Design and synthesis of selective inhibitors of Placental Alkaline Phosphatase

Article abstract of DOI:10.1016/j.bmc.2009.12.012

Placental Alkaline Phosphatase (PLAP) is a tissue-restricted isozyme of the Alkaline Phosphatase (AP) superfamily. PLAP is an oncodevelopmental enzyme expressed during pregnancy and in a variety of human cancers, but its biological function remains unknown. We report here a series of catechol compounds with great affinity for the PLAP isozyme and significant selectivity over other members of the AP superfamily. These selective PLAP inhibitors will provide small molecule probes for the study of the pathophysiological role of PLAP.

Full text of DOI:10.1016/j.bmc.2009.12.012

Bioorganic & Medicinal Chemistry 18 (2010) 573–579
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of selective inhibitors of Placental Alkaline Phosphatase
b
c
b
b
c
Marion Lanier a, , Eduard Sergienko , Ana Maria Simão , Ying Su , Thomas Chung , José Luis Millán ,
*
John R. Cashman a
a Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121-2804, USA
b Burnham Center for Chemical Genomics, Burnham Institute for Medical Research, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA
c Sanford Children’s Health Research Center, Burnham Institute for Medical Research, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Placental Alkaline Phosphatase (PLAP) is a tissue-restricted isozyme of the Alkaline Phosphatase (AP)
superfamily. PLAP is an oncodevelopmental enzyme expressed during pregnancy and in a variety of
human cancers, but its biological function remains unknown. We report here a series of catechol com-
pounds with great affinity for the PLAP isozyme and significant selectivity over other members of the
AP superfamily. These selective PLAP inhibitors will provide small molecule probes for the study of the
pathophysiological role of PLAP.
Received 8 October 2009
Revised 2 December 2009
Accepted 3 December 2009
Available online 11 December 2009
Keywords:
Ó 2009 Elsevier Ltd. All rights reserved.
Enzyme inhibitor
Placental Alkaline Phosphatase
Cancer
Oncodevelopmental
1. Introduction
and GCAP largely due to the fact that these genes evolved only re-
cently, having appeared during a late evolutionary duplication
Alkaline phosphatases (APs) are found in many organisms from
bacteria to humans.1,2 APs are a subgroup of a superfamily of
metalloenzymes with similar metal-binding sites and predicted
conserved protein structural folds.3 In humans, APs are encoded
by four genes traditionally named after the tissues where they
are predominantly expressed. The tissue-nonspecific AP (TNAP)
gene is expressed at greatest levels in liver, bone, kidney, in the
placenta during the 1st trimester of pregnancy, and at lower levels
in numerous other tissues.1,2 The other three isozymes, (i.e., pla-
cental AP (PLAP), germ cell AP (GCAP), and intestinal AP (IAP)),
show a much more restricted tissue expression, generally referred
to as tissue-specific APs. Tissue-specific PLAP, GCAP and IAP are
90–98% homologous while TNAP is only 50% identical with the
other three APs. Most of what we know about function comes from
studies of the biological roles of TNAP and more recently IAP. TNAP
is essential for proper skeletal mineralization. Its role is to hydro-
lyze inorganic pyrophosphate, a potent calcification inhibitor, via
its pyrophosphatase activity.4–7 Lack of TNAP leads to infantile
hypophosphatasia, a lethal form of rickets and osteomalacia in
mice, rescued by enzyme replacement therapy.8 IAP appears to
have a role in fat absorption in the gastrointestinal tract9,10 and
as a gut mucosal defense factor11 where its expression coincides
with the stage of colonization by commensal bacterial flora.12,13
In contrast, relatively little is known about the functions of PLAP
event that preceded the divergence of Old World Monkeys and
Apes.1
PLAP is expressed in large amounts in the syncytiotrophoblast
cells of the placenta from about the eighth week of gestation
throughout pregnancy.14 PLAP was one of the first enzymes recog-
nized as an oncofetal protein expressed in a variety of cancers.15,16
GCAP is also found expressed in the placenta although at levels 50-
fold lower than PLAP17 and it is re-expressed in testicular germ cell
tumors of the testis, particularly seminomas.18,19 PLAP and GCAP
are often co-expressed in ovarian cancer20 and it has been sug-
gested that transformation of normal to malignant trophoblast
might be associated with a switch from PLAP to GCAP expression.21
Thus, the ability to discriminate between these AP isozymes might
facilitate studies aimed at uncovering their pathophysiological
function.
Several isozyme-selective inhibitors of APs have been reported
(Fig. 1), including
some unrelated compounds, such as
L-Phe, L-Trp, L , L
-Leu22,23 -homoarginine24 and also
L
-leucinamide, levamisole, the
L
-stereoisomer of tetramisole25, and theophylline (a 1,3-dimethyl
derivative of xanthine).26 Inhibition by these agents is of a rare
uncompetitive type27 and the IC50 of these inhibitors are reported
to be above 5 l
M for all three APs and poorly selective.28,29 Be-
cause of the great homology between PLAP, GCAP and IAP, no
selective inhibitors of the PLAP enzymes have been reported until
now. In this report, we describe the development of potent PLAP
inhibitors that possess selectivity over TNAP and IAP and moderate
selectivity over GCAP.
* Corresponding author. Tel.: +1 858 458 9305; fax: +1 858 458 9311.
E-mail address: mlanier@hbri.org (M. Lanier).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.12.012

574
M. Lanier et al. / Bioorg. Med. Chem. 18 (2010) 573–579
COOH
NH2
L-Leu
COOH
NH2
COOH
NH2
N
H
L-Phe
L-Trp
O
H
N
H
N
H2N
N
COOH
NH2
L-HomoArg
N
N
O
N
NH
S
N
Levamisole
Theophilline
Figure 1. Examples of AP inhibitors.
