5, 54.0, 51.7, 27.4, 27.1; MS(E-
SI): 321 [M+H]+; HR ESIMS calcd for C15H16O2S2N2Na+ = 343.0551;
found = 343.05518.
Apparent inhibition constants (Ki) of porcine kidney TNAP activity determined at pH
7.8 and at 37 °C without DMSO
Inhibitors
Type
Ki (lM) SD
4.4.12. Compound 133
LevamisoleꢀHCl
129ꢀHCl
Uncompetitive
Uncompetitive
Uncompetitive
93 4 (n = 3)
85 6 (n = 3)
135 3 (n = 3)
A solution of 132 (0.63 mmol, 0.2 g) in 15 ml of chloroform was
added to 2 ml of thionyl chloride at 5 °C over a period of 30 min.
The mixture was stirred at room temperature for 2 h, 20 ml of
NaOH solution (1 M) was added, and the mixture was refluxed
for 1 h. Organic layer was dried by MgSO4, then filtered and the sol-
vent removed. The residue was purified by flash chromatography
(DCM/EtOAc = 1/1) to afford 133 (35 mg, 21.5%) as a milky solid.
Then 133 was dissolved in 2 ml methanol and 37% hydrochloric
acid in water was added. The mixture was stirred overnight and
the solvent was removed to leave a slight grey solid. After washing
with acetone and dried, the hydrochloride salt of 133 was ob-
tained. Data for compound 133ꢀHCl: 1H NMR (300 MHz, DMSO-
d6) d 7.86 (dd, 1H, J = 2.8 Hz, 7.0 Hz), 7.74 (dd, 1H, J = 2.1 Hz,
6.0 Hz), 7.46 (s, 1H), 7.40–7.32 (m, 2H), 5.85 (t, J = 8.7 Hz, 1H),
3.85 (t, J = 8.7, 1H), 3.74–3.56 (m, 2H), 3.44 (t, J = 8.7 Hz,1H),
3.23(q, J = 8.7 Hz, 2H); 13C NMR (75 MHz, DMSO-d6) d 138.6,
123.3, 123.2, 122.5, 121.4, 47.8, 33.6, 30.9, 28.7, 28.6, 28.4, 21.7,
13.1; MS(ESI): 261 [M+H]+; HR ESIMS calcd for C13H12S2N2 =
261.0520; found = 261.05207.
133ꢀHCl
was purified by flash chromatography (EtOAc) affording 129 (0.07 g,
43%) as a white solid. Then 129 was directly dissolved in 2 ml meth-
anol and 37% hydrochloric acid in water was added. The mixture was
stirred overnight and thesolvent was removed toleave a slight green
solid. After washing with acetone and dried, the hydrochloride salt
of 129 was obtained. Data for compound 129ꢀHCl: 1H NMR
(300 MHz, DMSO-d6) d 7.87 (dd, 1H, J = 2.1 Hz, 7.7 Hz), 7.70 (dd,
1H, J = 2.5 Hz, 6.6 Hz), 7.45 (s, 1H), 7.41–7.32 (m, 2H), 6.48 (d, 1H,
J = 4.5 Hz), 5.90 (t, J = 8.7 Hz, 1H), 5.77 (d, J = 8.7 Hz, 1H), 4.31 (t,
J = 8.7 Hz, 1H), 3.78 (t, J = 8.7 Hz 1H); 13C NMR (75 MHz, DMSO-d6)
d 170.6, 141.6, 138.2, 137.5 124.8, 124.5, 123.6, 123.2, 122.8,
121.9, 102.1, 71.1 53.2; MS(ESI): 259 [M+H]+; HR ESIMS calcd for
þ
C13H11S2N2 = 259.0364; found = 259.03637.
4.4.9. Compound 130
2-Aminothiazoline (11.8 mmol, 1.2 g) was dissolved in 25 ml
acetonitrile and small portions of 3-(2-bromoacetyl)benzo[b]thio-
phene 125 (11.8 mmol, 3 g) was added. The mixture was stirred
at rt for 2 h and filtered. The precipitate was macerated with a
10% Na2CO3 solution then filtered and dried. The crude product
was purified by flash chromatography (EtOAc) to afford 130
(2.88 g, 88.6%) as a white solid. Data for compound 130: 1H NMR
(300 MHz, DMSO-d6) d 9.08 (s, 1H), 8.59 (dd, 1H, J = 1.5 Hz,
7.7 Hz), 8.10 (dd, 1H, J = 1.1 Hz, 8.7 Hz), 7.55–7.44 (m, 2H), 4.85
(s, 2H), 3.72 (t, J = 8.7 Hz, 2H), 3.23 (t, J = 8.7 Hz, 2H); 13C NMR
(75 MHz, DMSO-d6) d 191.0, 161.9, 139.8, 139.5, 136.6, 132.5,
126.2, 125.8, 124.9, 123.3, 52.5, 52.0, 27.2; MS(ESI): 277 [M+H]+;
4.5. Screening test
To screen putative inhibitors, activity of BIAP as well as that of
porcine kidney TNAP were measured in 25 mM piperazine, 25 mM
glycylglycine, 5 mM MgCl2, 5
The mixtures containing the buffer, BIAP (0.1–0.3
TNAP (4–6
g mLꢁ1), and the inhibitors (0.1 mM for BIAP with final
l
M ZnCl2 at pH 10.4 and at 37 °C.61
l
g mLꢁ1), or
l
DMSO 1% (v/v), 0.4 mM for TNAP with final DMSO 4% (v/v)) were
incubated for 10 min at 37 °C without pNPP. Then, 0.05 mM pNPP
was added at the last minute to initiate the reaction. The activity
was quantified at 420 nm, using a molar absorption coefficient of
18.6 cmꢁ1 mMꢁ1 at pH 10.4. The activity of each sample containing
the inhibitor was compared with the control sample (without
inhibitor). A relative activity of 100 5% indicated that the inhibi-
tor has no effects. The experimental errors were 5% as determined
by the sample to sample assays. An higher relative activity (greater
than 105%) indicated that the tested compound has an activation
effect on the alkaline phosphatase activities, while a relative value
lower than 95% indicated the the tested compound is a inhibitor.
