Investigation of potential bioisosteric replacements for the carboxyl groups of peptidomimetic inhibitors of protein tyrosine phosphatase 1B: Identification of a tetrazole-containing inhibitor with cellular activity

Article abstract of DOI:10.1021/jm011100y

Protein tyrosine phosphatases (PTPs) constitute a diverse family of enzymes that, together with protein tyrosine kinases, control the level of intracellular tyrosine phosphorylation, thus regulating many cellular functions. PTP1B negatively regulates insu

Full text of DOI:10.1021/jm011100y

J . Med. Chem. 2002, 45, 1785-1798
1785
In vestiga tion of P oten tia l Bioisoster ic Rep la cem en ts for th e Ca r boxyl Gr ou p s of
P ep tid om im etic In h ibitor s of P r otein Tyr osin e P h osp h a ta se 1B: Id en tifica tion
of a Tetr a zole-Con ta in in g In h ibitor w ith Cellu la r Activity
Charlotta Liljebris,*,† Scott D. Larsen,‡ Derek Ogg,§ Barbara J . Palazuk,| and J ohn E. Bleasdale|
Departments of Medicinal Chemistry and Structural Chemistry, Biovitrum AB, SE-751 82 Uppsala and
SE-112 87 Stockholm, Sweden, and Departments of Medicinal Chemistry and Cell and Molecular Biology,
Pharmacia Corporation, Kalamazoo, Michigan 49007
Received November 12, 2001; Revised Manuscript Received J anuary 28, 2002
Protein tyrosine phosphatases (PTPs) constitute a diverse family of enzymes that, together
with protein tyrosine kinases, control the level of intracellular tyrosine phosphorylation, thus
regulating many cellular functions. PTP1B negatively regulates insulin signaling, in part, by
dephosphorylating key tyrosine residues within the regulatory domain of the â-subunit of the
insulin receptor, thereby attenuating receptor kinase activity. Inhibitors of PTP1B would
therefore have the potential of prolonging the phosphorylated (activated) state of the insulin
receptor and are anticipated to be a novel treatment of the insulin resistance characteristic of
type 2 diabetes. We previously reported a series of small molecular weight peptidomimetics as
competitive inhibitors of PTP1B, with the most active analogues having Ki values in the low
nanomolar range. Furthermore, we confirmed that the O-carboxymethyl salicylic acid moiety
is a remarkably effective novel phosphotyrosine mimetic. Because of the low cell permeability
of this compound class, it was important to investigate the possibility of replacing one or both
of the remaining carboxyl groups while maintaining PTP1B inhibitory activity. The analogues
described herein further support the importance of an acidic functionality at both positions of
the tyrosine head moiety. An important discovery was the ortho tetrazole analogue 29 (Ki )
2.0 µM), which was equipotent to the dicarboxylic acid analogue 2 (Ki ) 2.0 µM). Solution of
the X-ray cocrystal structure of the ortho tetrazole analogue 29 bound to PTP1B revealed that
the tetrazole moiety is well-accommodated in the active site and binds in a fashion similar to
the ortho carboxylate analogue 2 reported previously. This novel monocarboxylic acid analogue
revealed significantly higher Caco-2 cell permeability as compared to all previous compounds.
Furthermore, compound 29 exhibited modest enhancement of insulin-stimulated 2-deoxyglucose
uptake by L6 myocytes.
In tr od u ction
glucose homeostasis.11 Furthermore, PTP1B has been
implicated in the insulin resistance associated with
diabetes and obesity by the finding of correlations
between insulin resistance and the levels of PTP1B in
muscle and adipose tissue.12,13 This is further supported
by a variety of cellular and biochemical studies where
PTP1B has been shown to play a role in the dephos-
phorylation of the IR.14 Therefore, the use of specific
PTP1B inhibitors may enhance insulin action and
represents a novel strategy for the treatment of type 2
diabetes. A recent study with PTP1B knockout mice
supports this hypothesis by demonstrating that the loss
of PTP1B activity in vivo results in an enhancement of
insulin sensitivity and decreased susceptibility to diet-
induced obesity.15
Insulin resistance is central to type 2 diabetes and is
known to involve decreased tyrosine phosphorylation of
insulin receptors (IR) despite normal insulin levels.1,2
The biological actions of insulin are initiated when
insulin binds to the R-subunit of its receptor, resulting
in stimulation of intrinsic receptor tyrosine kinase
activity, autophosphorylation of the â-subunit, and the
subsequent phosphorylation of intracellular substrates.3
In the insulin resistant state in type 2 diabetes, insulin
binding to its receptor is usually normal but the insulin-
signaling cascade is attenuated, due to a defect probably
at the level of the IR itself.4
Protein tyrosine phosphatases (PTPs) constitute a
diverse family of enzymes and are responsible for the
selective dephosphorylation of tyrosine residues.5 Sev-
eral PTPs, including PTP1B, LAR, PTPR, and PTPꢀ, are
capable of dephosphorylating the IR, and thereby at-
tenuating tyrosine kinase activity.6-10 However, in one
case, LAR knockout mice have not shown altered
The development of specific PTP1B inhibitors to treat
type 2 diabetes has been the subject of intensive
research in recent years. Most progress toward specific
inhibitors has been made in the development of revers-
ible competitive inhibitors that mimic the natural
substrate, a phosphotyrosine-containing polypeptide.16,17
In general, most of these inhibitors suffer from low
bioavailability, even though a few investigators have
reported small molecules with in vivo activity in diabetic
mice.18 Because of the electrostatic properties of the
* To whom correspondence should be adressed. Tel.: +46 08
6973869. E-mail: charlotta.liljebris@biovitrum.com.
† Department of Medicinal Chemistry, Uppsala.
‡ Department of Medicinal Chemistry, Kalamazoo.
§ Department of Structural Chemistry, Stockholm.
| Department of Cell and Molecular Biology, Kalamazoo.
10.1021/jm011100y CCC: $22.00 © 2002 American Chemical Society
Published on Web 03/29/2002

1786 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Liljebris et al.
active site, it has proven difficult to develop an effective
phosphotyrosine mimetic with a formal charge of less
than -2 at physiological pH.
compounds 8 and 9. Catalytic hydrogenolysis of the
benzyl esters then afforded the pure target compounds
10 and 11. The compounds 10 and 11 were activated
with 1,1′-carbonyldiimidazole (CDI) in refluxing tet-
rahydrofuran (THF) and reacted with hydroxylamine,23
which generated the corresponding hydroxamates 12
and 13, respectively. Hydrolysis with aqueous LiOH
afforded the hydroxamic acid analogues 14 and 15. The
phenolic carboxyl group could be replaced with a
primary amide function by the same methodology. The
ortho benzyl ester 4 was alkylated with 2-bromoaceta-
mide to afford compound 16. Boc deprotection with TFA
and coupling with Boc-L-phenylalanine under standard
carbodiimide conditions produced compound 17. Final
hydrogenation generated pure primary amide 18.
Previously, we reported the discovery that a simple
tripeptide Ac-NH-Asp-Tyr(SO3H)-Nle-NH2 is a rela-
tively potent competitive inhibitor of PTP1B (Ki ) 5
µM).19,20 An analogue program around this compound
class was launched with the aim of attenuating the
peptidic character of the lead compound. This led to a
series of bisamides with the most potent compounds
having Ki values in the submicromolar range.20 An
important discovery was the novel phosphotyrosine
mimic, O-carboxymethyl salicylic acid,21 seen in com-
pound 1 (Ki ) 6.5 µM). This novel phosphotyrosine
bioisostere was independently reported by Burke et al.
who incorporated it into hexapeptides.22 Furthermore,
structure-activity relationship investigations of the
N-terminal side chain showed that the p-1 aspartic acid
could be successfully replaced by phenylalanine so that
compounds with neutral N termini could be developed,
which maintained good inhibitory activity, e.g., com-
pound 2 (Ki ) 2.0 µM).20
In this study, we have focused on the tyrosine head-
group. Because cell permeability is a key issue for these
compounds, it was important to investigate the pos-
sibility of replacing one or both of the remaining
carboxyl groups while maintaining PTP1B inhibitory
activity. This report describes the synthesis and inhibi-
tion activities of a series of analogues of 1 and 2,
wherein the carboxylic acids have been replaced with
various groups with the primary aim of increasing
permeability and potency.
An interesting carboxylic acid isostere is the 3-hy-
droxyisoxazole group.24 3-Hydroxy-isoxazole can be
synthesized from an acetylenic ester by reaction with
hydroxylamine.25 Introduction of the acetylenic ester
was performed on the O-alkylated intermediate 19 using
ethyl propiolate and copper(I) oxide as catalyst, which
afforded compound 20 (Scheme 2).26 Deprotection of the
Boc group and coupling with Boc-L-phenylalanine under
standard carbodiimide conditions generated compound
21. Treatment of 21 with hydroxylamine afforded a
mixture of the 3-hydroxyisoxazole compound and the
corresponding hydroxamic acid analogues. Separation
by reversed phase high-performance liquid chromato-
graph (HPLC) furnished pure target compound 22.
Hydrolysis of 21 with aqueous LiOH afforded the
acetylenic analogue 23, which was hydrogenated to
generate the saturated analogue 24.
Introduction of the lipophilic carboxylic acid bioisos-
tere tetrazole group at both positions is shown in
Schemes 3 and 4. Palladium-catalyzed cyanation reac-
tion between compound 3 and zinc cyanide produced the
nitrile analogue 25 in modest yield. Reported procedures
describe the use of sodium cyanide and the cocatalyst
CuI,27 although the use of these conditions did not
improve the yield for this reaction, probably because of
the electronic effect of the ortho hydroxy group, which
has a negative effect on the palladium coupling. Alky-
lation with methyl bromoacetate afforded compound 26,
which could be Boc deprotected and coupled with Boc-
L-phenylalanine to generate compound 27. Heating the
nitrile analogue 27 with trimethylsilyl azide in toluene
in the presence of catalytic amounts of dibutyltin oxide28
gave the tetrazole analogue 28 in low but readily
available yield. The low yield of this reaction is mostly
due to unreacted starting material, which was the only
detected side product. Hydrolysis with aqueous LiOH
afforded the target compound 29.
By similar methodology, the tetrazole group could be
introduced at the phenolic position, as outlined in
Scheme 4. Alkylation of the ortho methyl ester com-
pound 5 with 2-bromoacetonitrile afforded the nitrile
analogue 30. Heating with trimethylsilyl azide in tolu-
ene, in the presence of catalytic amounts of dibutyltin
oxide, afforded compound 31. The subsequent steps were
performed as previously described to generate the target
compound 33.
Ch em istr y
By a methodology similar to that previously de-
scribed,20 analogues of 2 could be prepared as shown in
Scheme 1. Carbonylative palladium-catalyzed coupling
of the ortho iodo tyrosine intermediate 3, in either
benzyl alcohol or methanol, generated the corresponding
esters 4 and 5. Alkylation with methyl bromoacetate or
benzyl bromoacetate afforded compounds 6 and 7,
respectively. Deprotection of the Boc group with trif-
luoroacetyl (TFA) and coupling with Boc-L-phenylala-
nine under standard carbodiimide conditions generated
The nitrile analogues of the succinic acid analogue 1
could be prepared by a similar methodology as described
in Schemes 3 and 4. Thus, starting from the nitrile