A library of 95,857 compounds30 was tested in a luminescent
O
O
R3
R1
HTS assay31 for discovery of chemical inhibitors of PLAP. Initially,
192 ‘hits’ (i.e., 0.2%) were identified as having at least 50% inhibi-
R1
Cl
X
a
R2
tion of PLAP functional activity in an assay at 20 lM. Eighty two
of these hits were confirmed in dose-response experiments for
inhibition of PLAP function.31 In parallel studies, the 82 confirmed
hits were also tested for inhibition selectivity against TNAP func-
tional activity. A few confirmed hits contained a catechol moiety
and were selected as a lead series. The catechol series was chemi-
cally stable and showed early trends of a structure activity rela-
1, 3-20
2
Scheme 1. Synthetic approach to explore the SAR of the LHS and RHS of screening
‘hit’ 1; preparation of analogs 3–20, X is nitrogen or sulfur. (a) 1 equiv HXR2R3,
dioxane.
tionship (SAR). Compound
inhibitor ‘hit’ from the PLAP inhibition screening campaign. 1 pos-
sessed an IC50 of 2.5 M but was weakly selective over TNAP
(IC50 = 8.6 M) and IAP (IC50 = 49.5 M) (Fig. 2).
A SAR was developed around 1 by varying all three regions
(LHS, RHS and linker) of the catechol ‘hit’ 1 (Fig. 2) to obtain rela-
tively selective PLAP inhibitors over the other members of the AP
family.
1 (Fig. 2) was the most potent
H
N
l
HO
HO
HO
HO
a
l
l
O
N
N
H
22
21
NH2
O
(CH2)n
HO
HO
(CH2)n
HO
HO
b
N
H
2. Chemistry
O
For variation of the LHS and RHS regions of the original hit 1,
compounds 3–20 were readily synthesized using a one step syn-
thesis by combining commercially available aryl substituted 2-
chloroacetophenones 2 with various amines or thiols as shown in
Scheme 1. Typically, the desired substituted 2-chloroacetophenone
2 was treated with 1 equiv of a nucleophile in dioxane either at
room temperature or by heating the mixture up to 80 °C. Products
1, 3–20 precipitated from the reaction mixture.
23 n=1
24 n=2
25 n=1
26 n=2
Scheme 2. Synthetic approach to explore the linker region of screening ‘hit’ 1:
preparation of analogs 22, 25 and 26. Reagents: (a) 2-amino 4,6-dimethyl
pyrimidine, NaBH(OAc)3, DCE; (b) 4,5-dimethyl 2-furoic acid, HBTU, DIEA, DMF.
3. Results and discussion
Exploration of the SAR of linker region of 1 (Fig. 2) necessitated
using different starting materials. Compound 22 (Scheme 2)
was prepared with standard reductive-amination conditions by
treating a mixture of 3,4-dihydroxybenzaldehyde 21 and 2-ami-
no-4,6-dimethylpyrimidine in the presence of triacetoxyborohy-
dride in dichloroethane (DCE). Compounds 25 and 26 (Scheme 2)
were obtained by treating 3,4-dihydroxybenzylamine 23 and
3,4-dihydroxyphenylethylamine 24, respectively, with 4,5-di-
methyl-2-furoic acid in the presence of standard peptide coupling
conditions (HBTU, DIEA).
To gain a quick understanding of the SAR requirements, analogs
27–33 (Fig. 3) were purchased and a sublibrary of 27 compounds
(Tables 1–4) was synthesized according to the procedure illus-
trated in Schemes 1 and 2. Chemical re-synthesis of compound 1
(Table 1) was done according to Scheme 1 to compare an authentic
sample with the commercially purchased compound in the bioas-
says used. Resynthesized 1 was found to have similar PLAP inhib-
itory potency as the library compound. Compounds 3–33 were
compared to 1 in the enzyme inhibition assays (i.e., PLAP, TNAP)
to characterize enzyme inhibition potency and selectivity. The
most active compounds in the PLAP assay were also tested against
IAP and GCAP.
Linker
O
Based on several purchased analogs (three representative
examples, 27, 31–33, are shown in Fig. 3), it was rapidly recognized
that the 3,4-dihydroxyl arrangement of the catechol in the LHS
seemed optimal for inhibition of alkaline phosphatase. Compound
HO
HO
S
N
PLAP IC50 = 2.5 µM
TNAP IC50 = 8.6 µM
N
1
IAP
IC50 = 49.5 µM
27 (Fig. 3) had an IC50 of 6.7
that did not have a 3,4-dihydroxyl group were not potent (IC50
>100 M) PLAP inhibitors.
lM whereas 28 (Fig. 3) and 29 (Fig. 3)
LHS
RHS
l
Figure 2. Depiction of the three regions of screening ‘hit’ 1 explored by chemical
synthesis: Left Hand Side (LHS), Right Hand Side (RHS) and Linker region
(encircled).
A more thorough exploration of the SAR of the LHS aryl substi-
tuent of compound 1 (Table 1) confirmed that the 3,4-dihydroxy

M. Lanier et al. / Bioorg. Med. Chem. 18 (2010) 573–579
575
O
OH
O
O
O
H
S
N
S
N
HO
HO
S
HO
HO
N
N
N
N
N
N
N
N
HO
H
NH2
30
PLAP IC50 = 39.3 4.3 µM
27
29
28
PLAP IC50 = 6.7 0.4 µM
PLAP IC50 >100 µM
PLAP IC50 >100 µM
O
O
O
HO
HO
S
N
HO
HO
S
N
N
O
O
O
N
N
N
HO
HO
O
O
O
31
PLAP IC50 = 2.2 0.1 µM
32
PLAP IC50 = 2.0 0.1 µM
33
PLAP IC50 = 3.0 0.2 µM
Figure 3. Examples of commercially available analogs 27–33 purchased and their IC50 values in the PLAP assay.
