Utilization of a peptide lead for the discovery of a novel PTP1B-binding motif

Article abstract of DOI:10.1021/jm010020r

Examination of the PTP1B inhibitory potency of an extensive series of phosphotyrosyl (pTyr) mimetics (Xxx) expressed in the EGFr-derived hexapeptide platform Ac-Asp-Ala-Asp-Xxx-Leu-amide previously led to the finding of high inhibitory potency when Xxx = 4-(phosphonodifluoromethyl)phenylalanyl (F₂Pmp) (K_i = 0.2 μM) and when Xxx = 3-carboxy-4-carboxy-methyloxyphenylalanyl (K_i = 3.6 μM). In the first instance, further work led from the F₂Pmp-containing peptide to monomeric inhibitor, 6-(phosphonodifluoromethyl)-2-naphthoic acid (K_i = 22 μM), and to the pseudo-dipeptide mimetic, N-[6-(phosphonodifluoromethyl)-2-naphthoyl]-glutamic acid (K_i = 12 μM). In the current study, a similar approach was applied to the 3-carboxy-4-carboxymethyloxyphenylalanyl-containing peptide, which led to the preparation of monomeric 5-carboxy-6-carboxymethyloxy-2-naphthoic acid (K_i = 900 μM). However, contrary to expectations based on the aforementioned F₂Pmp work, incorporation of this putative pTyr mimetic into the pseudo-dipeptide, N-[5-carboxy-6-carboxymethyloxy-2-naphthoyl]-glutamic acid, resulted in a substantial loss of binding affinity. A reevaluation of binding orientation for 5-carboxy-6-carboxymethyloxy-2-naphthoic acid was therefore undertaken, which indicated a 180° reversal of the binding orientation within the PTP1B catalytic site. In the new orientation, the naphthyl 2-carboxyl group, and not the o-carboxy carboxymethyloxy groups, mimics a phosphoryl group. Indeed, when 5-carboxy-2-naphthoic acid itself was examined at neutral pH for inhibitory potency, it was found to have K_i = 31 ± 7 μM, which is lower than parent 5-carboxy-6-carboxymethyloxy-2-naphthoic acid. In this fashion, 5-carboxy-2-naphthoic acid (or more appropriately, 6-carboxy-1-naphthoic acid) has been identified as a novel PTP1B binding motif.

Full text of DOI:10.1021/jm010020r

J . Med. Chem. 2001, 44, 2869-2878
2869
Expedited Articles
Utiliza tion of a P ep tid e Lea d for th e Discover y of a Novel P TP 1B-Bin d in g Motif†
Yang Gao,‡ J ohannes Voigt,‡ He Zhao,‡ Godwin C. G. Pais,‡ Xuechun Zhang,‡ Li Wu,§ Zhong-Yin Zhang,§ and
Terrence R. Burke, J r.*,‡
Laboratory of Medicinal Chemistry, Center for Cancer Research, National Cancer Institute, NCI-Frederick, Bldg. 376,
Boyles Street, Frederick, Maryland 21702, and Department of Molecular Pharmacology, Albert Einstein College of Medicine,
Bronx, New York 10461
Received J anuary 16, 2001
Examination of the PTP1B inhibitory potency of an extensive series of phosphotyrosyl (pTyr)
mimetics (Xxx) expressed in the EGFr-derived hexapeptide platform Ac-Asp-Ala-Asp-Xxx-
Leu-amide previously led to the finding of high inhibitory potency when Xxx ) 4-(phosphono-
difluoromethyl)phenylalanyl (F2Pmp) (Ki ) 0.2 µM) and when Xxx ) 3-carboxy-4-carboxy-
methyloxyphenylalanyl (Ki ) 3.6 µM). In the first instance, further work led from the F2Pmp-
containing peptide to monomeric inhibitor, 6-(phosphonodifluoromethyl)-2-naphthoic acid (Ki
) 22 µM), and to the pseudo-dipeptide mimetic, N-[6-(phosphonodifluoromethyl)-2-naphthoyl]-
glutamic acid (Ki ) 12 µM). In the current study, a similar approach was applied to the
3-carboxy-4-carboxymethyloxyphenylalanyl-containing peptide, which led to the preparation
of monomeric 5-carboxy-6-carboxymethyloxy-2-naphthoic acid (Ki ) 900 µM). However, contrary
to expectations based on the aforementioned F2Pmp work, incorporation of this putative pTyr
mimetic into the pseudo-dipeptide, N-[5-carboxy-6-carboxymethyloxy-2-naphthoyl]-glutamic
acid, resulted in a substantial loss of binding affinity. A reevaluation of binding orientation
for 5-carboxy-6-carboxymethyloxy-2-naphthoic acid was therefore undertaken, which indicated
a 180° reversal of the binding orientation within the PTP1B catalytic site. In the new
orientation, the naphthyl 2-carboxyl group, and not the o-carboxy carboxymethyloxy groups,
mimics a phosphoryl group. Indeed, when 5-carboxy-2-naphthoic acid itself was examined at
neutral pH for inhibitory potency, it was found to have Ki ) 31 ( 7 µM, which is lower than
parent 5-carboxy-6-carboxymethyloxy-2-naphthoic acid. In this fashion, 5-carboxy-2-naphthoic
acid (or more appropriately, 6-carboxy-1-naphthoic acid) has been identified as a novel PTP1B
binding motif.
In tr od u ction
substrate neighboring the pTyr residue, with features
outside the catalytic pocket.6 A principle focus of con-
siderable research to date has centered on interactions
within the pTyr-binding pocket7 and a number of small
molecule, non-phosphorus-containing structures which
bind to PTP1B with from reasonable to high affinity
have been discovered using either screening or computer-
assisted pharmacore-based methods.3
One approach toward inhibitor development is the
utilization of peptides as platforms for small molecule
lead discovery. Exemplary of this, display of pTyr
mimicking amino acid residues (Xxx) incorporated into
the epidermal growth factor receptor (EGFr988-993)-
derived sequence “Ac-Asp-Ala-Asp-Glu-Xxx-Leu-amide”,
which is a high affinity PTP1B substrate when Xxx )
pTyr (Km ) 3.2 µM),8 have been utilized to define phenyl
phosphate mimicking structures that replicate binding
interactions of pTyr itself.9,10 As a case in point, the high
PTP1B affinity of peptide 1a (Figure 1), when Xxx )
phosphono(difluoromethyl)phenylalanine (F2Pmp),11 pro-
vided initial evidence that aryldifluoromethylphospho-
nates such as (difluoro(2-naphthyl)methyl)phosphonic
acids are extremely good PTP-directed phenyl phos-
phate mimetics.12 More recently, a similar “peptide-
Development of small molecule protein-tyrosine phos-
phatase (PTP) inhibitors has important implications for
the treatment of a variety of diseases including certain
cancers and diabetese.2,3 Among PTPs, the human
nonreceptor PTP1B enzyme is important both because
it was the first PTP to have its X-ray crystal structure
reported4 and because it has been shown to potentially
contribute to some forms of diabetes.5 For these reasons,
it provides an ideal model system in which to explore
PTP inhibitor development. Binding of peptidyl phos-
photyrosyl (pTyr)-containing substrate to PTPs involves
two components: (1) critical recognition of the pTyr
“phenyl phosphate” moiety in a highly conserved (H/
V)CX5R(S/T) signature motif within the catalytic pocket
and (2) secondary interactions of amino acids in the
† For preliminary disclosures of this work, see ref 1.
* To whom correspondence should be addressed at Laboratory of
Medicinal Chemistry, Center for Cancer Research, National Cancer
Institute, Bldg. 376, Boyles Street, NCI-Frederick, P.O. Box B,
Frederick, MD 21702-1201. Tel: (301) 846-5906. Fax: (301) 846-6033.
E-mail: tburke@helix.nih.gov.
‡ National Cancer Institute.
§ Albert Einstein Medical School.
10.1021/jm010020r CCC: $20.00 © 2001 American Chemical Society
Published on Web 07/27/2001

2870 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Gao et al.
