Discovery and structure-activity relationship of oxalylarylaminobenzoic acids as inhibitors of protein tyrosine phosphatase 1B

Article abstract of DOI:10.1021/jm0205696

Protein Tyrosine phosphatase 1B (PTP1B) has been implicated as a key negative regulator of both insulin and leptin signaling pathways. Using an NMR-based screening approach with ¹⁵N- and ¹³C-labeled PTP1B, we have identified 2,3-dimethylphenyloxalylaminobenzoic acid (1) as a general, reversible, and competitive PTPase inhibitor. Structure-based approach guided by X-ray crystallography facilitated the development of 1 into a novel series of potent and selective PTP1B inhibitors occupying both the catalytic site and a portion of the noncatalytic, second phosphotyrosine binding site. Interestingly, oral biovailability has been observed in rats for some compounds. Furthermore, we demonstrated in vivo plasma glucose lowering effects with compound 12d in ob/ob mice.

Full text of DOI:10.1021/jm0205696

J . Med. Chem. 2003, 46, 2093-2103
2093
Discover y a n d Str u ctu r e-Activity Rela tion sh ip of Oxa lyla r yla m in oben zoic
Acid s a s In h ibitor s of P r otein Tyr osin e P h osp h a ta se 1B
Gang Liu,*,† Bruce G. Szczepankiewicz,† Zhonghua Pei,† David A. J anowick,†,§ Zhili Xin,† Philip J . Hajduk,‡
Cele Abad-Zapatero,‡ Heng Liang,‡ Charles W. Hutchins,‡ Stephen W. Fesik,‡ Steve J . Ballaron,†
Mike A. Stashko,† Tom Lubben,† Amanda K. Mika,† Bradley A. Zinker,† J ames M. Trevillyan,† and
Michael R. J irousek†,§
Metabolic Disease Research and Advanced Technology, Global Pharmaceutical Research and Development,
Abbott Laboratories, Abbott Park, Illinois 60064-6098
Received December 18, 2002
Protein Tyrosine phosphatase 1B (PTP1B) has been implicated as a key negative regulator of
both insulin and leptin signaling pathways. Using an NMR-based screening approach with
15N- and 13C-labeled PTP1B, we have identified 2,3-dimethylphenyloxalylaminobenzoic acid
(1) as a general, reversible, and competitive PTPase inhibitor. Structure-based approach guided
by X-ray crystallography facilitated the development of 1 into a novel series of potent and
selective PTP1B inhibitors occupying both the catalytic site and a portion of the noncatalytic,
second phosphotyrosine binding site. Interestingly, oral biovailability has been observed in
rats for some compounds. Furthermore, we demonstrated in vivo plasma glucose lowering effects
with compound 12d in ob/ob mice.
In tr od u ction
substrate of PTP1B.10,11 The evidence strongly suggests
that selective small-molecule inhibitors of PTP1B may
be effective therapeutics in treating insulin resistance,
type II diabetes, and obesity.
Type II diabetes is a progressive disease of metabolic
dysregulation characterized by insulin resistance in
peripheral tissues (liver, muscle, and adipose) and im-
paired insulin secretion by the pancreas. At the molec-
ular level, the mechanism of insulin resistance in type
II diabetes appears to involve defects in postreceptor
signal transduction1,2 and is not due to a structural
defect in the insulin receptor (IR) itself.3 Initial binding
of insulin to the IR evokes a cascade of phosphorylation
events, including the autophosphorylation of the IR on
multiple tyrosine residues,4 tyrosine phosphorylation of
IR substrate (IRS) proteins (IRS-1 to -4) and other
adaptor molecules.5 Eventually, the metabolic effect of
insulin signaling is manifested with the uptake of
glucose into the peripheral tissues.6
The activation of the IR by autophosphorylation is
reversed by the action of protein tyrosine phosphatases
(PTPases). To date, biochemical, “substrate-trapping”,
and in vivo studies favor PTPase 1B (PTP1B) as the
protein phosphatase directly dephosphorylating the IR.7
The most compelling data came from the targeted
disruption of the PTP1B gene in mice.8,9 Two laborato-
ries have independently generated PTP1B knockout
mice. These mice exhibit increased insulin sensitivity
and improved glucose tolerance. Unexpectedly, PTP1B
deficiency in these mice provided a protective effect on
diet-induced obesity, enhanced response toward leptin-
mediated weight loss, and suppression of feeding.
Subsequently, substrate-trapping experiments demon-
strate that leptin-activated J anus kinase 2 (J ak2) is a
Recent studies have provided important insight into
the basic structural requirements for PTP1B-substrate
and -inhibitor interactions and have suggested that
identification of highly selective, catalytic site-directed
PTP1B inhibitors are quite possible.12 However, despite
the significant progress made toward developing PTP1B
inhibitors, most of the specific inhibitors reported to date
do not possess oral bioavailability for potential clinical
use. In this communication, we now describe the dis-
covery of 2,3-dimethylphenyloxalylaminobenzoic acid (1)
as a novel general PTPase inhibitor scaffold through an
NMR-based screening. A detailed structure-activity
relationship (SAR) and the development of this series
of compounds into potent, selective, orally available
inhibitors of PTP1B with in vivo efficacy in ob/ob mice
are also reported.
Ch em istr y
Analogues varying the 2,3-dimethylaniline portion of
1 were prepared via two different routes. In method A
(Scheme 1), a Buchwald coupling between the o-bromo-
cinnamate 2 and naphthylamine 3, followed by the
oxidative cleavage of the resulting quinolone 4 provided
the final product 5.13 A more general route (method B)
started with a condensation between diphenyliodonium
carboxylate (DPIC) and the appropriate aniline (Scheme
2).14 The sterically hindered sec-aniline 6 could be
acylated with ethyloxalyl chloride through an intramo-
lecular acyl-transfer reaction. Subsequent saponification
of the monoester 7 provided the desired oxamic acid 8.
Scheme 3 described the synthesis of requisite p-
aminophenylalanine derivatives for condensation with
DPIC. Heck coupling of 4-bromoaniline derivatives with
methyl 2-acetamidoacrylate yielded the dehydroamino
* To whom correspondence should be addressed. Address: Metabolic
Disease Research, R4MC, AP-10, Abbott Laboratories, 100 Abbott Park
Road, Abbott Park, IL 60064-6098. Phone: 847-935-1224. Fax: 847-
938-1674. E-mail: gang.liu@abbott.com.
† Metabolic Disease Research.
§ Current Address: Pfizer Global R&D, La J olla Laboratories, 10770
Science Center Drive, San Diego, CA 92121-1187.
‡ Advanced Technology.
10.1021/jm0205696 CCC: $25.00 © 2003 American Chemical Society
Published on Web 04/12/2003

2094 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Sch em e 1. (Method A)a
Liu et al.
converted to the desired oxamic acid 12 following con-
densation with DPIC, acylation, and hydrolysis.
The enantiomerically pure analogue 17 was synthe-
sized from the readily available aniline 13 (Scheme 4).
Monoiodination of 13 with in situ generated ICl pro-
vided the iodide 14. The aniline 15 derived from DPIC
condensation with 14 was then coupled with acrylamide
to generate the diarylaniline 16. The standard acylation
and hydrolysis protocol yielded 17.
Resu lts a n d Discu ssion
NMR Scr een in g. To identify small molecule novel
phosphotyrosine (pTyr) mimetics that could serve as
inhibitors of PTP1B, an NMR-based screen was em-
ployed.15,16 With uniformly 15N-labeled PTP1B (residues
1-288), more than 10 000 low molecular weight com-
pounds (MW e 350 Da) were tested for binding to this
enzyme. From this screen, 2,3-dimethylphenyloxaly-
laminobenzoic acid (1) was identified as an active site
binder as evidenced by chemical shift perturbations for
the amide resonances of Val49, Gly220, and Gly218
residues in the catalytic site (see Figure 1). NMR
titrations yielded an estimated Kd value of 100 µM for
1 (Table 1).
a
Reagents and conditions: (a) Pd2(dba)3, 2-dicyclohexylphos-
phanyl-2′-dimethylaminobiphenyl, 60% NaH, toluene, 110 °C; (b)
KMnO4, Pyr/H2O, room temp.
Sch em e 2. (Method B)a
A detailed kinetic analysis of 1 was then conducted
using pNPP as the small-molecule substrate in a
continuously monitored colorimetry assay. Compound
1 exhibited classical kinetic behavior for a competitive,
reversible inhibitor with an inhibition constant (Ki) of
93 µM, consistent with the affinity measured by NMR
(Table 1). Unlike the character displayed by a previously
reported series of PTP1B inhibitors,17 the Ki value of 1
does not depend on the pH of the assay buffer system.
Kinetic analysis of 1 against other PTPases in a
selectivity panel indicted that 1 was a general phos-
phatase inhibitor with very little discrimination among
PTP1B, T-cell PTPase (TCPTP), leukocyte antigen-
related tyrosine phosphatase (LAR), CD45, and SH2-
domain-containing phosphotyrosine phosphatase-2 (SHP-
2). Therefore, oxalylarylaminobenzoic acid based com-
pounds, such as 1, represent a novel series of pTyr
mimetics.
a
Reagents and conditions: (a) Cu(OAc)2, i-PrOH, 85 °C; (b)
ClCOCO2Et, i-Pr2NEt, CH2Cl2, 0 °C to room temp; (c) NaOH,
MeOH, room temp.