Table 1
IC50 values for PLAP inhibition when the left hand side was varied
HO
HO
HO
O
O
O
O
N
S
O
HO
R4
N
HO
Compound number
PLAP potency (IC50
1
3
4
5
6
7
a
)
l
M
2.5 0.1
>100
>100
>100
>100
>100
a
Values are the mean SD of three experiments.
Table 2
IC50 values for PLAP inhibition when the linker region of 1 was varied.
O
O
O
HO
HO
HO
N
N
S
HO
HO
HO
N
N
N
O
H
N
H
N
O
H
HO
HO
Compound number
PLAP activity(IC50
1
22
25
26
a
)
lM
2.5 0.1
51.2 5.7
>100
>100
a
Values are the mean SD of three experiments.
Table 3
IC50 values for PLAP and TNAP inhibition: Variation of the right hand side of the lead
O
N
N
N
O
O
S
S
N
N
N
NH
N
N
N H
HO
HO
R
N
C N
S
N
N
N
O
HN
N
N
N
O
O
O
Compound number
1
30
39.3 4.3 2.2 0.1
>100 4.8 0.2
31
32
33
8
9
10
2.1 0.2 13.4 1.1 2.7 0.1
>100 >100 >100
11
9.1 0.6
93.8 16.2
PLAP inh. potency a(IC50
)
)
l
l
M
M
2.5 0.1
8.6 0.5
2.0 0.1
8.0 0.6
3.0 0.2
29.2 1.4
TNAP inh. potency a(IC50
a
Values are the mean SD of three experiments.
phenyl substituent was crucial for inhibition of PLAP functional
activity. Compared to lead compound 1, removing one of the
hydroxyl groups (i.e., 3, 4), capping one (i.e., 5, 7), or capping both
hydroxyl groups (i.e., 6) afforded compounds with no PLAP inhibi-
A brief SAR survey of the linker region was done by synthesis
and it was observed that synthetic modification of the linker region
of 1 led to a significant decrease of potency of PLAP inhibition.
Thus, shortening the linker by one atom spacer and removing the
ketone moiety afforded a 25-fold decrease in potency of inhibition
tory potency at 100 lM (Table 1).

576
M. Lanier et al. / Bioorg. Med. Chem. 18 (2010) 573–579
Table 4
IC50 values for inhibition of PLAP, TNAP and IAP: SAR of the right hand side of lead structure 1
O
N
N
N
N
N
N
N
N
N
N
HO
HO
R
N
N
N
N
N
N
N
N
Br
Br
N
N
Compound number
8
12
3.3 0.2
78.3 12.9 >100
(25) (>27)
13
14
15
5.8 0.2
41.1 4.1
(7)
16
15.8
47.5 5.5
(3)
17
18
19
20
1.2 0.1
26.3 2.8
(8)
a
PLAP potency IC50
l
M
2.1 0.2
>100
(>32)
4.2 0.3
17.2 1.3
>100
(>6)
1
0.8 0.05
18.0 1.7
(9)
5.8 0.6
>100
(>17)
4.9 0.6
8.7 0.7
(1.7)
a
TNAP (IC50
)
lM Selectivity
(TNAP/PLAP)
a
Values are the mean SD of three experiments.
(e.g., compound 22, Table 2). Compounds 25 and 26 (Table 2) that
had a 4,5-dimethylfuran in place of the 4,6-dimethylpyrimidine as
well as a different linker resulted in significant loss of inhibition
potency.
17 by introduction of an alkyl group (i.e., 5,6-dimethylbenzimida-
zole) to afford 18 decreased the potency by about threefold (i.e.,
IC50 of 18 = 5.8 lM). Addition of a methyl at the 2 position of the
benzimidazole of 17 to afford 20 maintained similar potency of
Structural variation of the RHS side chain provided SAR infor-
mation for further refinement of lead molecule 1. Compound 30
(Table 3) and other RHS alkyl-substituted side-chains (not shown)
suggested that an aromatic ring of the RHS was necessary for po-
tent PLAP inhibition. The isopropyl amine analog 30 was the most
inhibition compared with 17.
Within the imidazole series (Table 4), substitution at the 2-po-
sition did not seem to have a large effect on TNAP potency as the
TNAP IC50 for compounds 8, 12–14 was greater than 78 lM leading
to selectivity for PLAP inhibition over TNAP over 25-fold for an
unsubstituted imidazole (8) or imidazoles bearing a small alkyl
group such as methyl (12) or ethyl (13). Steric bulk at the 4- and
or 5-position of the imidazole with compounds such as 15, 16 or
19 and the benzimidazoles 17 and 20 showed a beneficial effect
on the compound’s ability to inhibit TNAP. The IC50 for 15–17, 19
potent of the alkyl-substituted side-chains with an IC50 of 39.3 lM
for PLAP inhibition (Table 3). Additional SAR studies with com-
pounds maintaining a catechol group in the LHS showed that com-
pounds tolerated a large number of RHS heteroaromatic rings (i.e.,
substituted thiopyridine 31, substituted tetrazole 32, benzofura-
none 33, imidazole 8, triazole 9, pyrazole 10, 2-amino benzimid-
azole 11, Table 3) and linkers (i.e., sulfur 1, 31, 32; carbon 33;
nitrogen 30, 8–11, Table 3) in place of the pyrimidine moiety. How-
ever, such heteroaromatic compounds were also generally poor
selective inhibitors of PLAP compared to TNAP (i.e., 2-, 4-, and
10-fold selectivity IC50 PLAP/IC50 TNAP for 31, 32, 33 and 11,
respectively, Table 3).
and 20 were 41.1, 47.5, 18, 8.7 and 26.3
mine being the optimum group size for the compounds tested (IC50
for 19 = 8.7 M). A larger group such as the 5,6-dimethylbenzimid-
azole (18) was not tolerated by the TNAP active site probably due
to steric factors. The IC50 for 18 was above 100 M.
lM, respectively, with bro-
l
l
In summary, of the compounds examined (Table 4), the unsub-
stituted imidazole 8 was a considerably more selective inhibitor of
PLAP compared with TNAP and had an IC50 TNAP/IC50 PLAP of over
32-fold.
