Identification of a monoacid-based, cell permeable, selective inhibitor of protein tyrosine phosphatase 1B

Article abstract of DOI:10.1016/j.bmcl.2003.08.064

Monoacid-based PTP1B inhibitors with improved physiochemical properties have been investigated. A (2-hydroxy-phenoxy) acetic acid-based phosphotyrosyl mimetic has been linked with an optimized second arylphosphate binding site ligand to produce compound 20 with low micromolar potency against PTP1B, good selectivity over TCPTP (20-fold) and high cell permeability in the Caco-2 system.

Full text of DOI:10.1016/j.bmcl.2003.08.064

Bioorganic & Medicinal Chemistry Letters 13 (2003) 3947–3950
Identification of a Monoacid-Based, Cell Permeable, Selective
Inhibitor of Protein Tyrosine Phosphatase 1B
Zhili Xin,a,* Gang Liu,a Cele Abad-Zapatero,b Zhonghua Pei,a
Bruce G. Szczepankiewicz,a Xiaofeng Li,a Tianyuan Zhang,a Charles W. Hutchins,b
Philip J. Hajduk,b Stephen J. Ballaron,a Michael A. Stashko,a Thomas H. Lubben,a
James M. Trevillyana and Michael R. Jirouseka,y
aMetabolicDiseaseResearch, GlobalPharmaceuticalResearchandDevelopment,AbbottLaboratories,AbbottPark,IL60064-6098, USA
bAdvanced Technology, Global Pharmaceutical Research and Development, Abbott Laboratories, Abbott Park, IL 60064-6098, USA
Received 4 June 2003; revised 28 August 2003; accepted 31 August 2003
Abstract—Monoacid-based PTP1B inhibitors with improved physiochemical properties have been investigated. A (2-hydroxy-
phenoxy) acetic acid-based phosphotyrosyl mimetic has been linked with an optimized second arylphosphate binding site ligand to
produce compound 20 with low micromolar potency against PTP1B, good selectivity over TCPTP (20-fold) and high cell perme-
ability in the Caco-2 system.
# 2003 Elsevier Ltd. All rights reserved.
Protein tyrosine phosphatase 1B (PTP1B) plays an
important role in insulin receptor signaling.1 There is
compelling evidence suggesting that PTP1B is primarily
responsible for the dephosphorylation of the insulin
receptor, and therefore acts to negatively regulate insulin
signaling.2 Inhibitors ofthis enzyme would be predicted
to enhance insulin-stimulated glucose transport and have
potential for the treatment of type II diabetes.3
bioavailability. It becomes a significant challenge to
develop both potent and cell permeable inhibitors
because ofthe nature ofthe enzyme. The electrostatic
properties ofthe PTP1B catalytic site are optimal ofr
binding phosphotyrosine, which bears two negative
charges at physiological pH. Charged ligands are there-
fore preferred for potent binding. There are a limited
number ofreports on cell permeable PTP1B inhibitors.
The enthusiasm for the development of PTP1B inhibi-
tors as therapeutic agents has led to intensive research
in recent years.4,5 Many efforts were focused on the
design ofphosphotyrosyl (pTyr) mimetics. Included
among these pTyr mimetics (Fig. 1) are difluoro-
methylene phosphonate 1,6 2-oxalylamino-benzoic acid
27 and O-carboxymethyl salicylic acid analogues 3.8
Unfortunately, most of these potent pTyr mimetics
contain at least two acids and led to inhibitors with
poor cellular permeability.
PTP1B inhibitors need to be cell permeable to reach the
intracellular target, and also to achieve desirable oral
*Corresponding author: Tel.: +1-847-935-7307; fax: +1-847-938-
1674; e-mail: zhili.xin@abbott.com
yCurrent address: Pfizer Global R&D, La Jolla Laboratories, 10770
Science Center Drive, San Diego, CA 92121-1187, USA.
Figure 1.
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.08.064