Sch em e 3a
In an effort to increase the potency of 1, analogues
varying the 2,3-dimethylaniline were prepared. These
new analogues were then evaluated for their ability to
inhibit PTP1B in both the NMR titration and colorim-
etry assay. To provide higher sensitivity and more
accurate Kd determination, selectively 13C-labeled PTP1B
was used for the NMR studies on these analogues.18 In
general, data from both NMR titration and colorimetry
assay agreed with each other very well. As shown in
Table 1, both regioisomers of the naphthalenylamine-
derived analogues 5 and 8a showed 3- to 4-fold improve-
ment over 1. More importantly, 7-hydroxynaphthalen-
1-ylamine-derived 8b showed further improvement of
affinity, according to both the NMR titration and Ki
analysis. The tetrahydronaphthalene analogue 8c was
only slightly better than 1, underlying the importance
of the aromaticity of the naphthalene ring.
a
Reagents and conditions: (a) methyl acetamidoacrylate,
Pd(OAc)2, P(o-tolyl)3, Et3N, DMF, 110 °C; (b) 10% Pd/C, MeOH/
EtOAc, atm of H2, 50 °C; (c) NaOH, MeOH, room temp; (d) EDCI,
HOBT, amyl amine, Et3N, DMF; (e) DPIC, Cu(OAc)2, DMF, 95
°C; (f) ClCOCO2Et, i-Pr2NEt, CH2Cl2, 0 °C to room temp; (g)
NaOH, MeOH, room temp.
X-r a y Cr ysta llogr a p h y. The improved potency of
analogues such as 5 and 8b facilitated the determina-
tion of their binding modes using X-ray crystallography.
Diffraction (resolution of 2.2 Å) of the PTP1B crystal
(1-322) soaked with 8b revealed an unusual binding
mode of this series of pTyr mimetics.19
ester 9. The double bond of 9 was hydrogenated to give
the p-aminophenylalanine methyl ester, which was then
hydrolyzed to give the acid 10. Coupling of 10 with
amylamine yielded the diamide 11, which was then

Oxalylarylaminobenzoic Acids as Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2095
Sch em e 4a
a
Reagents and conditions: (a) NaI, chloramine-T, AcOH, room temp; (b) DPIC, Cu(OAc)2, DMF, 95 °C; (c) acrylamide, Pd(OAc)2,
P(o-tolyl)3, Et3N, DMF, 110 °C; (d) ClCOCO2Et, i-Pr2NEt, CH2Cl2, 0 °C to room temp; (e) NaOH, MeOH, room temp.
benzoic acid of 8b acts as the physical barrier that
prevents the WPD loop from adopting the closed con-
formation. A unique hydrogen-bonding network is formed
in the catalytic site involving Arg221, Gly220, Ile219,
Ala217, and Ser216. The hydroxy group of the naph-
thalene resides in a hydrophilic pocket formed largely
by the hydroxy groups of Tyr46 and Ser216 and the
amino groups of Lys116 and Lys120. van der Waals
interaction is observed between the side chain of Gln262
and the aromatic portion of the benzoic acid. A π-π
interaction is also noted between the naphthalene ring
and Tyr46. The open conformation of the WPD loop
exposes additional hydrophobic residues, such as the
hydrophobic portions of Lys116, Lys120, and Asp181 in
the catalytic site, which have not been available to the
F igu r e 1. X-ray crystal structure (2.2 Å) of PTP1B soaked
inhibitors bound to PTP1B with the WPD loop in the
with compound 8b. Carbon is in orange and green for com-
closed conformation.20
pound 8b, oxygen is in red, nitrogen is in blue, and sulfur is
SAR Develop m en t. On the basis of the binding
space revealed by the X-ray structure of 8b, the ortho
substituents of the aryl group of 1 were further explored.
o-Ethyl- (8d), o-isopropyl- (8e), and o-benzyl- (8f) aniline-
based analogues showed 2- to 4-fold improvement in
binding affinity compared to 1. Further potency im-
provement was observed with the o-hydroxyethyl ana-
logue (8g, Ki ) 17 µM), which was designed to mimic
the binding of the 7-hydroxynaphthalene analogue 8b.
Further expansion of the hydrogen-bonding interaction
of the hydroxy group of 8g led to the synthesis of acryl-
amide-based 8h with slightly improved inhibitory po-
tency. Interestingly, an o-piperidine substituent yielded
an inhibitor 8i with a Ki value of 14 µM. These results
suggested a rather tolerant catalytic binding site for
both lipophilic and hydrophilic groups and opportunities
for further optimization of the binding elements in the
active site.
in yellow. Selected hydrogen bonds are indicated by dashed
lines.
All PTPases have a highly conserved catalytic pocket
with the “signature motif” (I/V)HCX5R(S/T)G, which
forms a semicircular cage around the pTyr phosphate.
Binding of phosphotyrosine into the catalytic pocket of
PTP1B causes a conformational change in the enzyme,
bringing a flexible WPD (tryptophan, proline, aspartic
acid) loop containing Asp181 into proximity with the
phosphotyrosine substrate. Formation of the thiolphos-
phoryl enzyme intermediate is facilitated by Asp181,
which acts as a general acid by providing a proton to
the leaving phenolic group and as a general base by
accepting a proton from water during the hydrolysis of
the thiolphosphoryl enzyme intermediate.
The most unusual feature revealed by the X-ray
crystal structure of the PTP1B/8b complex is the
conformation of the WPD loop (Figure 1). With 8b bound
to PTP1B, the flexible loop is in its native, presubstrate
binding conformation, i.e., the open conformation. The
An analysis of the binding surface near the catalytic
site of PTP1B revealed at least three binding pockets:
the catalytic site pTyr pocket, the second pTyr site, and

2096 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Liu et al.
Ta ble 1. SAR of the Catalytic Site-Directed
Oxalylarylaminobenzoic Acids
specificity for PTP1B over other closely related phos-
phatases is through interaction with the active site as
well as the neighboring, less homologous site 2.
A linker was then sought to branch out of the catalytic
site and reach into site 2 with the current template.
Since a number of PTP1B inhibitors have utilized an
alanine moiety as binding elements beyond the catalytic
site pTyr mimetic,24 an alanine-based linker appeared
attractive for initial exploration. The X-ray structure
of 8b suggests that the alanine-based chain could extend
out of the active site from the 4-position of the naph-
thalene, which would also give a system that resembled
a natural phosphotyrosine substrate. We therefore
prepared a prototype compound 18 according to the
route outlined in Scheme 2. The C-terminal pentylamide
of 18 was chosen by balancing the hydrophobicity and
chain length for reaching site 2. Compound 18 not only
exhibited reasonable binding affinity to PTP1B and
TCPTP (Table 2), but also established binding to both
the catalytic site and part of site 2 simultaneously,
according to the X-ray crystal structure (data not shown;
see Figure 2 for general binding mode). X-ray structure
of 18 also revealed two hydrogen-bonding interactions
with Asp48 of PTP1B that are critical for the enhanced
affinity. These interactions also position the pentyl
group in site 2 of PTP1B, which allows introduction of
site 2 ligands with this template. Since the tert-butyl
carbamate of 18 interacts with Asp48 of PTP1B, a basic
amino group in the same position was expected to
provide higher binding affinity. Surprisingly, compound
19 was completely inactive against PTP1B and TCPTP.
The exact cause for such a dramatic change of potency
is not clear. In contrast, compounds containing neutral
groups with hydrogen-bonding donors, such as aceta-
mido (20) and sulfonamido (21), were slightly more
potent than 18.
After establishment of a linker for bridging the cata-
lytic site and site 2, an effort toward optimization of this
series of compounds was undertaken. As shown in Table
2, naphthyl (12a ), o-ethyl (12b), o-isopropyl (12c),
o-hydroxyethyl (12d ), and o-piperidinyl (12e) based
analogues all displayed Ki values of ∼1 µM, with a 10-
fold improvement in binding affinity toward PTP1B and
TCPTP when compared to 18. The enantiomerically
pure analogue 17 exhibited a submicromolar inhibitory
constant (Ki ) 0.17 µM) for both PTP1B and TCPTP,
an over-50-fold improvement over 21.
The X-ray crystal structure of the PTP1B/17 complex
(resolution of 2.2 Å) as shown in Figure 2 illustrated
our structure-based inhibitor design approach.19 The
interactions established between PTP1B and the two
carboxylic acids of 8b remained largely intact for 17 in
the catalytic site. Additionally, the acrylamide of 17 was
in hydrogen bond contact with the amino group of
Lys120 (3.04 Å) and with the benzoic acid (3.09 Å)
intramolecularly. As indicated with 18, two critical
hydrogen bonds are observed between 17 and Asp48,
with the distance being 2.57 (main chain NH) and 2.44
Å (sulfonamido NH), respectively. The terminus of the
pentylamide located in the center of site 2 and is well
positioned for further functionalization.
a
Kd derived from 15N-labeled PTP1B.
the hexapeptide binding site.21 Zhang and co-workers
had identified the noncatalytic, cleftlike phosphotyro-
sine binding site (site 2) adjacent to the catalytic pocket,
which is not conserved among all PTPases and may
participate in substrate recognition.22 The X-ray crystal
structure of the insulin receptor peptide, D-pY-pY-R,
bound to PTP1B shows the N-terminal phosphotyrosine
bound in the catalytic pocket while the C-terminal
phosphotyrosine is bound in the second arylphos-
phate binding site.23 Our strategy for achieving high
Asp48 has been demonstrated to be a residue for
addressing selectivity because of the point mutation
between PTP1B/TCPTP and other phosphatases such

Oxalylarylaminobenzoic Acids as Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2097
Ta ble 2. SAR of Oxalylarylaminobenzoic Acids Occupying Both the Catalytic Site and a Portion of Site 2
Ki ( SEM (µM)
Ki ( SEM (µM)
compd
R1
R2
R3
PTP1B
(TCPTP)
18a
19a
20a
21a
12a b
H
H
H
H
H
H
H
H
t-BuOCO-
H
CH3CO-
CH3SO2-
CH3CO-
17.3 ( 1.9
>100
13.0
>100
10.9
ND
1.1 ( 0.9
9.8 ( 2.1
9.1
1.1 ( 0.5
12bb
12cb
12d b
12eb
17a
-CH2CH3
H
H
H
H
H
CH3CO-
CH3CO-
CH3CO-
CH3CO-
CH3SO2-
1.2 ( 0.3
1.2 ( 0.1
1.5 ( 0.7
2.0 ( 1.5
0.17 ( 0.07
0.9 ( 0.1
1.2 ( 0.3
1.5 ( 0.6
ND
-CH(CH3)2
-CH2CH2OH
1-piperidinyl
-(E)CH2dCH2CONH2
0.16 ( 0.06
a
b
(S)-Enantiomer. 1:1 racemic mixture.