The most potent inhibitors of PLAP (i.e., 1, 8, 11–13, 17 and 20,
Table 5) were also tested as inhibitors of IAP and GCAP, the two
other isozymes of the Alkaline Phosphatase family. Results are
summarized in Table 5. Substitution at the 2 position of the imid-
azole appeared to be critical for selectivity of IAP inhibition. When
the 2-substitutent was changed from a hydrogen to a methyl to an
ethyl group, the IC50 for IAP inhibition increased, leading to
Exploration of the SAR of the RHS region of compound 1 with
heterocycles such as imidazole, triazole or pyrazole directly at-
tached to the core structure led to moderately potent PLAP inhib-
itors (IC50 <14
lM) that possessed PLAP inhibition selectivity
over TNAP (IC50 >100
l
M) (i.e., compounds 8–10, Table 3). Encour-
aged by the large inhibition selectivity of 8–10 for inhibition of
PLAP compared to TNAP, a more thorough examination of the
SAR of the RHS was explored using substituted imidazoles. Keeping
the catechol moiety (LHS, Fig. 2) of compound 1 unchanged, a li-
brary of substituted imidazoles was prepared and tested as inhib-
itors of all three isozymes (i.e., PLAP, TNAP and IAP) for inhibition
potency and selectivity. Results are summarized in Table 4. The
presence of a hydrogen (8) or a small alkyl group (i.e., methyl, 12
or ethyl, 13) at the 2-position of the imidazole had little influence
on PLAP inhibitory potency. All three compounds, 8, 12 and 13, had
increasingly less potent compounds (IC50 = 30.2
lM, 64.6 lM,
>100 M respectively, for 8, 12, 13). While the number of com-
l
pounds examined was limited, a similar trend was also observed
for the benzimidazole analogs 17 and 20. Compound 17 had an
IC50 of 14.8
(i.e., compound 20), did not show any detectable inhibition in
the IAP assay (IC50 >100 M) that lead to a selectivity of over 80-
lM in the IAP assay while its 2-methylated version
IC50 values of approximately 3
ier group at the 2-position (i.e., phenyl, 14), potency decreased
fivefold (IC50 of 14 = 17.2 M). Substitution at the 4 position of
lM. With the introduction of a bulk-
l
fold for IAP over PLAP. Compounds shown in Table 5 were also
tested for their inhibitory potential against GCAP. All of them were
l
the imidazole ring with a small group such as methyl (e.g., 15,
Table 4), maintained similar inhibitory potency as the unsubstitut-
ed imidazole, 8. However, introduction of a larger group such as a
bromine atom into the 4 position of the imidazole (i.e., 16, Table 4)
moderately potent inhibitors of GCAP with IC50 values <10
IC50 value for 1, 8, 11 and 17 was 0.6 M, 1.2 M, 6.2
2.7
compounds 1, 8, 11 and 17 on PLAP and GCAP (Fig. 4), compounds
1, 8 and 11 can partially discriminate between PLAP and GCAP; the
l
l
M. The
M and
l
l
l
M, respectively. Also, compared to the inhibitory potency of
led to a decrease in potency by about sevenfold (i.e., IC50
15.8 M). Addition of a methyl group at the 2 position of com-
pound 16 (i.e., compound 19, Table 4) increased inhibitory potency
threefold. Compound 19 had an IC50 value of 5.0 M compared to
an IC50 of 15.8 M for 16. The 2-benzimidazole analog (i.e., 17)
was one of the most potent inhibitors with an IC50 of 0.8 M and
=
l
discrimination being optimal in the range around 1 lM while com-
pound 12, 13, 17 and 20 had the same inhibitory potency for both
isozymes at all inhibitor concentrations tested (graphs not shown
for 12, 13 and 20).
In summary, a series of 3,4-dihydroxy substituted catechols was
developed that maintained inhibitory potency of the original
screening hit 1 as an inhibitor of PLAP. Molecular modification of
l
l
l
this result suggested that aromatic pi–pi interaction with the en-
zyme may be important. Extending the steric bulk of compound

M. Lanier et al. / Bioorg. Med. Chem. 18 (2010) 573–579
577
Table 5
Inhibitory activity for IAP and GCAP enzymes for compounds 1, 8, 10–13, 17 and 20
O
N
N
N
N
N
NH
S
N
HO
HO
R
N
N
N
N
N
N
N
N
HN
N
Compound number
1
11
8
12
3.3 0.2
13
17
20
10
a
PLAP potency IC50
l
M
2.5 0.1
49.5 6.1
(20)
9.1 0.6
Nd
2.1 0.2
30.2 2.5
(15)
4.2 0.3
>100
0.8 0.05
14.8 6.8
(18)
1.2 0.1
>100
2.7 0.1
53.3 3.1
(25)
a
IAP (IC50
)
lM
64.6 7.5
Selectivity (IAP/PLAP)
(20)
(>24)
(>80)
a
*
*
*
GCAP (IC50
)
lM
0.6
6.2
1.2
2.7
Nd
Selectivity (GCAP/PLAP)
(0.2)
(0.7)
(0.6)
(2.1)
(2.1)
(3.4)
(1.4)
a
Values are the mean SD of three experiments.
