Design and synthesis of potent, non-peptidic inhibitors of HPTPβ

Article abstract of DOI:10.1016/j.bmcl.2006.05.074

The sulfamic acid phosphotyrosine mimetic was coupled with a previously known malonate template to obtain highly selective and potent inhibitors of HPTPβ. Potentially hydrolyzable malonate ester functionalities were replaced with 1,2,4-oxadiazoles without a significant effect on HPTPβ potency.

Full text of DOI:10.1016/j.bmcl.2006.05.074

Bioorganic & Medicinal Chemistry Letters 16 (2006) 4252–4256
Design and synthesis of potent, non-peptidic inhibitors of HPTPb
Kande K. D. Amarasinghe,a,* Artem G. Evidokimov,a Kevin Xu,a Cynthia M. Clark,a
Matthew B. Maier,a Anil Srivastava,b Anny-Odile Colson,a Gina S. Gerwe,a
George E. Stake,a Brian W. Howard,a Matthew E. Pokross,a
Jeffrey L. Graya and Kevin G. Petersa
aProcter & Gamble Pharmaceuticals, Health Care Research Center, 8700 Mason-Montgomery Road, Mason, OH 45040, USA
bChembiotek Research International, Block BN, Plot-7, Sec-V, Salt Lake City, Kolkata 700091, India
Received 3 April 2006; revised 22 May 2006; accepted 22 May 2006
Available online 12 June 2006
Abstract—The sulfamic acid phosphotyrosine mimetic was coupled with a previously known malonate template to obtain highly
selective and potent inhibitors of HPTPb. Potentially hydrolyzable malonate ester functionalities were replaced with 1,2,4-oxadia-
zoles without a significant effect on HPTPb potency.
Ó 2006 Elsevier Ltd. All rights reserved.
Protein tyrosine phosphorylation, regulated by the
opposing actions of protein tyrosine kinases (PTKs)
and protein tyrosine phosphatases (PTPases), plays a
key role in signal transduction. Disruption of the bal-
ance of the actions of PTKs and PTPases has been
implicated in a variety of disease processes and hence
the identification of small molecules capable of inhibit-
ing the specific kinases1 or phosphatases2 has emerged
as a new therapeutic intervention for a variety of diseas-
es such as cancer, atherosclerosis, and diabetes. Most of
the phosphatase inhibition programs have been focused
on developing selective inhibitors for PTP1B, a cytoplas-
mic PTP that negatively regulates insulin receptor ac-
tion, as new therapies for type-2 diabetes and obesity.3
role of HPTPb in vascular biology.5 Tie2 activation
has been implicated in maintenance of the adult vascula-
ture and in the development of collateral blood vessels
that restore blood flow to ischemic tissue.6 Thus, we
hypothesize that inhibition of HPTPb should enhance
Tie2 activation and thereby preserve vascular function
and enhanced blood flow to ischemic tissue. Consistent
with this hypothesis, we have recently shown that a
non-specific phosphatase inhibitor bis(maltolato)-
oxovanadium IV that inhibits HPTPb augments collat-
eral blood flow in a rat model of vascular insufficiency.7
These data provided the basis for initiating a program to
develop selective, small molecule HPTPb inhibitors for
the treatment of occlusive cardiovascular disease.
Despite the growing interest of PTP1B as a therapeutic
target, little attention has been given to the other phos-
phatases such as PTPa, HPTPb, CD45, LAR, and SHP-
1, which may also be attractive biological targets.4 We
became interested in developing non-peptidic, selective
inhibitors for HPTPb, a receptor-type phosphatase
which is expressed primarily in endothelial cells. HPTPb
is associated with and negatively regulates the activation
of Tie2, a receptor PTK for the angiopoietin family of
polypeptide growth factors, suggesting an important
Selectivity for the desired PTPase is a very important
goal in a phosphatase drug discovery program because
individual PTPases have specific and non-overlapping
functions in a variety of important biological processes.
Most phosphatase programs are hampered by the diffi-
culty of finding selective inhibitors for the specific PTP-
ases. Therefore, we reasoned that a set of highly selective
HPTPb inhibitors would not only be useful in providing
therapies for vascular diseases, but also would serve as
an important platform for other phosphatase inhibitor
discovery programs.8
Keywords: Protein tyrosine phosphatases; HPTPb; Sulfamic acid;
Malonates; Oxadiazoles.
Improving upon a HTS lead identified by screening of
the P&GP corporate repository, sulfamic acid function-
ality was identified as a potential phosphotyrosine
*
Corresponding author. Tel.: +1 513 622 1143; fax: +1 513 622
1433; e-mail: Amarasinghe.kk@pg.com
0960-894X/$ - see front matter Ó 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.05.074