(data not shown), some of them have been found to be
orally bioavailable in Sprague-Dawley rats. Following
a 5 mg/kg (mpk) oral dose, the nonselective inhibitor 5
achieved a high AUC (30.6 µg‚h/mL) and long half-life
(7.3 h) and had a bioavailability value of 17% (Table 4).
For the more selective analogues, such as 12b and 12d ,
oral AUC (0.94 and 0.55 µg‚h/mL, respectively) de-
creased and the volume of distribution (3.6 and 18.6
L/kg, respectively) increased dramatically following a
5 mpk dose, indicating a possible accumulation in the
peripheral tissues. Since PTP1B is an intracellular
protein target, the possible accumulation in insulin-
responsive tissues would be a desirable attribute for
small-molecule inhibitors. Bioavailability values of 23%
and 17% were calculated for 12b and 12d , respectively.
Compound 12d was then evaluated in vivo for its
ability to lower plasma glucose and insulin levels in the
genetically obese C57 BL/6J ob/ob diabetic mouse model.
To maximize compound exposure, 12d was given sub-
cutaneously via minipump to ob/ob mice at 30 and 120
mpk for 6 days. After 6 days of continuous infusion, the
plasma glucose levels of ob/ob mice from the 12d -treated
groups decreased dose-dependently when days 1 and 6
are compared. The plasma glucose level of the rosigli-
tazone-treated group (a positive control) reached the
level of lean control animals.
F igu r e 2. X-ray crystal structure (2.2 Å) of PTP1B soaked
with compound 17. Carbon is in orange and green for com-
pound 17, oxygen is in red, nitrogen is in blue, and sulfur is
in yellow. Selected hydrogen bonds are indicated by dashed
lines.
as LAR (Asn48) and SHP-2 (Asn48).17 Therefore, we
were gratified to find that compound 12a is selective
for PTP1B and TCPTP over other PTPases. As shown
in Table 3, compound 12a showed 6-, 30-, and greater
than 300-fold selectivity for PTP1B/TCPTP over LAR,
SHP-2, and CD45, respectively. Additionally, 12a also
exhibited excellent selectivity profiles over a dual
specificity phosphatase cdc25 (>300-fold) and a protein
serine/threonine phosphatase calcineurin (>300-fold).
TCPTP has the highest homology to PTP1B, with 74%
sequence identity in the catalytic domain, the catalytic
site being identical. Therefore, it is not surprising to
note that compound 12a and its closely related ana-
logues inhibited PTP1B and TCPTP equally well. PTP1B
inhibitors, such as 12a , are well positioned for further
exploring the less homologous site 2 for achieving
selectivity over TCPTP.
Figure 3 shows the reduction of the plasma glucose
level in ob/ob mice from this study. We observed an
average of 12% and 24% reduction of plasma glucose
levels with 30 and 120 mpk/day treatment of 12d ,
respectively, and 50% reduction with 3 mpk/day treat-
ment of rosiglitazone. However, results from the plasma
insulin levels were less clear. For the 30 mpk treatment
group, we observed a reduction of insulin level (from
35 ng/mL for the vehicle control group to 22 ng/mL),
which was consistent with the reduction of plasma
glucose level and increased insulin sensitivity. For the
120 mpk treatment group, the insulin level actually
remained about the same (37 ng/mL) as the vehicle
control. The rosiglitazone treatment group showed
In Vivo Stu d ies. Despite the fact that these dicar-
boxylic acid containing PTP1B inhibitors have low
cellular permeability in a Caco-2 permeability study

2098 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Ta ble 3. Phosphatase Selectivity Profile of Compound 12a
Liu et al.
Ki ( SEM (µM)
SHP-2
compd
PTP1B
TCPTP
LAR
CD45
cdc25
calcineurin
>300
12a
1.1 ( 0.5
1.1 ( 0.9
6.7 ( 4.7
29.6 ( 3.6
>300
>300
Ta ble 4. Pharmacokinetic Profiles of Selected PTP1B
Inhibitors Following Five mpk Doses in Rats
were accomplished on a Finnigan SSQ7000 GC/MS mass
spectrometer using electrospray ionization (ESI) technique.
Exact mass measurements were performed on a Finnigan
FTMS Newstar T70 mass spectrometer. The compound is
determined to be “consistent” with the chemical formula if the
exact mass measurement is within 5.0 ppm relative mass error
(RME) of the exact monoisotopic mass. The purity of the
compounds was determined on two analytic HPLC systems to
be of greater than 90% purity.
t1/2
(oral)
(h)
Cmax
(oral)
AUC
(oral)
CLp (iv)
compd (L/(h‚kg)) (L/kg)
Vâ
F
(µg/mL) µg‚hr/mL) (%)
5
12b
12d
0.03
1.21
1.53
0.29
3.59
18.6
7.3
1.2
9.0
1.72
0.69
0.09
30.6
0.94
0.55
16.8
22.6
16.7
Preparative HPLC was performed on an automated Gilson
HPLC system, using a SymmetryPrep Shield RP18 prep
cartridge, 25 mm × 100 mm i.d., S-7 µM, and a flow rate of 25
mL/min; λ ) 214, 245 nm; mobile phase A, 0.1% TFA in H2O;
mobile phase B, CH3CN; linear gradient 0-70% of B in 19 min.
The purified fractions were evaporated to dryness on a Savant
SpeedVac. Analytical HPLC experiments for selected com-
pounds were performed on two systems. For system A, samples
were analyzed by a Gilson analytical HPLC system, using a
YMC C-18 column (50 mm × 4.6 mm i.d., S-5 µM, 120 Å).
Solvent system used was acetonitrile/0.1% aqueous TFA,
gradient 0-70% over 15 min @ 2 mL/min. System B: Samples
were analyzed by HPLC/MS/ELSD on an Open Access Finni-
gan Navigator/Agilent 1100/Sedere Sedex 75 system using a
Phenomenex Luna C8 column (5 µm, 2.1 mm × 50 mm). The
solvent system used was acetonitrile/0.1% aqueous TFA, and
the gradient was 10-100% over 4.5 min at 1.5 mL/min. The
MS was operated in the +APCI mode.
F igu r e 3. Plasma glucose lowering effects of 12d following a
6-day treatment in ob/ob mice.
Gen er a l Syn th esis of Oxa lyla r yla m in oben zoic Acid s.
Meth od A, Exem p lified by Com p ou n d 5. 1-Na p h th a len -
1-yl-1H-qu in olin -2-on e (4). A mixture of 3-(2-bromophenyl)-
acrylic acid methyl ester (2, 135 mg, 0.56 mmol), 1-amino-
naphthalene (3, 80 mg, 0.56 mmol), tris(dibenzylideneacetone)-
dipalladium(0) (3 mg, 0.003 mmol), 2-dicyclohexylphosphino-
2′-(N,N-dimethyl)aminobiphenyl (4 mg, 0.01 mmol), and 60%
NaH dispersion in mineral oil (50 mg, 1.2 mmol) in toluene (2
mL) was heated to reflux for 5.5 h, diluted with water (10 mL)
and 1 N HCl (5 mL), and extracted with ethyl acetate. The
combined extracts were washed with water and brine, dried
(MgSO4), filtered, and concentrated. The concentrate was
purified by flash column chromatography on silica gel with
1:1 ethyl acetate/hexanes to provide 97 mg (0.36 mmol, 64%
substantial reduction of plasma insulin level as expected
(6 versus 2 ng/mL for the lean control). Also, it is
important to note that the same degree of body weight
increase was seen across the compound-treated groups
and the control groups following the 6-day study (data
not shown). So we were cautiously encouraged by the
initial in vivo results with 12d , which seemed to support
further optimization of this series of compounds.
In conclusion, a novel oxalylarylaminobenzoic acid
based pTyr mimetic was discovered through an NMR-
based fragment screening, and the unique binding mode
of this series of inhibitors to PTP1B was determined by
X-ray crystallography. The structure-based approach
facilitated the development of this series of potent and
selective PTP1B inhibitors, which occupy both the
catalytic site and part of the less homologous, second
phosphotyrosine binding site. Although the core struc-
ture of these inhibitors is charged, we have observed
reasonable bioavailability in rats for some compounds
and demonstrated plasma glucose lowering effects with
compound 12d in ob/ob mice. These inhibitors set the
stage for screening novel site 2 ligands and achieving
selectivity against the most homologous phosphatase,
TCPTP, which will be reported in due course.