*
Compounds 12, 13 and 20 were tested for GCAP and compared to PLAP on a different occasion. The enzyme concentrations were different. IC50 values for PLAP and GCAP
were 14.3, 30.5
l
M for 12; 0.8, 1.7
lM for 13 and 6.6, 9.4
l
M for 20, respectively.
Cmpd 1
Cmpd 8
100
100
80
60
40
20
0
80
60
40
20
0
1E-8
1E-7
1E-6
1E-5
1E-4
1E-3
1E-8
1E-7
1E-6
1E-5
1E-4
1E-3
Log [inhibitor] (M)
Log [inhibitor] (M)
Cmpd 11
Cmpd 17
100
100
80
60
40
20
80
60
40
20
0
0
1E-8
1E-7
1E-6
1E-5
1E-4
1E-3
1E-8
1E-7
1E-6
1E-5
1E-4
1E-3
Log [inhibitor] (M)
Log [inhibitor] (M)
Figure 4. Effect of increasing concentrations of compound 1, 8, 11 and 17 on the inhibition of PLAP (d) and GCAP (s).
1 provided PLAP inhibitors with greater inhibition selectivity over
two other related isozymes, TNAP and IAP (Tables 4 and 5). Com-
pound 13, possessing a 2-ethylimidazole substituent, was over
27-fold more selective as an inhibitor of PLAP compared to TNAP
and IAP and maintained reasonable PLAP inhibitory potency
compounds (i.e., 10, 13 and 20) were more potent inhibitors of
PLAP than previously reported isozyme-selective inhibitors of
APs (Fig. 1) and considerably more selective. We were also able
to improve upon the selectivity of the screening hit 1, especially
with regard to TNAP selectivity. Compounds 1, 8 and 11 are also
somewhat selective for PLAP over GCAP in the one micromolar
range. Because of their inhibitory selectivity, these catechol com-
pounds may be useful as tools to understand the physiological role
of PLAP in biology and pharmacology and may have applications
(IC50 = 4.2 lM). Compound 10 was more than 50- and 25-fold
selective as an inhibitor of PLAP over TNAP and IAP, respectively
(Fig. 5). Finally, compound 20 was 10- and 40-fold more selective
an inhibitor of PLAP over IAP and TNAP, respectively. All three

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M. Lanier et al. / Bioorg. Med. Chem. 18 (2010) 573–579
O
O
O
O
N
N
HO
HO
N
HO
HO
N
HO
HO
S
N
HO
HO
N
N
N
10
20
1
13
PLAP IC50 = 2.1 µM
TNAP IC50 >100 µM
PLAP IC50 = 2.5 µM
TNAP IC50 = 20 µM
PLAP IC50 = 2.5 µM
TNAP IC50 = 5.6 µM
PLAP IC50 = 3.7 µM
TNAP IC50 >100 µM
IAP
IC50 = 53.3 µM
IAP
IC50 > 100 µM
IAP
IC50 = 49 µM
IAP
IC50 >100 µM
Figure 5. Summary of the IC50 values for selective PLAP inhibitors 10, 13 and 20.
for treating cancer or in cancer diagnostics because elevated levels
of TNAP are found in ovarian and testicular cancer patients.
methane/methanol, 90:10, v/v) and the reaction product that pre-
cipitated from the solution was filtered, washed with a minimum
amount of dioxane and then dried under high vacuum to afford
the product as a solid in 30–50% yield.
4. Experimental section
Compound 1: 1H NMR (CD3OD) d 7.55 (dd, J = 8.3 and 2.3 Hz,
1H), 7.47 (d, J = 2.3 Hz, 1H), 7.12 (s, 1H), 6.84 (d, J = 8.3 Hz, 1H),
4.77 (s, 2H), 2.38 (s, 6H); LC/MS-1: tR = 1.41 (100%). MS: m/z
290.9 [M+H]+, expected 291 [M+H]+.
4.1. General methods and materials
Reagents, starting materials, and solvents were purchased from
commercial suppliers in the highest quality available and were
used as received. To concentrate refers to evaporation under vac-
uum using a Büchi rotory evaporator. Reaction products were puri-
Compound 3: 1H NMR (DMSO) d 7.95 (d, J = 3 Hz, 2H), 7.10 (s,
1H), 6.85 (d, J = 3 Hz, 2H), 4.57 (s, 2H), 2.32 (s, 6H); LC/MS-1:
tR = 1.83 (100%). MS: m/z 275.4 [M+H]+, expected 275.4 [M+H]+.