F igu r e 1. Rationale for the design of monomeric pTyr mimetics 4 and 5 and dipeptide mimetic 6 mimetics using peptide 1b as
a lead, based on a similar approach in going from 1a to 2 to 3.25,26
Sch em e 1a
Sch em e 2a
a
Reagents and conditions: (i) methyl bromoacetate, K2CO3,
DMF, 100%; (ii) CuCN, DMF, 46%; (iii) a. NaOH, H2O; b. HCl,
100%.
a
Reagents and conditions: (i) methyl bromoacetate, K2CO3,
DMF, 88%; (ii) CuCN, DMF, 56%; (iii) a. NaOH, H2O; b. HCl,
100%.
based” approach has been applied to the discovery of
high affinity nonphosphonate-based pTyr mimetics.13
Reported herein is the utilization of one of these lead
pTyr mimetics, 3-carboxy-4-carboxymethyloxyphenyl-
alanyl residue 1b,14 as a starting point for the discovery
of a new naphthyl-based motif which displays good
PTP1B affinity.
Sch em e 3a
Syn th esis
The synthesis of conformationally constrained ana-
logue 4 (Figure 1) from commercially available 1,6-
dibromo-2-naphthol (7) was readily achieved by initial
O-alkylation (methyl bromoacetate) to provide 8,15 fol-
lowed by transformation to the dicyano compound 9
using CuCN as a coupling reagent,16 and finally hy-
drolysis of ester and cyano functionality to provide the
target compound 4 (Scheme 1).17
a
Reagents and conditions: (i) a. CuCN, DMF; b. NaOH, EtOH-
Treatment of commercially available (Aldrich) naph-
thol AS BI ((7-bromo-3-hydroxy(2-naphthyl))-N-benz-
amide, 10) in a similar fashion provided isomeric
analogue 5 (Scheme 2).
H2O; c. HCl, 98%; (ii) a. BBr3, CH2Cl2; b. MeOH, 99%; (iii) NaI,
chloramine-T, 99%; (iv) tert-butyl bromoacetate, K2CO3, DMF,
91%: (v) TMS-CH2CH2-OH, Pd(OAc)2, CO, Ph2PCH2CH2PPh2.
By analogy to the synthesis of peptide 1b,14 compound
18 was initially sought as a variant of constrained
analogue 4 bearing carboxyl protection at positions 5
and 6 which rendered it compatible with peptide cou-
pling of the free 2-carboxyl to ultimately provide pseudo-

Peptide Lead for a PTP1B-Binding Motif
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2871
Sch em e 4a
a
Reagents and conditions: (i) allyl bromide, NaH, DMSO, 91%; (ii) Cl2CHOCH3, TiCl4, CH2Cl2, 77%; (iii) sulfamic acid, NaClO, H2O-
acetone, 100%; (iv) tert-butyl 2,2,2-trichloroacetimide, BF3‚OEt2, 58%; (v) NaBH4, Pd(PPh3)4, THF; (vi) tert-butyl bromoacetate, K2CO3,
63% for two steps; (vii) LiOH, THF-H2O, 100%.
Sch em e 5a
dipeptide 6 (Figure 1). The approach to 18 (Scheme 3)
began with carbonylation of commercially available
2-bromo-6-methoxynaphthol (13) to yield 6-methoxy-2-
naphthoic acid (15) which was O-demethylated with
18
BBr3 and esterified in a one-pot sequence to yield
methyl 6-hydroxy-2-naphthoate (15). Introduction of
iodine at the 5-position using NaI and chloramine T19
provided 16, which upon O-alkylation (tert-butyl bromo-
acetate) gave key intermediate 17. With 17 in hand,
several conditions were examined to effect carboxylation
to desired 18 without success.
Failure to prepare 18 was potentially attributable to
a
Reagents and conditions: (i) ethyl bromoacetate, K2CO3, 68%
steric hindrance at the crowded 5-position. The high
degree of steric crowding at the 5-position suggested
that carboxyl functionality situated at this position
could exhibit reduced reactivity under conditions envi-
sioned for eventual coupling of the 2-carboxyl, thereby
eliminating the formal need for carboxyl protection at
the 5-position. Therefore, target 25 was devised, bearing
a free 5-carboxyl group. Synthesis of 25 started with
intermediate 15, which was initially protected as its
allyl ether 19,20 then the carboxyl group was introduced
in a two-step fashion21 via aldehyde 20 which was then
oxidized22 to acid 21 (Scheme 4). Temporary protection23
of the newly generated 5-carboxy as its tert-butyl ester
(22), followed by removal of the 6-O-allyl protection,24
gave 23, which was alkylated to 24 in 63% yield for two
steps. Treatment with LiOH selectively hydrolyzed both
the 2- and 5-esters in the presence of the 6-ester,
providing desired target 25 suitably protected for cou-
pling of the 2-carboxyl group.
or CF2BrCO2Et, K2CO3, 40%; (ii) LiOH, 97% (for 26), 95% (for 27).
Sch em e 6a
a
Reagents and conditions: (i) Cl2CHOCH3, TiCl4, CH2Cl2, 54%;
(ii) sulfamic acid, NaClO, H2O-acetone, 100%; (iii) NaOH, EtOH-
H2O; (iv) BBr3, CH2Cl2, 100%.
Analogues 28 and 29, which lacked carboxyl func-
tionality ortho to the 6-position, were prepared in
straightforward fashion from common intermediate 15
as shown in Scheme 5.
To prepare 33 and 34 (Scheme 6), methyl 6-methoxy-
2-naphthoate was subjected to the two-step approach
of Scheme 4, whereby carboxyl functionality was first
introduced at the aldehyde level (compound 31) followed
by oxidation (compound 32).
phosphate-mimicking motifs which can be recognized
with high affinity in the PTP1B catalytic site. This
approach is conceptually similar to our previous study,
where the initial lead11 provided by the F2Pmp-contain-
ing peptide (1a , Figure 1), resulted in the development
of 6-(phosphonodifluoromethyl)-2-naphthoic acid (2) as
a moderately potent small molecule PTP1B inhibitor
which functioned as a monomeric pTyr mimetic.25,26 The
starting point for the current study was the previously
reported 3-carboxy-4-carboxymethyloxyphenylalanyl resi-
due (1b), which was among the highest affinity non-
phosphonate-containing pTyr mimetics against PTP1B
in a peptide context.14 On the basis of the above-
mentioned transformation of 1a to 2 in the F2Pmp
study, it was envisioned that 1b could be translated to
isomeric 5-carboxy- and 7-carboxy 6-(carboxymethyl-
oxy)-2-naphthoic acids 4 and 5, respectively. Two iso-
Resu lts a n d Discu ssion
Ra tion a le. The current study was intended as a
peptide-lead approach to non-phosphonate-containing
small molecule PTP1B inhibitor discovery. Its rationale
was predicated on the use of pTyr mimicking amino acid
residues (Xxx), displayed in the sequence “Ac-Asp-Ala-
Asp-Glu-Xxx-Leu-amide” (1, Figure 1), to define phenyl

2872 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Ta ble 1. Inhibition of PTP1B by Naphthyl-Based Inhibitors
Gao et al.
mers were required, because unlike F2Pmp, which is
symmetrical about its phenyl axis of rotation and can
therefore be represented by a single 6-substituted
2-naphthoic acid (2), m-carboxy residue 1a can exhibit
conformational isomerism about its phenyl axis of
rotation (A and B, Figure 1). Accordingly, 5-carboxy-
and 7-carboxy 2-naphthoic acids (4 and 5, respectively)
were designed as conformationally constrained repre-
sentations of these rotamers.
