Selective protein tyrosine phosphatase 1B inhibitors: Targeting the second phosphotyrosine binding site with non-carboxylic acid-containing ligands

Article abstract of DOI:10.1021/jm034088d

Protein tyrosine phosphatase (PTPase) 1B (PTP1B) has been implicated as a key negative regulator of both insulin and leptin signaling cascades. We identified several salicylic acid-based ligands for the second phosphotyrosine binding site of PTP1B using a NMR-based screening. Structure-based linking with a catalytic site-directed oxalylarylaminobenzoic acid-based pharmacophore led to the identification of a novel series of potent PTP1B inhibitors exhibiting 6-fold selectivity over the highly homologous T-cell PTPase (TCPTP) and high selectivity over other phosphatases.

Full text of DOI:10.1021/jm034088d

J . Med. Chem. 2003, 46, 3437-3440
3437
Letters
Selective P r otein Tyr osin e P h osp h a ta se
1B In h ibitor s: Ta r getin g th e Secon d
P h osp h otyr osin e Bin d in g Site w ith
Non -Ca r boxylic Acid -Con ta in in g Liga n d s
Gang Liu,*,† Zhili Xin,† Heng Liang,‡
Cele Abad-Zapatero,‡ Philip J . Hajduk,‡
David A. J anowick,†,§ Bruce G. Szczepankiewicz,†
Zhonghua Pei,† Charles W. Hutchins,‡
Stephen J . Ballaron,† Michael A. Stashko,†
Thomas H. Lubben,† Cathy E. Berg,†
Cristina M. Rondinone,† J ames M. Trevillyan,† and
Michael R. J irousek†,§
F igu r e 1. Known PTP1B inhibitors with selectivity over
TCPTP.
Zhang and co-workers first identified a second phos-
photyrosine (pTyr) binding site (site 2) in the vicinity
of PTP1B catalytic site. They also pioneered the notion
of achieving selectivity among PTP1B, TCPTP, and
other phosphatases by targeting the less homologous
site 2.8 We recently reported a series of oxamic acid-
based PTP1B inhibitors which for the first time vali-
dated such strategy. A highly potent, oxamic acid-based
PTP1B inhibitor occupying both the catalytic site and
site 2 was discovered using a linked-fragment ap-
proach.9 More recently, the discovery of a highly potent
PTP1B inhibitor 1 (Figure 1, Ki ) 76 nM) with 5-fold
TCPTP selectivity identified the region of site 2 impor-
tant for imparting TCPTP selectivity.10
Metabolic Disease Research and Advanced Technology,
Global Pharmaceutical Research and Development, Abbott
Laboratories, Abbott Park, Illinois 60064-6098
Received April 23, 2003
Abstr a ct: Protein tyrosine phosphatase (PTPase) 1B (PTP1B)
has been implicated as a key negative regulator of both insulin
and leptin signaling cascades. We identified several salicylic
acid-based ligands for the second phosphotyrosine binding site
of PTP1B using a NMR-based screening. Structure-based
linking with a catalytic site-directed oxalylarylaminobenzoic
acid-based pharmacophore led to the identification of a novel
series of potent PTP1B inhibitors exhibiting 6-fold selectivity
over the highly homologous T-cell PTPase (TCPTP) and high
selectivity over other phosphatases.
Zhang and co-workers demonstrated another ap-
proach for achieving TCPTP selectivity with the dis-
covery of a difluorophosphonic acid-based, highly potent
PTP1B inhibitor 2 (Figure 1, Ki ) 2.4 nM) with 10-fold
TCPTP selectivity.11 X-ray crystallography of PTP1B
complexed with a derivative of 2 revealed that targeting
the area defined by residues Lys41, Arg47, and Asp48
can also be exploited to enhance potency and selectivity
of PTP1B inhibitors.12
Protein tyrosine phosphatase (PTPase) 1B (PTP1B)
has been implicated as a major negative regulator of
the insulin and leptin signaling pathway.1 The most
compelling data came from two independent studies on
the targeted disruption of PTP1B gene in mice.2,3 These
PTP1B knock out mice exhibit phenotypes of increased
insulin sensitivity, improved glucose tolerance, resis-
tance to diet-induced obesity, enhanced response toward
leptin-mediated weight loss, and suppression of
feeding.2-5 These observations provide a high level of
validation for PTP1B as a therapeutic target for the
treatment of both type II diabetes and obesity.
