Design and synthesis of pyrazole derivatives as potent and selective inhibitors of tissue-nonspecific alkaline phosphatase (TNAP)

Article abstract of DOI:10.1016/j.bmcl.2008.10.107

Tissue-nonspecific alkaline phosphatase (TNAP) plays a central role in regulating extracellular matrix calcification during bone formation and growth. High-throughput screening (HTS) for small molecule TNAP inhibitors led to the identification of hits in

Full text of DOI:10.1016/j.bmcl.2008.10.107

Bioorganic & Medicinal Chemistry Letters 19 (2009) 222–225
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of pyrazole derivatives as potent and selective inhibitors
of tissue-nonspecific alkaline phosphatase (TNAP)
Shyama Sidique a,b, , Robert Ardecky a,b, , Ying Su a,b, Sonoko Narisawa b, Brock Brown a,b, José Luis Millán b,
Eduard Sergienko a,b, Nicholas D. P. Cosford a,b,
*
a Burnham Center for Chemical Genomics, Burnham Institute for Medical Research, 10901 N. Torrey Pines Road, La Jolla, CA 92037, USA
b 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:
Tissue-nonspecific alkaline phosphatase (TNAP) plays a central role in regulating extracellular matrix cal-
cification during bone formation and growth. High-throughput screening (HTS) for small molecule TNAP
inhibitors led to the identification of hits in the sub-micromolar potency range. We report the design,
synthesis and in vitro evaluation of a series of pyrazole derivatives of a screening hit which are potent
TNAP inhibitors exhibiting IC50 values as low as 5 nM. A representative of the series was characterized
in kinetic studies and determined to have a mode of inhibition not previously observed for TNAP
inhibitors.
Received 29 August 2008
Revised 22 October 2008
Accepted 27 October 2008
Available online 31 October 2008
Keywords:
Pyrazoles
Alkaline phosphatases
Competitive inhibitors
Hit-to-probe optimization
Published by Elsevier Ltd.
The alkaline phosphatase isozyme family in mammals is com-
prised of two groups, the tissue-specific alkaline phosphatases
(placental, intestinal and germ cell) and tissue-nonspecific alkaline
phosphatase (TNAP).1 The major function of TNAP in bone tissue is
the degradation of extracellular inorganic pyrophosphate (PPi), a
potent inhibitor of calcification, to inorganic phosphate. In this
way a controlled steady state level of PPi is maintained, thus sus-
taining normal bone mineralization. Increased expression of TNAP
accelerates calcification in bovine vascular smooth muscle cells
(VSMCs),2 and macrophages can induce a calcifying phenotype in
IC50 data for compounds were deposited to PubChem under
AID 1056 (http://pubchem.ncbi.nlm.nih.gov/assay/assay.cgi?aid =
1056).
High-throughput screening (HTS) of 66,000 compounds using a
luminescence-based assay9,10 (see PubChem link to AID 1056 for
details) developed in the Burnham Center for Chemical Genomics
(BCCG) led to the identification of the pyrazole derivative CID-
646303 (1 in Fig. 1). Preliminary hit follow up was accomplished
by performing similarity searches on databases of commercially
human VSMCs by activating TNAP in the presence of IFN
c and
1,25(OH)2D3.3 Small molecule inhibitors of TNAP therefore have
the potential to probe the causative mechanisms, or treat the
pathology, of diseases caused by medial calcification such as idio-
pathic infantile arterial calcification, end-stage renal disease and
diabetes.4-6
NH
O
NH
N
N
O
Cl
OH
OMe
Cl
Cl
Until now, levamisole and theophilline were the only available
1, CID-646303
IC50 = 0.98 μM
2, IC50 = 0.5 μM
inhibitors of TNAP with Ki values of 16 and 82 l
M, respectively.7
We recently reported the discovery of novel potent and selective
small molecule inhibitors of TNAP using high-throughput screen-
ing (HTS).8 Herein we report our efforts on the hit-to-lead optimi-
zation of a pyrazole TNAP inhibitor screening hit with micromolar
potency to provide novel derivatives with low nanomolar in vitro
potency and excellent selectivity for TNAP. The structures and
NH
N
NH
N
OH
N
O
O
O
O
3, IC50 = 1.3 μM
4, IC50 = 0.5 μM
* Corresponding author.