In itia l Resu lts. Analogues 28 and 29 (Table 1)
represent naphthyl-based mimetics of 4-carboxymethyl-
oxyphenylalanine and 4-carboxy(difluoromethyl)oxy-
phenylalanine, respectively.27 The low inhibitory po-
tency is consistent with the reported poor PTP1B
affinity of the former pTyr mimetic when expressed in
the model peptide 1.13 Compounds 4 and 5 exhibited
enhanced potencies, with the 5-carboxy analogue 4 (Ki
) 900 µM) showing approximately 10-fold higher affin-
ity than 28. This indicated the importance of the added
5-carboxyl group. The position of the carboxyl also
affected affinity, since the isomeric 7-carboxy isomer 5
(Ki ) 250 µM) was approximately 3-fold more potent
than 4.
Exa m in a tion of Dip ep tid e Mim etic 6, a n d Re-
eva lu a t ion of Bin d in g Mod e. From preliminary
results, it appeared that either compound 4 or com-
pound 5 could be utilized as a naphthyl-based mimetic
of 1b in a manner similar to the previous use of 2 as a
mimetic of F2Pmp-containing 1a (Figure 1).25 In that
previous study, coupling of 2 to a Glu residue resulted
in pseudo-dipeptide 3, whose approximate 2-fold in-
crease in affinity was attributable to interaction of the

Peptide Lead for a PTP1B-Binding Motif
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2873
F igu r e 2. Binding interactions within the PTP1B signature motif. (a) Binding of a pTyr phenyl phosphate as indicated by X-ray
crystallography.6 (b) Molecular modeling prediction of 3-carboxy-4-carboxymethyloxyphenyl binding as reported in ref 14. (c) Binding
of a 2-(oxalylamino)-benzoic acid moiety as indicated by X-ray crystallography.28 (d) Preferred docking mode of compound 4 utilizing
unbiased techniques as indicated in the Experimental Section.
Glu γ-carboxyl with residues outside the pTyr binding
pocket. By analogy, pseudo-dipeptide 6 was designed as
a further elaboration of 4. Surprisingly, however, rather
dipeptide mimetic 6 prompted a docking study using
three different methods: Flexidock,29 the low mode
conformational search in Macromodel 6.5 according to
Kolossavary,30 and QMD-docking31 (see Experimental
Section).32 In all cases, the protein atoms were kept
fixed. Consistently, all methods gave a binding mode
reversed by 180° to that which had been expected based
in Figure 2b. In other words, the 6-carboxymethyloxy
group was oriented away from the catalytic cleft, with
the 2-carboxyl occupying a “phosphate-mimicking” posi-
tion within the catalytic cleft (Figure 2d). It was also
observed in this model that the 5-carboxyl group made
key binding interactions as well.
Th e 2,5-Dica r boxyn a p h th yl Moiety a s a New
P h en yl P h osp h a te Mim etic. To further explore this
unexpected binding mode, the inhibitory potencies in a
series of 12 mono-, di-, and tricarboxy/amido/sulfono-
substituted naphthyls were determined (compounds
32-41, Table 1). Of these, only 2,5-dicarboxy-substi-
tuted analogue 35 (Ki ) 31 ( 7 µM, Table 1) exhibited
good affinity.33 The poor affinities of isomeric 2,6- and
2,7-dicarboxy analogues (compounds 37 and 36) indicate
the importance of the “2,5-dicarboxynaphthyl” motif.
Additionally, loss of affinity upon introduction of func-
than exhibiting enhanced potency, compound 6 (IC50
)
3800 µM, Table 1) displayed a significant decrease in
affinity relative to 4. This necessitated a reevaluation
of the originally hypothesized binding mode of 4.
Comparison of the suggested mode of binding for
peptide 1b (Figure 2b)14 with the binding of pTyr, as
indicated by X-ray crystallography (Figure 2a),6 shows
that the carboxymethyl group of 1b interacts with the
signature motif loop in a manner similar to several of
the interactions displayed by the phosphate group in
pTyr; however, its 3-carboxyl group participates in
additional hydrogen bonding to residues Tyr 46, Lys
120, Ser 216, and Asp 181 (through a bridging H2O
molecule), which does not occur in the case of pTyr. A
recently published crystal structure of a related 2-(ox-
alylamino)-benzoic acid PTP1B inhibitor also shows the
benzoyl carboxy group interacting with Tyr 46, Lys 120,
and directly with Asp 181 (Figure 2c),28 supporting this
suggested mode of binding for 1b. The same mode of
binding was assumed initially for compound 4; however,
the extremely deleterious effect of incorporating 4 into

2874 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Gao et al.
o-carboxy-carboxymethyloxyphenyl portion was in-
tended to replicate the interactions of phenyl phosphate.
When incorporation of one of these monomeric inhibitors
(4) into dipeptide 6 resulted in significant loss of PTP1B
affinity, a reevaluation of binding orientation for 4 was
undertaken. This indicated that the naphthyl 2-carboxyl
group, and not the o-carboxy carboxymethyloxy groups,
was mimicking a phosphoryl group. A reevaluation of
binding orientation for 5-carboxy-6-carboxymethyloxy-
2-naphthoic acid was therefore undertaken, which
indicated a 180° reversal of the binding orientation
within the PTP1B catalytic site. In the new orientation,
the naphthyl 2-carboxyl group and not the o-carboxy
carboxymethyloxy groups mimics a phosphoryl group.
Indeed, when 5-carboxy-2-naphthoic acid itself was
examined for inhibitory potency, it was found to have a
Ki value at neutral pH of 31 ( 7 µM, which is lower
than parent 4. This therefore identifies 5-carboxy-2-
naphthoic acid (or more appropriately, 6-carboxy-1-
naphthoic acid) as a new binding motif for PTP1B
inhibitors, wherein one acidic group accommodates the
position of pTyr phosphoryl oxygen atoms and the other
interacts with residues Tyr 46, Lys 120, and Ser 216.
The enzyme-ligand interactions displayed by this bind-
ing mode are similar to the originally proposed binding
mode of 2-(carboxymethyloxy)-benzoic acid type PTP1B
inhibitors (1b),14 which formed the conceptual basis of
the study. The proposed binding is also quite similar to
the actual binding of 2-(oxalylamino)-benzoic acid, as
evidenced by a recently reported X-ray structure,28
although the binding affinity of 5-carboxy-2-naphthoic
acid is greater than this latter monomeric phenyl
phosphate mimetic under physiologically relevant con-
ditions [2-(oxalylamino)-benzoic acid Ki ) 200 µM at pH
7.0].28 It should be noted, however, that in translating
a flexible 3-carboxy-4-carboxymethyloxyphenylalanyl
residue into the rigid planar naphthyl system the spatial
distribution of carboxyl groups about the naphthyl ring
was radically different than anticipated. The 2,5-dicar-
boxy naphthalene motif reported herein provides a new
approach toward phenyl phosphate mimicking function-
ality, which may be of use in the design of carboxy-based
PTP1B inhibitors.
F igu r e 3. Docking of bis-carboxynaphthyls into the PTP1B
signature motif as described in the Experimental Section:
white, 2,5-dicarboxy (35); blue, 2,6-dicarboxy (37); and red, 2,7-
dicarboxy (36).
tionality into the 6-position (compounds 33 and 34)
further supports the sensitivity of the environment
surrounding the 5-carboxyl group.
To gain a clearer understanding of the effect of the
naphthyl-substitution pattern on binding affinity, all 12
ligands were docked by the QMD-dock method allowing
flexibility for selected residues within the catalytic site.
Comparing the binding modes of 35 with 36 and 37
(Figure 3), it was evident that, while maintaining
interactions of the 2-carboxyl within the phosphate-
binding loop, for 36 and 37 the positions of the 7- and
6-carboxyl groups respectively, are less favorable for
interactions with Tyr 46, Lys 120, and Ser 216 (Figure
3). Additionally, it was observed for compound 34, which
contains an added hydroxyl at the 6-position, that the
hydroxyl group does not undergo hydrophilic interac-
tions, and the same was true for the third sulfonate
group of 41 (not shown). Therefore, although 34 and 41
include the parent “2,5-diacidic” arrangement of 35, the
desolvation energy expended on binding is not compen-
sated for by additional hydrogen bonds.