T-cell PTPase (TCPTP), a phosphatase implicated in
regulating T-cell activation,6 has the highest homology
to PTP1B, with 74% sequence identity to PTP1B in the
catalytic domain and 100% sequence identity in the
catalytic site. Selective inhibition of PTP1B over TCPTP
is therefore highly desirable and represents one of the
most challenging aspects for drug discovery. Indeed,
only two groups have addressed the TCPTP selectivity
in a structure-based fashion, despite the large number
of PTP1B inhibitors reported in the recent years.7
Site 2 ligands discovered so far for achieving TCPTP
selectivity are all carboxylic acids.9,10 Given that most
of the catalytic site-directed PTP1B inhibitors are highly
charged molecules, non-carboxylic acid-containing ligands
targeting site 2 are highly desirable for improving the
overall druglike properties. Herein, we report the
discovery of first nonacid-containing, salicylate-based
ligands for site 2 of PTP1B, and a structure-based
linking approach for identifying potent PTP1B inhibi-
tors with selectivity over TCPTP.
To identify site 2 ligands for PTP1B, NMR-based
screening was developed using a selectively 13C-labeled
PTP1B (residues 1-288).13 The unique signal of Met258
in site 2 provided an unambiguous marker for inter-
action with any potential site 2 ligands. Approximately
10 000 compounds in the SAR-by-NMR library (MW <
200 Da) were screened.14 From this screening, a variety
of small organic acids were identified as potential site
2 ligands. Figure 2 shows two salicyclic acid (3, 4)
derivatives and a quinaldic acid (5) derivative with
dissociation constants (Kds) in the submillimolar to low
millimolar range. The salicylic acid-based ligands be-
* To whom the correspondence should be addressed: R4MC, AP10,
100 Abbott Park Road, Abbott Park, IL 60064-6098. Tel: 847-935-1224.
Fax: 847-938-1674. E-mail: gang.liu@abbott.com.
† Metabolic Disease Research.
‡ Advanced Technology.
§ Current Address: Pfizer Global R&D, La J olla Laboratories, 10770
Science Center Drive, San Diego, CA 92121-1187.
10.1021/jm034088d CCC: $25.00 © 2003 American Chemical Society
Published on Web 06/25/2003

3438 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
Letters
Sch em e 1.a Preparation of Oxalylaminobenzoic
Acid-Based PTP1B Inhibitors with Salicylate-Based Site
2 Ligands
F igu r e 2. PTP1B site 2 ligands identified using NMR
screening with 13C-labeled protein and their respective dis-
sociation constants.
F igu r e 3. First PTP1B inhibitor with salicylate-based site 2
ligand for achieving selectivity over TCPTP.
came the focus of linking efforts because of their binding
affinity and structural diversity.
The weak affinity of salicylic acids 3 and 4 prevented
direct determination of their binding modes using X-ray
crystallography. The X-ray structure of PTP1B com-
plexed with 1 provided critical guidance for incorporat-
ing the new site 2 ligands.10 The methionine amide could
be replaced by an ether linkage to provide the same
hydrogen bond between 1 and Gln262. To preserve the
free hydroxyl group of site 2 ligands, a C2 symmetrical
methyl 2,6-dihydroxybenzoate was coupled to our cata-
lytic site inhibitor. To our delight, the resulting inhibitor
6 (Figure 3) exhibited good inhibitory potency and
phosphatase selectivity. The kinetic analysis of 6 using
pNPP as substrate in a colorimetric assay confirmed
compound 6 as a competitive and potent PTP1B inhibi-
tor (Ki ) 120 nM) with 4-fold selectivity over TCPTP
(Ki ) 470 nM). Much greater specificity (>300-fold) over
less homologous phosphatases was also observed (Table
2).