Inhibitors of Protein Tyrosine Phosphatase 1B
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1787
Sch em e 1a
a
Reagents: (a) CO(g), Pd(OAc)2, DPPF, Et3N, methanol or benzylalcohol, DMF, 80 °C. (b) Methyl 2-bromoacetate or benzyl
2-bromoacetate or 2-bromoacetamide, K2CO3, acetone, 50 °C. (c) TFA, CH2Cl2. (d) Boc-L-Phe-OH, EDC, HOBT, CH2Cl2. (e) H2, 10% Pd/C,
MeOH. (f) NH2OH HCl, CDI, DMF, 80 °C. (g) LiOH, THF.
intermediates 26 and 30, respectively, Boc deprotection
and acylation with succinic anhydride and the subse-
quent hydrolysis with LiOH afforded the corresponding
mononitrile analogues 34 and 35 (Table 2).
Compound 39 could be transformed to the succinic acid
analogue 42 as previously described.
Resu lts a n d Discu ssion
An analogue where the carboxylic acid in the phenolic
position of 1 was replaced by a hydroxyl group could be
prepared as outlined in Scheme 5. Initial attempts to
alkylate the phenolic position of 5 by refluxing with
2-bromoethanol in the precence of base failed, and only
starting materials could be recovered. The same reaction
was performed in a tightly sealed Pyrex tube (Heck
vial), as ethyleneoxide is probably formed during the
reaction, which gave the seven-membered lactone ana-
logue 36. Similar conditions starting from the benzy-
lester 4 gave the same product, which supports the
structure of the intermediate lactone. Boc deprotection
and the subsequent acylation with succinic anhydride
formed the lactone succinic acid analogue 37, which
could be transferred to the hydroxyl analogue 38 by
hydrolysis using LiOH as shown in Scheme 5.
Competitive inhibition of PTP1B by the compounds
was measured using pNPP as substrate, as previously
described.19 For the most active compounds, inhibition
kinetics was determined, and from plots of V vs S at
various concentrations of inhibitor, Ki values were
computed using the direct linear method of Cornish-
Bowden. Compounds were also tested for inhibition of
other recombinant, purified PTP enzymes (LAR and
SHP-2) as an intitial assessment of their specificity.
Results are summarized in Tables 1 and 2. Activity of
compounds against TC-PTP, the known PTP that is
structurally most similar to PTP1B, was also deter-
mined. For clarity, only inhibition data for LAR and
SHP-2 are presented here. In general, however, none
exhibited significant selectivity between PTP1B and TC-
PTP.
A methyl keto analogue of compound 1 could be
prepared as outlined in Scheme 6. Palladium-catalyzed
cross-coupling reaction between the aryl halide 3 and
butyl vinyl ether and the subsequent hydrolysis of the
resulting enol ether generated the methyl ketone 39.29
The monoester compounds 10 and 11, and also the
primary amide 18, showed little or no inhibition of
PTP1B. This verifies earlier findings that an acidic
function at both positions is required for activity.20 This
is further supported by the inhibition exhibited by the

1788 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Liljebris et al.
Sch em e 2a
a
Reagents: (a) Methyl bromoacetate, K2CO3, acetone, 50 °C (89%). (b) Ethyl propiolate, Cu2O, DMF, 110 °C (47%). (c) TFA, CH2Cl2.
(d) Boc-Phe-OH, HOBT, EDC, CH2Cl2 (84%). (e) NH2OH HCl, NaOH, ethanol/THF (19%). (f) LiOH, THF (99%). (g) H2, Pd/C, MeOH
(98%).
analogues of compound 1 shown in Table 2. The nitrile
analogues 34 and 35, and also the keto and hydroxy
analogues 38 and 42, are all inactive as inhibitors of
PTP1B.
droxyisoxazole has been reported to be a good carboxylic
acid bioisostere group.24 However, the ortho 3-hydroxy-
isoxazole analogue 22 was inactive as a competitive
inhibitor of PTP1B.
Extension of the ortho carboxylic acid group by two
carbons (propynoic acid 23 and propanoic acid 24)
resulted in loss of inhibitory activity. This is most
probably due to steric effects in the active site cleft.
Interestingly, the ortho hydroxamic acid derivative 14
showed modest activity as a competitive inhibitor of
PTP1B, whereas compound 15, with hydroxamic acid
at the phenolic position, was nearly inactive. This
indicates that the ortho position apparently accom-
modates carboxyl replacements more easily.
The highlight of this work was the discovery of the
ortho tetrazole analogue 29, which was equipotent to
the dicarboxylic acid derivative 1 as a PTP1B inhibitor
(Table 1). This is the first monocarboxylic acid analogue
in this series with high affinity for the PTP1B enzyme.
The analogous derivative, with the tetrazole group at
the phenolic position (33), could not be accommodated
by the active site cleft and was nearly inactive. 3-Hy-
Tetrazole 29 indicated some increased permeability
into Caco-2 cells30 when compared to the dicarboxylic
acid analogues, even though the values are still in the
low permeability range. Compound 29 was thus tested
for its effects on insulin-stimulated 2-deoxyglucose (2-
DOG) uptake into intact cells (L6 myocytes). In the
presence of insulin at a concentration (10 nM) that
supported half-maximal 2-DOG uptake by L6 myocytes,
compound 29 (100 µM) showed modest cell activity by
augmenting insulin-stimulated uptake of 2-DOG to 147
( 8% of that of the vehicle (0.1% dimethyl sulfoxide
(DMSO)) control. However, basal uptake (without in-
sulin) in the presence of compound 29 was 99 ( 2% that
of the control cells.31 Because of the polar properties of
these compound series, we have not been able to
demonstrate reproducible cell activities with previous
tested analogues, the exception being prodrug tri- and
diesters, which are likely to be hydrolyzed intracellu-

Inhibitors of Protein Tyrosine Phosphatase 1B
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1789
Sch em e 3a
As observed with 2, the binding of 29 is accompanied
by closure of the mobile WPD loop (comprised of
residues 179-189) of the PTP1B catalytic site. The
peptide backbone portion of both bound inhibitors forms
an extended â-strand conformation closely resembling
the conformation of peptidic substrates.32 Two conserved
hydrogen bonds are maintained between the side chain
of Asp48 and the main chain nitrogens on either side
of the central tyrosine phosphate mimic (P and P + 1
positions) and a third between the main chain nitrogen
of Arg47 and a main chain carbonyl of the inhibitor at
the P - 2 position. The phenyl ring of the phosphoty-
rosine (pTyr) mimic also makes hydrophobic packing
interactions with the side chains of Tyr46, Val49,
Ala217, Ile219, Gln262, and Phe181. The P - 1 phenyl
ring of both compounds 2 and 29 makes hydrophobic
interactions with the side chains of Arg47 and Asp48.
This substituent is also observed to make a crystal
contact with the side chain of Phe280 from the neigh-
boring PTP1B molecule. In addition, the C-terminal
pentyl chain (P + 1) of both compounds lies in a shallow
hydrophobic pocket and forms hydrophobic contacts
with the side chains of Val49, Ile219, and Gln262
(Figure 2).
The terminal O-methylcarboxy of the tyrosine head-
group makes a series of four direct hydrogen bonds to
the main chain nitrogen of Phe182, the side chain amide
of Gln266, the side chain NE1, and the main chain
nitrogen of Arg221. In addition, two water molecules
are hydrogen-bonded to the ether oxygen. These waters
are positioned similarly to the phosphate oxygen atoms
of the peptide substrate crystal structures and make
similar hydrogen bond interactions with the backbone
amides of Ser216, Ala217, Ile219, and Gly220.32
The ortho tetrazole substituent on the tyrosine head-
group is directed toward Lys120, but it is unable to
make the hydrogen bond observed by the ortho carboxy-
late of compound 2. The loss of this hydrogen bond,
however, is at least partially compensated by more
extensive hydrophobic and van der Waals interactions
with Phe182, Tyr46, Asp181, Ser216, and Lys120.
a
Reagents: (a) Zn(CN)2, Pd(PPh3)4, DMF, 80 °C (38%). (b)
Methyl bromoacetate, K2CO3, acetone, 50 °C (94%). (c) TFA,
CH2Cl2. (d) Boc-L-Phe-OH, EDC, HOBT, CH2Cl2 (84%). (e) TMS-
N3, Bu2SnO (catalytic), toluene, 110 °C (19%). (f) LiOH, THF (90%).
larly.20 The modest cell activity of compound 29 is
consistent with the evidence that this analogue has
increased permeability properties and is able to pen-
etrate into cells to exert its effects. It also supports the
hypothesis that PTP1B inhibitors can be developed into
antihyperglycemic therapy.
St r u ct u r e Solu t ion of P TP 1B-Com p ou n d 29
Com p lex. The mode of binding of the ortho tetrazole
analogue 29 was determined by solving an X-ray coc-
rystal structure with a C-terminal truncated form of
PTP1B (Figures 1 and 2). As with the crystal structure
of PTP1B complexed with 2,20 only one of the two
protein molecules in the asymmetric unit was observed
to have ligand bound. The binding site of the second
PTP1B molecule was effectively blocked by crystal
contacts from a symmetry-related PTP1B molecule. In
the binding site that does contain bound ligand, how-
ever, the overall structure of the PTP1B-compound 29
complex is very similar to that previously reported for
220 (Figures 1 and 2). This was not unexpected though
as the two compounds differ only in the ortho substitu-
ent of the tyrosine headgroup with the replacement of
a carboxylic acid by the tetrazole moiety.
Con clu sion s
Modification of the tyrosine head moiety of 1 and 2
by the introduction of carboxylic acid bioisosteres has
verified the importance of acidic functionality at both
positions. Nonacidic replacements such as ester, alcohol,
keto, and carboxamide gave inactive compounds. Re-
placement by less acidic groups with greater lipophilic-
ity resulted in compounds with varying degrees of
Sch em e 4a
a
Reagents: (a) Bromoacetonitrile, K2CO3, acetone, 16 h (93%). (b) TMS-N3, Bu2SnO (catalytic), toluene, 110 °C (37%). (c) TFA, CH2Cl2.
(d) Boc-L-Phe-OH, EDC, HOBT, CH2Cl2 (32%). (e) LiOH, THF (99%).

1790 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Liljebris et al.
Ta ble 1. Tyrosine Carboxyl Replacements of the N-Terminal Boc-Phe Analogue 2
a
Described in ref 19.
Sch em e 5a
a
Reagents: (a) 2-Bromoethanol, K2CO3, DMF, 80 °C, sealed Heck vial (21%). (b) TFA, CH2Cl2. (c) Succinic acid anhydride, TEA, CH2Cl2
(42%). (d) LiOH, THF (75%).
activity. Modification of the carboxylic acid appended
to the phenol of the tyrosine headgroup resulted in
inactive compounds, whereas the intermediate activity
of the ortho hydroxamic acid analogue 14 indicated that