3948
Z. Xin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3947–3950
Liljebris et al. investigated bioisosteric replacements for
the carboxyl group of O-carboxymethyl salicyclic acid,
and discovered that an ortho tetrazole analogue 4
(Ki=2.0 mM) was equipotent to the corresponding
dicarboxylic acid derivative and this compound
these potential active site ligands with our optimized
salicylate-based, neutral site 2 ligand11 offered opportu-
nities to identify cell permeable, potent and selective
PTP1B inhibitors.
9
achieved a low level ofcell membrane permeability.
The representative syntheses ofmonoacids 8 and 17 are
outlined in Scheme 1. Amine 6 was prepared by mono-
alkylation ofmethyl 2,6-dihydroxybenzoate with N-
Boc-4-aminobutanol and subsequent deprotection. 4-
nitro-N-Boc-phenylalanine was coupled with amine 6 to
generate amide 7. The nitro group was then reduced to
the corresponding aniline by catalytic hydrogenation.
This aniline was acylated and the resulting ethyl ester
was selectively hydrolyzed in the presence ofa methyl
salicylate under basic conditions to provide oxamic acid
analogue 8. Due to steric hindrance and deprotonation
Considering minimization ofcharge as a means of
enhancing cell membrane diffusion, Burke and co-
workers examined monocarboxy-based pTyr mimetics
and reported poor PTP1B inhibitory potency for these
analogues.10 The need for more potent and selective
PTP1B inhibitors with improved physiochemical prop-
erties remains a critical issue.
PTP1B inhibitor 5 has been previously reported from
this laboratory.11 Compound 5 exhibited good inhibi-
tory potency against PTP1B (Ki=18 nM) and good
selectivity over other phosphatases, including the most
homologous TCPTP (4-fold). It contains a diaryl
oxamic acid benzoic acid, a catalytic site binding phar-
macophore, and a salicylate-based second arylphos-
phate binding site (site 2)12 binder. However, both
Caco-2 permeability and rat pharmacokinetic studies
suggest a major obstacle for future development with
this series ofcompounds. The low cell permeability of 5
as indicated by Papp less than 1ꢀ10ꢁ6 cm/s is most
likely due to the presence ofthe two carboxylic acids,
which both retain negative charges at physiological pH
(7.4).
ofthe phenolic hydroxy group, the methyl ester of
8
remained intact. For the synthesis ofmonoacid 17, N-
Boc-tyrosine benzyl ester was O-alkylated with ethyl
bromoacetate, and the benzyl group was removed by
hydrogenation to afford acid 16. The acid was coupled
with amine 6 and subsequent saponification yielded
phenoxy acetic acid 17.
2-Hydroxy substituted analogue 20 was prepared as
illustrated in Scheme 2. Bromination of2-(benzyloxy)
phenol was followed by alkylation of the bromophenol
with ethyl bromoacetate. It was then coupled via Heck
reaction with benzyl 2-acetamidoacrylate to provide
ester 18. Hydrogenation of 18 reduced the double bond
and released the acid and the phenol at the same time.
Acid 19 was coupled with amine 6, and saponification
ofthe ethyl ester then afforded (2-hydroxy-phenoxy)
acetic acid 20.
In our efforts to identify PTP1B inhibitors with
improved permeability, many phosphotyrosine replace-
ments bearing either one or no charge were linked with
a site 2 ligand. The pTyr mimetics generally have little
measurable inhibitory activity on their own, but linking
Early SAR studies showed that monoacid analogues
having a phenylalanine backbone showed no preference
for N-Boc or N-acetyl substitution group (8 vs 8a in
Table 1). These groups were assumed to be equivalent
for our subsequent SAR investigation. Compound 8
was identified as a low micromolar PTP1B inhibitor
(Ki=8.8 mM). Oxamic acids have been previously
reported as effective phosphate replacements for SH2
domain ligands.13 They were also found to be useful for
PTP1B inhibition. Compound 8 was a reasonably
potent and selective PTP1B inhibitor (16-fold over
Scheme 1. Reagents and conditions: (a) N-Boc-butanol, Ph3P, DEAD,
THF, 54%; (b) 4 N HCl in dioxane, 100%; (c) amine 6, TBTU, i-
Pr2NEt, DMF, 79%; (d) 10% Pd/C, MeOH, H2, rt, 100%; (e) ClCO-
COOEt, i-Pr2NEt, CH2Cl2, 0 ꢂCꢁrt, 84%; (f) NaOH, EtOH, H2O,
98%; (g) ethyl bromoacetate, K2CO3, CH3CN, reflux, 94%; (h) iso-
butyllchloroformate, Et3N, amine 6, 95%.
Scheme 2. Reagents and conditions: (a) Br2, HOAc, CH2Cl2, rt,
78%; (b) Ethyl bromoacetate, K2CO3, DMF, 90 ꢂC, 100%; (c) 2-
acetamidoacrylic acid benzyl ester, Pd(OAc)2, (o-Tol)3P, Et3N,
MeCN, reflux, 53%; (d) 10% Pd/C, H2, rt, 100%; (e) amine 6,
TBTU, i-Pr2NEt, DMF, 84%; (f) NaOH, MeOH, H2O, 91%.