Compound 4: 1H NMR (CD3OD) d 7.57 (d, J = 7.8 Hz, 1H), 7.44 (s,
1H), 7.39 (d, J = 7.8 Hz, 1H). 7.34 (br s, 1H), 7.10 (dd, J = 6.9 and
0.9 Hz, 1H), 4.95 (s, 2H), 2.52 (s, 6H); LC/MS-1: tR = 2.55 (100%).
MS: m/z 274.9 [M+H]+, expected 275 [M+H]+.
fied, when necessary, by chromatography on silica gel (40–63 lm)
with the solvent system indicated. Proton nuclear magnetic reso-
nance data were recorded on a Varian Mercury 300 MHz Spec-
trometer using TMS as the internal standard. Carbon nuclear
magnetic resonance data were recorded on a Brucker 300 MHz
Spectrometer using TMS as the internal standard. Compounds
were assessed for purity by HPLC–MS and were at least 85% pure
at two wavelengths (224 nM and 254 nM). All tested compounds
were characterized by 1H NMR and purity assessed by LC/MS-1
(Platform: Agilent: equipped with an auto-sampler, an UV detector
(220 nm and 254 nm), a MS detector (APCI); HPLC column: Waters
XTerraMS C18, 3.0 Â 250 mm; HPLC gradient: 1.0 mL/min, from
10% acetonitrile in water to 90% acetonitrile in water in 46 min
maintaining 90% for 7.0 min. Both acetonitrile and water have
0.025% TFA.) or LC/MS-2 (Platform: Agilent 1100 series: equipped
with an auto-sampler, an UV detector (230 nm and 254 nm), a
MS detector (APCI); HPLC column: Phenomenex Synergi-Max RP,
2.0 Â 50 mm column; HPLC gradient: Solvent C is 6 mM Ammo-
nium Formate in water, solvent D is 25% acetonitrile in methanol.
The gradient runs from 5% D (95% C) to 95% D (5% C) in 6.43 min
with a 1.02 min hold at 95% D followed by a return and hold at
5% D for 1.52 min) (tR is retention time (TIC% purity).
Compound 5: 1H NMR (CDCl3) d 7.72 (dd, J = 8.1 and 1.8 Hz, 1H),
7.58 (d, J = 1.8 Hz, 1H), 6.96 (d, J = 8.1 Hz, 1H), 6.68 (s, 1H), 4.59 (s,
2H), 3.94 (s, 3H), 2.34 (s, 6H); LC/MS-1: tR = 1.57 (100%). MS: m/z
304.9 [M+H]+, expected 305 [M+H]+.
Compound 6: 1H NMR (DMSO) d 7.77 (dd, J = 9.0 and 2.0 Hz,
1H), 7.51 (d, J = 2.0 Hz, 1H), 6.95 (d, J = 9.0 Hz, 1H), 6.07 (s, 1H),
4.61 (s, 2H), 2.30 (s, 6H); LC/MS-1: tR = 3.72 (100%). MS: m/z
303.4 [M+H]+, expected 303.5 [M+H]+.
Compound 7: 1H NMR (CDCl3) d 8.05 (d, J = 3 Hz, 2H), 6.95 (d,
J = 3 Hz, 2H), 6.71 (s, 1H), 4.57 (s, 2H), 3.87 (s, 3H), 2.32 (s, 6H); LC/
MS-1: tR = 3.45 (100%). MS: m/z 288.9 [M+H]+, expected 289 [M+H]+.
Compound 8: LC/MS-1: tR = 0.36 (100%). MS: m/z 219.3 [M+H]+,
expected 219 [M+H]+.
Compound 9: 1H NMR (CD3OD) d 8.31 (s, 2H), 7.45–7.39 (m,
2H), 6.83 (d, J = 9 Hz, 1H), 4.80 (s, 2H). LC/MS-2: tR = 0.65 (100%).
MS: m/z 220.1 [M+H]+, expected 220 [M+H]+.
Compound 10: 1H NMR (CD3OD) d 7.48 (dd, J = 8.1 and 1.8 Hz,
1H), 7.42 (d, J = 2.1 Hz, 1H), 7.40 (s, 1H), 7.32 (s, 1H), 6.86 (d,
J = 8.1 Hz, 1H), 5.56 (s, 2H), 2.10 (s, 3H); 13C NMR (DMSO) d
189.6, 151.4, 145.6, 138.9, 132.7, 126.0, 121.8, 116.5, 115.2,
114.7, 58.9, 8.4;); LC/MS-1: tR = 2.45 (100%). MS: m/z 234.5
[M+H]+, expected 234 [M+H]+.
4.2. TNAP, PLAP and IAP Luminescent assays31
Briefly, the TNAP assay was performed in 384-well white plates
Compound 11: 1H NMR (CD3OD) d 7.46–7.41 (m, 2H), 7.29–7.22
(m, 2H), 7.11–7.06 (m, 2H), 6.83 (d, J = 9 Hz, 1H). MS (ESI+) m/z
284.2 (M+H)+.
(784075, Greiner, Monroe, NC) at 50
say buffer containing 100 mM DEA–HCl (pH 9.8), 1 mM MgCl2, and
20 M ZnCl2. The luminescence signal was measured after a 30-
lM CDP-star substrate in as-
l
Compound 12: 1H NMR (CD3OD) d 7.55–7.40 (m, 2H), 7.14 (s,
1H), 7.09 (dd, J = 15 and 1.5 Hz, 1H), 6.83 (d, J = 9 Hz, 1H), 4.80 (s,
2H), 2.34 (s, 3H); LC/MS-1: tR = 0.55 (100%). MS: m/z 233.5
[M+H]+, expected 233 [M+H]+.
min incubation at room temperature on an EnVision plate reader
(PerkinElmer, Waltham, MA). Analogous luminescent assays were
optimized and used for PLAP, IAP and GCAP with the CDP-star con-
centration adjusted to their respective Km values, 85
and 10 M, respectively.
lM, 177 lM
Compound 13: 1H NMR (CD3OD) d7.45–7.38 (m, 2H), 7.00 (s, 2H),
6.83 (d, J = 9 Hz, 1H), 4.80 (s, 2H), 2.76 (q, J = 7.8 Hz, 2H), 1.29 (t,
J = 7.8 Hz, 3H); 13C NMR (DMSO) d 190.5, 151.8, 148.6, 145.4,
125.9, 122.8, 121.8, 119.1, 115.3, 114.8, 66.3, 19.4, 11.8; LC/MS-2:
tR = 0.74 (100%). MS: m/z 247.1 [M+H]+, expected 247 [M+H]+.