Inhibitors of Protein Tyrosine Phosphatase 1B
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1791
Sch em e 6a
a
Reagents: (a) Butyl vinyl ether, Pd(OAc)2, DPPP, TlOAc, DMF, 90 °C, 16 h. (b) HCl, H2O (41%). (c) Methyl 2-bromoacetate, K2CO3,
acetone (36%). (d) TFA, CH2Cl2. (e) Succinic anhydride, Et3N, CH2Cl2 (64%). (f) LiOH, THF/MeOH/H2O (59%).
Ta ble 2. Tyrosine Carboxyl Replacements of the N-Terminal
Succinamic Acid Analogue 1
Exp er im en ta l Section
Ch em ist r y. Gen er a l Com m en t s. All experiments were
carried out under an N2 atmosphere, except the hydrogenation
and carbonylation reactions. Melting points were determined
in open glass capillaries on a Gallenkamp apparatus and were
1
not corrected. H nuclear magnetic resonance (NMR) and 13C
NMR were recorded on a Bruker Advance DPX 400 spectrom-
eter at 400.1 and 100.6 MHz, respectively, or on a Bruker DRX
500 at 500 MHz and 125.7 MHz, respectively. 1H NMR and
% inhibition of
13C NMR spectra were referenced to internal tetramethylsi-
phosphatases (Ki)a
lane. IR spectra were recorded on a Perkin-Elmer Spectrum
compd
R1
R2
PTP1B
87
LAR SHP-2
1000 FT-IR spectrophotometer. Ionspray mass spectrometry
(MS) spectra were obtained on a Perkin-Elmer API 150EX
mass spectrometer or a Micromass Platform-LCZ instrument.
Preparative HPLC were performed on a Gilson HPLC 305,
column: YMC-Pack AQ (50 mm × 4.6 mm; 2 µM 120 Å 3);
eluent: A-MilliQ/0.1% TFA B-CH3CN. Analytical HPLC were
performed on a Shimadzu LC-6A liquid chromatograph using
HiChrom Nucleosil C-18 column (HCl; size, 100 ,, × 4.6 mm)
and J our C8 precolumn; mobile phase was CH3CN/water (0.1%
TFA) at 1 mL/min. Elementary analyses were performed by
Mikro Kemi AB, Uppsala, Sweden.
1b
COOH
COOH
0
5
(6.5 µM)
34
35
38
37
CN
COOH
COOH
COOH
CN
CH2OH
4
3
4
1
0
0
0
0
0
0
0
0
(lactone)
COOCH2CH2
COCH3
42
COOH
8c
-2
1
Assayed at 100 µM. Described in refs 18 and 19. c Inhibition
measured on PTP1 (rat homologue of PTP1B).
a
b
Ben zyl 5-[(2S)-2-[(ter t-Bu toxyca r bon yl)a m in o]-3-oxo-
3-(p en tyla m in o)p r op yl]-2-h yd r oxyben zoa te (4). Triethy-
lamine (1.71 mL, 12.5 mmol) and benzyl alcohol (6.45 mL, 62
mmol) were added to a suspension of 3 (2.97 g, 6.23 mmol),
palladium(II) acetate (42 mg, 0.19 mmol), and 1,1′-bis(diphe-
nylphosphino)ferrocene (DPPF, 207 mg, 0.37 mmol) in dim-
ethyl formamide (DMF; 15 mL). The mixture was saturated
with CO (1 atm) and stirred at 70 °C for 16 h. The mixture
was allowed to reach room temperature and extracted with
EtOAc (40 mL). The organic layer was washed with 10%
aqueous HCl (20 mL) and brine (20 mL), dried (Na2SO4), and
concentrated. The residue was purified by column chromatog-
raphy (SiO2, EtOAc/n-hexane 1:2), which furnished 5.7 g of a
yellow oil. This crude material still contained some benzyl
alcohol. Crystallization in EtOAc/n-hexane gave 0.82 g (27%)
the ortho position might tolerate replacements more
easily. This was confirmed by the important discovery
of the ortho tetrazole analogue 29, which was equipotent
to the dicarboxylic acid analogue 2. Solution of the X-ray
cocrystal structure of the ortho tetrazole analogue 29
bound to PTP1B indeed revealed that the tetrazole
moiety is well-accommodated in the active site and binds
in a similar fashion to the previously reported ortho
carboxylate analogue 2. Of particular importance, this
new monocarboxylic acid analogue revealed significantly
higher Caco-2 cell permeability as compared to all
previous compounds. The increased permeability prop-
erties of compound 29 may account for its modest
augmentation of insulin-stimulated 2-DOG uptake into
L6 myocytes. This result represents the first positive
cell activity data from nonprodrug analogues of this
compound class and demonstrates the potential of these
analogues to be developed into drug therapy for type 2
diabetes.
1
of pure 4 as a white solid. H NMR 400 MHz (CDCl3): δ 0.84
(t, 3H, J ) 7.1, 14.4), 1.13 (m, 2H), 1.23 (m, 2H,), 1.35 (m,
2H), 1.39 (s, 9H), 2.95 (d, 2H, J ) 6.7), 3.11 (m, 2H), 4.18 (m,
1H), 5.07 (br m, 1H), 5.37 (d, 2H, J ) 1,7), 5.77 (br m, 1H),
6.91 (d, 1H, J ) 8.5), 7.30 (dd, 1H, J ) 2.2, 8.5), 7.35-7.47
(m, 5H), 7.69 (d, 1H, J ) 2.2), 10.69. 13C NMR (CDCl3): δ 13.9,
22.3, 28.2, 28.4, 28.9, 29.0, 37.7, 39.4, 56.8, 67.1, 80.3, 112.2,
117.9, 127.5, 128.5, 128.6, 128.7, 130.3, 135.1, 136.9, 155.8,

1792 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Liljebris et al.
160.7, 169.8, 170.7. MS (ESI) m/z 485 (M + H). Anal.
phenylalanine (0.20 g, 0.77 mmol), 1-hydroxybenzotriazole
(0.10 g, 0.77 mmol), and 1-(3-dimethylaminopropyl)-3-ethyl-
carbodiimide hydrochloride (EDC, 0.15 g, 0.77 mmol) were
added to the solution, which was then stirred at room tem-
perature overnight. The reaction mixture was diluted with
CH2Cl2 (5 mL) and washed with saturated aqueous NaHCO3
(5 mL), brine (5 mL), and 10% aqueous HCl (5 mL). The
organic layer was dried (Na2SO4) and concentrated. The
residue was purified by column chromatography (SiO2, EtOAc).
(C27H36N2O6) C, H, N.
Meth yl 5-[(2S)-2-[(ter t-Bu toxyca r bon yl)a m in o]-3-oxo-
3-(p en tyla m in o)p r op yl]-2-h yd r oxyben zoa te (5). Triethy-
lamine (0.61 mL, 4.41 mmol) was added to a stirring suspen-
sion of 3 (1.05 g, 2.20 mmol), palladium(II) acetate (14 mg,
0.066 mmol), and DPPF (73 mg, 0.13 mmol) in DMF/MeOH
4:1 (5 mL). A carbon monoxide atmosphere was established
in the reaction vessel, and the mixture was stirred at 70 °C
for 16 h. After the mixture was cooled to ambient temperature,
the mixture was extracted with EtOAc (5 mL), and the organic
layer was washed with 10% aqueous HCl (2 × 2 mL), dried
(MgSO4), and concentrated. The residue was purified by flash
chromatography (SiO2, EtOAc/n-hexane 1:2), which furnished
Ben zyl 5-[(2S)-2-({(2S)-2-[(ter t-Bu toxycar bon yl)am in o]-
3-ph en ylpr opan oyl}am in o)-3-oxo-3-(pen tylam in o)pr opyl]-
2-(2-m eth oxy-2-oxoeth oxy)ben zoa te (8). Compound 8 was
prepared according to the general procedure for Boc depro-
tection and carbodiimide coupling, yielding 0.46 g (86%) as a
1
1
0.54 g (60%) of 5 as a white solid. H NMR 500 MHz (CDCl3):
white solid. H NMR 400 MHz (MeOH-d4): δ 0.86 (t, 3H, J )
δ 0.86 (t, 3H, J ) 7.2, 14.6), 1.17 (m, 2H), 1.25 (m, 2H), 1.36
(m, 2H), 1.41 (s, 9H), 2.97 (d, 2H, J ) 7.2), 3.12-3.21 (m, 2H),
3.92 (s, 3H), 4.21 (dd, 1H, J ) 14.7, 7.2), 5.11 (br s, 1H), 5.81
(br m, 1H), 6.91 (d, 1H, J ) 8.5), 7.30 (dd, 1H, J ) 8.5, 2.1),
7.67 (d, 1H, J ) 2.1). 13C NMR (CDCl3): δ 13.86, 22.24, 28.26
(3C), 28.90, 29.05, 37.77, 39.47, 52.23, 54.69, 80.37, 112.25,
117.82, 127.54, 130.34 (2C), 136.76, 160.56, 170.32, 170.74.
Anal. (C21H32O6N2) C, H.
Gen er a l Alk yla tion P r oced u r e. The general procedure
for alkylation of the phenolic position is exemplified by the
preparation of compound 6. Methyl bromoacetate (0.35 mL,
3.75 mmol) and freshly ground K2CO3 (0.52 g, 3.75 mmol) were
added to a solution of 4 (0.61 g, 1.25 mmol) in acetone (15 mL).
The mixture was stirred at 50 °C overnight. After the mixture
was cooled to ambient temperature, H2O (10 mL) was added
and the mixture was extracted with EtOAc (10 mL). The
organic layer was dried (Na2SO4) and concentrated. The
residue was purified by column chromatography (SiO2, EtOAc/
n-hexane 1:1).
7.2, 14.5), 1.18 (m, 2H), 1.24 (m, 2H), 1.34 (s, 9H), 1.38 (m,
2H), 2.72 (dd, 1H, J ) 9.4, 13.8), 2.89-3.11 (m, 3H), 3.71 (s,
3H), 4.22 (m, 1H), 4.49 (m, 1H), 4.74 (s, 2H), 5.31 (s, 2H), 6.94
(d, 1H, J ) 8.6), 7.14-7.46 (m, 6H), 7.65 (d, 1H, J ) 1.7). 13C
NMR (MeOH-d4): δ 14.6, 23.6, 28.9, 30.2, 30.4, 38.3, 39.3, 40.7,
52.9, 56.1, 58.0, 67.4, 68.1, 81.1, 115.8, 122.3, 128.0, 129.5,
129.5, 129.7, 129.9, 130.6, 131.8, 133.8, 136.0, 137.9, 138.7,
158.2, 167.8, 171.0, 172.7, 174.3. MS (ESI) m/z 702 (M - H).
Anal. (C39H49N3O9) C, H, N.
Meth yl 2-[2-(Ben zyloxy)-2-oxoeth oxy]-5-[(2S)-2-({(2S)-
2-[(t er t -b u t oxyca r b on yl)a m in o]-3-p h e n ylp r op a n oyl}-
a m in o)-3-oxo-3-(p en tyla m in o)p r op yl]ben zoa te (9). The
reaction was carried out with 7 (0.78 g, 1.41 mmol) according
to the general Boc deprotection and carbodiimide reaction
procedure, yielding 0.85 g (86%) of 9 as a white solid. 1H NMR
400 MHz (MeOH-d4): δ 0.88 (t, 3H, J ) 7.1, 14.4), 1.18 (m,
2H), 1.28 (m, 2H), 1.34 (s, 9H), 1.37 (m, 2H), 2.74 (dd, 1H, J )
9.4, 13.8), 2.93 (dd, 1H, J ) 7.5, 13.8), 2.97-3.05 (m, 3H), 3.12
(m, 1H), 3.82 (s, 3H), 4.23 (m, 1H), 4.50 (m, 1H), 4.79 (s, 2H),
5.18 (s, 2H), 6.69 (br d, 0.6H), 6.91 (d, 1H, J ) 8.6). 13C NMR
(MeOH-d4): δ 14.6, 23.7, 28.7, 28.9, 30.2, 30.4, 38.3, 39.3, 40.8,
40.9, 52.9, 56.1, 58.0, 67.4, 68.2, 81.1, 115.7, 122.2, 128.0, 129.7,
129.7, 129.9, 130.6, 131.7, 133.8, 135.9, 137.3, 138.8, 158.1,
168.4, 170.4, 172.7, 172.8, 174.3. MS (ESI) m/z 702 (M - H).
Anal. (C39H49N3O9) C, H, N.
Ben zyl 5-[(2S)-2-[(ter t-Bu toxyca r bon yl)a m in o]-3-oxo-
3-(p en tyla m in o)p r op yl]-2-(2-m eth oxy-2-oxoeth oxy)ben -
zoa te (6). The reaction was carried out with methyl 2-bro-
moacetate (0.35 mL, 3.75 mmol) and 4 (0.61 g, 1.25 mmol),
1
yielding 0.46 g (66%) of 6 as a white solid. H NMR 400 MHz
(MeOH-d4): δ 0.87 (t, 3H, J ) 7.1, 14.3), 1.17-1.40 (m, 6H),
1.35 (s, 9H), 2.79 (dd, 1H, J ) 8.4, 13.6), 2.98-3.14 (m, 3H),
3.74 (s, 3H), 4.19 (m, 1H), 4.75 (s, 2H), 5.33 (s, 2H), 6.95 (d,
1H, J ) 8.5), 7.32-7.39 (m, 5H), 7.46 (dd, 1H, J ) 1.9, 8.5),
7.67 (d, 1H, J ) 1.9). 13C NMR (MeOH-d4): δ 14.6, 23.7, 28.9,
30.2, 30.4, 38.7, 40.6, 52.9, 57.7, 67.4, 68.1, 80.9, 115.8, 122.2,
129.5, 129.5, 129.9, 132.2, 133.7, 136.0, 137.9, 157.8, 158.1,
167.8, 171.1, 174.0. MS (ESI) m/z 555 (M - H). Anal.
(C30H40N2O8) C, H, N.
5-[(2S )-2-({(2S )-2-[(t er t -Bu t oxyc a r b on yl)a m in o]-3-
p h en ylp r op a n oyl}a m in o)-3-oxo-3-(p en tyla m in o)p r op yl]-
2-(2-m eth oxy-2-oxoeth oxy)ben zoic Acid (10). A mixture
of 8 (0.41 g, 0.58 mmol) and 10% Pd/C (80 mg) in methanol
(25 mL) was hydrogenated at atmospheric pressure for 2 h.
The mixture was filtered through Celite, and the solvent was
removed under reduced pressure to afford 0.34 g (96%) of 10
1
as a white solid. H NMR 400 MHz (MeOH-d4): δ 0.89 (t, 3H,
Meth yl 2-[2-(Ben zyloxy)-2-oxoeth oxy]-5-[(2S)-2-[(ter t-
Bu t oxyca r bon yl)a m in o]-3-oxo-3-(p en t yla m in o)p r op yl]-
ben zoa te (7). The reaction was carried out with benzyl
2-bromoacetate (0.84 mL, 5.3 mmol) and 5 (0.72 g, 1.77 mmol)
according to the general alkylation procedure, yielding 0.82 g
J ) 7.1, 14.4), 1.19-1.42 (m, 6H), 1.36 (s, 9H), 2.73 (dd, 1H, J
) 9.5, 13.8), 2.93-3.07 (m, 3H), 3.14 (m, 1H), 3.75 (s, 2H), 4.23
(m, 1H), 4.51 (br m, 1H), 4.81 (s, 2H), 6.96 (d, 1H, J ) 8.5),
7.17-7.27 (m, 5H), 7.35 (dd, 1H, J ) 1.8, 8.5), 7.72 (d, 1H, J
) 1.8), 7.84 (br m, 0.5H), 7.98 (br m, 0.2H). 13C NMR (MeOH-
d4): δ 14.6, 23.7, 28.9, 30.2, 30.2, 30.4, 38.3, 39.3, 40.8, 53.0,
56.1, 58.0, 67.4, 81.1, 115.6, 122.7, 128.0, 129.7, 130.6, 132.0,
134.2, 135.9, 138.8, 157.9, 158.2, 169.7, 171.2, 172.7, 172.8,
174.3. MS (ESI) m/z 612 (M - H). Anal. (C32H43N3O9) C, H, N.
1
(84%) of 7 as a white solid. H NMR 400 MHz (MeOH-d4): δ
0.89 (t, 3H, J ) 6.8, 13.9), 1.22 (m, 2H), 1.29 (m, 2H), 1.37 (s,
9H), 1.39 (m, 2H), 2.81 (dd, 1H, J ) 8.5, 13.3), 3.00 (dd, 1H, J
) 7.4, 13.3), 3.07-3.15 (m, 2H), 3.82 (s, 3H), 4.20 (m, 1H), 4.79
(s, 2H), 5.21 (s, 2H), 6.91 (d, 1H, J ) 8.5), 7.31-7.35 (m, 6H),
7.66 (s, 1H). 13C NMR (MeOH-d4): δ 14.6, 23.7, 28.9, 30.3, 30.4,
38.7, 40.7, 52.8, 57.7, 67.5, 68.2, 80.9, 115.6, 122.1, 129.6, 129.7,
129.9, 132.2, 133.7, 135.8, 137.3, 158.0, 168.4, 170.5, 174.1.
MS (ESI) m/z 555 (M - H). Anal. (C30H40N2O8) C, H, N.
2-[4-[(2S)-2-({(2S)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-3-
p h en ylp r op a n oyl}a m in o)-3-oxo-3-(p en tyla m in o)p r op yl]-
2-(m eth oxyca r bon yl)p h en oxy]a cetic Acid (11). A mixture
of 9 (0.66 g, 0.94 mmol) and 10% Pd/C (100 mg) in methanol
(30 mL) was hydrogenated at atmospheric pressure for 2 h.
The mixture was filtered through Celite, and the solvent was
removed under reduced pressure to afford 0.57 g (97%) of 11
Gen er a l Boc Dep r otection a n d Ca r bod iim id e Cou -
p lin g P r oced u r e. The general procedure for Boc deprotection
and carbodiimide coupling is exemplified by the preparation
of compound 8. Trifluoroacetic acid (0.90 mL) was carefully
added to a solution of 6 (0.44 g, 0.78 mmol) in CH2Cl2 (8 mL)
at 0 °C. The mixture was stirred for 4 h allowing the solution
to warm to ambient temperature. The volatiles were removed
in vacuo, and the residue was partitioned between EtOAc (15
mL) and saturated aqueous NaHCO3 (2 × 10 mL). The organic
layer was dried (Na2SO4) and concentrated, which gave 0.35
g (98%) of the crude amine as a yellowish oil. The amine was
dissolved in CH2Cl2 (7 mL) and cooled with ice. Boc-L-
1
as a white solid. H NMR 400 MHz (MeOH-d4): δ 0.89 (t, 3H,
J ) 7.1, 14.4), 1.21 (m, 2H), 1.29 (m, 2H), 1.35 (s, 9H), 1.37
(m, 2H), 2.75 (dd, 1H, J ) 9.3, 13.8), 2.94 (dd, 1H, J ) 7.4,
13.8), 2.98-3.05 (m, 3H), 3.13 (m, 1H), 3.86 (s, 3H), 4.24 (m,
1H), 4.50 (m, 1H), 4.69 (s, 2H), 6.95 (d, 1H, J ) 8.6), 7.17-
7.27 (m, 5H), 7.35 (dd, 1H, J ) 1.5, 8.6), 7.64 (d, 1H, J ) 1.5),
7.83 (br m, 1H), 7.98 (br d, 0.6H). 13C NMR (MeOH-d4): δ 14.6,
23.7, 28.7, 28.9, 30.2, 30.4, 38.3, 39.3, 40.7, 40.9, 52.9, 56.2,
58.0, 67.5, 81.1, 115.8, 121.9, 128.0, 129.7, 130.6, 131.6, 133.8,