Z. Xin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3947–3950
3949
Table 1. SAR on modification ofacid as pTyr mimetics
Table 2. Monoacid-based PTP1B inhibitors and their respective cell
permeability
Compd
Ki (mM) (PTP1B)
Ki (mM)
(TCPTP)
Cell pemeability
(Pappꢀ10ꢁ6 cm/s)
8
17
20
8.8 (ꢄ1.4)
220 (ꢄ30)
9.0 (ꢄ1.2)
141
>200
182 (ꢄ59)
Low (<1)
Moderate (1–10)
High (>10)
#
R1
R2
H
R3
Kib (mM) (PTP1B)
8.8 (ꢄ1.4)
8
NHBoc
8aa
H
H
NHAc
NHAc
NHAc
NHAc
NHAc
NHBoc
NHBoc
NHBoc
NHBoc
NHAc
NHAc
NHAc
6.1 (ꢄ0.2)
>200
9a
Figure 2.
10a
11a
12a
13
H
>200
new pTyr mimetic should be designed with no more
than one carboxylic acid, and the acid should have a
higher pKa than the oxamic acid ofcompound 8.
H
>200
The design ofnew pTyr mimetics turned out to be a
difficult task. A few analogues with no ionizable group
at physiological pH were investigated (Table 1, com-
pounds 9–12). Despite the potential for some of these
entities to participate in hydrogen bond interactions
within the catalytic pocket, none ofthe compounds
showed any inhibitory activity. It seemed inevitable to
include at least a mono-charged functionality to obtain
an electrostatic interaction that could anchor a ligand
into the catalytic site. Among the acids evaluated, com-
pound 13, closely related to oxamic acid analogue 8,
showed no inhibitory activity when tested at 200 mM
concentration. Phenoxy acetic acid has been evaluated
as a pTyr mimetic by other groups, but showed no
effective inhibition.8,10 However, the phenoxy acetic
acid containing analogue 17 provided weak inhibitory
activity against PTP1B (Ki=220 mM) when linked with
our salicylate-based site 2 ligand. Compound 17
(pKaꢃ3) also had improved Caco-2 cell permeability
compared to oxamic acid analogue 8 (Table 2).
H
>200
H
>200
14
H
>200
15
H
>200
17
H
220 (ꢄ30)
9.0 (ꢄ1.2)
2.2(ꢄ0.4)
>200
20a
21a
22a
OH
COOH
NH2
Further SAR on the phenoxy acetic acid series identified
2-hydroxy substituted compound 20 with low micro-
molar potency against PTP1B (Ki=9.0 mM), only a 4-
fold decrease from di-acid analogue 21 (Ki=2.2 mM). 2-
Amino substituted analogue 22, however, did not exhi-
bit any inhibitory activity. Compound 20 also exhibited
good selectivity over most ofother phopatases and it
was 20-fold selective for PTP1B over TCPTP.
a1:1 racemic mixture.
bThe kinetic analysis was conducted using pNPP as the small molecule
substrate in a continuously-monitored colorimetry assay. Values are
means ofmore than two experiments with the number in the paren-
theses being the standard deviation.
TCPTP, Table 2) bearing only one negative charge at
physiological pH. Unfortunately, this monoacid-based
inhibitor still has low cell permeability, presumably due
to the oxamic acid, which has a calculated pKa of2.1.
This concern was further confirmed by the fact that N-
phenyl oxamic acid fails to cross the cell membrane in a
Caco-2 permeability assay. The poor permeability of
oxamic acids limits their potential to be developed into
orally active PTP1B inhibitors. It was decided that the
Ofparticular note was that compound 20 demonstrated
a high level ofmembrane penetration ( >10ꢀ10ꢁ6 cm/s).
The 2-hydroxy group makes formation of a lactone
possible with compound 20. The equilibrium between
the lactone and acid forms (Fig. 2, 20a and 20b) has
potential utility in circumventing the poor cell perme-
ability ofthe existing PTP1B inhibitors. However, the
existence ofthis equilibrium under various physiological
conditions has yet to be determined.