Exp er im en ta l Section
Recom bin a n t Hu m a n P TP 1B. The cDNA encoding the
catalytic domain of human PTP1B (amino acids 1 to 321) was
obtained using the polymerase chain reaction (PCR) from a
human fetal brain cDNA library (Stratagene). The PCR
primers used were 5′-AGCTGGATCCATATGG AGATGG-
AAAAGGAGTT (encoding both a BamHI and a NdeI site) and
3′-ACGCGAATTCTTAATTGTGTGGCTCCAGGATTCG (en-
coding an EcoRI site). The PCR product was digested with
BamHI and EcoRI and subcloned into a pUC118 vector. The
PTP1B coding sequence was confirmed by DNA sequencing.
The coding region for PTP1B was then cut from pUC118-
PTP1B with BamHI and EcoRI and ligated to the correspond-
ing sites of plasmid pT7-7. The PTP1B coding sequence was
placed in frame downstream of the phage T7 RNA polymerase
promoter at the NdeI site of pT7-7 to provide the translational
initiation at Met 1 of PTP1B. The resulting plasmid pT7-7/
PTP1B was used to transform Escherichia coli BL21(DE3). An
overnight culture of BL2(DE3) cells with the pT7-7 expression
vector containing the mutant PTP1B was diluted 1:100 into 1
L of 2 × YT medium containing 100 µg/mL ampicillin. The
culture was grown at 37 °C until absorbance at 600 nm
reached 0.6, at which point the cells were induced with 0.4
mM isopropyl â-D-thiogalatoside for 6 h. The cells were
Con clu sion s
We have previously examined the PTP1B inhibitory
potencies of a series of pTyr mimetics expressed in a
hexapeptide platform, with the intent of defining phenyl
phosphate-mimicking functionality which could be uti-
lized in non-peptide, small molecule PTP1B inhibitors.
Although the initial direction of this work resulted in
aryl difluorophosphonate-containing inhibitors, it was
of considerable interest to define nonphosphonate-
containing, carboxy-based phenyl phosphate mimetics
for use in small molecule inhibitor design. In the present
study, we have taken as a starting point the o-carboxy-
carboxymethyloxyphenyl motif, which was one of the
most potent phenyl phosphate mimetics in the peptide
platform. On the basis of our approach with difluoro-
phosphonomethyl inhibitors, two isomeric 2-naphthoic
acid analogues (4 and 5) were examined as conforma-
tionally constrained variants of the parent 3-carboxy-
4-carboxymethyloxyphenylalanyl residue, in which the

Peptide Lead for a PTP1B-Binding Motif
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2875
harvested by centrifugation and stored at -20 °C. The frozen
cell pellet was thawed at room temperature and resuspended
in 30 mL of ice-cold buffer A (100 mM 2-(4-morpholino)-ethane
sulfonic acid, pH 6.5, 1 mM DTT) and lysed by two passes
through a French press at 1300 psi. All of the following steps
were then carried out at 4 °C. The lysate was centrifuged at
15 000 rpm (Dupont SS-34 rotor) for 30 min. The supernatant
was incubated with 50 mL of CM-Sephadex C50 equilibrated
with buffer A and shaken gently for 40 min. The resin was
washed three times with the same volume of buffer A, loaded
onto a column, and washed again with 10 bed volumes of buffer
A. The protein was then eluted from the column by a linear
gradient from 0 to 0.5 M NaCl in 200 mL of buffer A. The
positions of the peak fractions were assessed by Coomassie
Blue staining of SDS-PAGE. Peak fractions which contain
homogeneous PTP1B were combined into a final pool of
protein, which was then concentrated to 30 mg/mL.
P h osp h a ta se Assa y. The PTP1B phosphatase activity was
assayed at 30 °C in a reaction mixture (0.2 mL) containing
appropriate concentrations of p-nitrophenyl phosphate (pNPP)
as substrate. The buffer used was pH 7.0, 50 mM 3,3-
dimethylglutarate, 1 mM EDTA. The ionic strength of the
solution was kept at 0.15 M using NaCl. The reaction was
initiated by addition of enzyme and quenched after 2-3 min
by addition of 1 mL of 1 N NaOH. The nonenzymatic hydroly-
sis of the substrate was corrected by measuring the control
without the addition of enzyme. The amount of product
p-nitrophenol was determined from the absorbance at 405 nm
using a molar extinction coefficient of 18 000 M-1 cm-1. Steady
state kinetic parameters were evaluated by fitting directly the
v vs [S] data to the Michaelis-Menten equation using KIN-
ETASYST (IntelliKinetics, State College, PA).
and fast atom bombardment mass spectra (FABMS) were
acquired with a VG Analytical 7070E mass spectrometer under
the control of a VG 2035 data system. Where indicated,
FABMS matrixes used were glycerol (Gly) or nitrobenzoic acid
(NBA). 1H NMR data are reported in ppm relative to TMS and
referenced to the solvent in which they were run. Solvent was
removed by rotary evaporation under reduced pressure, and
silica gel chromatography was performed using Merck silica
gel 60 with a particle size of 40-63 µm. Anhydrous solvents
were obtained commercially and used without further drying.
HPLCs were conducted using a Waters Prep LC4000 system
having photodiode array detection and binary solvent systems
as indicated where A ) 0.1% aqueous TFA and B ) 0.1% TFA
in acetonitrile and either Vydac C18 (10 µ) Peptide & Protein
or Advantage C18 (5 µm) columns (preparative size, 20 mm
dia. × 250 mm long with a flow rate of 10 mL/min; semi-
preparative size, 10 mm dia. × 250 mm long, with a flow rate
of 2 mL/min.).
Meth yl 2-(1,6-Dibr om o-2-n a p h th yloxy)a ceta te (8). To
a solution of 1,6-dibromo-2-naphthol (7) (3 g, 10 mmol) in
anhydrous DMF (100 mL) was added methyl bromoacetate (5.8
g, 38 mmol) and potassium carbonate (3.3 g, 25 mmol), and
the mixture was heated to 60∼70 °C and stirred (7 h). Solvent
was removed under vacuum, H2O (20 mL) was added to quench
the reaction, and the mixture was subjected to an extractive
workup, dried (Na2SO4), and concentrated. Purification by
silica gel flash chromatography afforded 8 (3.78 g, 100%
yield): mp 138-140 °C; H NMR (CDCl3) δ 8.13 (1H, d, J )
9.3 Hz), 7.96 (1H, d, J ) 1.7 Hz), 7.70 (1H, J ) 9.0 Hz), 7.64
(1H, dd, J ) 2.0, 9.3 Hz), 7.18 (1H, d, J ) 9.0 Hz), 4.85 (2H,
s), 3.83 (3H, s). FABMS (+VE) m/z 372 (M + H)+.
Meth yl 2-(1,6-Dicya n o-2-n a p h th yloxy)a ceta te (9). To a
solution of 8 (0.74 g, 2.0 mmol) in anhydrous DMF (10 mL)
was added copper(I) cyanide (430 mg, 4.8 mmol). The mixture
was refluxed (4 h), then cooled to room temperature, and
poured into a solution of FeCl3‚6H2O (800 mg) and HCl (37%,
2 mL) in H2O (12 mL). After being maintained at 60∼70 °C
(10 min), the mixture was subjected to an extractive workup,
dried (Na2SO4), and concentrated. Purification by silica gel
flash chromatography afforded 9 (247 mg, 46%): mp 193-195
°C; H NMR (CDCl3) δ 8.25∼8.21 (2H, m), 8.10 (1H, d, J ) 9.3
Hz), 7.81 (1H, dd, J ) 1.5, 8.8 Hz), 7.25 (1H, d, J ) 9.3 Hz),
4.10 (2H, s), 3.84 (3H, s). CIMS (+VE, NH3) m/z 284 (M +
NH4+).