K. K. D. Amarasinghe et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4252–4256
4253
mimetic.9 In order to obtain novel inhibitors of HPTPb,
the sulfamic acid phosphotyrosine mimetic was coupled
to known non-peptidic PTP backbones. A variety of
non-peptidic scaffolds including malonates and deoxy-
benzoin, benzotriazoles, and 1,2-oxazines combined
with various phosphotyrosine mimetics have been
reported as PTPase inhibitors.3c,10,11 One of these back-
bones, dimethyl malonate ester, was chosen as a tem-
plate, and sulfamic acid 3 was selected as an initial
target (Fig. 1). Alkylation of the commercially available
dimethyl 2-(4-nitrobenzyl) malonate with benzyl bro-
mide followed by the reduction and sulfamation of the
nitro group furnished 3 (Scheme 1).12 Compound 3
proved to be a competitive, reversible inhibitor of
HPTPb with a Ki of 0.20 lM.13 It also showed good
selectivity versus a representative panel of PTPases
including PTP1B, TC-PTP, and HPTP A (Fig. 1).
which face opposite directions, make hydrogen-bonding
interactions with Asn1735. The benzyl group attached
to the 2-position of the malonate did not seem to make
any significant interaction with HPTPb.
Optimization of the newly found HPTPb inhibitor be-
gan with substituting the phenyl ring of the benzyl group
in hopes of achieving additional interactions with the
enzyme. Synthesis was carried out as outlined in Scheme
1 using appropriately substituted benzyl bromides. No
significant potency gain was achieved by substituting
the phenyl ring at 3- or 4-positions by sulfonamides
(4a and 4b), esters (4c and 4d), acids (4e) or ethers (4f
and 4g) (Table 1). However, the doubly charged sym-
metric bis-sulfamic acid 4h showed an IC50 of 60 nM
for HPTPb making it the most potent inhibitor of the
series.
The X-ray crystal structure of 3 bound to HPTPb re-
veals that the sulfamic acid functionality binds in the
P0 pocket making it a good phosphotyrosine mimetic
(Fig. 2a).14,15 The two ester groups of the malonate,
Additional diversity at the 2-position of the malonate es-
ter was introduced either by alkylation of 4-nitrobenzyl
bromide with diethyl 2-(tert-butoxycarbonyl)malonate
or by alkylation of dimethyl 2-(4-nitrobenzyl) malonate
O
NH
O
O
O
S
HN
O
O
O
O
HO
N
H
1
O
O
O
S
PTP1B IC50 = 39.6 μM
HO
N
H
3
O
O
PTP1B IC50 = 6.1 μM
HPTPβ IC50 = 0.32 μM
TC-PTP IC50 = 19.4 μM
HPTPA IC50 = 0.9 μM
H2O3P
O
O
X
F
2
F
PTP1B IC50 = (0.94-0.04) μM
Figure 1. Design of non-peptidic inhibitors of HPTPb.
O
O
O
O
O
O
O
a, b
O
S
O
NH4
N
H
3
O2N
O
O
Scheme 1. Reagents and conditions: (a) NaH, benzyl bromide, THF, 0 °C to rt. (b) i—H2, Pd/C, MeOH; ii—Pyr–SO3, Pyr then NH4OH.
Figure 2. Crystal structures of 3 (a), 5m (b), and 12 (c) bound to HPTPb. Carbon atoms of the compounds are shown in blue, those of the key
residues are colored green.