1
yield) of 4. H NMR (300 MHz, DMSO-d6) δ 8.16 (d, J ) 9.5
Hz, 2H), 8.12 (d, J ) 8.1 Hz, 1H), 7.84 (dd, J ) 7.6, 1.5 Hz,
1H), 7.74 (dd, J ) 8.1, 7.1 Hz, 1H), 7.60 (dd, J ) 8.1, 1.0 Hz,
1H), 7.57 (dt, J ) 7.3, 1.4 Hz, 1H), 7.46 (ddd, J ) 8.4, 7.0, 1.3
Hz, 1H), 7.32 (ddd, J ) 8.5, 7.1, 1.4 Hz, 1H), 7.25 (dd, J ) 7.5,
1.4 Hz, 1H), 7.20 (dd, J ) 7.6, 1.0 Hz, 1H), 6.78 (d, J ) 9.5 Hz,
1H), 6.30 (d, J ) 8.5 Hz, 1H); MS (ESI) (M + H)+ at m/z 272.
2-(Na p h th a len -1-yloxa lyla m in o)ben zoic Acid (5). A
solution of 4 (97 mg, 0.36 mmol) in 1.2 mL of pyridine at room
temperature was treated with 1.2 mL of water, cooled to 0 °C,
treated with KMnO4 (210 mg, 1.3 mmol), warmed to room
temperature, and stirred for 17 h. The mixture was treated
with methanol (0.2 mL), stirred for 5 min, treated with 1 N
NaOH (4 mL), and filtered through diatomaceous earth
(Celite). The filter cake was washed with water (15 mL), and
the combined filtrates were washed with diethyl ether, cooled
to 0 °C, adjusted to less than pH 3 with 12 N HCl, and
extracted with ethyl acetate. The combined extracts were
washed with brine, dried (MgSO4), filtered, and concentrated.
The concentrate was purified by reverse-phase HPLC to
provide 5 (35 mg, 0.10 mmol, 29% yield) as a 3:2 mixture of
rotamers. 1H NMR (3:2 mixture of rotamers) (500 MHz,
DMSO-d6) δ 8.37 (d, J ) 8.7 Hz, 1H), 8.05-8.03 (m, 1H), 8.01-
7.95 (m, 1H), 7.87 (dd, J ) 1.8, 7.7 Hz, 1H), 7.68-7.73 (m,
1H), 7.63-7.59 (m, 1H), 7.57-7.49 (m, 2H), 7.45-7.38 (m, 1H),
7.34 (dt, J ) 7.5, 1.1 Hz, 1H), 7.21 (d, J ) 7.7 Hz, 1H), 6.89 (d,
J ) 7.3 Hz, 1H); MS (ESI) (M + H)+ at m/z 336; HRMS calcd
Exp er im en ta l Section
Gen er a l. Unless otherwise specified, all solvents and
reagents were obtained from commercial suppliers and used
without further purification. All reactions were performed
under nitrogen atmosphere unless specifically noted. Flash
chromatography was performed using silica gel (230-400
mesh) from E. M. Science. Proton NMR spectra were recorded
on a Varian Mercury 300 NMR spectrometer or a Varian
UNITY 400/500, as specified for individual compounds. NMR
spectral experiments were run with Me4Si as an internal
standard, and spectra are reported as shift (multiplicity,
coupling constants, proton counts). Mass spectral analyses

Oxalylarylaminobenzoic Acids as Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2099
for (M + H)+ C19H14N1O5 336.0872, found 336.0881; analytical
HPLC tR ) 6.05 min (A), tR ) 1.80 min (B).
(br m, 3H), 1.62-1.54 (m, 1H); MS (ESI) (M + H)+ at m/z 340;
HRMS calcd for (M + H)+ C19H17N1O5 340.1185, found
340.1196.
Gen er a l Syn th esis of Oxa lyla r yla m in oben zoic Acid s.
Meth od B, Exem p lified by Com p ou n d 1. 2-(2,3-Dim eth -
ylp h en yla m in o)ben zoic Acid (6; R ) 2,3-Dim eth yl). To a
mixture of 2,3-dimethylaniline (1.21 g, 10.0 mmol), diphenyl-
iodonium 2-carboxylate monohydrate (3.42 g, 10.0 mmol), and
copper(II) acetate (73 mg, 0.40 mmol) was added 40 mL of
2-propanol. The mixture was stirred at reflux under N2 for 23
h and then concentrated in vacuo. The solid residue was taken
up in 100 mL of ethyl acetate with gentle heating to aid
dissolution. The solution was extracted with water (2 × 25 mL)
and brine (1 × 25 mL), dried over MgSO4, filtered, and
concentrated to a solid. This was recrystallized from 40 mL of
ethyl acetate to give 1.3 g (54%) of colorless crystals. 1H NMR
(400 MHz, DMSO-d6) δ 12.95 (br s, 1H), 9.44 (br s, 1H), 7.89
(d, J ) 7.1 Hz, 1H), 7.30 (t, J ) 7.2 Hz, 1H), 7.11 (m, 2H),
7.03 (t, J ) 4.5 Hz, 1H), 6.69 (m, 2H), 2.29 (s, 3H), 2.10 (s,
3H); MS (ESI) (M + H)+ at m/z 242.
2-[(2,3-Dim eth ylp h en yl)oxa lyla m in o]ben zoic Acid (1).
To an ice-cooled solution of 6 (R ) 2,3-dimethyl) (343 mg, 1.55
mmol) and triethylamine (503 µL, 3.56 mmol) in dichlo-
romethane (5 mL) was added ethyloxalyl chloride (401 µL, 3.56
mmol) over 30 min. The reaction mixture was warmed to
ambient temperature and stirred for 16 h. The reaction
mixture was then treated with 1 M HCl (10 mL) and extracted
with dichloromethane (2 × 15 mL). The combined extracts
were washed with brine, dried over Na2SO4, filtered, and
concentrated in vacuo. The residue was dissolved in 5 mL of
methanol, and 4.5 mL (4.5 mmol) of 1 N NaOH(aq) was added.
After being stirred for 2 h at ambient temperature, the basic
reaction mixture was diluted with 1 M HCl (10 mL). The
product was isolated via reverse-phase HPLC to provide 235
mg (48%) of 1 as a light-brown solid. 1H NMR (300 MHz,
DMSO-d6, a 1:1 mixture of rotamers) δ [7.94 (d, J ) 7.5 Hz),
7.81 (dd, J ) 7.8, 1.7 Hz), 1H in total], [7.55 (td, J ) 7.8, 1.7
Hz), 7.48 (td, J ) 7.8, 1.7 Hz), 1H in total], [7.41 (td, J ) 7.8,
1.1 Hz), 7.35 (td, J ) 7.4, 1.4 Hz), 1H in total], [7.29 (dd, J )
7.8, 1.4 Hz), 7.24 (dd, J ) 6.1 Hz), 1H in total], [7.19 (d, J )
7.8 Hz), 7.08 (d, J ) 7.8 Hz), 1H in total], 7.14 (d, J ) 7.5 Hz,
1H), [7.03 (d, J ) 7.1 Hz), 6.83 (d, J ) 7.1, 1.0 Hz), 1H in total];
MS (ESI) (M + H)+ at m/z 314; HRMS calcd for (M + H)+
2-[(2-E t h ylp h en yl)oxa lyla m in o]b en zoic Acid (8d )
(Meth od B). 1H NMR (400 MHz, DMSO-d6, a mixture of
rotamers) δ [7.92 (d, J ) 8.0 Hz), 7.82 (d, J ) 7.8 Hz), 1H in
total], [7.56 (t, J ) 7.6 Hz), 7.49 (t, J ) 7.6 Hz), 1H in total],
7.44-7.27 (m, 4H), [7.21-7.09 (m), 6.84 (d, J ) 6.8 Hz), 2H in
total], 2.74-2.60 (m, 2H), 0.94 (t, J ) 7.3 Hz, 3H). MS (ESI)
(M + H)+ at m/z 314, (M + NH4)+ at m/z 331; HRMS calcd for
(M + H)+ C17H16N1O5 314.1028, found 314.1021.
2-[(2-Isop r op ylp h en yl)oxa lyla m in o]ben zoic Acid (8e)
(Meth od B). 1H NMR (400 MHz, DMSO-d6, a mixture of
rotamers) δ [7.96 (d, J ) 8.0 Hz), 7.82 (d, J ) 8.0 Hz), 1H in
total], 7.62-7.14 (m, 6H), [7.06 (d, J ) 8.0 Hz), 6.80 (d, J )
8.0 Hz), 1H in total], 3.19 (br m, 1H), 1.16 (d, J ) 8.0 Hz, 1H).
MS (ESI) (M - H)- at m/z 326; HRMS calcd for (M - H)-
C18H16N1O5 326.1029, found 326.1024.
2-[(2-Ben zylp h en yl)oxa lyla m in o]b en zoic Acid (8f)
(Meth od B). 1H NMR (400 MHz, DMSO-d6, a mixture of
rotamers) δ [7.92 (d, J ) 4.0 Hz), 7.80 (dd, J ) 7.8, 4.0 Hz),
1H in total], 7.50-7.39 (m, 2H), 7.36-7.02 (m, 9H), [6.95 (d, J
) 8.0 Hz), 6.79 (d, J ) 8.0 Hz), 1H in total], 4.23 (d, J ) 15.9
Hz, 1H), 3.87 (d, J ) 15.9 Hz, 1H); MS (ESI) (M + NH4)+ at
m/z 393; HRMS calcd for (M + H)+ C22H18N1O5 376.1185, found
376.1177.
2-{[2-(2-H yd r oxyet h yl)p h en yl]oxa lyla m in o}b en zoic
Acid (8g) (Meth od B). 1H NMR (300 MHz, DMSO-d6) δ [7.98
(dd, J ) 7.5, 1.8 Hz), 7.85 (dd, J ) 7.5, 1.8 Hz), 1H in total],
7.58-7.22 (m, 6H), [7.18 (d, J ) 7.5 Hz) and 6.83 (d, J ) 7.5
Hz), 1H in total], 4.44 (t, J ) 6.9 Hz, 2H), 3.12-2.92 (m, 2H);
MS (ESI) (M + H)+ at m/z 330; HRMS calcd for (M + H)+
C17H16N1O6 330.0977, found 330.0986.