þ
HR ESIMS calcd for C13H13OS2N2 = 277.0469; found = 277.04684.
4.4.10. Compound 131
To a mixture of 130 (10.4 mmol, 2.88 g) and 5 ml of pyridine in
50 ml of chloroform was added 2 ml of acetic anhydride. The mix-
ture was refluxed for 1.5 h and the chloroform was removed to
leave an oil. Then the residue was washed by ethyl ether and puri-
fied by flash chromatography (EtOAc) to afford 131 (2.52 g, 76%) as
a brown solid. Data for compound 131: 1H NMR (300 MHz, DMSO-
d6) d 9.15 (s, 1H), 8.57 (dd, 1H, J = 1.5 Hz, 8.7 Hz), 8.12 (dd, 1H,
J = 1.3 Hz, 8.5 Hz), 7.55–7.46 (m, 2H), 5.20 (s, 2H), 3.76 (t,
J = 8.7 Hz, 2H), 3.22 (t, J = 8.7 Hz, 2H); 13C NMR (75 MHz, DMSO-
d6) d 188.8, 180.7, 170.0, 140.6, 139.5, 136.5, 132.0, 126.3, 125.9,
124.8, 123.4, 54.0, 50.4, 27.5, 27.0; MS(ESI): 319 [M+H]+; HR ESIMS
4.6. Inhibition of the best benzothiophene inhibitor on BIAP
and matrix-vesicle TNAP activity
From the screening test, the best benzothiophene inhibitor was
selected. Its activity was measured in 25 mM piperazine, 25 mM
glycylglycine, 5 mM MgCl2, 5
þ
calcd for C15H15O2S2N2 = 319.0575; found = 319.05751.
l
M ZnCl2 at pH 10.4 and at 37 °C in
the presence of either 0.2
l
g mLꢁ1 BIAP or 10
l
g mLꢁ1 matrix ves-
4.4.11. Compound 132
icles (MVs) with 0.1 mM pNPP. The concentrations of inhibitor (0–
1.2 mM) are indicated in Figure 1. MV-protein concentration was
determined by the method of Bradford.62 MV extracellular organ-
elles produced by chondrocytes, osteoblasts and odontoblasts47,63
initiate normal skeletal calcification and are characterized by high
TNAP activity.13,14 Collagenase released MVs were isolated from
bone and epiphyseal cartilage slices of 17-day-old chicken em-
bryos according to Balcerzak et al.64
To a solution of 131 (4.7 mmol, 1.5 g) in 25 ml of methanol
maintained at 10 °C was added in small portions 5 mmol of NaBH4.
The solution was stirred at room temperature for 2 h, solvent was
removed under vacuum, and the residue was suspended in water
and extracted with DCM. The DCM layer was dried with MgSO4
and the solvent was removed to leave a solid which was purified
by flash chromatography (EtOAc/MeOH = 95/5) to afford 132
(0.6 g, 40%) as a yellowish solid. Data for compound 132: 1H
NMR (300 MHz, DMSO-d6) d 8.31 (d, J = 7.2 Hz, 1H), 7.99 (d,
J = 7.2 Hz, 1H), 7.63 (s, 1H), 7.45–7.35 (m, 2H), 5.89 (d, J = 7.2 Hz,
1H), 5.35 (t, br, 1H), 4.12 (dd, J = 2.4, 13.5 Hz 1H), 3.95 (m, 1H)
3.70 (m, 1H), 3.47 (m 1H) 3.15–3.06 (m, 2H), 2.18 (s, 3H); 13C
NMR (75 MHz, DMSO-d6) d 180.4, 169.9, 140.5, 138.8, 137.5,
4.7. Determination of the inhibition constant in physiological
pH
To determine the inhibition constant Ki of the soluble inhibitors,
porcine kidney TNAP activity was measured in 0.1 M Tris–HCl buffer

7300
L. Li et al. / Bioorg. Med. Chem. 17 (2009) 7290–7300
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l
M ZnCl2 at pH 7.8 and at 37 °C. The mix-
tures containing the buffer, TNAP (6
(from 100 to 500 M) were incubated for 10 min at 37 °C without
pNPP. Then, pNPP was added at the last minute to initiate the reac-
tion. The concentrations of pNPP were 10 M, 20 M, 40 M,
100 M and 1000 M, respectively. The change in absorbance of
l
g mlꢁ1), and the inhibitors
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Acknowledgements
We thank Dr. John Carew for the English correction. Ms. Lina Li
was a recipient of a scholarship awarded by the China-France Doc-
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