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K. K. D. Amarasinghe et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4252–4256
Table 2. HPTPb inhibition data for compounds 5a-m
Table 1. HPTPb inhibition data for compounds 4a–i
O
OR
R1
O
O
O
O
O
O
O
S
X
O
NH4
N
RO
O
S
H
O
NH4
N
O
H
5a-m
4a-h
Compound
R1
R
HPTPb IC50 (lM)
Compound
X
HPTPb IC50 (lM)
5a
–H
Me
1.0
4a
4b
4c
4d
4e
4f
4-SO2NH2
3-SO2NH2
4-CO2Me
3-CO2Me
4- CO2H
4-OMe
0.43
0.30
0.41
0.29
0.14
0.39
0.33
0.06
0.10
5b
5c
5d
Et
0.45
0.40
0.23
NHBoc
Et
NHCbz
O
Me
4g
4h
4i
3-OMe
4-NHSO3H
3-NHSO3H
OEt
5e
5f
Me
Me
Me
0.57
0.15
0.27
N
with the appropriate alkyl halide followed by simple
functional group transformations. Substitution of the
2-position of the malonate ester by hydrogen (5a) or
protected amines (5b and 5c) lowered the potency (Table
2). An aromatic group attached to the 2-position via a 3-
or 4-atom linker (5f and 5g) did not have a meaningful
effect on the HPTPb potency. Molecular modeling stud-
ies suggested that a hydrogen-bonding accepting group
attached to the 2-position via a 2-atom linker would
grab some additional interactions. Thus, compound
5m with 2-Boc-amino ethane group at the 2-position
of malonate was synthesized. Compound 5m showed
an IC50 of 90 nM for HPTPb. The X-ray crystal struc-
ture of 5m shows that the two ester groups of the malon-
ate interact with Asn1735 in a similar fashion as 3
(Fig. 2b). In addition, the carbonyl oxygen atom on
the Boc group makes an extra hydrogen bond with
His1871. This partially explains why 5m is more potent
than other compounds of the series. Compounds 5c, 5d,
and 5f were screened in a panel of 10 PTPases (Table 3).
All these compounds show excellent selectivity against
other PTPases such as PTP 1B, HPTPA, CD-45, etc.
O
5g
N
N
N
N
5h
5i
Me
Me
0.20
0.08
N
S
S
Ph
N
N
N
O O
Ph
S
O O
5j
Me
Me
Me
Et
0.42
0.15
0.38
0.09
Ph
S
O O
5k
5l
O
O
With highly potent and selective malonate-based
HPTPb inhibitors in hand, we decided to replace the
potentially hydrolyzable ester functionalities with the
ester isostere 1,2,4-oxadiazole. Ester groups were
converted to two isomeric oxadiazole rings to obtain
compounds 8 and 12 as outlined in Scheme 2. The bis-
amidoxime obtained by the reaction of bis-nitrile 6 with
hydroxyl amine in refluxing methanol was acylated with
acetic anhydride and cyclized to obtain the bis-oxadia-
zole 7. The nitro group was then reduced with SnCl2
and the resulting aniline was combined with pyridine–
SO3 complex in pyridine to obtain the sulfamic acid 8.
Bis-oxadiazole 10, obtained by the reaction of 9 with
acetamide oxime and K2CO3 in refluxing toluene,16
was alkylated with 4-nitrobenzyl bromide to obtain 11.
The bis-oxadiazole 11 was converted to the required sul-
famic acid 12 in the usual manner. Both compounds
were equipotent to their diester counterpart 3. The bind-
ing pose of 12 in PTPb is very similar to that of malon-
ate-derived compound 3 (Fig. 2c). Only one oxadiazole
NHBoc
5m
Table 3. Comparison of phosphatase inhibition potency [IC50 (lM)] of
5c, 5d, and 5f
Phosphatase
Compound
5c
5d
5f
HPTPb
PTP 1B
HCPTPA
HPTPe
LAR-1
LAR-2
PTPc
TC-PTP38
HPTPl
CD45
0.40
12.8
82.2
>500
227
0.23
16.2
4.8
0.15
2.3
7.7
>500
288
>500
409
>500
93
472
196
>500
200
109
78
>500
280
15.3
>500
29
>500
126

K. K. D. Amarasinghe et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4252–4256
4255
O
O
N
N
N
N
N
N
CN
b
a
O O
S
CN
O2N
O
N
O2N
N
N
H
6
7
O
O
8
NH4
N
O
N
O
O
N
N
O
N
N
O
OEt
c
b
N
d
O
O
S
O
OEt
O
NH4
N
O
N
N
O
N
O2N
N
H
9
12
10
11
N
Scheme 2. Reagents and conditions: (a) i—NH2OH, MeOH, reflux; ii—(CH3CO)2O, rt; iii—CH3COOH, reflux; (b) i—SnCl2Æ2H2O, EtOH, reflux;
ii—Pyr–SO3, Pyr then NH4OH; (c) acetamide oxime, K2CO3, toluene, reflux; (d) NaH, 4-nitrobenzyl bromide, THF, 0 °C to rt.
Table 4. HPTPb inhibition data for compounds 13a–e
In conclusion, a structure-based approach was taken to
prepare a series of novel malonate ester derived aryl sul-
famic acids as inhibitors of HPTPb. Most of the com-
pounds turned out to be highly potent inhibitors of
HPTPb with good selectivity over several other phos-
phatases. Diester groups of the malonate were replaced
by 1,2,4-oxadiazole, a common isostere of the ester, that
retained the HPTPb potency.
N
O
N
R5
O
O
S
O
NH4
N
N
O
N
H
Compound
R5
HPTPb IC50 (lM)
Acknowledgment
O
13a
0.23
Authors thank Dr. Jennifer Tinsley for helpful
discussions.
13b
13c
0.34
0.21
O
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CN
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Compound 12 was selected to introduce further diversity
at the 2-position due to its slightly higher potency com-
pared to 8 and its ease of synthesis. Compounds 13a–e
were synthesized in a similar fashion to 12 according
to Scheme 2 using the appropriate alkylating agent. It
is important to note that both malonate and bis-oxadia-
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substitutions at the 2-position (Table 4). Compound 13e
with a 3-phenylpropyl group in the 2-position showed
the highest HPTPb potency of the series.

4256
K. K. D. Amarasinghe et al. / Bioorg. Med. Chem. Lett. 16 (2006) 4252–4256
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the Advanced Photon Source, Argonne National Lab-
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supported by the U.S. Department of Energy, Office of
Science, Office of Basic Energy Science, under Contract
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