2-Ca r boxym eth oxy)n a p h th a len e-1,6-d ica r boxylic Acid
(4). A solution of bis-nitrile 9 (120 mg, 0.45 mmol) in EtOH-
H2O (1:1; 5 mL) containing sodium hydroxide (200 mg; 5 mmol)
was refluxed (12 h) and then cooled to room temperature.
Ethanol was removed by evaporation, and the resulting
aqueous solution was acidified with 1.0 M aqueous HCl,
subjected to an extractive workup, dried (Na2SO4), and taken
to dryness to provide crude product (143 mg). Purification by
HPLC gave final product 4 as a white solid: mp 266 °C (dec);
H NMR (DMSO-d6) δ 8.56 (1H, s), 8.1 (1H, d, J ) 9.3 Hz),
7.97 (1H, dd, J ) 1.5, 8.8 Hz), 7.87 (1H, d, J ) 9.0 Hz), 7.64
(1H, d, J ) 9.3 Hz), 4.95 (2H, s). Anal. (C14H10O7‚1.29H2O) C,
H.
In h ibition Con sta n t Deter m in a tion . Inhibition con-
stants for the small PTP inhibitors were determined for PTP1B
in the following manner. The initial rate at eight different
pNPP concentrations (0.2 Km to 5 Km) was measured at three
different fixed inhibitor concentrations.34 The inhibition con-
stant and inhibition pattern were evaluated using a direct
curve-fitting program KINETASYST (IntelliKinetics, State
College, PA).
Molecu la r Mod elin g. All force field simulations were
performed with the Insight II 98.0/Discover 3.0 modeling
package31 using the cff91 force field (running on SGI comput-
ers). A dielectric constant of 1.00 and the cell multipole method
with fine accuracy were used for all nonbonding interactions
except for the high-temperature quenched molecular dynamics
(QMD) simulations. The crystal structure (PDB code 1BZJ )26
of [1,1-difluoro-1-(6′-carboxy-naphth-2′-yl)]methylphosphonate
bound to PTP1B was used as the starting geometry for PTP1B.
For ligand 4, 10 quenched molecular dynamics (QMD) cycles
were performed with different seeds for the random number
generator for each cycle. Each QMD cycle consisted of 60 ps
MD at 2000 K in an NVT ensemble, keeping all protein atoms
fixed except the hydrogen atoms of Tyr46, Lys120, and Ser216.
A distance constraint between the C-4 atom of the ligand
naphthyl ring and the nitrogen atom of Ser222 of PTP1B was
applied using a flat bottom potential with a force constant of
0.0 for the distance range of 0-12 Å and 1000.0 for longer
distances. The vdW interactions were scaled to 2%, and the
Coulombic interactions to 20% during the high-temperature
MD simulations. The coordinates were saved every 200 fs and
subsequently minimized by 300 steps of the Polak-Ribiere
conjugated gradient algorithm (CG-PR). For each of the 10
runs, the lowest energy conformation out of the 300 minimized
structures was stored. The frame with lowest energy among
the 10 stored structures is depicted in Figure 2d. The QMD-
docking for ligands 35, 36, and 37 was performed in a similar
way which only differed in allowing side chain flexibility for
Tyr46, Arg47, Asp48, Lys120, Asp181, Phe182, Ser215, Ser216,
Ala217, Ile219, and Arg221, and minimizing the lowest energy
complex of the 10 QMD runs by 1000 steps of CG-PR using
the esff-force field. The results are depicted in Figure 3.
Meth yl 2-{6-Br om o-3-[N-(2-m eth oxyph en yl)car bam oyl]-
2-n a p h th yloxy}a ceta te (11). To a solution of Naphthol AS
BI (10) (1.28 g, 3.4 mmol) in anhydrous DMF (20 mL) was
added methyl bromoacetate (2.0 g, 1.31 mmol) and potassium
carbonate (1.1 g, 8.6 mmol), and the mixture was heated to
60∼70 °C with stirring (5 h). Solvent was removed under
vacuum, H2O (20 mL) was added to quench the reaction, and
the mixture was subjected to an extractive workup, dried (Na2-
SO4), and concentrated. Purification by silica gel flash chro-
matography afforded 11 (1.34 g, 88% yield): mp 150-152 °C;
H NMR (CDCl3) δ 10.30 (1H, s), 8.73 (1H, s), 8.59 (1H, dd, J
) 2.0, 7.6 Hz), 8.08 (1H, s), 7.62 (2H, s), 7.14 (1H, s), 7.12∼7.01
(2H, m), 6.94 (1H, dd, J ) 1.7, 7.8 Hz), 5.00 (2H, s), 3.95 (3H,
s), 3.85 (3H, s). Anal. (C21H18BrNO5) C, H, N.
Meth yl 2-{6-Cyan o-3-[N-(2-m eth oxyph en yl)car bam oyl]-
2-n a p h th yloxy}a ceta te (12). To a solution of 11 (889 mg,
2.0 mmol) in anhydrous DMF (10 mL) was added copper(I)
Syn th esis. Gen er al Syn th etic Meth ods. Elemental analy-
ses were obtained from Atlantic Microlab Inc., Norcross, GA,

2876 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Gao et al.
cyanide (214 mg, 2.4 mmol). The mixture was refluxed (4 h),
then cooled to room temperature, and poured into a solution
of FeCl3‚6H2O (800 mg) and HCl (37%, 2 mL) in H2O (12 mL).
After being maintained at 60∼70 °C (10 min), the mixture was
subjected to an extractive workup, dried (Na2SO4), and con-
centrated. Purification by silica gel flash chromatography
afforded 12 (450 mg, 56% yield): mp 163-165 °C; H NMR
(CDCl3) δ 10.23 (1H, s), 8.87 (1H, s), 8.58 (1H, dd, J ) 1.7, 7.6
Hz), 8.31 (1H, s), 7.83 (1H, d, J ) 8.8 Hz), 7.66 (1H, dd, J )
1.7, 8.6 Hz), 7.20 (2H, s), 7.13∼7.05 (2H, m) 6.95 (1H, dd, J )
1.7, 8.0 Hz), 5.02 (2H, s), 3.92 (3H, s), 3.85 (3H, s). FABMS
(+VE) m/z 391.2 (M + H)+. Anal. (C22H18N2O5‚1/4H2O) C, H, N.
3-Ca r boxym eth oxy)n a p h th a len e-2,7-d ica r boxylic Acid
(5). A solution of nitrile 12 (100 mg, 0.26 mmol) in EtOH-
H2O (1:1; 5 mL) containing sodium hydroxide (200 mg; 5 mmol)
was refluxed (12 h), and then cooled to room temperature.
Ethanol was removed by evaporation, and the resulting
aqueous solution was acidified with 1.0 M aqueous HCl,
subjected to an extractive workup, dried (Na2SO4), and taken
to dryness to provide crude product (75 mg). Purification by
HPLC gave final product 5 as white solid: mp 278 °C (dec); H
NMR (CD3OD + D2O) δ 7.44 (1H, s), 7.90 (1H, dt, J ) 9.0, 1.7
Hz), 8.07 (1H, dt, J ) 8.5, 0.5 Hz), 8.48 (1H, d, J ) 0.7 Hz),
8.62 (1H, s). FABMS (-VE) m/z 289 (M - H)-. Anal.
(C14H10O7‚0.15CF3CO2H‚H2O) C, H.
Dip ep tid e Mim etic 6. A total of 1.0 g (0.42 mmol) of
N-Fmoc Rink amide resin (Bachem, Lot 513049) was swollen
with NMP (3 × 5 mL), then deblocked with 20% piperidine in
NMP (5 mL wash then 5 mL for 20 min), and washed with
NMP (10 × 5 mL). An HOBt active ester, prepared by reacting
Fmoc-L-Glu (Ot-Bu) (893 mg, 2.1 mmol), HOBt (283 mg, 2.1
mmol), and DIPCDI (265 mg, 2.1 mmol) in NMP (5 mL) (10
min), was added to the washed and deblocked resin, and the
mixture was rocked at room temperature (10 h). The resin was
washed with NMP (10 × 5 mL) and CH2Cl2 (10 × 5 mL), and
then dried overnight under high vacuum to provide Fmoc-L-
Glu (Ot-Bu)-Rink amide resin (1.21 g, 97% yield). A portion of
the resin (250 mg, 0.10 mmol) was swollen with NMP (3 ×
1.5 mL), deblocked with 20% piperidine in NMP (1.5 mL wash
then 1.5 mL, 20 min), and washed with NMP (10 × 1.5 mL).