2-{[2-(2-Ca r b a m oylvin yl)p h e n yl]oxa lyla m in o}b e n -
zoic Acid (8h ) (Meth od B). 1H NMR (300 MHz, DMSO-d6, a
mixture of rotamers) δ 8.05-7.92 (m, 1H), 7.76 (d, J ) 15.6
Hz, 1H), 7.86-7.23 (m, 7H), 7.17-7.08 (m, 1H), 7.05-6.79 (m,
1H), 6.72-6.53 (three sets of d, J ) 15.6 Hz, 1H in total); MS
(ESI) (M + H)+ at m/z 355; HRMS calcd for (M + H)+
C18H15N2O6 355.0930, found 355.0921.
2-[Oxa lyl(2-p ip er id in -1-ylp h en yl)a m in o]ben zoic Acid
1
(8i) (Meth od B). H NMR (400 MHz, DMSO-d6, a mixture of
rotamers) δ 7.83 (d, J ) 7.2 Hz, 1H), 7.53 (t, J ) 7.2 Hz, 1H),
7.43-7.30 (m, 2H), 7.38 (t, J ) 8.4 Hz, 1H), 7.24-7.08 (m, 2H),
6.99 (br m, 1H), 2.86 (br m, 4H), 1.68-1.38 (m, 6H); MS (ESI)
(M + H)+ at m/z 369; HRMS calcd for (M + H)+ C20H20N2O5
369.1450, found 369.1466.
C17H15N1O5 314.1028, found 314.1037; analytical HPLC tR
6.16 min (A), tR ) 1.75 min (B).
)
2-(Na p h t h a len -2-yloxa lyla m in o)b en zoic Acid (8a )
1
(Meth od A). H NMR (300 MHz, DMSO-d6) δ 7.98-7.82 (m,
4H), 7.72-7.62 (m, 3H), 7.58-7.45 (m, 4H); MS (ESI) (M +
H)+ at m/z 336; HRMS calcd for (M + H)+ C19H14N1O5
336.0872, found 336.0879.
Gen er a l Syn th esis of p-Am in op h en yla la n in e Der iva -
t ives, E xem p lified b y 12a . 2-Acet yla m in o-3-(4-a m in o-
n a p h th a len -1-yl)a cr ylic Acid Meth yl Ester (9, Ar
)
2-[(7-H yd r oxyn a p h t h a len -1-yl)oxa lyla m in o]b en zoic
Acid (8b). A solution of 2-[(7-benzyloxy-1-naphthyl)(carboxy-
carbonyl)amino]benzoic acid (method A from 7-benzyloxy-1-
naphthalenamine) (74 mg, 0.17 mmol) in dioxane (1 mL) at
room temperature was treated with 10% Pd/C (10 mg) and
60% HClO4 (1 drop), stirred under H2 (1 atm) for 4 h, and fil-
tered through diatomaceous earth (Celite). The filter cake was
washed with ethyl acetate, and the combined filtrates were
concentrated. The concentrate was purified by reverse-phase
2-Na p h th a len e). To a mixture of 4-bromo-1-naphthylamine
(2.5 g, 11.3 mmol), Pd(OAc)2 (140 mg, 0.63 mmol), and P(o-
tolyl)3 (570 mg, 1.87 mmol) in anhydrous N,N-dimethylforma-
mide (10 mL) in a pressure tube was added methyl 2-aceta-
midoacrylate (2.1 g, 14.7 mmol) and triethylamine (5.3 mL,
37.5 mmol). The mixture was flushed with N2 for 3 min and
then sealed and heated at 110 °C for 4 h. The reaction mixture
was cooled to ambient temperature and then partitioned be-
tween ethyl acetate and water. The aqueous layer was ex-
tracted once with ethyl acetate, and the combined organic
layers were washed with brine, dried (Na2SO4), filtered, con-
centrated in vacuo. The crude residue was purified via silica
gel chromatography, eluting with ethyl acetate to provide 2.5
1
HPLC to provide 8b as a mixture of rotamers. H NMR (300
MHz, DMSO-d6) δ 10.04 (br s, 1H), 9.91 (br s, 1H), 7.92-7.75
(m, 3H), 7.64-7.14 (m, 6H), [7.11 (dd, J ) 8.8, 2.4 Hz), 6.92
(d, J ) 7.8 Hz), 1H in total]; MS (ESI) (M + Na)+ at m/z 374;
HRMS calcd for (M + Na)+ C19H13N1O6 374.0641, found
374.0628; analytical HPLC tR ) 5.78 min (A), tR ) 1.53 min
(B).
1
g (76%) of compound 9 as a yellow solid. H NMR (400 MHz,
DMSO-d6) δ 9.34 (s, 1H), 8.15 (d, J ) 8.0 Hz, 1H), 7.90 (d, J
) 8.0 Hz, 1H), 7.78 (s, 1H), 7.63 (d, J ) 8.0 Hz, 1H), 7.51 (t, J
) 7.6 Hz, 1H), 7.42 (t, J ) 7.6 Hz, 1H), 6.72 (d, J ) 8.0 Hz,
1H), 6.29 (s, 2H), 3.73 (s, 3H), 1.94 (s, 3H); MS (ESI) (M +
H)+ at m/z 285.
2-Acet yla m in o-3-(4-a m in on a p h t h a len -1-yl)p r op ion ic
a cid (10). To a solution of 9 (2.5 g, 8.8 mmol) in 1:1 (v/v) ethyl
acetate/methanol (50 mL) under N2 was added Pd/C (10%, 250
mg). The reaction flask was capped with a hydrogen balloon
and heated at 60 °C for 18 h. The mixture was filtered through
2-[Oxa lyl-(5,6,7,8-t et r a h yd r on a p h t h a len -1-yl)a m in o]-
1
ben zoic Acid (8c) (Meth od A). H NMR (500 MHz, DMSO-
d6, a mixture of rotamers) δ [7.92 (d, J ) 7.5 Hz), 7.81 (dd, J
) 7.8, 1.9 Hz), 1H in total], [7.54 (dt, J ) 7.8, 1.6 Hz), 7.48
(dt, J ) 7.5, 1.6 Hz), 1H in total], [7.41 (dt, J ) 7.8, 1.5 Hz),
7.35 (dt, J ) 7.5, 1.3 Hz), 1H in total], 7.24-7.03 (m, 3H), [6.96
(d, J ) 7.2 Hz), 6.84 (d, J ) 8.1 Hz), 1H in total], 3.11 (td, J
) 16.8, 5.0 Hz, 1H), 2.81 (br s, 1H), 2.75 (m, 2H), 1.89-1.63

2100 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Liu et al.
diatomaceous earth, and the filter bed was washed with 1:1
(v/v) ethyl acetate/methanol (2 × 25 mL). The filtrate was
concentrated in vacuo to provide the methyl propionate (2.5
g, 100%). The methyl ester was dissolved in methanol (50 mL),
and then 3 N NaOH (4.75 mL, 14.3 mmol) was added dropwise.
After the reaction mixture was stirred for 3 h at ambient
temperature, the methanol was removed in vacuo and the
remaining aqueous solution was acidified to pH ∼4.5 with 3
N HCl. The mixture was concentrated to dryness in vacuo,
taken up in 10% (v/v) methanol/dichloromethane (25 mL), and
filtered through diatomaceous earth. The filter cake was
washed with an additional 10% (v/v) methanol/dichloromethane
(25 mL). The filtrate was concentrated under reduced pressure
to provide acid 10 as a brown solid (1.75 g, 74%). 1H NMR
(400 MHz, DMSO-d6) δ 8.17 (d, J ) 8.0 Hz, 1H), 8.08 (d, J )
8.0 Hz, 1H), 7.99 (d, J ) 8.0 Hz, 1H), 7.48 (t, J ) 7.8 Hz, 1H),
7.38 (t, J ) 7.8 Hz, 1H), 7.12 (d, J ) 7.8 Hz, 1H), 6.60 (d, J )
7.8 Hz, 1H), 1.77 (s, 3H), 4.45 (td, J ) 9.2, 4.8 Hz, 1H), 3.43
(dd, J ) 14.0, 4.8 Hz, 1H), 3.05 (dd, J ) 14.0, 9.2 Hz, 1H); MS
(ESI) (M + H)+ at m/z 273.
2-Acet yla m in o-3-(4-a m in on a p h t h a len -1-yl)-N-p en t yl-
p r op ion a m id e (11). A solution of 10 (500 mg, 1.84 mmol),
1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochlo-
ride (EDCI) (493 mg, 2.57 mmol), 1-hydroxybenzotriazole
hydrate (360 mg, 2.21 mmol), and n-pentylamine (320 µL, 2.75
mmol) in anhydrous N,N-dimethylformamide (10 mL) was
adjusted to pH ∼6 by the addition of triethylamine, and then
the reaction mixture was stirred for 5 h at ambient temper-
ature. The reaction mixture was diluted with water and
extracted with ethyl acetate (3 × 15 mL). The combined ethyl
acetate layers were washed with water and brine, dried over
Na2SO4, filtered, and concentrated in vacuo. The product was
purified via silica gel chromatography, eluting with 5% (v/v)
MeOH/EtOAc to provide amide 11 as a yellow solid (552 mg,
88%). 1H NMR (400 MHz, DMSO-d6) δ 8.15 (d, J ) 8.0 Hz,
1H), 8.07 (s, 1H), 8.04 (s, 1H), 7.87 (t, J ) 5.9 Hz, 1H), 7.45 (t,
J ) 8.0 Hz, 1H), 7.36 (t, J ) 8.0 Hz, 1H), 7.08 (d, J ) 8.0 Hz,
1H), 6.58 (d, J ) 8.0 Hz, 1H), 5.52 (s, 2H), 4.27 (q, J ) 5.9 Hz,
1H), 3.28 (dd, J ) 14.0, 5.9 Hz, 1H), 2.94-3.06 (m, 3H), 1.77
(s, 3H), 1.18-1.39 (m, 4H), 1.14 (heptet, J ) 7.8 Hz, 2H), 0.84
(t, J ) 7.5 Hz, 3H); MS (ESI) (M + H)+ at m/z 342.