a
Reagents and conditions: (a) DEAD, Ph3P, 2,6-dihydroxyben-
zoates, THF, rt; (b) 4 N HCl/dioxane, rt; (c) TBTU, 7, Et3N, DMF,
rt; (d) TFA, anisole, CH2Cl2, rt; (e) H2, 10% Pd/C, MeOH.
Inhibitor 6 and related analogue 10 were synthesized
as shown in Scheme 1. Briefly, a Mitsunobu and
deprotection sequence from tert-butyl 4-hydroxybutyl-
carbamate and methyl 2,6-dihydroxybenzoate yielded
salicylate ether 7. The readily available acid cores S-8-I
and 8-II (see Supporting Information) were coupled with
7 to give amides S-9-I and 9-II. Removal of the acid-
labile protecting groups yielded 6 and 10. Surprisingly,
due to steric hindrance and deprotonation of the phe-
nolic hydroxy group, the methyl ester of 6 could not be
hydrolyzed under basic conditions. Instead, decarboxy-
lation took place after prolonged heating. The salicylic
acid analogue 11 was secured through hydrogenation
of a benzyl ester intermediate. Other related analogues
(12-17) were prepared in the same fashion using
readily available resorcinol derivatives for Mitsunobu
ether formation (see Supporting Information).
F igu r e 4. X-ray crystal structure of PTP1B (resolution 2.2
Å) in complex with 6. Carbon is in orange for PTP1B and green
for compound 6, oxygen is in red, nitrogen is in blue, sulfur is
in yellow.
4, diamide linker of 6 makes critical hydrogen bond
contacts with Asp48 to position the salicylate in site 2.
The ether oxygen of 6 forms a hydrogen bond with
Gln262 (3.05 Å) to hold the salicylate in place. Forced
by the two ortho-substituents, the salicylate carboxylate
group adapts an out of plane conformation and is in
hydrogen bond contact with Tyr20 (2.84 Å) and Arg254
(3.07 Å). The hydroxyl group of 6 hydrogen bonds with
Arg24 (2.92 Å) and Arg254 (2.84 Å). Because of the
diminished electron-withdrawing effect of the carboxy-
late group, the hydroxyl group of the salicylate is
expected to remain as un-ionized under physiological
pH (∼7.4). The aromatic portion of the salicylate lies
Diffraction of the PTP1B crystals (residues 1-322)
soaked with 6 (resolution of 2.2 Å) revealed the usual
binding mode associated with this series of pTyr mi-
metics in the active site (Figure 4).15 However, the X-ray
structure of the complex did not offer clear clues for the
origin of TCPTP selectivity in site 2. As shown in Figure

Letters
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16 3439
Ta ble 1. SAR of the Salicylate-Based Site 2 Ligands for
PTP1B Inhibitors
Ta ble 2. Phosphatase Selectivity Profile of PTP1B Inhibitors 6
and 17
Ki (µM)
PTP1 B LAR SHP-2
CD45
cdc25 C calcineurin
6
17
0.12
0.040
33
96
>200
>3.0a
178
>200
>3.0a
>3.0a >3.0a
>3.0a
a
Highest concentration tested.
catalytic site ethyl-substituted analogue 10 is 6-fold
more potent (Ki ) 18 nM) with 4-fold selectivity over
TCPTP (Ki ) 65 nM). Compounds 11 and 12 exemplified
the importance of the methyl ester and the hydroxyl
group of the salicylate. Salicylic acid 11 showed both
reduced PTP1B potency and TCPTP selectivity. The
carbonyl of the methyl salicylate hydrogen bonds with
PTP1B, while the methoxy part points to the solvent
space (Figure 4). The entropy gain from the fixed
position of the carbonyl group might explain the potency
and selectivity difference between ester 10 and acid 11.