E-mail address: ncosford@burnham.org (N.D.P. Cosford).
These authors contributed equally to this work.
Figure 1. Initial hit from screening and commercial analogues.
0960-894X/$ - see front matter Published by Elsevier Ltd.
doi:10.1016/j.bmcl.2008.10.107

S. Sidique et al. / Bioorg. Med. Chem. Lett. 19 (2009) 222–225
223
H
H
N
O
O
N
O
N
N
O
O
O
R1
R1
R1
R1
a
b
c
O
OH
O
8
7
5
6
Scheme 1. Reagents and conditions: (a) i—NaOMe, Et2O, dimethyl oxalate, 25 °C, 4ꢀ12 h; ii—AcOH (75–90%); (b) N2H2, AcOH, 100 °C, 12 h (50–85%); (c) LiOH, THF, MeOH,
reflux (90–95%).
Table 1
available analogues. In this initial phase, 50 commercial analogues
were identified, purchased and tested for their ability to inhibit
TNAP. This allowed us to define some important features of the
structure–activity relationships (SAR). For example, the potency
Summary of in vitro data from first focused library
NH
Cl
N
O
NH
Cl
O
N
in this series was improved from IC50 = 0.98
zole 1 to IC50 = 0.50 M for the 2,4-dichlorophenyl ester derivative
2 (Fig. 1). Furthermore, conversion of the tricyclic derivative 3,
with an IC50 value of 1.33 M, to the pyrrolidine amide analogue
4 led to a 3-fold improvement in potency (IC50 = 0.50 M). Encour-
aged by these results we designed and synthesized two focused li-
braries of substituted pyrazole amide analogues. In order to
optimize the potency of the hit structure the pyrazole acid scaffold
8 was selected as the key synthon for the preparation of amide
analogues (Scheme 1).
The synthetic chemistry used for the preparation of the pyra-
zole acid scaffolds is shown in Scheme 1. Reaction of acetophenone
derivatives 5 with sodium methoxide and dimethyl oxalate yielded
the 1,3-diketone derivatives 6 in excellent yields (75–90%). Com-
pound 6 was then reacted with hydrazine to give the correspond-
ing pyrazole ester 7. Saponification of the methyl ester provided
access to the pyrazole acids 8.
The synthetic chemistry used for hit optimization is shown in
Scheme 2. The pyrazole acid 8 was treated with HOBT, EDC and
DIEA to produce the amides 911 or the desired hydrazide derivative
10.
lM for the lead pyra-
OH
NR
HOBT, EDC, DMF
RNH2
l
Cl
Cl
R1
R1
l
l
Compound
R1
H
RNH2
IC50 (lM)
9a
NH2CH2CH2OH
0.044
0.051
9b
9c
9d
H
H
H
H2N
HN
0.119
0.398
H2N
9e
9f
H
H
0.828
0.488
N
NH
N
NH
N
NH
9g
9h
H
H
0.375
0.574
HO
In light of the preliminary data generated from the HTS hits and
commercial analogues our goal was to determine the key compo-
nents of the SAR required for potency. For the focused library syn-
HN
O
thesis we selected
a 2,4-dichloro and 2,4-dichloro-5-fluoro
substitution pattern for the phenyl ring based on the initial SAR
data. In the first library, twenty six compounds were synthesized
and tested in the in vitro assay. This led to the identification of four
analogues with potency values of 100 nM or better (Table 1). The
incorporation of a hydroxyl group on the amide generally in-
creased potency (9a and 9j). In all cases the 2,4-dichloro analogues
were more potent than the corresponding 2,4-dichloro-5-fluoro
analogues (Table 1). A second generation set of pyrazoles consist-
ing of a library of twenty eight compounds were synthesized next
(Table 2). In this series we found that branching of the amides gen-
erally decreased potency in the in vitro assay, especially when the
chain length was greater than three carbon atoms. We also ob-
served that amides with chain lengths of three carbons or less were
the most active (Tables 1 and 2).