Inhibitors of Protein Tyrosine Phosphatase 1B
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1793
F igu r e 1. Stereo representation of the crystal structure (2.3
Å resolution) of 29 bound to PTP1B. The enzyme backbone is
represented as a ribbon diagram with key residues presented
in detail and labeled. Heteroatoms are colored by type (carbon,
green for PTP1B and purple for 29; oxygen, red; nitrogen, blue;
sulfur, yellow). Selected hydrogen-bonding interactions with
the enzyme are depicted with dashed lines.
136.1, 138.8, 158.1, 158.4, 168.5, 172.7, 172.8, 174.3. MS (ESI)
m/z 612 (M - H). Anal. (C32H43N3O9) C, H, N.
Gen er a l P r oced u r e for th e In tr od u ction of Hyd r ox-
a m ic Acid . The general procedure for introducing hydroxamic
acid is exemplified by the preparation of 12. To a solution of
10 (116 mg, 0.19 mmol) in THF/DMF (2.5:0.2 mL) was added
CDI (61 mg, 0.38 mmol), and the mixture was refluxed at 80
°C for 1 h. The mixture was cooled to ambient temperature,
and hydroxylamine hydrochloride (39 mg, 0.57 mmol) was
added. The reaction mixture was refluxed at 80 °C for 4 h.
After the mixture was cooled to room temperature, the mixture
was partitioned between EtOAc (3 mL) and 3 M aqueous HCl
(3 mL), and the organic layer was dried (Na2SO4) and
concentrated. The residue was purified by column chromatog-
raphy (SiO2, 5% MeOH in CH2Cl2) and further purified by
crystallization in EtOAc.
Met h yl 2-{4-[(2S)-2-({(2S)-2-[(ter t-Bu t oxyca r b on yl)-
a m in o]-3-p h en ylp r op a n oyl}a m in o)-3-oxo-3-(p en tyla m i-
n o )p r o p y l]-2-[(h y d r o x y a m in o )c a r b o n y l]p h e n o x y }-
a ceta te (12). Compound 12 was repared according to the
general procedure for introducing hydroxamic acid, yielding
34 mg (28%) of 12 as a white solid; mp 165.4-166.9 °C. 1H
NMR 400 MHz (MeOH-d4): δ 0.89 (t, 3H, J ) 7.1, 14.4), 1.20
(m, 2H), 1.29 (m, 2H), 1.35 (s, 9H), 1.38 (m, 2H), 2.70 (dd, 1H,
J ) 9.4, 13.3), 2.91-3.17 (m, 5H), 3.81 (s, 3H), 4.21 (m, 1H),
4.54 (m, 1H), 4.86 (s, 2H, obscured by solvent peak), 6.98 (d,
1H, J ) 8.1), 7.16-7.27 (m, 5H), 7.34 (br d, 1H, J ) 8.1), 7.84
(br s, 1H). 13C NMR (MeOH-d4): δ 14.6, 23.7, 28.9, 30.2, 30.4,
38.4, 39.2, 40.7, 56.1, 58.1, 67.3, 81.1, 114.7, 121.9, 128.0, 129.7,
130.6, 132.5, 133.4, 135.3, 135.8, 138.8, 156.2, 158.1, 165.7,
F igu r e 2. Schematic representation of the interactions
between PTP1B and compound 29. Green dotted lines repre-
sent hydrogen bonds or electrostatic interactions, and red
dotted lines represent hydrophobic interactions. Atom colors
are as follows: carbon, black; nitrogen, blue; oxygen, red; and
sulfur, yellow. Distances are shown in Ångstro¨ms.
28.9, 30.2, 30.4, 38.4, 39.3, 40.7, 53.1, 56.1, 58.0, 69.3, 81.1,
116.1, 120.9, 128.1, 129.7, 130.6, 132.2, 134.1, 136.9, 138.7,
158.4, 167.7, 168.0, 172.7. 174.3. MS (ESI) m/z 627 (M - H).
Anal. (C32H44N4O9) C, H, N.
2-{4-[(2S)-2-({(2S)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-3-
p h en ylp r op a n oyl}a m in o)-3-oxo-3-(p en tyla m in o)p r op yl]-
2-[(h yd r oxya m in o)ca r bon yl]p h en oxy}a cetic Acid (14).
To a solution of 12 (10 mg, 0.0165 mmol) in THF (200 µL) was
added a 2.5 M aqueous solution of LiOH (9.9 µL, 0.0248 mmol),
and the mixture was stirred at ambient temperature for 2 h.
The reaction mixture was then acidified with 3 M aqueous HCl
and extracted with EtOAc (2 mL). The organic layer was dried
(Na2SO4) and concentrated to afford 8.0 mg (79%) of 14 as a
1
white solid. H NMR 400 MHz (MeOH-d4): δ 0.88 (t, 3H, J )
7.1, 14.5), 1.19 (m, 2H), 1.29 (m, 2H), 1.35 (s, 9H), 1.38 (m,
2H), 2.71 (dd, 1H, J ) 9.5, 13.5), 2.94 (m, 1H), 2.98-3.07 (m,
3H), 3.12 (m, 2H), 4.22 (dd, 1H, J ) 5.1, 9.5), 4.54 (m, 1H),
4.81 (s, 2H), 6.99 (d, 1H, J ) 8.5), 7.17-7.27 (m, 5H), 7.35
(dd, 1H, J ) 1.8, 8.5), 7.85 (d, 1H, J ) 1.8). 13C NMR (MeOH-
d4): δ 14.6, 23.7, 28.9, 30.2, 30.4, 38.4, 39.2, 40.7, 56.1, 58.0,
67.3, 81.1, 114.8, 121.9, 128.0, 129.7, 130.6, 132.4, 133.4, 135.4,
138.8, 156.3, 158.1, 165.7, 172.4, 172.8, 174.3. MS (ESI) 613
(M - H). Anal. (C31H42N4O9‚1/2H2O) C, H, N.
171.3, 172.8, 174.6. MS (ESI) m/z 627 (M-H). Anal. (C32H44
N4O9‚1/2H2O): C, H, N.
-
Meth yl 5-[(2S)-2-({(2S)-2-[(ter t-Bu toxycar bon yl)am in o]-
3-ph en ylpr opan oyl}am in o)-3-oxo-3-(pen tylam in o)pr opyl]-
2-[2-(h yd r oxya m in o)-2-oxoeth oxy]ben zoa te (13). The re-
action was carried out with 11 (103 mg, 0.17 mmol) according
to the general procedure for introducing hydroxamic acid,
although column chromatography (SiO2, 5% MeOH in CH2-
Cl2) was sufficient to afford 46 mg (44%) of pure title compound
5-[(2S )-2-({(2S )-2-[(t er t -Bu t oxyc a r b on yl)a m in o]-3-
p h en ylp r op a n oyl}a m in o)-3-oxo-3-(p en tyla m in o)p r op yl]-
2-[2-(h yd r oxya m in o)-2-oxoeth oxy]ben zoic Acid (15). To
a solution of 13 (10 mg, 0.016 mmol) in THF (400 µL) was
added a 2.5 M aqueous solution of LiOH (19 µL, 0.048 mmol),
and the mixture was stirred at ambient temperature for 2 h.
The reaction mixture was then acidified with 3 M aqueous HCl
and extracted with EtOAc (2 mL). The organic layer was dried
(Na2SO4) and concentrated to afford 8 mg (83%) of 15 as a
1
13 as a white solid; mp ) 160.9-163.5 °C. H NMR 400 MHz
(MeOH-d4): δ 0.88 (t, 3H, J ) 7.1, 14.5), 1.19 (m, 2H), 1.28
(m, 2H), 1.35 (s, 9H), 1.37 (m, 2H) 2.77 (dd, 1H, J ) 9.2, 13.7),
2.93 (dd, 1H, J ) 7.5), 2.98-3.06 (m, 3H), 3.13 (m, 1H), 3.90
(s, 3H), 4.22 (dd, 1H, J ) 5.3, 9.1), 4.50 (app t, 1H), 4.66 (s,
2H), 7.06 (d, 1H, J ) 8.5), 7.17-7.27 (m, 5H), 7.43 (dd, 1H, J
) 1.8, 8.5), 7.75 (d, 1H, 1.8). 13C NMR (MeOH-d4): δ 14.6, 23.7,