2-{[4-(2-Acet yla m in o-2-p en t ylca r b a m oylet h yl)n a p h -
th a len -1-yl]oxa lyla m in o}ben zoic Acid (12a ). To a stirred
suspension of 11 (552 mg, 1.62 mmol) and diphenyliodonium
2-carboxylate monohydrate (580 mg, 1.78 mmol) in N,N-
dimethylformamide (10 mL) was added anhydrous Cu(OAc)2
(14.6 mg, 0.081 mmol). The mixture was heated at 95 °C for
1.5 h. The reaction mixture was then concentrated in vacuo,
and the crude residue was purified by reverse-phase prepara-
tive HPLC to give 2-[4-(2-acetylamino-2-pentylcarbamoyleth-
yl)naphthalen-1-ylamino]benzoic acid as a light-brown solid
(619 mg, 83%).
To an ice cooled, stirred solution of above benzoic acid (619
mg, 1.34 mmol) and triethylamine (680 µL, 5.13 mmol) in
dichloromethane (10 mL) was added ethyloxalyl chloride (452
µL, 4.04 mmol) slowly over 30 min. The reaction mixture was
warmed to ambient temperature and stirred for 16 h. After
this time, 1 N HCl (10 mL) was added and the mixture was
extracted with dichloromethane (2 × 20 mL). The combined
dichloromethane layers were washed with brine, dried over
Na2SO4, filtered, and concentrated in vacuo to give the crude
2-{[4-(2-acetylamino-2-pentylcarbamoylethyl)naphthalen-1-yl]-
ethoxyoxalylamino}benzoic acid.
1H), 3.67-2.82 (m, 6H), [(1.81, s), (1.78, s) (1.75, s), 3H in total],
1.39-1.00 (m, 4H), 0.92-0.69 (m, 3H); MS (ESI) (M + H)+ at
m/z 534; HRMS calcd for (M + H)+ C29H32N3O7 534.2240, found
534.2246; analytical HPLC tR ) 6.66 min (A), tR ) 1.84 min
(B).
2-{[4-(2-Ac e t y la m in o -2-p e n t y lc a r b a m o y le t h y l)-2-
et h ylp h en yl]oxa lyla m in o}b en zoic Acid (12b ). Light-
brown solid; 1H NMR (300 MHz, DMSO-d6, a mixture of rota-
mers) δ 8.17-8.01 (m, 1H), 7.98-7.74 (m, 2H), 7.60-6.93 and
6.83-6.71 (m, 6H), 4.52-4.36 (m, 1H), 3.07-2.84 (m, 4H),
2.83-2.53 (m, 2H), 1.87 (s, 3H), 1.42-1.01 (m, 9H), 0.93 (t, J
) 7.7 Hz, 2H), 0.88-0.74 (m, 3H); MS (ESI) (M + H)+ at m/z
512; HRMS calcd for (M + H)+ C27H34N3O7 512.2397, found
512.2416; analytical HPLC tR ) 6.49 min (A), tR ) 1.82 min
(B).
N-Acetyl-4-[(ca r boxyca r bon yl)(2-ca r boxyp h en yl)a m i-
n o]-3-isop r op yl-N-p en tylp h en yla la n in a m id e (12c). Light-
brown solid; 1H NMR (500 MHz, DMSO-d6, a mixture of
rotamers) δ 7.90-8.16 (m, 2H), 6.73-7.60 (m, 4H), 4.40-4.52
(m, ca. 0.6 H), 2.7-3.3 (m, ca. 5.4H), 1.77 and 1.74 (s, 3H),
1.1-1.41 (m, 11H), 0.62-0.90 (m, 3H); MS (ESI) (M + H)+
at m/z 526; HRMS calcd for (M + H)+ C28H35N3O7 526.2553,
found 526.2579; analytical HPLC tR ) 6.86 min (A), tR ) 1.89
min (B).
N -Ace t yl-4-[(ca r b oxyca r b on yl)(2-ca r b oxyp h e n yl)-
a m in o]-N -p e n t yl-3-(2-h yd r oxye t h a n e )p h e n yla la n in -
a m id e (12d ). 1H NMR (300 MHz, DMSO-d6, a mixture of
rotamers) δ 8.14-7.58 (m, 3H), 7.52-6.85 (m, 6H), 4.80-4.30
(m, 3H), 3.60-3.79 (m, 2H), 3.04-2.64 (m, 6H), 1.82-1.73
(multiple s, 3H in total), 1.40-1.10 (m, 4H), 0.84 (t, J ) 7.4
Hz, 3H); MS (ESI) (M + H)+ at m/z 528; HRMS calcd for (M +
Na)+ C27H33N3O8Na 550.2165, found 550.2187; analytical
HPLC tR ) 5.60 min (A), tR ) 1.58 min (B).
2-{[4-(2-Acet yla m in o-2-p en t ylca r b a m oylet h yl)-2-p ip -
er id in -1-ylp h en yl]oxa lyla m in o}ben zoic Acid (12e). Light-
brown solid; 1H NMR (500 MHz, DMSO-d6, a mixture of
rotamers) δ 7.90-8.16 (m, 2H), 6.73-7.60 (m, 4H), 4.40-4.52
(br m, ca. 0.6 H), 2.7-3.3 (m, ca. 8.4H), 1.77 and 1.74 (s, 3H),
1.19-1.62 (m, 18H); MS (ESI) (M + H)+ at m/z 567; HRMS
calcd for (M + H)+ C30H39N4O7 567.2819, found 567.2844;
analytical HPLC tR ) 5.52 min (A), tR ) 1.52 min (B).
3-(4-Am in o-3-iod op h en yl)-2-m et h a n esu lfon yla m in o-
N-p en tylp r op ion a m id e (14). To a stirred solution of 3-(4-
aminophenyl)-2-(S)-methanesulfonylamino-N-pentylpropiona-
mide (13) (1.1 g, 3.4 mmol) in acetic acid (5 mL) was added
NaI (0.59 g, 3.9 mmol) followed by the addition of chloram-
ines-T trihydrate (1.1 g, 3.9 mmol). The solution was stirred
for 1 h, concentrated under reduced pressure, diluted with
aqueous Na2S2O4 solution, and partitioned between EtOAc and
aqueous NaHCO3. The organic layer was washed with brine,
dried (Na2SO4), filtered, concentrated under reduced pressure,
and purified on silica gel, eluting with 20% EtOAc/hexanes to
provide 0.78 g (1.7 mmol, 50%) of 14 as a light-yellow solid.
1H NMR (300 MHz, DMSO-d6) δ 7.83 (t, J ) 5.6 Hz, 1H), 7.32
(d, J ) 2.2 Hz, 1H), 6.85 (dd, J ) 7.8, 2.2 Hz, 1H), 6.63 (d, J
) 7.8 Hz, 1H), 4.18 (q, J ) 7.5 Hz, 1H), 3.04 (t, J ) 6.3 Hz,
1H), 2.94 (t, J ) 6.3 Hz, 1H), 2.82 (s, 3H), 2.66 (dd, J ) 13.8,
6.0 Hz, 1H), 2.54 (dd, J ) 13.8, 6.0 Hz, 1H), 1.40-1.10 (m,
6H), 0.68 (t, J ) 7.4 Hz, 3H); MS (ESI) (M + H)+ at m/z 454.
2-[2-(2-Ca r b a m oylvin yl)-4-(2-m et h a n esu lfon yla m in o-
2-p en tylca r ba m oyleth yl)p h en yla m in o]ben zoic Acid (16).
To a stirred suspension of 14 (214 mg, 0.47 mmol) and DPIC
(168 mg, 0.49 mmol) in N,N-dimethylformamide (5 mL) was
added anhydrous Cu(OAc)2 (7.3 mg, 0.040 mmol). The resulting
mixture was heated to 95 °C for 90 min. The reaction mix-
ture was then concentrated under reduced pressure. The
residue was further concentrated to a constant weight on an
oil pump to give 2-[2-iodo-4-(2-methanesulfonylamino-2-pen-
tylcarbamoylethyl)phenylamino]benzoic acid (15) as a light-
brown solid (306 mg), which was used directly without further
purification.
To a solution of above crude ester (950 mg) in methanol (7
mL) at ambient temperature was added 1 N NaOH (3.9 mL).
The basic mixture was stirred at ambient temperature for 2
h, and then 1 N HCl (10 mL) was added. The product was
purified by reverse-phase preparative HPLC to provide 210
mg of compound 12a as a light-brown solid (0.39 mmol, 29%
yield over two steps). 1H NMR (400 MHz, DMSO-d6, a mixture
of rotamers) δ 13.13 (s, 1H), 8.43 (t, J ) 7.8 Hz, 1H), 8.32 (d,
J ) 8.4 Hz, 1H), 8.22 (dd, J ) 8.4, 2.8 Hz, 1H), [8.18 (d, J )
8.4 Hz), 8.08 (d, J ) 8.4 Hz), 1H in total], 8.01-7.83 (m, 3H),
7.75-7.12 (m, 5H), 6.86-6.80 (m, 1H), 4.58 (q, J ) 7.4 Hz,
To a mixture of 15 (306 mg, 0.51 mmol), Pd(OAc)2 (6 mg,
0.026 mmol), P(o-tolyl)3 (23 mg, 0.80 mmol) in anhydrous N,N-
dimethylformamide (10 mL) in a pressure tube was added

Oxalylarylaminobenzoic Acids as Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11 2101
acrylamide (66 mg, 0.93 mmol) and triethylamine (0.25 mL,
1.79 mmol). The mixture was flushed with N2 for 3 min before
it was sealed and heated to 90 °C for 16 h. The reaction
mixture was cooled to ambient temperature, and the solvent
was removed on a SpeedVac. The residue was taken up in
methanol and purified on a Gilson preparative HPLC to
highlighted in yellow), along with four additional N-terminal
residues (GFSH) that remain after cleavage of the GST fusion.