9i
H
1.450
HN
9j
F
F
NH2CH2CH2OH
0.100
0.324
9k
HN
9l
F
4.850
0.044
H2N
NH2NH2
10
H
ond hydroxyethyl side chain was added (9p), these changes did not
affect the potency of the initial hit 9a (Tables 1 and 2). The most
potent compound in this series was the 2,3,4-trichlorophenyl ana-
logue 9v (Table 3). This compound showed exceptional activity
with an in vitro IC50 of 5 nM (Table 3). Furthermore, compound
In the second series, when the hydroxyethyl chain was in-
creased by one or two additional carbon atoms (9m, 9n), or a sec-
9v was inactive (IC50 > 10 lM) against both the related placental
alkaline phosphatase (PLAP) isozyme and the house keeping en-
zyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH), indi-
cating a selectivity for TNAP of at least 2000-fold.
We next performed a series of experiments to elucidate the
mechanism of action (MOA) of the novel TNAP inhibitors. The cat-
alytic mechanism by which TNAP degrades PPi consists of rapid
phosphorylation of the active site in the presence of the phos-
pho–donor substrate and a rate-limiting dephosphorylation by
the phospho-acceptor substrate, either water or amino-containing
alcohols (Fig. 2). All the TNAP inhibitors known to date are uncom-
H
H
O
N
O
N
N
N
R1
R1
a
OH
NX
9, X = R3R4 (alkylamine)
10, X = NH2 (hydrazine)
8
Scheme 2. Reagent and condition: (a) EDC, HOBT, DMF, DIEA, NH2X (85–95%).

224
S. Sidique et al. / Bioorg. Med. Chem. Lett. 19 (2009) 222–225
Table 2
CDP* vs [9v] at 6.25 mM DEA
a
Summary of in vitro data from second focused library
1000
750
500
250
NH
N
O
Cl
NH
N
O
Cl
1/V (0.5 uM)
OH
1/V (0.25 uM)
1/V (0.125 uM)
1/V (0.0625 uM)
1/V (0.0313 uM)
1/V (0.0156 uM)
NR
HOBT, EDC, DMF
RNH2
Cl
Cl
R1
R1
Compound
R1
RNH2
IC50 (lM)
9m
9n
9o
9p
H
H
H
H
NH2CH2CH2CH2OH
NH2CH2CH2CH2CH2OH
NH2CH2CH2OMe
0.031
0.035
0.046
0.047
-100
0
100
200
300
400
500
NH2(CH2CH2OH)2
1/[CDP-star] (uM)
H2N
9q
9r
H
H
0.082
0.459
DEA at varied [9v] (18.75 uM CDP*)
150
b
H2N
HN
NH2CH2CH2CH2OH
NH(CH2CH2OH2)
1/V (1 uM)
1/V (0.5 uM)
1/V (0.25 uM)
1/V (0.125 uM)
1/V (0.0625 uM)
1/V (0.0313 uM)
9s
H
0.285
100
50
9t
9u
F
F
0.056
0.127
Table 3
Summary of in vitro data for trichloro- and trifluorophenyl derivatives
-25 -15 -5
5
15
25
35
45
R1
N
NH
NH
O
R1
N
O
1/[DEA] (mM-1)
R2
R3
R2
R3
OH
NR
HOBT, EDC, DMF
RNH2
Figure 3. Mechanism of action studies for compound 9v: Lineweaver–Burke plots.
TNAP kinetic data were obtained in the presence of (a) 6.25 mM DEA or (b) 18.8 uM
CDP-star. The concentration of the second substrate was varied in the presence of
different concentrations of 9v. Closed circle, 1000 nM; closed square, 500 nM; open
square, 250 nM; closed triangle, 125 nM; open triangle, 62.5 nM; closed upside-
down triangle, 31 nM; open upside-down triangle, 15.6 nM.
R4
R4
Compound
R1
R2
R3
R4
RNH2
IC50 (lM)
9v
9w
9x
Cl
F
F
Cl
H
H
Cl
F
F
H
F
H
NH2CH2CH2OH
NH2CH2CH2OH
NH2CH2CH2OH
0.005
0.134
0.035
This is the first time that a competitive MOA has been established
for an inhibitor of TNAP.