3950
Z. Xin et al. / Bioorg. Med. Chem. Lett. 13 (2003) 3947–3950
Acknowledgements
The authors thank the Abbott Analytical Department
1
for assistance in acquiring H NMR and mass spectra
for the compounds discussed in this report.
References and Notes
1. (a) Saltiel, A. R.; Kahn, C. R. Nature 2001, 414, 799. (b)
Evans, J. L.; Jallal, B. Exp. Opin. Invest. Drugs 1999, 8, 139.
2. Cheng, A.; Dube, N.; Gu, F.; Tremblay, M. L. Eur. J.
Biochem. 2002, 269, 1050.
3. Blaskovich, M. A.; Kim, H.-O. Exp. Opin. Ther. Pat. 2002,
12, 871.
4. Ripka, W. C. In Annual Reports in Medicinal Chemistry;
Doherty, A. M., Ed.; Academic: San Diego, CA, 2000; Vol.
35, p 231.
5. Johnson, T. O.; Ermolieff, J.; Jirousek, M. Nat. Rev. Drug
Discov. 2000, 1, 696.
6. Burke, T. R., Jr.; Kole, H. K.; Roller, P. P. Biochem. Bio-
phys. Res. Commun. 1994, 204, 129.
˚
Figure 3. X-ray structure (2.2 A resolution) ofPTP1B soaked with 20.
Color scheme: carbons in orange and green for compound 20, oxygen
in red, nitrogen in blue, and sulfur in yellow.
7. Andersen, H. S.; Iversen, L. F.; Jeppesen, C. B.; Branner,
S.; Norris, K.; Rasmussen, H. B.; Møller, K. B.; Møller,
N. P. H. J. Biol. Chem. 2000, 275, 7101.
8. Larsen, S. D.; Barf, T.; Liljebris, C.; May, P. D.; Ogg, D.;
O’Sullivan, T. J.; Palazuk, B. J.; Schostarez, H. J.; Stevens,
F. C.; Bleasdale, J. E. J. Med. Chem. 2002, 45, 598.
9. Liljebris, C.; Larsen, S. D.; Ogg, D.; Palazuk, B. J.; Bleas-
dale, J. E. J. Med. Chem. 2002, 45, 1785.
10. Gao, Y.; Wu, L.; Luo, J. H.; Guo, R.; Yang, D.; Zhang,
Z.-Y.; Burke, T. R., Jr. Bioorg. Med. Chem. Lett. 2000, 10,
923.
11. Liu, G.; Xin, Z.; Liang, H.; Abad-Zapatero, C.; Hadjuk,
P. J.; Janowick, D. A.; Szczepankiewicz, B. G.; Pei, Z.;
Hutchins, C. W.; Ballaron, S. J.; Stashko, M. A.; Lubben, T.;
Berg, C. E.; Rondinone, C. M.; Trevillyan, J. M.; Jirousek,
M. R. J. Med. Chem. 2003, 46, 3437.
Compound 20 has been crystallized with PTP1B. The
X-ray crystal structure of 20 bound to PTP1B14 (Fig. 3)
revealed that it exists as the hydroxy acid form. It binds
very tightly in the catalytic pocket, with the WPD flap
down. The molecule also extends to the second phos-
photyrosine binding site. The salicylate-based ligand
binds as the same fashion as previously described11 in
site 2. The carboxylic acid moiety in the catalytic site
has a similar binding mode to the other acids reported
with the WPD loop in the closed conformation.8 The
improved potency and selectivity against TCPTP (20-
fold) of compound 20 might be due to the effect ofthe
phenol hydroxyl group, which interacts with Lys120
and Tyr46 via a water molecule (not shown).
12. Puius, Y. A.; Zhao, Y.; Sullivan, M.; Lawrence, D. S.;
Almo, S. C.; Zhang, Z.-Y. Proc. Natl. Acad. Sci. U.S.A. 1997,
94, 13420.
13. Beaulieu, P. L.; Cameron, D. R.; Ferland, J.-M.; Gau-
thier, J.; Ghiro, E.; Gillard, J.; Gorys, V.; Poirier, M.; Ran-
court, J.; Wernic, D.; Llinas-Brunet, M. J. Med. Chem. 1999,
42, 1757.
14. Refined crystallographic coordinates for the structure of
hPTP1B complexed with compound 20 have been deposited
with the Protein Data Bank (www.rcsb.org) with entry code
1QXK.
In summary, we have discovered monoacid-based,
selective PTP1B inhibitors with low micromolar inhibi-
tory activity. Ofparticular interest, compound
20
achieved significantly higher Caco-2 cell permeability as
compared to all previous compounds. This compound
also exhibited 20-fold selectivity over TCPTP, the high-
est degree ofselectivity reported to date. These results
provided us with opportunities for designing more
selective, drug-like PTP1B inhibitors.