Compound 14: 1H NMR (CD3OD) d 7.84 (d, J = 7.2 Hz, 2H), 7.46–
7.34 (m, 5H), 7.14 (s, 2H), 6.83 (d, J = 9 Hz, 1H), 4.78 (s, 2H); LC/MS-
1: tR = 1.71 (100%). MS: m/z 295.4 [M+H]+, expected 295.5 [M+H]+.
l
4.3. Synthesis of compounds 1, 3–20
In a typical procedure, 2-chloro-acetophenone (0.27 mmol) in
dioxane (1 mL) was added to the desired substituted amine or thiol
(0.27 mmol). The solution was stirred overnight in a sealed vial at
room temperature. The reaction was monitored by TLC (dichloro-

M. Lanier et al. / Bioorg. Med. Chem. 18 (2010) 573–579
579
Compound 15: 1H NMR (CD3OD) d 7.58–7.37 (m, 3H), 7.09 (br s,
Acknowledgments
1H), 6.84 (d, J = 9 Hz, 1H), 4.80 (s, 2H), 2.32 (s, 3H); MS: m/z 233.3
[M+H]+, expected 233.5 [M+H]+.
This work was supported by NIH Roadmap Initiatives grant
U54HG003916. Ana Maria Simão and José Luis Millán were sup-
ported by NIH grants DE12889 and 1X01MH077602-01.
Compound 16: 1H NMR (CD3OD) d 7.62 (s, 1H), 7.48–7.39 (m,
2H), 7.11 (s, 1H), 6.83 (d, J = 9 Hz, 1H), 4.80 (s, 2H); LC/MS-1:
tR = 1.53 (100%). MS: m/z 299.6 [M+H]+, expected 299.5 [M+H]+.
Compound 17: 1H NMR (CD3OD) d 7.67–7.25 (m, 6H), 6.98–6.90
(m, 1H), 6.93 (d, J = 9 Hz, 1H), 6.83 (d, J = 9 Hz, 1H), 4.79 (s, 2H);
MS: m/z 269.3 [M+H]+, expected 269 [M+H]+.
References and notes
1. Millán, J. L. Mammalian Alkaline Phosphatases. From Biology to Applications in
Medicine and Biotechnology; Wiley-VCH Verlag GmbH
& Co: Weinheim,
Compound 18: 1H NMR (CD3OD) d 8.20 (s, 1H), 7.45–7.36 (m,
4H), 6.83 (d, J = 9 Hz, 1H), 4.77 (s, 2H), 2.35 (s, 6H); MS: m/z
297.3 [M+H]+, expected 297.5 [M+H]+.
Germany, 2006. pp 1–322.
2. McComb, R. B.; Bowers, G. N., Jr.; Posen, S. Alkaline Phosphatase; Plenum: New
York, 1979.
3. Galperin, M. Y.; Bairoch, A.; Koonin, E. V. Protein Sci. 1998, 7, 1829.
4. Hessle, L.; Johnson, K. A.; Anderson, H. C.; Narisawa, S.; Sali, A.; Goding, J. W.;
Terkeltaub, R.; Millán, J. L. Proc. Natl. Acad. Sci. U.S.A. 2002, 99, 9445.
5. Harmey, D.; Hessle, L.; Narisawa, S.; Johnson, K. A.; Terkeltaub, R.; Millán, J. L.
Am. J. Pathol. 2004, 164, 1199.
6. Harmey, D.; Johnson, K. A.; Zelken, J.; Camacho, N. P.; Hoylaerts, M. F.; Noda,
M.; Terkeltaub, R.; Millán, J. L. J. Bone Miner. Res. 2006, 21, 1377.
7. Murshed, M.; Harmey, D.; Millán, J. L.; McKee, M. D.; Karsenty, G. Genes Dev.
2005, 19, 1093.
8. Millán, J. L.; Narisawa, S.; Lemire, I.; Loisel, T. P.; Boileau, G.; Leonard, P.;
Gramatikova, S.; Terkeltaub, R.; Pleshko Camacho, N.; McKee, M. D.; Crine, P.;
Whyte, M. P. J. Bone Miner. Res. 2008, 23, 777.
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Cell. Biol. 2003, 23, 7525.
10. Nakano, T.; Inoue, I.; Koyama, I.; Kanazawa, K.; Nakamura, K.; Narisawa, S.;
Tanaka, K.; Akita, M.; Masuyama, T.; Seo, M.; Hokari, S.; Katayama, S.; Alpers, D.
H.; Millán, J. L.; Komoda, T. Am. J. Physiol. Gastrointest. Liver Physiol. 2007, 292,
1439.
11. Goldberg, R. F.; Austen, W. G., Jr.; Zhang, X.; Munene, G.; Mostafa, G.; Biswas, S.;
McCormack, M.; Eberlin, K. R.; Nguyen, J. T.; Tatlidede, H. S.; Warren, H. S.;
Narisawa, S.; Millán, J. L.; Hodin, R. A. Proc. Natl. Acad. Sci. U.S.A. 2008, 105,
3551.
12. Bates, J. M.; Akerlund, J.; Mittge, E.; Guillemin, K. Cell Host Microbe 2007, 2, 371.
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J. L. Am. J. Physiol. Gastrointest. Liver Physiol. 2007, 293, 1068.
14. Fishman, L.; Miyayama, H.; Driscoll, S. G.; Fishman, W. H. Cancer Res. 1976, 36,
2268.