To gain some structural insight into how the inhibitors interact
with TNAP protein, we performed in silico docking studies to
investigate the potential binding mode of 9a in the active site of
TNAP using the Schrodinger Glide program.14
DOH
E + DO-P
E·DO-P
E-Pi·DOH
AOH
E-Pi
E·Pi
E + Pi
i
i
Since the TNAP 3-dimensional crystal structure is not available,
a homology model, built using the placental alkaline phosphatase
(PLAP) crystal structure (PDB code: 1EW2) as the template, was
used for docking.15 The sequence identity of TNAP and PLAP is
56% according to the sequence alignment algorithm blastp pro-
vided by NCBI BLAST (http://blast.ncbi.nlm.nih.gov/), while the
homology is 74%. The binding mode suggested by Glide is shown
in Figure 4. In this model, the carbonyl of the amide chain coordi-
nates to the Zn2+ ion and the 2,4-dichlorophenyl ring of compound
9a contributes to the binding through a stacking interaction with
the side chain of Tyr371.
E-Pi·AOH
E·AO-P
E +AO-P
i
i
Figure 2. The catalytic mechanism of the alkaline phosphatase reaction.12 The
initial alkaline phosphatase (E) catalyzed reaction consists of a substrate (DO–Pi)
binding step, phosphate-moiety transfer to Ser-93 (in the TNAP sequence of its
active site) and product alcohol (DOH) release. In the second step of the reaction,
phosphate is released through hydrolysis of the covalent intermediate (E–Pi) and
the non-covalent complex (EꢁPi) of the inorganic phosphate in the active site. In the
presence of nitrogen-containing alcohol molecules (AOH), such as the buffer
diethanolamine (DEA), phosphate is also released via
reaction.
a transphosphorylation
In agreement with the importance of the stacking interaction,
replacement of the phenyl group by an acyl group results in com-
plete loss of activity (data not shown). Compound 9a was docked
into the catalytic site either with serine 93 phosphorylated or with
free serine 93. Interestingly, the binding modes are the same, sug-
gesting that compound 9a functions as a competitive inhibitor.
This predicted inhibition mechanism is consistent with the ki-
netic/mechanistic studies.
In summary, we have described the design, synthesis and chem-
ical optimization of a series of pyrazole amide derivatives that are
potent inhibitors of TNAP. The hit-to-lead optimization of the
screening hit 1 led to compound 9v, which is approximately 200
times more potent against TNAP, and shows a high degree of selec-
tivity against the related PLAP isozyme. Mechanistic studies
petitive with respect to phospho-donors7,8 and are likely to be non-
or uncompetitive with diethanolamine (DEA). The latter conclusion
is based on the fact that the majority of the alkaline phosphatase
assays are performed in the presence of saturating concentrations
of DEA or other phosphor-acceptors.9
To characterize the mechanism of action (MOA) of the novel
TNAP inhibitor series we selected compound 9v for additional
studies. By performing detailed kinetic studies,13 we demonstrated
that 9v is competitive with respect to both substrates, the water-
soluble 1,2-dioxetane reagent disodium 2-chloro-5-(50-chloro-4-
methoxyspiro[1,2-dioxetane-3,20-tricyclo[3.3.1.13,7]decan]-4-
yl)-phenol-1-(dihydrogen phosphate) (CDP-star) and DEA (Fig. 3).

S. Sidique et al. / Bioorg. Med. Chem. Lett. 19 (2009) 222–225
225
5. Addison, W. N.; Azari, F.; Sorensen, E. S.; Kaartinen, M. T.; McKee, M. D. J. Biol.
Chem. 2007, 282, 15872.
6. Kaunitz, J. D.; Yamaguchi, D. T. J. Cell. Biochem. 2008, 105, 655.
7. Kozlenkov, A.; Hoylaerts, M. F.; Ny, T.; Le Du, M.-H.; Millán, J. L. J. Bone Miner.
Res. 2004, 19, 1862.