Uniformly 15N-labeled and 13CH3(IVLM)-labeled PTP1B was
prepared by growing the bacteria on a minimal medium
containing 1 g/L of 15NH4Cl (Cambridge Isotopes) or by adding
100:100:50 mg/L of 13C-γ-methionine/3,3′-13C-R-ketovalerate/
3-13C-R-ketobutyrate18 1 h before induction with 1 mM IPTG.
Carbenicillin (0.1 mg/mL) was used as the selection agent. In
addition to the standard salts, metals, and vitamins, the media
contained the following additives (g/L of culture): Ala (0.5),
Arg (0.4), Asp (0.4), Cys (0.05), Glu (0.65), Gln(0.4), Gly (0.55),
His (0.1), Lys (0.42), Phe (0.13), Pro (0.1), Ser (2.1), Thr (0.23),
Tyr (0.17), adenine (0.5), guanosine (0.65), thymine (0.2), uracil
(0.5), cytosine (0.2), sodium acetate (1.5), and succinic acid
(1.5). The cell pellets were resuspended in a buffer containing
10 mM sodium phosphate (pH 7.4) and 150 mM NaCl (PBS
buffer). The cells were then lysed using a microfluidizer
(Microfluidics International), and the resulting lysate was
clarified via centrifugation at 76000g for 10 min. The clarified
cell lysate was loaded onto a glutathione Sepharose 4B column
(Pharmacia), and the resin was washed with 20 column
volumes of PBS buffer. The protein was eluted with 5 column
volumes of a buffer containing 50 mM Tris (pH 8.0) and 10
mM reduced glutathione. The N-terminal GST fusion was
cleaved with thrombin (1 U of thrombin/mg of PTP1B) at 4 °C
for 72 h. Cleavage was monitored by SDS-PAGE analysis.
The resulting solution was then concentrated and dialyzed to
remove the glutathione (final dilution factor of 10-10) and
loaded onto a glutathione Sepharose 4B column to remove the
GST. The flow-through (containing PTP1B) was collected and
dialyzed against NMR buffer (see below). Typical protein yields
were 10-20 mg of PTP1B per liter of culture.
The NMR samples were composed of uniformly 15N-labeled
or 13CH3(IVLM) PTP1B in an H2O/D2O (9/1) Tris-buffered
solution (25 mM, pH 7.5) containing DTT (10 mM). Ligand
binding was detected by acquiring sensitivity-enhanced 1H/
15N- or 1H/13C-HSQC spectra25 on 400 µL of PTP1B in the
presence and absence of added compound. Protein concentra-
tions were 300 and 25 µM for the 15N- and 13CH3(IVLM)-
labeled samples, respectively. A Bruker sample changer was
used on a Bruker AMX500 spectrometer equipped with a
cryoprobe.26 Compounds were individually tested at concentra-
tions of 0.1-15.0 mM, and binding was determined by moni-
toring changes in the HSQC spectra. Dissociation constants
were obtained for selected compounds by monitoring the
chemical shift changes of the protein resonances as a function
of ligand concentration. Data were fit using a single binding
site model. A least-squares grid search was performed by
varying the values of Kd and the chemical shift of the fully
saturated protein.
1
provide 153 mg of 16 (0.28 mmol, 60%) as a yellow solid. H
NMR (300 MHz, DMSO-d6) δ 9.53 (s, 1H), 8.00 (t, J ) 5.6 Hz,
1H), 7.90 (dd, J ) 8.1, 1.8 Hz, 1H), 7.64-7.53 (m, 2H), 7.54
(d, J ) 15.6 Hz, 1H), 7.33 (dd, J ) 7.2, 1.8 Hz, 1H), 7.31 (d, J
) 7.2 Hz, 1H), 7.09 (br s, 1H), 6.75 (td, J ) 7.5, 0.9 Hz, 1H),
6.70 (d, J ) 8.7 Hz, 1H), 6.61 (d, J ) 15.6 Hz, 1H), 4.02 (q, J
) 7.2 Hz, 1H), 3.15-2.76 (m, 4H), 2.65 (s, 3H), 1.36-1.16 (m,
6H), 0.81 (t, J ) 7.2 Hz, 1H); MS (ESI) (M + H)+ at m/z 517.
2-{[2-(2-Ca r b a m oylvin yl)-4-(2-(S )-m e t h a n e su lfon yl-
a m in o-2-p en t ylca r b a m oylet h yl)p h en yl]oxa lyla m in o}-
ben zoic Acid (17). Meth od B fr om 16. Light-yellow solid;
1H NMR (300 MHz, DMSO-d6, a mixture of rotamers) δ 8.05-
7.92 (m, 1H), 7.79 (d, J ) 15.6 Hz, 1H), 7.86-7.23 (m, 7H),
7.17 (d, J ) 7.8 Hz, 1H), 7.05 and 6.79 (d, J ) 8.4 Hz, 1H in
total), 6.74-6.53 (three sets of d, J ) 15.6 Hz, 1H in total),
4.38 (m, 1H), 3.10-2.69 (m, 4H), 2.43 (s, 3H), 1.11-1.42 (m,
6H), 0.90-0.77 (m, 3H); MS (ESI) (M + H)+ at m/z 589; HRMS
calcd for (M + H)+ C27H33N4O9S 589.1968, found 589.1979;
analytical HPLC tR ) 4.89 min (A), tR ) 1.35 min (B).
2-{[4-((S )-2-t er t -B u t o x y c a r b o n y la m i n o -2-p e n t y l-
ca r ba m oyleth yl)p h en yl]oxa lyla m in o}ben zoic Acid (18).
White solid; 1H NMR (300 MHz, DMSO-d6, a mixture of
rotamers) δ 7.86 (s, 1H), 7.55-7.41 (m, 2H), 7.38 (br d, J )
6.9 Hz, 1H), 7.17 (d, J ) 8.1 Hz, 2H), 7.09 (d, J ) 8.1 Hz, 2H),
6.71 (d, J ) 8.7 Hz, 1H), 4.00-4.10 (br m, 1H), 3.06-2.95 (br
m, 2H), 2.89-2.80 (br m, 2H), 1.42-1.14 (m, 6H), 1.28 (s, 9H),
0.85 (t, J ) 6.9 Hz, 3H); MS (ESI) (M + H)+ at m/z 542; HRMS
calcd for (M + H)+ C28H35N3O8 542.2502, found 542.2510.
2-{[4-(2-(S)-Am in o-2-p en t ylca r b a m oylet h yl)p h en yl]-
1
oxa lyla m in o}ben zoic Acid (19). Light-brown oil; H NMR
(300 MHz, DMSO-d6, a mixture of rotamers) δ 8.35-8.07 (m,
3H), 7.96-7.85 (m, 1H), 7.68-7.36 (m, 3H), 7.25-7.17 (m, 2H),
3.95-3.84 (m, 1H), 3.13-2.87 (m, 4H), 1.40-1.08 (m, 6H),
0.90-0.77 (m, 3H); MS (ESI) (M + H)+ at m/z 442.
2-{[4-(2-(S )-Ace t yla m in o-2-p e n t ylca r b a m oyle t h yl)-
p h en yl]oxa lyla m in o}ben zoic Acid (20). White solid; 1H
NMR (300 MHz, DMSO-d6, a mixture of rotamers) δ 8.12-
8.03 (m, 1H), 7.97-7.82 (m, 2H), 7.66-7.32 (m, 3H), 7.28-
7.14 (m, 3H), 4.42 (q, J ) 6.0 Hz, 1H), 3.06-2.85 (m, 3H), 2.80-
2.66 (m, 1H), 1.75 (s, 3H), 1.40-1.10 (m, 6H), 0.86-0.78 (m,
3H); MS (ESI) (M + H)+ at m/z 484; HRMS calcd for (M + H)+
C25H30N3O7 484.2084, found 484.2079.
2-{[4-(2-(S )-M e t h a n e s u lfo n y la m i n o -2-p e n t y lc a r -
bam oyleth yl)ph en yl]oxalylam in o}ben zoic Acid (21). White
solid; 1H NMR (300 MHz, DMSO-d6, a mixture of rotamers) δ
7.86 (s, 1H), 7.55-7.41 (m, 2H), 7.38 (br d, J ) 6.9 Hz, 1H),
7.17 (d, J ) 8.1 Hz, 2H), 7.09 (d, J ) 8.1 Hz, 2H), 6.71 (d, J )
8.7 Hz, 1H), 4.00-4.10 (br m, 1H), 3.06-2.95 (br m, 2H), 2.89-
2.80 (br m, 2H), 2.84 (s, 3H), 1.42-1.14 (m, 6H), 0.85 (t, J )
6.9 Hz, 3H); MS (ESI) (M + H)+ at m/z 520; HRMS calcd for
(M + H)+ C24H30N3O8S 520.1753, found 520.1746.