1794 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Liljebris et al.
1
white solid. H NMR 400 MHz (MeOH-d4): δ 0.88 (t, 3H, J )
n yl]-2-p r op yn oa te (20). Ethyl propiolate (1.94 mL, 19.2
mmol) was added to a suspension of copper(I) oxide (0.61 g,
6.39 mmol) in anhydrous DMF (3 mL) under a nitrogen
atmosphere. A solution of 19 (4.38 g, 7,99 mmol) in DMF (40
mL) was added. The reaction flask was flushed with nitrogen,
tightly sealed, and stirred at 110 °C for 16 h. The reaction
mixture was filtered through a short pad of SiO2 and washed
with EtOAc. The organic layer was washed with 1 M aqueous
HCl (20 mL), brine (20 mL), and saturated aqueous NaHCO3
(20 mL), dried (Na2SO4), and concentrated. The residue was
purified by column chromatography (SiO4, EtOAc/hexane 1:2
to 1:1), which afforded 1.93 g (47%) of 20 as a white solid; mp
7.1, 14.5), 1.19 (m, 2H), 1.29 (m, 2H), 1.35 (s, 9H), 1.38 (m,
2H), 2.78 (dd, 1H), 2.95 (dd, 1H), 3.14 (m, 1H), 4.23 (dd, 1H,
5.2, 9.2), 4.51 (m, 1H), 4.68 (s, 2H), 7.04 (d, 1H, J ) 8.5), 7.17-
7.27 (m, 5H), 7.42 (dd, 1H, J ) 2.1, 8.5), 7.78 (d, 1H, J ) 2.1).
13C NMR (MeOH-d4): δ 14.3, 23.4, 286, 29.9, 30.1, 38.1, 39.0,
40.5, 55.8, 57.7, 68.9, 80.9, 115.8, 121.2, 127.7, 129.4, 130.3,
131.9, 134.2, 136.4, 138.4, 158.2, 167.6, 169.0, 172.4, 172.5,
174.0. MS (ESI) m/z 613 (M - H). Anal. (C31H42N4O9‚1/2H2O)
C, H, N.
Ben zyl 2-(2-Am in o-2-oxoeth oxy)-5-[(2S)-2-[(ter t-bu toxy-
ca r bon yl)a m in o]-3-oxo-3-(p en tyla m in o)p r op yl]ben zoa te
(16). The reaction was carried out with compound 4 (295 mg,
0.61 mmol) and 2-bromoacetamide (168 mg, 1.22 mmol)
according to the general alkylation procedure, yielding 273 mg
1
133.9-135.1 °C. H NMR 400 MHz (CDCl3): δ 0.87 (t, 3H, J
) 7.1, 14.4), 1.22 (m, 2H), 1.29 (m, 2H), 1.35 (t, 3H, J ) 7.1),
1.41 (s and m, 11H), 2.97 (app t, 2H, J ) 6.7, 13.2), 3.17 (m,
2H), 3.80 (s, 3H), 4.21 (m, 1H), 4.29 (q, 2H, J ) 7.1), 4.72 (s,
2H), 5.06 (br s, 1H), 5.91 (m, 1H), 6.70 (d, 1H, J ) 8.6), 7.22
(dd, 1H, J ) 2.1, 8.6), 7.38 (d, 1H, J ) 2.1). 13C NMR (CDCl3):
δ 13.9, 14.1, 22.2, 28.2, 28.9, 29.1, 37.4, 39.5, 52.3, 55.9, 62.0,
65.9, 80.3, 82.1, 85.0, 109.9, 112.6, 130.3, 133.0, 135.7, 154.0,
155.3, 158.8, 168.7, 170.6. MS (ESI) m/z 517 (M - H). Anal.
(C27H38N2O8‚H2O) C, H, N.
1
(83%) of 16 as a white solid. H NMR 400 MHz (MeOH-d4): δ
0.88 (t, 3H, J ) 7.1, 14.4), 1.18 (m, 2H), 1.27 (m, 2H), 1.37 (s,
9H), 1.40 (m, 2H, partly obscured), 2.82 (dd, 1H, J ) 8.4, 13.5),
3.00-3.09 (m, 2H), 3.13 (m, 1H), 3.85 (s, 0.5H), 4.21 (br m,
1H), 4.56 (s, 2H), 5.36 (s, 2H), 7.07 (d, 1H, J ) 8.5), 7.36-7.49
(m, 6H), 7.81 (s, 1H). 13C NMR (MeOH-d4): δ 14.7, 23.8, 29.0,
30.4, 30.5, 38.9, 40.7, 57.8, 68.2, 69.1, 81.0, 115.7, 120.7, 129.8,
129.9, 130.1, 132.4, 134.2, 137.0, 137.8, 157.9, 158.4, 167.2,
174.0, 174.1. MS (ESI) m/z 542 (M + H). Anal. calcd for
Eth yl 3-[5-[(2S)-2-({(2S)-2-[(ter t-Bu toxycar bon yl)am in o]-
3-ph en ylpr opan oyl}am in o)-3-oxo-3-(pen tylam in o)pr opyl]-
2-(2-m et h oxy-2-oxoet h oxy)p h en yl]-2-p r op yn oa t e (21).
Compound 21 was prepared from 20 (1.01 g, 1.95 mmol)
according to the general Boc deprotection and carbodiimide
coupling procedure, yielding 1.09 g (84%) of 21 as a white solid;
mp 121.8-123.1 °C. 1H NMR 400 MHz (CDCl3): δ 0.87 (t, 3H,
J ) 7.1, 14.4), 1.14-1.41 (m, 6H), 1.34 (t, 3H, J ) 7.1), 1.35
(s, 9H), 2.81 (m, 1H), 3.01-3.09 (m, 4H), 3.15 (m, 1H), 3.77 (s,
3H), 4.27 (m, 1H), 4.29 (q, 2H, J ) 7.1), 4.56 (m, 1H), 4.69 (s,
2H), 4.97 (d, 1H, J ) 6.3), 6.17 (br m, 1H), 6.41 (br m, 1H),
6.66 (d, 1H, J ) 8.6), 7.10-7.19 (m, 4H), 7.25-7.34 (m, 3H).
13C NMR (CDCl3): δ 13.9, 14.1, 22.2, 28.1, 28.9, 36.4, 37.7,
39.6, 52.2, 53.6, 56.1, 62.0, 65.8, 80.6, 82.0, 85.0, 109.8, 112.6,
127.2, 128.8, 129.2, 129.9, 133.1, 135.5, 136.0, 153.9, 158.8,
168.6, 169.6, 170.9. MS (ESI) m/z 664 (M - H). Anal.
(C36H47N3O9) C, H, N.
2-[4-[(2S)-2-({(2S)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-3-
p h en ylp r op a n oyl}a m in o)-3-oxo-3-(p en tyla m in o)p r op yl]-
2-(3-h yd r oxy-5-isoxa zolyl)p h en oxy]a cetic Acid (22). A
amount of 2.5 M aqueous NaOH (670 µL, 1.68 mmol) was
added to hydroxylamine hydrochloride (61 mg, 0.87 mmol).
This mixture was added to a solution of 21 (446 mg, 0.67 mmol)
in ethanol/THF (1 mL:2 mL). The clear yellow solution was
stirred overnight at ambient temperature and then acidified
with 1 M aqueous HCl. The reaction mixture was extracted
with EtOAc (2 × 4 mL), and the organic layer was washed
with brine (4 mL), dried (Na2SO4), and concentrated, which
afforded 418 mg of a crude material as a yellowish solid. This
material was a mixture of the target compound and the
corresponding hydroxamic acid analogues. Separation by
reversed phase HPLC furnished 82 mg (19%) of pure title
compound 22 as a white solid; mp, sublimed above 260 °C. 1H
NMR 500 MHz (MeOH-d4): δ 0.87 (t, 3H, J ) 6.9, 14.4), 1.18
(m, 2H), 1.26 (m, 2H), 1.35 (s, 9H), 1.38 (m, 2H), 2.78 (m, 1H),
3.00-3.07 (m, 4H), 3.16 (dd, 1H), 4.25 (dd, 1H, J ) 5.0, 9.1),
4.53 (m, 1H), 4.77 (s, 2H), 6.83 (s, 1H), 7.01 (d, 1H, J ) 8.5),
7.16-7.30 (m, 6H), 7.74 (br s, 1H). 13C NMR (MeOH-d4): δ
14.3, 23.3, 28.6, 29.9, 30.1, 38.2, 38.9, 40.5, 55.9, 57.7, 66.3,
80.9, 97.0, 113.6, 118.1, 127.7, 128.8, 129.4, 130.3, 131.3, 133.4,
138.4, 155.0, 157.8, 166.8, 171.6, 172.5, 172.7, 174.0. HRMS
(EI) for C33H42N4O9: calcd, 638.2952; found, 638.2957. MS
(ESI) m/z 637 (M - H). Anal. (C33H42N4O9 H2O) C, H, N.
C
29H39N3O7‚2H2O: C, 60.30; H, 6.80; N, 7.27. Found: C, 60.0;
H, 6.6; N, 7.9.
Ben zyl 2-(2-Am in o-2-oxoeth oxy)-5-[(2S)-2-({(2S)-2-[(ter t-
b u t oxyca r b on yl)a m in o]-3-p h en ylp r op a n oyl}a m in o)-3-
oxo-3-(p en tyla m in o)p r op yl]ben zoa te (17). The reaction
was carried out with 16 (72 mg, 0.13 mmol) according to the
general procedure for Boc deprotection and carbodiimide
coupling, yielding 44 mg (49%) of 17 as a white solid. 1H NMR
400 MHz (MeOH-d4): δ 0.85 (t, 3H, J ) 7.2, 14.5), 1.13 (m,
2H), 1.23 (m, 2H), 1.34 (s and m, 9H and 2H), 2.78 (dd, 1H, J
) 9.5, 13.5), 2.9-3.13 (m, 5H), 4.21 (dd, 1H), 4.49 (br t, 1H),
4.53 (s, 2H), 5.34 (s, 2H), 7.03 (d, 1H, J ) 8.5), 7.15-7.26 (m,
5H), 7.33-7.46 (m, 6H), 7.78 (s, 1H). 13C NMR (MeOH-d4): δ
14.7, 23.8, 29.0, 30.3, 30.5, 38.5, 39.4, 40.8, 56.2, 58.1, 68.3,
69.1, 81.2,115.7, 120.8, 128.1, 129.8, 129.9, 130.1, 130.7, 131.9,
134.3, 137.1, 137.8, 138.8, 158.1, 158.5, 167.1, 172.7, 174.0,
174.4. MS (ESI) m/z 687 (M - H). Anal. (C38H48N4O8) C, H, N.
2-(2-Am in o-2-oxoet h oxy)-5-[(2S)-2-({(2S)-2-[(ter t-b u -
t o x y c a r b o n y l)a m in o ]-3-p h e n y lp r o p a n o y l}a m in o )-3-
oxo-3-(p en tyla m in o)p r op yl]ben zoic Acid (18). A mix-
ture of 17 (38 mg, 0.056 mmol) and 10% Pd/C (12 mg) in
methanol (2.2 mL) was hydrogenated at atmospheric pressure
for 3 h. The mixture was filtered through Celite, and the
solvent was removed under reduced pressure to afford 30 mg
1
(90%) of 18 as a white solid. H NMR 400 MHz (MeOH-d4): δ
0.88 (t, 3H, J ) 7.1, 14.5), 1.19 (m, 2H), 1.29 (m, 2H), 1.35 (s,
9H), 1.39 (m, 2H, partly obscured), 2.73 (dd, 1H, J ) 9.6, 13.6),
2.92-3.06 (m, 4H), 3.13 (m, 1H), 4.23 (m, 1H), 4.52 (m, 1H,
partly obscured), 4.55 (s, 2H), 6.75 (br d, 0.5H), 6.98 (d, 1H, J
) 8.5), 7.18-7.27 (m, 5H), 7.31 (br d, 1H), 7.67 (br s, 1H), 7.82
(br m, 0.5H). 13C NMR (MeOH-d4): δ 14.6, 23.7, 28.9, 30.2,
30.4, 38.4, 39.2, 40.7, 56.1, 58.0, 69.2, 81.1, 115.4, 128.0, 129.7,
130.6, 131.7, 133.8, 135.1, 138.8, 157.6, 172.8, 174.3, 174.5.
MS (ESI) m/z 597 (M - H). Anal. (C31H42N4O8‚1.5H2O) C, H,
N.
Met h yl 2-{4-[(2S)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-3-
oxo-3-(p en tyla m in o)p r op yl]-2-iod op h en oxy}a ceta te (19).
Compound 19 was prepared from 3 (2.24 g, 4.70 mmol),
according to the general alkylation procedure, which generated
2.29 g (89%) of compound 19 as a white solid. 1H NMR 400
MHz (CDCl3): δ 0.88 (t, 3H, J ) 7.1, 14.4), 1.20 (m, 2H), 1.29
(m, 2H), 1.38 (m, 2H), 1.43 (s, 9H), 2.96 (m, 2H), 3.17 (m, 2H),
3.81 (s, 3H), 4.19 (m, 1H), 4.67 (s, 2H), 5.06 (br s, 1H), 5.79
(br s, 1H), 6.63 (d, 1H, J ) 8.4), 7.12 (dd, 1H, J ) 2.1, 8.4),
7.64 (d, 1H, J ) 2.1). 13C NMR (CDCl3): δ 14.0, 22.3, 28.3,
28.9, 29.1, 37.2, 39.5, 52.4, 56.0, 66.3, 80.3, 86.6, 112.3, 130.4,
132.3, 140.5, 155.4, 155.7, 168.8, 170.6. MS (ESI) m/z 544 (M
- H). Anal. (C22H33N2O6I) C, H.
3-[5-[(2S)-2-({(2S)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-3-
p h en ylp r op a n oyl}a m in o)-3-oxo-3-(p en tyla m in o)p r op yl]-
2-(2-h yd r oxy-2-oxoeth oxy)p h en yl]-2-p r op yn oic Acid (23).
To a solution of 21 (128 mg, 0.19 mmol) in THF (3 mL) was
added a 2.5 M aqueous solution of LiOH (309 µL, 0.77 mmol).
The mixture was stirred at ambient temperature for 2.5 h at
which time TLC showed that still much starting material was
left. Additional LiOH (78 µL, 0.19 mmol) was added, and the
mixture was stirred for another 2 h. The mixture was acidified
Eth yl 3-[5-[(2S)-2-[(ter t-Bu toxyca r bon yl)a m in o]-3-oxo-
3-(p en tyla m in o)p r op yl]-2-(2-m eth oxy-2-oxoeth oxy)p h e-