8. Narisawa, S.; Harmey, D.; Yadav, M. C.; O’Neill, W. C.; Hoylaerts, M. F.; Millán, J.
L. J. Bone Miner. Res. 2007, 22, 1700.
9. Sergienko, E. A.; Millán, J. L., unpublished results.
10. TNAP activity was measured on an EnVision plate reader in the presence of
100 mM diethanolamine–HCl, pH 9.8, 1 mM MgCl2, 20
CDP-star.
lM ZnCl2, and 50 lM
11. Representative synthesis of 9v:
A mixture of 2,3,4-trichloroacetophenone
(4.46 g, 0.02 mol) and dimethyl oxalate (2.36 g, 0.02 mol) was dissolved in Et2O
(100 mL) and stirred at room temperature. To this solution was added a freshly
prepared solution of NaOMe, prepared from sodium metal (0.55 g, 0.024 mol)
dissolved in MeOH (10 mL). A yellow precipitate immediately formed and the
reaction was stirred for 1 h at room temperature. The precipitate was filtered
and dissolved in water (200 mL) and the solution was adjusted to pH 3 with
HOAc. The resulting precipitate was filtered and dried to give 5.20 g (84%) of
methyl 2,4-dioxo-4-(2,3,4-trichlorophenyl)butanoate (6). A mixture 6 (5.20 g,
0.018 mol) and HOAc (100 mL) was stirred at room temperature. To this
solution was added hydrazine (7.5 mL, 0.24 mol) and the reaction mixture was
stirred overnight. The precipitate was filtered, washed with hexane and dried
to give 5.19 g (95%) of methyl 3-(2,3,4-trichlorophenyl)-1H-pyrazole-5-
Figure 4. Proposed binding mode of 9a in the catalytic site of the enzyme.
Compound 9a is displayed in ball and stick mode and the protein residues within
4 Å of the ligand are displayed in stick mode. Dashed lines highlight the potential
key interactions between protein and 9a. The zinc ions are shown in gold,
magnesium ion in magenta.
carboxylate (7).
A mixture of 7 (5.19 g, 0.017 mol), MeOH (100 mL), THF
(100 mL) and LiOH solution (2 M, 10 mL) was heated at reflux overnight. The
reaction was cooled and the solvents removed under reduced pressure. The
residue was dissolved in water (100 mL) and EtOAc (100 mL) and the pH of the
solution was adjusted to neutral with 1 M HCl. The organic layer was
separated, dried and the solvents were removed under reduced pressure to
give 5.01 g (96%) of 3-(2,3,4-trichlorophenyl)-1H-pyrazole-5-carboxylic acid
(8). A mixture of 8 (0.29 g, 0.001 mol), EDC (0.19 g, 0.001 mol), HOBt (0.14,
0.001 mol) and DMF (10 mL), was stirred for 1 h. To this solution was added
ethanolamine (1 mL, 0.017 mol). The reaction was stirred overnight and the
solvents were removed under reduced pressure. The residue was dissolved in
EtOAc (25 mL) and water (25 mL). The organic layer was separated, and
washed with HCl (1 M) followed by sat. sodium bicarbonate solution and the
organic layer was dried over magnesium sulfate and evaporated. The residue
was purified using automated medium pressure silica gel chromatography
(ISCO) eluting with DCM:EtOAc 100:0–70:30 gradient to yield 0.27 g (80%) of
9v. 1H NMR (300 MHz, DMSO-d6,TMS) d 8.48 (s, 1H), 7.71 (s, 2H), 7.34 (s, 1H),
4.82 (s, 1H), 3.54–3.35 (m, 4H); 13C NMR (75 MHz, DMSO-d6,TMS) d 160.3,
145.1, 142.0, 133.2, 132.4, 131.9, 131.7, 130.0, 129.7, 106.9, 60.4, 42.2. HRMS
Calcd for C12H10Cl3N3O2: 333.9911. Found: 333.9919.
demonstrated a novel MOA for 9v, and these data were supported
by in silico docking studies. Compound 9v should prove to be an
extremely useful small molecule tool to facilitate investigations
into the biochemistry and pharmacology of TNAP.
Acknowledgments
This work was supported by NIH Grants U54HG003916 and
DE12889.
References and notes
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varied concentrations of substrate at different concentrations of test
compound. Parallel studies were performed for DEA and CDP-star substrates.
The concentration of the second substrate was maintained constant (see Fig. 2
legend for details).
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