Removal of the hydroxyl group (12) led to an almost 100-
fold decrease of inhibitory potency with deterioration
of TCPTP selectivity as well. Replacing the methyl ester
with a nitro group (13) resulted in a 10-fold loss of
potency and complete loss of selectivity over TCPTP.
Replacement of the methyl ester with a methyl amide
(14) resulted in a 20-fold loss of binding potency and a
2-fold decrease in TCPTP selectivity.
The X-ray structure of 6 indicated that Phe52 in site
2 was readily accessible to the salicylate. Since this
residue is not conserved between PTP1B and TCPTP,
interaction with Phe52 had the potential to impart
greater selectivity to this series of PTP1B inhibitors. We
attempted to extend off the 5-position of the salicylate
with either a bromine (15) or a phenyl group (16) to
interact with Phe52. However, such substitutions were
not well tolerated, with over 10-fold decrease of inhibi-
tory potency for both analogues, compared to 10. There
seem to be real differences in TCPTP selectivity for
these two analogues. Bromine substitution (15) gave
6-fold selectivity over TCPTP, while the phenyl analogue
16 showed no TCPTP selectivity. Interestingly, com-
pared to 10, a 4-chloro substituent (17) maintained the
inhibitory potency (Ki ) 40 nM) and gave almost 6-fold
selectivity over TCPTP. The additional hydrophobic
interaction between the chlorine of 17 and Met258 of
PTP1B could partially explain the slightly improved
selectivity profiles.
Compound 17 was profiled more extensively against
other phosphatases in our selectivity panel. As shown
in Table 2, 17 showed greater than 75-fold selectivity18
over other PTPases, including leukocyte antigen-related
tyrosine phosphatase (LAR), SH2-domain-containing
phosphotyrosine phosphatase-2 (SHP-2), and CD45,
respectively (Table 2). Additionally, 17 also exhibited
excellent selectivity profiles (>75-fold)18 over a dual
specificity phosphatase cdc25C, and a protein serine/
threonine phosphatase calcineurin.
a
1:1 mixture of enantiomers.
on top of the hydrophobic side chain of Met258, provid-
ing complimentary van der Waals interaction.
Despite the high homology in the catalytic domain,
PTP1B and TCPTP clearly fulfill different biological
functions, as demonstrated by the knock out mice.2-3,6
Fine structural differences not readily identifiable by
primary sequence analyses may account for the ob-
served differences in inhibitor recognition by PTP1B and
TCPTP. The recently published TCPTP crystal struc-
ture16 has identified two areas that could confer differ-
ent substrate recognition capacities between PTP1B and
TCPTP. In particular, the area defined by Cys32/His34,
Lys39/Glu41, and Phe52/Tyr54 (TCPTP residues in
italics) in site 2 is in very close proximity to the
salicylate-binding region. Thus, the subtle differences
between PTP1B and TCPTP in site 2 could partly
explain the different inhibitor recognition for 6.
Encouraged by the superior in vitro profiles of 6, we
then sought to expand the structure-activity relation-
ship (SAR) of the salicylate-based site 2 ligands. Since
the 3-ethylphenylalanine derivatives as PTP1B inhibi-
tors were generally 5-10 times more potent than the
unsubstituted ones,17 we prepared all the new analogues
with this new template as shown in Table 1. The SAR
of the salicylate was first explored. Compared to 6, the
Even with the non-carboxylic acid-containing site 2
ligand, compound 10 is still a highly charged molecule
with limited ability to cross a cell membrane, as
indicated by low permeability across Caco-2 cell mono-
layers (Papp < 1 × 10-6 cm/s).19 Therefore, when 10 was
tested for augmentation of insulin-stimulated protein
kinase B (PKB) phosphorylation (Ser473) in FAO cells,20

3440 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 16
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Liang, H.; Huang, F.; Hutchins, C. W.; Abad-Zapatero, C.;
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(10) Xin, Z.; Oost, T.; Abad-Zapatero, C.; Hajduk, P. J .; Pei, Z.;
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no augmenting effect was observed, possibly as the
result of low permeability of 10 via passive diffusion.