Inhibitors of Protein Tyrosine Phosphatase 1B
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1795
with 3 M aqueous HCl and extracted with EtOAc (5 mL). The
organic layer was dried (Na2SO4) and concentrated to afford
119 mg (99%) of 23 as a white solid; mp 124.5-130.1 °C. H
40.5, 52.7, 55.6, 57.6, 66.4, 80.8, 102.9, 113.9, 117.0, 127.7,
129.4, 130.3, 132.2, 135.6, 136.8, 138.4, 157.7, 160.0, 170.1,
172.2, 174.0. MS (ESI) m/z 593 (M - H). Anal. (C32H42N4O7)
C, H, N.
1
NMR 400 MHz (MeOH-d4): δ 0.90 (t, 3H, J ) 7.1, 14.5), 1.19-
1.42 (m, 6H), 1.36 (s, 9H), 2.75 (dd, 1H), 2.86 (dd, 1H), 2.90-
3.05 (m, 3H), 3.14 (m, 1H), 4.23 (dd, 1H, J ) 5.3, 9.2), 4.49
(m, 1H), 4.76 (s, 2H), 6.87 (d, 1H, J ) 8.7), 7.18-7.29 (m, 6H),
7.37 (s, 1H). 13C NMR (MeOH-d4): δ 14.3, 23.4, 28.6, 30.0, 30.1,
30.9, 37.8, 39.0, 40.5, 40.6, 55.7, 57.7, 66.4, 80.9, 83.6, 86.0,
110.6, 113.6, 127.7, 129.4, 130.3, 131.5, 134.3, 136.7, 138.4,
156.8, 160.4, 171.9, 172.4, 174.0. MS (ESI) m/z 622 (M - H).
Anal. calcd for C33H41N3O9: C, 63.55; H, 6.63; N, 6.74. Found:
C, 63.0; H, 5.7; N, 6.7.
3-[5-[(2S)-2-({(2S)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-3-
p h en ylp r op a n oyl}a m in o)-3-oxo-3-(p en tyla m in o)p r op yl]-
2-(ca r boxym eth oxy)p h en yl]p r op a n oic Acid (24). A mix-
ture of 23 (76 mg, 0.12 mmol) and 10% Pd/C (20 mg) in
methanol (4 mL) was hydrogenated at atmospheric pressure
for 4 h. The mixture was filtered through Celite, and solvent
was removed under reduced pressure to afford 75 mg (98%) of
compound 24 as a white solid; mp 99.8-103.2 °C. 1H NMR
400 MHz (MeOH-d4): δ 0.89 (t, 3H, J ) 7.1, 14.4), 1.17-1.40
(m, 6H), 1.34 (s, 9H), 2.62 (m, 2H), 2.79 (m, 1H), 2.98 (m, 4H),
3.02 (m, 2H), 3.10 (m, 1H), 4.24 (dd, 1H, J ) 5.1, 9.0), 4.49 (br
m, 1H), 4.64 (s, 2H), 6.73 (d, 1H, J ) 8.2), 6.97 (m, 2H), 7.20-
7.28 (m, 5H). 13C NMR (MeOH-d4): δ 14.3, 23.4, 27.2, 28.6,
29.9, 30.1, 35.1, 38.1, 38.8, 40.5, 55.9, 57.7, 60.2, 66.5, 80.9,
112.5, 127.7, 129.4, 129.4, 130.4, 130.6, 130.7, 132.3, 138.4,
156.4, 157.8, 172.7, 172.8, 173.9, 177.2. MS (ESI) m/z 626 (M
- H). Anal. (C33H45N3O9) C, H, N.
Met h yl 2-[4-[(2S)-2-({(2S)-2-[(ter t-Bu t oxyca r b on yl)-
a m in o]-3-p h en ylp r op a n oyl}a m in o)-3-oxo-3-(p en tyla m i-
n o)p r op yl]-2-(1(2)H-t et r a zol-5-yl)p h en oxy]a cet a t e (28).
To a suspension of 27 (0.45 g, 0.75 mmol) in toluene (4 mL),
in a heavy-walled Pyrex tube fitted with a screw cap, were
added trimethylsilyl azide (0.30 mL, 2.25 mmol) and dibutyltin
oxide (19 mg, 0.075 mmol). The mixture was flushed with
nitrogen, tightly sealed, and stirred at 110 °C for 16 h. Upon
cooling, the volatiles were evaporated under reduced pressure.
The residue was purified by column chromatography (SiO2,
EtOAc), which afforded 90 mg (19%) of compound 28 as a white
solid; mp 189.5-192.8 °C. 1H NMR 400 MHz (MeOH-d4): δ
0.85 (t, 3H, J ) 6.9, 14.1), 1.16-1.41 (m, 6H), 1.33 (s, 9H),
2.69 (dd, 1H, J ) 9.4, 13.7), 2.95-3.17 (m, 5H), 3.79 (s, 3H),
4.22 (dd, 1H, J ) 4.9, 9.4), 4.58 (m, 1H), 4.99 (s, 2H), 7.09-
7.24 (m, 6H), 7.42 (d, 1H, J ) 1.7, 6.9), 8.09 (d, 1H, J ) 1.7).
13C NMR (MeOH-d4): δ 14.3, 23.4, 28.6, 29.9, 30.1, 38.1, 39.0,
40.5, 53.0, 55.7, 57.7, 66.8, 80.8, 114.0, 114.6, 127.7, 129.4,
130.2, 131.6, 132.7, 135.2, 138.5, 155.6, 157.8, 171.4, 172.4,
174.0. MS (ESI) m/z 636 (M - H). HRMS (EI) calcd for
C
32H43N7O7, 637.3224; found, 637.3234. Anal. calcd for
C32H43N7O7: C, 60.27; H, 6.80; N, 15.37. Found: C, 60.7; H,
7.0; N, 14.3.
2-[4-[(2S)-2-({(2S)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-3-
p h en ylp r op a n oyl}a m in o)-3-oxo-3-(p en tyla m in o)p r op yl]-
2-(1(2)H-tetr a zol-5-yl)p h en oxy]a cetic Acid (29). To a so-
lution of 28 (19 mg, 0.029 mmol) in THF (0.6 mL) was added
a 2.5 M aqueous solution of LiOH (35 µL, 0.087 mmol), and
the mixture was stirred at ambient temperature for 2 h. The
reaction mixture was then acidified with 3 M aqueous HCl
and extracted with EtOAc (2 mL). The organic layer was dried
(Na2SO4) and concentrated to afford 16 mg (90%) of compound
ter t-Bu t yl (1S)-1-(3-Cya n o-4-h yd r oxyb en zyl)-2-oxo-2-
(p en tyla m in o)eth ylca r ba m a te (25). To a solution of 3 (5.94
g, 12.47 mmol) in DMF (anhydrous, 30 mL) in a heavy-walled
Pyrex tube fitted with a screw cap was added Pd(PPh3)4 (0.58
g, 0.50 mmol) and zinc cyanide (1.61 g, 13.72 mmol). The vial
was flushed with nitrogen, tightly sealed, and stirred at 80
°C for 16 h. Upon cooling, the mixture was partitioned between
EtOAc (50 mL) and 2 M aqueous ammonium hydroxide (50
mL). The organic layer was dried (Na2SO4) and concentrated.
The residue was purified by column chromatography (SiO2,
EtOAc/n-hexane 1:1) to afford 1.76 g (38%) of compound 25 as
1
29 as a white solid. H NMR 400 MHz (MeOH-d4): δ 0.85 (t,
3H, J ) 7.0, 14.2), 1.18 (m, 2H), 1.24 (m, 2H), 1.33 (s, 9H),
1.38 (m, 2H), 2.70 (dd, 1H, J ) 9.4, 13.8), 2.96-3.08 (m, 3H),
3.14 (m, 2H), 4.22 (dd, 1H, J ) 5.1, 9.4), 4.58 (m, 1H), 4.95 (s,
2H), 7.12-7.29 (m, 6H), 7.44 (dd, 1H, J ) 1.9, 8.5), 7.86 (br
m, 0.5H), 8.01 (br m, 1H), 8.10 (d, 1H, J ) 1.9). 13C NMR
(MeOH-d4): δ 14.3, 23.4, 28.7, 30.0, 30.2, 38.2, 39.0, 40.6, 55.8,
67.0, 80.9, 114.0, 114.9, 127.8, 129.5, 130.3, 131.5, 132.8, 135.4,
138.5, 155.9, 157.9, 172.5, 173.1, 174.1. MS (ESI) m/z 622 (M
- H). HRMS (EI) calcd for C31H41N7O7, 623.3068; found,
623.3071. Anal. calcd for C31H41N7O7: C, 59.70; H, 6.63; N,
15.72. Found: C, 59.5; H, 6.7; N, 14.0.
1
a white solid. H NMR 400 MHz (MeOH-d4): δ 0.91 (t, 3H, J
) 7.3, 14.4), 1.18-1.48 (m, 6H), 1.39 (s, 9H), 2.77 (dd, 1H, J )
8.6, 13.7), 2.98 (dd, 1H, J ) 6.45, 13.7), 3.08-3.21 (m, 2H),
4.20 (m, 1H), 6.88 (d, 1H, J ) 8.5), 7.34 (dd, 1H, J ) 2.2, 8.5),
7.38 (d, 1H, 2.2). 13C NMR (MeOH-d4): δ 14.7, 23.8, 29.0, 30.4,
30.5, 38.6, 40.8, 57.7, 81.1, 100.8, 117.4, 118.3, 130.6, 135.3,
137.2, 157.9, 160.9, 174.1. MS (ESI) m/z 376 (M + H). Anal.
(C20H29N3O4) C, H, N.
Meth yl 5-[(2S)-2-[(ter t-Bu toxyca r bon yl)a m in o]-3-oxo-
3-(p en tyla m in o)p r op yl]-2-(cya n om eth oxy)ben zoa te (30).
The reaction was carried out with bromoacetonitrile (103 µL,
1.48 mmol) and compound 5 (0.30 g, 0.74 mmol) according to
the general alkylation procedure, which afforded 0.36 g (93%)
Met h yl 2-{4-[(2S)-2-[(ter t-Bu t oxyca r b on yl)a m in o]-3-
oxo-3-(pen tylam in o)pr opyl]-2-cyan oph en oxy}acetate (26).
Compound 26 was prepared from 25 (0.61 g, 1.63 mmol) by
the general alkylation procedure, which afforded 0.69 g (94%)
1
1
of compound 26 as a white solid. H NMR 400 MHz (CDCl3):
of compound 30 as a white solid. H NMR 400 MHz (CDCl3):
δ 0.90 (t, 3H, J ) 7.1, 14.4), 1.22-1.45 (m, 6H), 1.38 (s, 9H),
2.79 (dd, 1H, J ) 8.8, 13.7), 3.01 (m, 1H), 3.11-3.18 (m, 2H),
3.77 (s, 3H), 4.87 (s, 2H), 7.00 (d, 1H, J ) 8.7), 7.46 (dd, 1H,
J ) 2.2, 8.7), 7.51 (d, 1H, J ) 2.2). 13C NMR (CDCl3): δ 14.7,
23.7, 28.9, 30.3, 30.4, 38.4, 40.7, 53.0, 57.5, 66.6, 81.0, 103.1,
114.1, 117.4, 132.9, 135.9, 137.1, 157.8, 160.2, 170.4, 173.8.
MS (ESI) m/z 448 (M + H). Anal. (C23H33N3O6) C, H, N.
Met h yl 2-{4-[(2S)-2-({(2S)-2-[(ter t-Bu t oxyca r b on yl)-
a m in o]-3-p h en ylp r op a n oyl}a m in o)-3-oxo-3-(p en tyla m i-
n o)p r op yl]-2-cya n op h en oxy}a ceta te (27). The reaction was
carried out with 26 (0.55 g, 1.23 mmol) according to the general
Boc deprotection and carbodiimide reaction procedure, yielding
0.61 g (84%) as a white solid; mp 121.4-123.0 °C. 1H NMR
400 MHz (MeOH-d4): δ 0.90 (t, 3H, J ) 7.2, 14.5), 1.21 (m,
2H), 1.29 (m, 2H), 1.37 (s, 9H), 1.40 (m, 2H), 2.75 (dd, 1H, J )
9.2, 13.5), 2.91 (dd, 1H, J ) 7.7, 13.5), 2.97-3.06 (m, 3H), 3.12
(m, 1H), 3.75 (s, 3H), 4.24 (dd, 1H, J ) 5.3, 9.2), 4.51 (app t,
1H, J ) 7.1, 14.1), 4.87 (s, 2H), 6.98 (d, 1H, J ) 8.6), 7.14-
7.27 (m, 5H), 7.44 (dd, 1H, J ) 1.9, 8.6), 7.49 (d, 1H, J ) 1.9).
13C NMR (MeOH-d4): δ 14.3, 23.4, 28.6, 30.0, 30.1, 37.7, 39.0,
δ 0.87 (3H, t, J ) 7.0, 14.3), 1.18-1.38 (6H, m), 1.41 (9H, s),
3.00 (1H, dd, J ) 6.8, 13.8), 3.08 (1H, dd, J ) 7.6, 13.8), 3.17
(2H, m), 3.89 (3H, s), 4.26 (1H, m), 4.85 (2H, s), 5.11 (1H, br
s), 5.94 (1H, br m), 7.06 (1H, d, J ) 8.4), 7.39 (1H, dd, J ) 2.2,
8.4), 7.72 (1H, d, J ) 2.2). 13C NMR (CDCl3): δ 13.9, 22.3, 28.2,
28.9, 29.0, 37.6, 39.5, 52.3, 55.7, 80.4, 115.0, 116.8, 122.1, 132.6,
133.0, 134.7, 155.1, 156.0, 165.5, 170.6. MS (ESI) m/z 446 (M
- H). Anal. (C23H33N3O6) C, H.
Meth yl 5-[(2S)-2-[(ter t-Bu toxyca r bon yl)a m in o]-3-oxo-
3-(p en tyla m in o)p r op yl]-2-(1(2)H-tetr a zol-5-yl-m eth oxy)-
ben zoa te (31). To a suspension of 30 (0.21 g, 0.48 mmol) in
toluene (1.1 mL), in a heavy-walled Pyrex tube fitted with a
screw cap, were added trimethylsilyl azide (127 µL, 0.96 mmol)
and dibutyltin oxide (12 mg, 0.05 mmol). The mixture was
flushed with nitrogen, tightly sealed, and stirred at 110 °C
for 16 h. Upon cooling, the volatiles were evaporated under
reduced pressure. The residue was purified by column chro-
matography (SiO2, gradient: 5% MeOH in CH2Cl2 to 5%
MeOH and 1% HOAc in CH2Cl2), which afforded 86 mg (37%)
1
of compound 31 as a white solid. H NMR 400 MHz (MeOH-