An HOBt active ester, prepared by reacting 2-tert-butoxycar-
bonylmethoxy-naphthalene-1,6-dicarboxylic acid (25) (87 mg,
0.25 mmol), HOBt (34 mg, 0.25 mmol), and DIPCDI (40 mL,
0.25 mmol) in NMP (1.5 mL) (10 min), was added, and the
resin was rocked at room temperature (overnight). The resin
was washed with NMP (10 × 1.5 mL) and CH2Cl2 (10 × 1.5
mL), and then cleaved by treatment with a solution of TFA
(1.85 mL):H2O (0.1 mL):triethylsilane (50 µL) (1 h). Superna-
tant was collected by filtration, and the resin was washed with
TFA (2 × 2 mL) and CH2Cl2 (2 × 2 mL). The combined filtrate
was evaporated, 5 mL of H2O was added, and the mixture was
taken to dryness. This procedure was repeated two times.
Residue was purified by HPLC to provide 6 (14 mg, 33% yield).
H NMR (DMSO-d6) δ 8.60 (1H, s), 8.23 (1H, d, J ) 9.3 Hz),
8.09 (1H, d, J ) 8.1 Hz), 8.02 (1H, dd, J ) 1.7, 8.8 Hz), 7.87
(1H, d, J ) 8.8 Hz), 7.52 (1H, d, J ) 9.0 Hz), 4.86 (2H, s), 4.27
(1H, m), 2.20 (2H, t, J ) 7.81 Hz), 1.98 (1H, m), 1.77 (1H, m).
FABMS (-VE) m/z 417 (M - H)-. HR-FABMS calcd for
C19H18N2O9: 417.0934. Found: 417.0947.
6-Meth oxy-2-n a p h th oic Acid (14). To a solution of 2-bro-
mo-6-methoxynaphthalene (13) (10.0 g, 42.2 mmol) in anhy-
drous DMF (120 mL) was added copper(I) cyanide (4.53 g, 50.4
mmol, 1.2 equiv). The mixture was refluxed (4 h), then cooled
to room temperature, and poured into a solution of FeCl3‚6H2O
(15.8 g) and HCl (37%, 40 mL) in H2O (200 mL). After being
maintained at ∼70 °C (20 min), the mixture was subjected to
an extractive workup, dried (Na2SO4), and concentrated.
Concentration under vacuum afforded crude 2-cyano-6-meth-
oxynaphthalene (7.78 g; 100% yield). The nitrile was dissolved
in EtOH-H2O (1:1; 100 mL) containing (8.0 g, 0.2 mol) of
sodium hydroxide; the resulting solution was refluxed over-
night. After cooled to room temperature, ethanol was evapo-
rated, and the residue was acidified with concentrated HCl
and subjected to an extractive workup, dried (Na2SO4), and
taken to dryness to provide crude 14, which was recrystallized
from ethyl acetate to give 6.00 g of pure product, with an
additional 2.4 g being recovered from the filtrate (98%
combined yield): mp 107-108 °C; H NMR (DMSO-d6) δ 8.56
(1H, s), 8.18 (1H, dd, J ) 1.7, 8.5 Hz), 7.91 (1H, d, J ) 8.8
Hz), 7.72 (1H, d, J ) 8.6 Hz), 7.19∼7.10 (2H, m), 3.90 (3H, s).
FABMS (-VE) m/z 201.1 (M - H)-. Anal. (C12H10O3) C, H.
Meth yl 6-Hyd r oxy-2-n a p h th oa te (15). To 6-methoxy-2-
naphthoic acid (14) (4.044 g, 20.0 mmol) in anhydrous CH2Cl2
(100 mL) was added dropwise 1.0 M BBr3 in CH2Cl2 (40 mL,
40 mmol) at -78 °C under argon, and the mixture was stirred
at -78 °C (1 h), then raised to room temperature and stirred
overnight. To the mixture at -78 °C was added anhydrous
MeOH (10 mL), and then the mixture was warmed to room
temperature and stirred (30 min). Solvent was evaporated to
dryness, additional MeOH (10 mL) was added, and the mixture
taken to dryness and purified (silica gel flash chromatography)
to give 15 (3.91 g, 99% yield): mp 168-170 °C; H NMR
(DMSO-d6) δ 8.50 (1H, d, J ) 1.7 Hz), 7.98 (1H, d, J ) 8.6
Hz), 7.87 (1H, dd, J ) 1.7, 8.8 Hz), 7.77 (1H, d, J ) 8.6 Hz),
7.19∼7.17 (2H, m), 3.88 (3H, s). FABMS (+VE) m/z 202.1 (M+).
Anal. (C12H10O3‚0.1H2O) C, H.
Meth yl 6-Hyd r oxy-5-iod o-2-n a p h th oa te (16). Sodium
iodide (2.23 g, 14.9 mmol) was dissolved in boiling acetic acid
(19 mL), and added carefully to a solution of chloramine-T
trihydrate (4.20 g, 14.9 mmol) in acetic acid (3.75 mL) with
stirring and cooling. To a solution of methyl 6-hydroxy-2-
naphthoate (15) (2.87 g, 14.2 mmol) in acetic acid (7.5 mL) at
80 °C was added rapidly with stirring the in situ generated
solution of ICl, and the solution was maintained at 80 °C (1
h), then poured into crushed ice, subjected to an extractive
workup, and dried (Na2SO4). Concentration and purification
by silica gel flash chromatography afforded 16 (4.61 g, 99%
yield): mp 165-166 °C; H NMR (CD3OD) δ 8.47 (1H, s), 8.11
(1H, d, J ) 8.8 Hz), 8.02 (1H, dd, J ) 1.7, 9.0 Hz), 7.87 (1H,
d, J ) 8.79 Hz), 7.22 (1H, J ) 8.8 Hz), 3.95 (3H, s). FABMS
(+VE) m/z 328 (M+). HR-FABMS calcd for C12H10O3I (MH+):
328.9675. Found: 328.9676.
ter t-Bu tyl 2-[1-Iod o-6-(m eth oxyca r bon yl)-2-n a p h th yl-
oxy]a ceta te (17). To a solution of 16 (2.624 g, 6.9 mmol) in
anhydrous DMF (48 mL) were added tert-butyl bromoacetate
(5.10 g, 26.2 mmol) and potassium carbonate (2.27 g, 17.2
mmol), and the mixture was heated to 60∼70 °C and stirred
overnight. The reaction mixture was cooled to room temper-
ature, acetone (50 mL) was added, and precipitated solid was
filtered off. The resulting solution was evaporated to dryness,
and the residue was extracted with ethyl acetate, washed with
water and brine, then dried (Na2SO4) and concentrated.
Purification by silica gel flash chromatography afforded 17
(3.22 g, 91% yield). H NMR (CDCl3) δ 8.51 (1H, d, J ) 1.71
Hz), 8.21 (1H, d, J ) 8.79 Hz), 8.10 (1H, dd, J ) 1.7, 9.0 Hz),
7.92 (1H, d, J ) 8.79 Hz), 7.10 (1H, d, J ) 9.0 Hz), 4.76 (2H,
s), 3.99 (3H, s), 1.49 (9H, s).
Meth yl 6-Allyloxy-2-n a p h th oa te (19). To a solution of
sodium hydride (851 mg, 21.3 mmol) in DMSO (10 mL) was
added a solution of methyl 6-hydroxy-2-naphthoate (15) (3.91
g, 19.5 mmol) in DMSO (10 mL) at 0 °C under argon. The
solution was stirred at 0 °C (20 min), then a solution of allyl
bromide (2.57 g, 21.3 mmol) in DMSO (5 mL) was added, and
the mixture was stirred at ambient temperature overnight.