To circumvent this problem, a prodrug approach using
the diester of 10 was explored. The ester prodrug 18
(Scheme 1) exhibited significantly increased penetration
of Caco-2 cell monolayers (Papp > 1 × 10-6 cm/s). More
importantly, 18 showed augmentation of submaximal
insulin (0.025 nM)-stimulated PKB phosphorylation in
a dose dependent manner at concentrations of 100 µM
(65% increase) and 300 µM (150% increase), respec-
tively, when compared to the level of PKB phosphory-
lation in vehicle treated cells (see Supporting Informa-
tion). The conversion of ester 18 to the parent compound
10 was confirmed by LC-MS when 18 was incubated
with lysate of FAO cells (see Supporting Information).
Similar prodrug strategies for improving cellular per-
meability of PTP1B inhibitors have been reported by
Bleasdale et al.21 and Andersen et al.,22 respectively.
In summary, a novel series of salicylate-based ligands
for the second phosphotyrosine binding site of PTP1B
was discovered through NMR-based fragment screening
and structure-based assembly strategy. Linking with
the oxalylarylaminobenzoic acid-based catalytic site
pharmacophore resulted in a series of highly potent
PTP1B inhibitors with selectivity over TCPTP. The
unique binding mode of this series of PTP1B inhibitors
was determined by X-ray crystallography. The identi-
fication of the salicylate-based site 2 ligands offers
opportunities for linking with other novel, less charged,
active site-directed pTyr mimetics to yield highly potent,
selective, and cell permeable PTP1B inhibitors.
PTP1B Complexed with
a Potent and Selective Bidentate
Inhibitor. J . Biol. Chem. 2003, 278, 12406-12414.
(13) Hajduk, P. J .; Augeri, D. J .; Mack, J .; Mendoza, R.; Yang, J .;
Betz, S. F.; Fesik, S. W. NMR-Based Screening of Proteins
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(14) Shuker, S. B.; Hajduk, P. J .; Meadows, R. P.; Fesik, S. W.
Discovering High-Affinity Ligands for Proteins: SAR By NMR.
Science 1996, 274, 1531-1534.
Ack n ow led gm en t. We thank Drs. Elizabeth Everitt
and Mark Schurdak for determining the Caco-2 perme-
ability of 10 and 18.
(15) Refined crystallographic coordinates for the structure of PTP1B
complexed with compound 6 have been deposited with the
Protein Data Bank (www.rcsb.org) with entry codes 1PH0.
(16) Iversen, L. F.; Møller, K. B.; Pedersen, A. K.; Peters, G. H.;
Petersen, A. S.; Andersen, H. S.; Branner, S.; Mortensen, S. B.;
Møller, N. P. H. Structure Determination of T Cell Protein-
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fold since 3 µM is the highest concentration tested.
(19) Yee, S. In Vitro Permeability Across Caco-2 Cells (Colonic) Can
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M. L.; Wang, X.-Y.; Thompson, D. P.; Conradi, R. A.; Mathews,
W. R.; Laborde, A. L.; Stuchly, C. W.; Heijbel, A.; Bergdahl, K.;
Bannow, C. A.; Smith, C. W.; Svensson, C.; Liljebris, C.;
Schostarez, H. J .; May, P. D.; Stevens, F. C.; Larsen, S. D. Small
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ment Insulin Action. Biochemistry 2001, 40, 5642-5654.
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Mortensen, S. B.; Christensen, M. S.; Branner, S.; Hansen, T.
K.; Lau, J . F.; J eppesen, L.; Moran, E. J .; Su, J .; Bakir, F.; J udge,
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Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails and analytical and spectral characterization data of key
compounds. This material is available free of charge via the
Internet at http://pubs.acs.org.
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