1796 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9
Liljebris et al.
d4): δ 0.88 (t, 3H, J ) 7.0, 14.2), 1.23 (m, 2H,), 1.31 (m, 2H),
1.35 (s, 9H), 1.42 (m, 2H), 2.82 (dd, 1H), 3.05 (dd, 1H), 3.12
(m, 2H), 3.83 (s, 3H), 4.21 (br m, 1H), 5.51 (s, 2H), 7.17 (d,
1H, J ) 8.6), 7.42 (dd, 1H, J ) 2.2, 8.6), 7.69 (dd, 1H, J )
2.2), 7.93 (br m, 1H). 13C NMR (MeOH-d4): δ 15.7, 24.7, 29.9,
31.3, 31.4, 39.8, 41.7, 54.0, 58.7, 63.7, 81.9, 117.8, 123.5, 134.1,
134.9, 137.3, 156.5, 158.7, 158.8, 169.3, 175.0. MS (ESI) m/z
491 (M + H). Anal. (C23H34N6O6) C, H, N.
174.4, 174.9, 176.7. MS (ESI) m/z 446 (M - H). Anal.
(C22H29N3O7‚1.5H2O) C, H, N.
[2-(5-Oxo-2,3-d ih yd r o-5H-ben zo[e][1,4]d ioxep in -7-yl)-
(1S)-1-p en tylca r ba m oyl-eth yl]ca r ba m ic Acid ter t-Bu tyl
Ester (36). To a solution of 4 (359 mg, 0.74 mmol) (can also
use 5) were added 2-bromoethanol (157.8 µL, 2.23 mmol) and
K2CO3 in DMF (3 mL) in a Heck vial. The vial was tightly
sealed, and the mixture was stirred at 80 °C overnight. The
reaction mixture was cooled and diluted with EtOAc (5 mL).
The organic layer was washed with 10% aqueous HCl, dried
(Na2SO4), and concentrated. Purification was by flash chro-
matography (SiO2, EtOAc/n-hexane 1:1), which furnished 65
Meth yl 5-[(2S)-2-({(2S)-2-[(ter t-Bu toxycar bon yl)am in o]-
3-ph en ylpr opan oyl}am in o)-3-oxo-3-(pen tylam in o)pr opyl]-
2-(1(2)H-tetr a zol-5-yl-m eth oxy)ben zoa te (32). The reaction
was carried out with 31 (40 mg, 0.10 mmol) according to the
general Boc deprotection and carbodiimide reaction procedure.
The crude product was purified by chromatography (SiO2,
gradient: 5% MeOH in CH2Cl2 to 5% MeOH and 1% HOAc in
CH2Cl2), which gave 21 mg (32%) of compound 32 as a white
solid. 1H NMR 400 MHz (MeOH-d4): δ 0.88 (t, 3H, J ) 7.1,
14.4), 1.20 (m, 2H), 1.28 (m, 2H), 1.35 (s, 9H), 1.37 (m, 2H),
2.75 (dd, 1H), 2.90-3.08 (m, 2H), 3.12 (m, 2H), 3.82 (s, 3H),
4.22 (dd, 1H, J ) 5.1, 9.3), 4.50 (br m, 1H), 5.47 (s, 2H), 7.17-
7.25 (m, 6H), 7.40 (d, 1H), 7.64 (d, 1H). 13C NMR (MeOH-d4):
δ 13.0, 22.1, 27.3, 28.2, 28.6, 28.8, 36.7, 37.7, 39.2, 51.3, 54.5,
56.5, 61.3, 79.5, 115.1, 121.0, 126.4, 128.0, 128.1, 128.2, 129.0,
129.1, 130.7, 132.3, 134.6, 137.1, 154.8, 156.4, 166.8, 171.1.
172.7. MS (ESI) m/z 636 (M - H). HRMS (EI) calcd for
1
mg (21%) of 36 as a white solid. H NMR 400 MHz (CDCl3):
δ 0.87 (t, 3H), 1.23 (m, 2H), 1.28 (m, 2H), 1.40 (s, 9H), 1.41
(partly obscured by singlett, m, 2H), 2.97 (dd, 1H), 3.09 (dd,
1H), 3.19 (m, 2H), 4.28 (m, 1H), 4.47 and 4.47 (two m, 2H +
2H, coupling according to COSY), 5.12 (broad m, 1H), 6.06
(broad t, 1H), 6.94 (d, 1H, J ) 8.4 Hz), 7.36 (dd, 1H, J ) 2.1,
8.4 Hz), 7.71 (d, 1H, J ) 2.1 Hz). 13C NMR (CDCl3): δ 13.9,
22.3, 28.9, 29.1, 37.5, 39.5, 55.7, 65.6, 70.8, 118.8, 121.3, 131.4,
134.1, 136.1, 153.7, 155.4, 169.0, 170.7. MS (ESI) m/z 421 (M
+ H). Anal. (C22H32N2O6) C, H, N.
N-[2-(5-Oxo-2,3-d ih yd r o-5H -b en zo[e][1,4]d ioxep in -7-
yl)-(1S)-1-p en tylca r ba m oyl-eth yl]su ccin a m ic Acid (37).
Compound 37 was prepared from 36 (43 mg, 0.13 mmol)
according to the general method described for the Boc depro-
tection and carbodiimide coupling using succinic anhydride.
Purification of the crude product by flash chromatograpy (SiO2,
gradient: MeOH/CH2Cl2 5:95 to HOAc/MeOH/CH2Cl2 1:5:94)
afforded 26 mg (42%) of 37 as a white solid. 1H NMR 400 MHz
(MeOH-d4): δ 0.91 (t, 3H), 1.25 (m, 2H), 1.32 (m, 2H), 1.45
(m, 2H), 2.4-2.6 (2m, 2H+2H), 2.88 (dd, 1H, J ) 8.8, 13.8 Hz),
3.1-3.22 (m, 3H), 4.50 (s, 4H), 4.53 (m, 1H), 6.99 (d, 1H, J )
8.4 Hz), 7.45 (dd, 1H, J ) 2.3, 8.4 Hz), 7.70 (d, 1H, J ) 2.3
Hz), 7.96 (broad m, 1H). 13C NMR (MeOH-d4): δ 16.6, 25.6,
32.2, 32.3, 32.4, 33.6, 40.1, 42.7, 58.3, 69.3, 74.5, 122.8, 124.6,
135.4, 137.0, 139.4, 157.5, 173.8, 175.2, 176.8, 178.6. MS (ESI)
m/z 421 (M + H). Anal. (C21H28N2O7) C, H, N.
C
32H43N7O7 + Na, 660.3122; found, 660.3100.
5-[(2S )-2-({(2S )-2-[(t er t -Bu t oxyc a r b on yl)a m in o]-3-
p h en ylp r op a n oyl}a m in o)-3-oxo-3-(p en tyla m in o)p r op yl]-
2-(1(2)H-tetr a zol-5-yl-m eth oxy)ben zoic Acid (33). To a
solution of 32 (6.7 mg, 0.01 mmol) in THF (0.2 mL) was added
a 2.5 M aqueous solution of LiOH (13 µL, 0.03 mmol), and the
mixture was stirred at ambient temperature for 2 h. The
reaction mixture was then acidified with 3 M aqueous HCl
and extracted with EtOAc (2 mL). The organic layer was dried
(Na2SO4) and concentrated to afford 6 mg (99%) of compound
1
33 as a white solid. H NMR 400 MHz (MeOH-d4): δ 0.88 (t,
3H, J ) 7.1, 14.4), 1.2-1-4 (m, 6H), 1.35 (s, 9H), 2.78 (m,
1H), 2.95-3.18 (m, 5H), 4.22 (dd, 1H), 4.52 (br m, 1H), 5.53
(s, 2H), 7.16-7.27 (m, 6H), 7.42 (dd, 1H), 7.72 (d, 1H). 13C NMR
(MeOH-d4): δ 14.6, 23.6, 28.9, 29.8, 30.2, 30.4, 31.2, 38.3, 39.3,
40.8, 56.1, 58.0, 62.8, 81.2, 116.7, 123.0, 128.0, 128.2, 129.6,
129.7, 129.8, 130.6, 130.7, 132.7, 134.3, 136.2, 138.7, 157.9,
169.6, 172.7, 174.3. MS (ESI) m/z 622 (M - H). HRMS (EI)
calcd for C31H41N7O7 + Na, 646.2965; found, 646.2951.
5-[(1S)-2-(3-Car boxy-pr opion ylam in o)-2-pen tylcar bam -
oyl-eth yl]-2-(2-h yd r oxyeth oxy)ben zoic Acid (38). To a
solution of 37 (20 mg, 0.048 mmol) in THF (0.6 mL) was added
a 2.5 M aqueous solution of LiOH (38 µL, 0.095 mmol). The
mixture was stirred at ambient temperature for 2.5 h. The
mixture was diluted with EtOAc (3 mL) and extracted with
saturated aqueous NaHCO3 (2 × 3 mL). The water layer was
acidified with 3 M aqueous HCl and extracted with EtOAc (2
× 2 mL). The organic layer was dried (Na2SO4) and concen-
(S)-N-[2-(4-Ca r boxym eth oxy-3-cya n o-p h en yl)-1-p en tyl-
ca r ba m oyl-eth yl]su ccin a m ic Acid (34). Compound 34 was
prepared from 26 according to the general method described
for the Boc deprotection and carbodiimide coupling using
succinic anhydrid. Hydrolysis of the methylester using LiOH
in THF afforded the target compound 34 in 82% yield as a
1
trated to afford 16 mg (75%) of 38 as a white solid. H NMR
400 MHz (MeOH-d4): δ 0.91 (t, 3H), 1.23 (m, 2H), 1.31 (m,
2H), 1.43 (m, 2H), 2.4-2.62 (2m, 2H+2H), 2.88 (dd, 1H), 3.05-
3.2 (m, 3H), 3.89 (m, 2H), 4.21 (m, 2H), 4.53 (m, 1H), 7.10 (d,
1H, J ) 8.5 Hz), 7.42 (dd, 1H, J ) 2.3, 8.5 Hz), 7.76 (d, 1H, J
) 2.3 Hz), 7.93 (broad m, 1H). 13C NMR (MeOH-d4): δ 16.8,
25.8, 31.9, 32.2, 32.4, 32.5, 33.8, 40.3, 42.9, 58.7, 63.3, 74.9,
117.8, 123.5, 133.8, 136.2, 138.4, 161.2, 171.9, 175.5, 176.9,
178.7. MS (ESI) m/z 439 (M + H). Anal. (C21H30N2O8) C, H, N.
[(1S)-2-(3-Acetyl-4-h ydr oxy-ph en yl)-1-pen tylcar bam oyl-
eth yl]ca r ba m ic Acid ter t-Bu tyl Ester (39). In an oven-
dried, 25 mL, heavy-walled, and thin-necked Pyrex tube fitted
with a screw cap were combined 3 (440 mg, 0.92 mmol),
palladium(II) acetate (6.2 mg, 3 mol %), 1,3-bis(diphenylphos-
phino)propane (DPPP, 23 mg, 6 mol %), and thallium(I) acetate
(263 mg, 0.998 mmol). The mixture was suspended in DMF
(2 mL), and butyl vinyl ether (0.26 mL, 1.85 mmol) was added.
The flask was purged with nitrogen and tightly sealed, and
the reaction mixture was stirred 90 °C for 16 h. The mixture
was to cooled to room temperature, diluted with THF (2 mL),
and treated with 10% aqueous HCl (2 mL). After it was stirred
for 30 min, the mixture was extracted with EtOAc (2 × 2 mL)
and washed with brine (3 × 2 mL). The organic layer was dried
(MgSO4) and concentrated. The residue was purified by flash
chromatography (SiO2, EtOAc/n-hexane 1:2), which furnished
150 mg (41%) of 39 as a colorless oil. 1H NMR 400 MHz
(CDCl3): δ 0.86 (t, 3H, J ) 7.1, 14.4), 1.16 (m, 2H), 1.26 (m,
1
white solid. H NMR 400 MHz (MeOH-d4): δ 0.92 (t, 3H, J )
7.0, 14.3 Hz), 1.24 (m, 2H), 1.33 (m, 2H), 1.45 (m, 2H), 2.43-
2.61 (m, 4H), 2.87 (dd, 1H, J ) 8.5, 13.9 Hz), 3.08-3.17 (m,
3H), 4.53 (dd, 1H, J ) 6.3, 8.5), 4.85 (s, 2H), 7.00 (d, 1H, J )
8.7 Hz), 7.49 (dd, 1H, J ) 2.1, 8.7 Hz), 7.53 (1H, d, J ) 2.1
Hz). 13C NMR (MeOH-d4): δ 14.7, 23.8, 30.4, 30.4, 30.5, 31.7,
37.8, 40.9, 56.4, 66.6, 103.2, 114.2, 117.5, 132.7, 135.8, 137.0,
160.5, 171.8, 173.2, 175.0, 176.7. MS (ESI) m/z 432 (M - H).
Anal. calcd for C21H27N3O7‚1/2H2O: C, 57.01; H, 6.38; N, 9.49.
Found: C, 57.0; H, 6.4; N, 8.7.
5-[(2S)-2-(3-Car boxy-pr opion ylam in o)-2-pen tylcar bam -
oyl-eth yl]-2-cya n om eth oxy-ben zoic Acid (35). Compound
35 was prepared from 30 according to the general method
described for the Boc deprotection and carbodiimide coupling
using succinic anhydrid. Hydrolysis of the methylester using
LiOH in THF afforded the target compound 35 in 82% yield
1
as a white solid. H NMR 400 MHz (MeOH-d4): δ 0.90 (t, 3H,
J ) 7.2, 14.5 Hz), 1.22 (m, 2H), 1.31 (m, 2H), 1.42 (m, 2H),
2.40-2.60 (m, 4H), 2.89 (dd, 1H, J ) 8.5, 13.8 Hz), 3.08-3.19
(m, 3H), 4.52 (m, 1H), 4.60 (s, 2H), 7.05 (d, 1H, J ) 8.5 Hz),
7.46 (dd, 1H, J ) 2.3, 8.5 Hz), 7.83 (d, 1H, J ) 2.3 Hz). 13C
NMR (MeOH-d4): δ 14.7, 23.8, 30.3, 30.5, 30.5, 31.8, 38.3, 40.8,
56.6, 69.2, 115.7, 121.5, 132.3, 134.5, 136.5, 158.4, 169.3, 173.4,