NMR-Ba sed Scr een in g. Human PTP1B (residues 1-288)
was cloned into the pGEX-5X vector (Amersham Pharmacia)
and expressed in E. coli BL21(DE3) cells. The final protein
construct used for the NMR studies was 292 residues compris-
ing the following amino acid sequence:
Biologica l Assa y. The procedure for the pNPP assay has
been described elsewhere.27
X-r a y Cr ysta llogr a p h ic Stu d ies: P r otein P r od u ction ,
Cr ysta lliza tion , a n d Da ta Collection . The final concentra-
tion of the purified PTP1B protein (1-322) was 3-4 mg/mL
and contained various amounts of reducing agents, normally
2-4 mM DTT. Crystals of PTP1B belonging to space group
P3121 (a ) b ) 88.4 Å, c ) 104.5 Å) were grown routinely using
the protocol described by Puius et al.22 with the following
changes. The buffers used in protein crystallization were
degassed for approximately 30 min and then sparged with
argon gas prior to their use for crystallization. This simple
precaution extended the lifetime of the reactive cysteine
(Cys215) in the active site and provided a more stable and
structurally homogeneous conformation of the active site. This
protocol also allowed the direct soaking of all the PTP1B
inhibitors into the crystals. Soaking times ranged from 1 to 5
h and were normally no more that 2 h. Soaking was performed
using 50 µL of the reservoir in the crystallization well and
mixed with
a 2-4 µL of stock (DMSO) solution of the
compound at 100 µM. The soaking protocol was validated with
a control compound, 2-(oxalylamino)-4,5,6,7-tetrahydrothieno-
[2,3-c]pyridine-3-carboxylic acid.17 No significant differences
were observed within an extended area (∼20 Å) around the
Two point mutations exist in the construct (E252D and D265E,

2102 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 11
Liu et al.
(5) Czech, M. P.; Corvera, S. Signaling Mechanisms That Regulate
Glucose Transport. J . Biol. Chem. 1999, 274, 1865-1868.
(6) Olefsky, J . M. Insulin-Stimulated Glucose Transport, Minireview
Series. J . Biol. Chem. 1999, 274, 1863.
active site when the structure obtained was compared with
the one reported. The soaking protocol permitted the rapid
structure determination of PTP1B/inhibitor complexes.
After the soaking period, crystals were transferred to a
cryoprotectant solution for 5-10 s with rayon loops and
exposed to the X-rays. The cryoprotectant solution consisted
of 100 mM HEPES and 0.2 M magnesium acetate adjusted to
pH 7.1 with NaOH at a 16% w/v concentration of PEG8000
with a 35% v/v concentration of glycerol. Crystallographic data
were collected routinely in a conventional, in-house protein
crystallography laboratory, and occasionally at sector 17 at
the Advanced Photon Source (ID and BM lines of IMCA-CAT).
The data collection hardware was a Mar Research image plate
(180 mm), a Mar Research 345 mm image plate, or a Mar
Research 165 mm CCD detector, all driven by the manufac-
turer’s software. For a complete data set, 80 1° frames were
collected at a crystal to detector distance of 80-90 mm.
Exposure times were typically 10 min/frame. This strategy
resulted in >95% completeness in the vast majority of the data
sets with reducing R factors between 5% and 9%. Data were
processed with HKL2000 and scaled with SCALEPACK or
within HKL2000. Phase and map calculations were performed
using XPLOR or CNX. The modeling and electron-density
fitting software QUANTA was used to manipulate the models.
(7) For reviews on PTP1B target validation, see the following. (a)
Goldstein, B. J . Protein-Tyrosine Phosphate 1B (PTP1B):
A
Novel Therapeutic Target for Type 2 Diabetes Mellitus, Obesity
and Related States of Insulin Resistance. Curr. Drug Targets:
Immune, Endocr. Metab. Disord. 2001, 1, 265-276. (b) Zhang,
Z.-Y. Protein Tyrosine Phosphatases: Prospects for Therapeu-
tics. Curr. Opin. Chem. Biol. 2001, 5, 416-423. (c) Ukkola, O.;
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for the Treatment of Obesity and Associated Co-Morbidities. J .
Intern. Med. 2002, 251, 467-475. (d) Cheng, A.; Dube, N.; Gu,
F.; Tremblay, M. L. Coordinated Action of Protein Tyrosine
Phosphatases in Insulin Signal Transduction. Eur. J . Biochem.
2002, 269, 1050-1059.
(8) Elchebly, M.; Payette, P.; Michaliszyn, E.; Cromlish, W.; Collins,
S.; Loy, A. L.; Normandin, D.; Cheng, A.; Himms-Hagen, J .;
Chan, C. C.; Ramachandran, C.; Gresser, M. J .; Tremblay, M.
L.; Kennedy, B. P. Increased Insulin Sensitivity and Obesity
Resistance in Mice Lacking the Protein-Tyrosine Phosphatase-
1B Gene. Science 1999, 283, 1544-1548.
(9) Klaman, L. D.; Boss, O.; Peroni, O. D.; Kim, J . K.; Martino, J .
L.; Zabotny, J . M.; Moghal, N.; Lubkin, M.; Kim, Y.-B.; Sharpe,
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5489.
P h a r m a cok in et ic An a lysis. Experimental details for
pharmacokinetic analysis were previously described.28
In Vivo Stu d y w ith ob/ob Dia betic Mou se Mod el. To
evaluate the effects of PTP1B inhibitors in ob/ob mice,
compound 12d was administered subcutaneously via osmotic
minipumps. Briefly, male ob/ob mice and their lean litter
mates of 5-6 weeks (average age) (The J ackson Laboratories)
were acclimated to the animal research facilities for 5 days.
They were weighed, and tail snip glucose levels were deter-
mined by the glucose oxidase method (Precision G glucose
meter, Abbott Laboratories, North Chicago). The animals were
randomized into five treatment groups based on their plasma
glucose levels and body weight (animals with glucose levels
between 250 and 400 mg/dL were used). Baseline plasma
insulin samples were taken from a subset of the animals
representing each treatment group randomized once (n ) 10
ob/ob and n ) 6 lean ob/+ litter mates; ELISA, ALPCO
Diagnostics, Windham, NH). The five treatment groups were
as follows: vehicle control, minipump (n ) 10); 12d , 30 mg/
kg minipump (n ) 10); 12d , 120 mg/kg minipump (n ) 10);
rosiglitazone, 3 mg/kg minipump (n ) 10); lean control, no
minipump (n ) 10).
(10) Cheng, A.; Uetani, N.; Simoncic, P. D.; Chaubey, V. P.; Lee-Loy,
A.; McGlade, C. J .; Kennedy, B. P.; Tremblay, M. L. Attenuation
of Leptin Action and Regulation of Obesity by Protein Tyrosine
Phosphatase 1B. Dev. Cell 2002, 2, 497-503.
(11) Zabolotny, J . M.; Bence-Hanulec, K. K.; Stricker-Krongrad, A.;
Haj, F.; Wang, Y.; Minokoshi, Y.; Kim, Y. B.; Elmquist, J . K.;
Tartaglia, L. A.; Kahn, B. B.; Neel, B. G. PTP1B Regulates
Leptin Signal Transduction in Vivo. Dev. Cell 2002, 2, 489-495.
(12) For recent reviews on PTP1B inhibitors, see the following. (a)
Burke, T. R., J r.; Zhang, Z.-Y. Protein Tyrosine Phosphatases:
Structure, Mechanism, and Inhibitor Discovery. Biopolymers
1998, 47, 225-241. (b) Moller, N. P. H.; Iversen, L. F.; Andersen,
H. S.; McCormack, J . G. Protein Tyrosine Phosphatases (PTPs)
as Drug Targets: Inhibitors of PTP1B for the Treatment of
Diabetes. Curr. Opin. Drug Discovery Dev. 2000, 3, 527-540.
(c) Blaskovich, M.; Kim, H.-O. Recent Discovery and Develop-
ment of Protein Tyrosine Phosphatase Inhibitors. Expert Opin.
Ther. Pat. 2002, 12, 871-905. (d) J ohnson, T. O.; Ermolieff, J .;
J irousek, M. R. Protein Tyrosine Phosphatase 1B Inhibitors for
Diabetes. Nat. Rev. Drug Discovery 2002, 1, 696-709.
(13) Robert, A. S. Isatoic Anhydride Derivatives. U.S. Patent 3,238,-
201, 1966.
(14) Scherrer, R. A.; Beatty, H. R. Preparation of o-Substituted Ben-
zoic Acids by the Copper(II)-Catalyzed Reaction of Diphen-
yliodonium-2-Carboxylate with Anilines and Other Nucleophiles.
J . Org. Chem. 1980, 45, 2127-2131.
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Science 1996, 274, 1531-1534.
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T.; Shuker, S. B.; Meadows, R. P.; Steinman, D. H.; Carrera, G.
M.; Marcotte, P. A.; Severin, J .; Walter, K.; Smith, H.; Gubbins,
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(17) Iversen, L. F.; Andersen, H. S.; Branner, S.; Mortensen, S. B.;
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On day 1, the Alzet minipumps (model 2001, 1 µL/h, 7 days,
200 µL reservoir) was primed and loaded with vehicle control
(20% DMSO/80% PEG400) and drugs. Animals were then
implanted with Alzet minipumps. The animals were continu-
ously dosed sc from days 1-6. After the day 6 dosing, tail bleed
glucose and insulin (ob/ob and ob/+ only) levels, as well as
body weight, were determined under nonfasting conditions by
10:00 a.m.
Ack n ow led gm en t. We thank Elizabeth Everitt for
determining the Caco-2 permeability, Bach Hickman
and Kennan Marsh for obtaining the rat pharmacoki-
netic data, and J amey Mack and Flora Huang for
preparing the isotopically labeled proteins for the NMR-
based screening.
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