15. Fishman, W. H.; Ghosh, N. K.; Inglis, N. R.; Green, S. Enzymologia 1968, 34, 317.
16. Fishman, W. H.; Inglis, N. R.; Green, S.; Anstiss, C. L.; Gosh, N. K.; Reif, A. E.;
Rustigian, R.; Krant, M. J.; Stolbach, L. L. Nature 1968, 219, 697.
17. Povinelli, C. M.; Knoll, B. J. Placenta 1991, 12, 663.
18. Wahren, B.; Holmgren, P. A.; Stigbrand, T. Int. J. Cancer 1979, 24, 749.
19. Wahren, B.; Hinkula, J.; Stigbrand, T.; Jeppsson, A.; Andersson, L.; Esposti, P. L.;
Edsmyr, F.; Millán, J. L. Int. J. Cancer 1986, 37, 595.
20. Smans, K. A.; Ingvarsson, M. B.; Lindgren, P.; Canevari, S.; Walt, H.; Stigbrand,
T.; Bäckström, T.; Millán, J. L. Int. J. Cancer 1999, 83, 270.
21. Ovitt, C. E.; Strauss, A. W.; Alpers, D. H.; Chou, J. Y.; Boime, I. Proc. Natl. Acad. Sci.
U.S.A. 1986, 83, 3781.
22. Fishman, W. H.; Sie, H. G. Enzymologia 1971, 41, 141.
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30. The compound library was supplied by the National Institutes of Health (NIH)
Molecular Libraries Small Molecule Repository (MLSMR, http://
www.mli.nih.gov/mlsmr).
31. Sergienko, E.; Su, Y.; Chan, X.; Brown, B.; Hurder, A.; Narisawa, S.; Millán, J. L. J.
Biomol. Screen. 2009, 14, 824.
Compound 19: 1H NMR (CD3OD) d 7.45–7.40 (m, 2H), 6.93 (s,
1H), 6.83 (d, J = 9 Hz, 1H), 4.79 (s, 2H), 2.32 (s, 3H); LC/MS-1:
tR = 1.48 (100%). MS: m/z 313.0 [M+H]+, expected 313 [M+H]+.
Compound 20: 1H NMR (CD3OD) d 7.90–7.39 (m, 5H), 6.98–6.90
(m, 1H), 6.93 (d, J = 6 Hz, 1H), 6.83 (d, J = 9 Hz, 1H), 4.79 (s, 2H),
2.68 (s, 3H); 13C NMR (DMSO) d 189.6, 152.7, 151.4, 151.2, 145.4,
135.4, 126.1, 122.9, 122.3, 121.9, 117.3, 115.2, 113.9, 110.2,66.3,
13.5; MS: m/z 283.3 [M+H]+, expected 283 [M+H]+.
Compound 22: 50 mg (0.36 mmol) of 3,4-dihydroxybenzalde-
hyde and 44 mg (0.36 mmol) of 2-amino-4,6-dimethylpyridine
was stirred in 1 mL DCE in the presence of 153 mg (0.72 mmol) of so-
dium triacetoxyborohydride and 1 drop of acetic acid at rt overnight.
The reaction was quenched with 1 mL water. The product was ex-
tracted with ethyl acetate and purified by PTLC (ethyl acetate/meth-
anol, 90:10, v/v) to give 25 mg of the product (28% yield). 1H NMR
(CD3OD) d 7.29 (s, 1H), 6.88 (d, J = 9 Hz, 1H), 6.80–6.62 (m, 2H),
4.41 (s, 2H), 2.25 (s, 3H), 1.96 (s, 3H); LC/MS-1: tR = 2.33 (85.13%).
MS: m/z 246.4 [M+H]+, expected 246.5 [M+H]+.
Compound 25: 50 mg (0.23 mmol) of 3,4-dihydroxybenzylamine
23 was dissolved in 1 mL THF/1 mL DMF. 100 mg (0.27 mmol) of
HBTU, 128 lL (0.69 mmol) of DIEA and 32 mg (0.23 mmol) of 1,5-di-
methyl-3-furoic acid were added and the mixture was stirred at rt
overnight. The crude material was purified by PTLC (DCM/MeOH,
90:10, v/v) to give a yellow solid (60 mg, 98% yield)). 1H NMR
(CD3OD) d 6.88 (s, 1H), 6.76 (s, 1H), 6.69 (d, J = 7.8 Hz, 1H), 6.63 (d,
J = 7.8 Hz, 1H), 6.27 (s, 1H), 4.35 (s, 2H), 2.25 (s, 3H), 1.96 (s, 3H);
LC/MS-1: tR = 2.33 (100%). MS: m/z 262.5 [M+H]+, expected 262.5
[M+H]+.
Compound 26: 44 mg (0.23 mmol) of dopamineÁHCl 24 was dis-
solved in 1 mL THF/1 mL DMF. 100 mg (0.27 mmol) of HBTU,
128 lL (0.69 mmol) of DIEA and 32 mg (0.23 mmol) of 4,5-di-
methyl-2-furoic acid were added and the mixture was stirred at
rt overnight. The crude material was purified by PTLC (DCM/MeOH,
90:10, v/v) to give an off-white solid (30 mg, 47% yield)). 1H NMR
(CD3OD) d 6.68 (d, J = 8 Hz, 1H), 6.66 (s, 1H), 6.54 (d, J = 9.4 Hz,
1H), 6.18 (s, 1H), 3.39–3.49 (m, 2H), 2.69 (t, J = 8 Hz, 2H), 2.44 (s,
3H), 2.21 (s, 3H); LC/MS-1: tR = 2.23 (100%). MS: m/z 275.9
[M+H]+, expected 276 [M+H]+.
Compounds 27–30 and 32–33 were purchased from Chem-
Bridge and 31 from ChemDiv.