The reaction mixture was poured into ice-cold H2O (50 mL)
and extracted with ethyl acetate, washed with water and brine,
then dried (Na2SO4) and concentrated. Purification by silica
gel flash chromatography afforded 19 (4.30 g, 91% yield): mp
80-82 °C; H NMR (CDCl3) δ 8.61 (1H, s), 8.03 (1H, dd, J )
1.7, 8.8 Hz), 7.86 (1H, d, J ) 9.0 Hz), 7.75 (1H, d, J ) 8.8 Hz),
7.24 (1H, dd, J ) 2.4, 9.0 Hz), 7.17 (1H, d, J ) 2.4 Hz),
6.21∼6.06 (1H, m), 5.54∼5.33 (2H, m), 4.70∼4.67 (2H, m), 3.97
(3H, s). FABMS (+VE) m/z 242.2 (M+). Anal. (C13H14O3) C, H.
Meth yl 6-Allyloxy-5-for m yl-2-n a p h th oa te (20). To a
solution of TiCl4 (2.07 g, 10.9 mmol) and R,R-dichloromethyl
methyl ether (645 mg, 5.6 mmol) in anhydrous CH2Cl2 (8 mL)
at 0 °C was added a solution of methyl 6-allyloxy-2-naphthoate
(19) (1.21 g, 5.0 mmol) in anhydrous CH2Cl2 (8 mL), and the

Peptide Lead for a PTP1B-Binding Motif
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18 2877
solution was stirred at 0 °C (1 h), then raised to room
temperature and stirred overnight. The reaction was quenched
by addition of dilute aqueous HCl, then subjected to an
extractive workup, dried (Na2SO4), and concentrated. Purifica-
tion by silica gel flash chromatography afforded 20 (1.045 g,
77.3% yield: mp 108-109.5 °C) with 107 mg of starting
material being recovered. H NMR (CDCl3) δ 10.95 (1H, s), 9.32
(1H, d, J ) 9.28 Hz), 8.52 (1H, d, J ) 1.7 Hz), 8.18 (1H, dd, J
) 1.7, 9.0 Hz), 8.14 (1H, d, J ) 9.0 Hz), 7.34 (1H, d, J ) 9.3
Hz), 6.20∼6.04 (1H, m), 5.54∼5.36 (2H, m), 4.86∼4.83 (2H,
m), 3.98 (3H, s). FABMS (+VE) m/z 271.1. (M + H)+. Anal.
(C16H14O4) C, H.
2-Allyloxy-6-(m et h oxyca r b on yl)n a p h t h oic Acid (21).
Sulfamic acid (870 mg, 8.95 mmol) was added at 0 °C over 20
min to a solution of methyl ester 20 (1.045 g, 4.05 mmol) in
acetone:H2O (2:1, 33 mL). Sodium chlorite (80%, 524 mg, 4.63
mmol) was then added, and the solution was stirred at 0 °C
(30 min), then concentrated, extracted with CH2Cl2, washed
with brine, and dried over MgSO4. Solvent was evaporated
under vacuum to afford 21 (1.12 g, 97%). H NMR (CDCl3) δ
8.56 (1H, d, J ) 1.5 Hz), 8.31 (1H, d, J ) 9.0 Hz), 8.13 (1H,
dd, J ) 1.7, 9.0 Hz), 8.04 (1H, d, J ) 9.3 Hz), 7.35 (1H, d, J )
9.0 Hz), 6.19∼6.04 (1H, m), 5.56∼5.34 (2H, m), 4.86∼4.83 (2H,
m), 3.99 (3H, s).
ter t-Bu tyl 2-Allyloxy-6-(m eth oxyca r bon yl)n a p h th oa te
(22). To a solution of methyl ester 21 (1.43 g, 5.0 mmol) in
anhydrous CH2Cl2 (8 mL) were added tert-butyl 2,2,2-trichlo-
roacetimide (1.79 mL, 10.0 mmol) in cyclohexane (17 mL) at 0
°C followed by BF3‚etherate (100 µL), and the reaction mixture
was stirred at room temperature (16 h). Solid NaHCO3 was
added, solid precipitate was filtered off, and the filtrate was
taken to dryness. Purification by silica gel flash chromatog-
raphy provided 22 (1.04 g, 58% yield): mp 105-106 °C; H
NMR (CDCl3) δ 8.55 (1H, d, J ) 1.5 Hz), 8.07 (1H, dd, J )
1.7, 9.0 Hz), 7.95 (1H, d, J ) 9.0 Hz), 7.83 (1H, d, J ) 8.8 Hz),
7.03 (1H, d, J ) 9.0 Hz), 6.15∼6.00 (1H, m), 5.52∼5.43 (1H,
m), 5.34∼5.28 (1H, m), 4.76∼4.72 (2H, m), 3.98 (3H, s), 1.68
(9H, s). FABMS (+VE) m/z 343 (M + H)+. Anal. (C20H22O5) C,
H.
ter t-Bu tyl 2-{1-[(ter t-Bu tyl)oxyca r bon yl]-6-(m eth oxy-
ca r bon yl)-2-n a p h th yloxy}a ceta te (24). To a solution of
methyl ester 22 (342 mg, 1.00 mmol) in anhydrous THF (8
mL) was added Pd(PPh3)4 (23 mg, 0.02 mmol), and the solution
was stirred at room temperature (5 min). A solution of NaBH4
(58 mg, 1.5 mmol) in THF (1.5 mL) was added dropwise at 0
°C with monitoring by TLC. When all starting material had
disappeared, unreacted NaBH4 was destroyed by addition of
acetone (10 mL), then solvent was evaporated, and residue
placed under high vacuum to yield compound 23 as a solid
(mp 106-107 °C) which was sufficiently pure for further use.
[H NMR (CDCl3) δ 10.83 (1H, s), 8.55 (1H, s), 8.10 (1H, d, J )
8.8 Hz), 7.99 (1H, dd, J ) 1.7, 9.0 Hz), 7.85 (1H, d, J ) 8.8
Hz), 7.28 (1H, dd, J ) 1.7, 9.0 Hz), 3.89 (3H, s), 1.61 (9H, s).
FABMS (+VE) m/z 303.2 (M + H)+.Anal. (C17H18O5) C, H.]
Crude 23 was dissolved in DMF (10 mL), and to this solution
were added K2CO3 (346 mg, 2.5 mmol) and tert-butyl bromo-
acetate (742 mg, 3.8 mmol), and the reaction mixture was
stirred at room temperature overnight. To the mixture was
added acetone (30 mL), and precipitated solid was filtered off.
The filtrate was evaporated, and the resulting residue was
purified by silica gel flash chromatography to yield 24 as a
white solid (271 mg, 63% yield): mp 67-68 °C; H NMR (CDCl3)
δ 8.64 (1H, d, J ) 1.7 Hz), 8.24 (1H, d, J ) 8.8 Hz), 8.03 (1H,
dd, J ) 1.7, 8.8 Hz), 7.74 (1H, d, J ) 8.8 Hz), 7.47 (1H, d, J )
9.0 Hz), 4.89 (2H, s), 3.91 (3H, s), 1.60 (9H, s), 1.43 (9H, s).
FABMS (+VE) m/z 416.2 (M + H)+. Anal. (C23H28O7) C, H.
25 (203 mg, 100% yield): H NMR (DMSO-d6) δ 8.65 (1H, d, J
) 1.5 Hz), 8.26 (1H, d, J ) 9.3 Hz), 8.07 (1H, dd, J ) 1.7, 8.8
Hz), 7,77 (1H, d, J ) 8.8 Hz), 7.53 (1H, d, J ) 9.3 Hz), 4.96
(2H, s), 1.65 (9H, s).
Eth yl 2-[6-(Meth oxyca r bon yl)(2-n a p h th yloxy)]a ceta te
(26). To a solution of methyl 6-hydroxy-2-naphthoate (15) (600
mg, 3.0 mmol) in anhydrous DMF (20 mL) was added K2CO3
(1.0 g, 7.5 mmol) followed by ethyl bromoacetate (1.8 mL, 11.4
mmol), and the mixture was stirred with heating (60-70 °C,
overnight). After cooling to room temperature, the mixture was
diluted with EtOAc (300 mL), the resulting white precipitate
was filtered off, and the residual solution was washed with
brine and dried (Na2SO4). Evaporation to dryness and purifi-
cation by silica gel chromatography (EtOAc:hexane) provided
26 as a white solid (584 mg, 68% yield): mp 76-79 °C; H NMR
(CDCl3) δ 8.52 (1H, s), 8.01 (1H, s, J ) 8.6 Hz), 7.85 (1H, d, J
) 8.9 Hz), 7.72 (1H, d, J ) 8.6 Hz), 7.29-7.24 (1H, m), 7.07
(1H, s), 4.74 (2H, s), 4.28 (2H, t, J ) 7.1 Hz), 3.94 (3H, s), 1.29
(3H, q). FABMS (+VE) m/z 288.1 (M + H)+. Anal. (C16H16O5‚
3/4H2O) C, H.
Eth yl 2,2-Diflu or o-2-[6-(m eth oxyca r bon yl)(2-n a p h th yl-
oxy)]a ceta te (27). Reaction of methyl 6-hydroxy-2-naphthoate
(15) as described for the synthesis of 26, with the use of ethyl
bromodifluoroacetate instead of ethyl bromoacetate, provided
27 as a white solid in 40% yield: mp 216.5-217.5 °C; H NMR
(CDCl3) δ 8.58 (1H, s), 8.08(1H, dd, J ) 8.6, 1.6 Hz), 7.95 (1H,
d, J ) 8.9 Hz), 7.84 (1H, d, J ) 8.6 Hz), 7.68 (1H, s), 7.4 (1H,
dd, J ) 8.9, 2.2 Hz), 4.4 (2H, s), 3.97 (3H, s), 1.35 (3H, q).
FABMS (+VE) m/z 324 (M + H)+. Anal. (C16H14O5F2‚5/4H2O)
C, H.
6-(Ca r b oxym et h oxy)n a p h t h a len e-2-ca r b oxylic Acid
(28). A mixture of 26 (115 mg, 0.4 mmol) and LiOH‚H2O (80
mg, 2.0 mmol) in H2O (15 mL) was stirred at room temperature
(overnight). The mixture was adjusted with 3 N HCl to ∼pH
2, then MeOH was removed under reduced pressure, and
residue was subjected to an extractive workup (EtOAc).
Evaporation of solvent provided 28 as a white solid (95 mg,
97% yield): mp 306-309 °C; H NMR (DMSO-d6) δ 12.95 (1H,
br s), 8.52 (1H, s), 8.04 (1H, d, J ) 8.9 Hz), 7.90-7.87 (2H,
m), 7.35-7.26 (m, 2H), 4.84 (2H, s). FABMS (-VE) m/z 245
(M - H)-. Anal. (C13H10O5‚1/4H2O) C, H.
6-(Ca r b oxyd iflu or om et h oxy)n a p h t h a len e-2-ca r b ox-
ylic Acid (29). Treatment of 27 as described above for the
preparation of 28 provided product 29 as a white solid in 95%
yield: mp > 360 °C; H NMR (CD3OD) δ 6.95 (1H, s), 6.51 (1H,
d, J ) 8.6 Hz), 6.38 (1H, d, J ) 9.0 Hz), 6.24 (1H, d, J ) 8.6
Hz), 6.13 (1H, s), 5.84 (1H, dd, J ) 8.9, 1.8 Hz). FABMS (-VE)
m/z 281 (M - H-). HR-FABMS calcd for C13H8O5F2: 281.0273
(M - H). Found: 281.0262.
Meth yl 5-F or m yl-6-m eth oxy-2-n a p h th oa te (31). Formy-
lation of methyl 6-methoxy-2-naphthoate (30) by a procedure
similar to that employed for the conversion of compound 19
to 20 provided product 31 in 54% yield. H NMR (CDCl3) δ
10.90 (1H, s), 9.23 (1H, d, J ) 9.0 Hz), 8.64 (1H, d, J ) 1.7
Hz), 8.19 (2H, d, J ) 9.0 Hz), 7.39 (1H, d, J ) 9.3 Hz), 4.11
(3H, s), 3.99 (3H, s). FABMS (+VE) m/z 245 (M + H)+.
2-Me t h o x y -6-(m e t h o x y c a r b o n y l)n a p h t h a le n e c a r -
boxylic Acid (32). Treatment of 31 in a manner similar to
that used to prepare compound 21 from 20 yielded product 32
in quantitative yield. H NMR (CDCl3) δ 8.58 (1H, d, J ) 1.7
Hz), 8.44 (1H, d, J ) 9.0 Hz), 8.14 (1H, dd, J ) 2.0, 9.0 Hz),
8.11 (1H, d, J ) 9.3 Hz), 7.49 (1H, d, J ) 9.3 Hz), 4.12 (3H, s),
3.99 (3H, s). FABMS (-VE) m/z 259 (M - H)-. HR-FABMS
calcd for C14H11O5 (M - H): 259.0606. Found: 259.0605.
2-Met h oxyn a p h t h a len e-1,6-d ica r b oxylic Acid (33).
Treatment of a solution of 32 in in EtOH:H2O (1:1) with excess
sodium hydroxide, followed by evaporation of EtOH and
acidification with aqueous HCl (1.0 N), provided 33 as a white
precipiate in quantitative yield. H NMR (DMSO-d6) δ 13.11
(2H, s, br), 8.61 (1H, d, J ) 1.5 Hz), 8.25 (1H, d, J ) 9.0 Hz),
8.00 (1H, dd, J ) 1.7, 8.8 Hz), 7.75 (1H, d, J ) 8.8 Hz), 7.60
(1H, d, J ) 9.3 Hz). 3.96 (3H, s). FABMS (-VE) m/z 245 (M -
H)-. HR-FABMS calcd for C13H9O5 (M - H): 245.0450.
Found: 245.0469.
2-{[(ter t-Bu tyl)oxyca r bon yl]m eth oxy}n a p h th a len e-1,6-
d ica r boxylic Acid (25). To an ice-cold solution of 24 (238 mg,
0.55 mmol) in THF (14 mL) was added aqueous LiOH‚H2O
(0.2 M, 8.25 mL, 1.65 mmol), and the mixture was allowed to
warm to room temperature and stirred (15 h). The solution
was diluted with EtOAc (35 mL) and washed with ice-cold 0.2
N HCl brine (35 mL), and the aqueous phase subjected to an
extractive workup, dried (Na2SO4), and concentrated to afford

2878 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 18
Gao et al.
phorus-containing phosphotyrosyl mimetics and their use in the
preparation of tyrosine phosphatase inhibitory peptides. Tetra-
hedron 1998, 54, 9981-9994.
2-H yd r oxyn a p h t h a len e-1,6-d ica r b oxylic Acid (34).
Treatment of a suspension of 33 in anhydrous CH2Cl2 with a
solution of BBr3 in CH2Cl2 (1.0 M) at -78 °C and stirring at
room temperature (overnight), followed by pouring into ice-
cold H2O, provided a precipitated solid which was collected
by filtration. Crystallization from acetonitrile-H2O gave 34
in quantitative yield. H NMR (DMSO-d6) δ 8.53 (1H, d, J )
1.5 Hz), 8.42 (1H, d, J ) 9.0 Hz), 8.17 (1H, d, J ) 9.0 Hz),
8.01 (1H, dd, J ) 1.7, 9.0 Hz), 7.27 (1H, d, J ) 9.0 Hz). FABMS
(-VE) m/z 231 (M - H)-. HR-FABMS calcd for C12H7O5 (M -
H): 231.0293. Found: 231.0316.
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