Inhibitors of Protein Tyrosine Phosphatase 1B
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 9 1797
2H), 1.40 (m, 2H), 1.42 (s, 9H), 2.62 (s, 3H), 3.01 (m, 2H), 3.16
(m, 2H), 4.25 (m, 1H), 5.05 (br s, 1H), 5.81 (br m, 1H), 6.91 (d,
1H, J ) 8.5), 7.32 (dd, 1H, J ) 8.5, 2.0), 7.56 (d, 1H, J ) 2.0).
13C NMR (CDCl3): δ 13.9, 22.3, 26.7, 28.3, 28.9, 29.1, 37.7,
39.5, 56.0, 80.0, 118.6, 119.6, 127.2, 131.3, 131.3, 137.5, 161.3,
170.7, 204.4. Anal. (C21H32O5N2) C, H.
Ack n ow led gm en t. We are grateful to Tuulikki
Lindmark for performing the Caco-2 cell permeability
analyses on selected compounds.
Su p p or tin g In for m a tion Ava ila ble: Combustion analy-
ses for new compounds. Crystallography materials and meth-
ods for PTP1B-compound 29 complex. This material is
available free of charge via the Internet at http://pubs.acs.org.
[(1S)-2-Acetyl-4-(2-ter t-bu toxyca r bon yla m in o-2-p en tyl-
ca r ba m oyl-eth yl)p h en oxy]a cetic Acid Meth yl Ester (40).
A mixture of 39 (294 mg, 0.75 mmol), methyl bromoacetate
(142 µL, 1.50 mmol), and freshly ground K2CO3 (207 mg, 1.50
mmol) was suspended in acetone (4 mL). The mixture was
stirred at room temperature for 48 h, after which time H2O (2
mL) was added and the mixture was extracted with EtOAc (3
mL). The organic layer was washed with brine (2 mL), dried
(MgSO4), and concentrated. The residue was purified by flash
chromatography (SiO2, EtOAc/n-hexane 1:1), which furnished
126 mg (36%) of 40 as a white solid. 1H NMR 400 MHz
(CDCl3): δ 0.87 (t, 3H, J ) 7.1, 14.3), 1.21 (m, 2H), 1.28 (m,
2H), 1.39 (m, 2H), 1.40 (s, 9H), 2.69 (s, 3H), 2.98 (m, 1H), 3.04
(m, 1H), 3.17 (m, 2H), 3.81 (s, 3H), 4.23 (m, 1H), 4.71 (s, 2H),
5.03 (br s, 1H), 5.86 (br m, 1H), 6.75 (d, 1H, J ) 8.6), 7.31 (dd,
1H, J ) 2.3, 8.6), 7.59 (d, 1H, J ) 2.3). 13C NMR (CDCl3): δ
13.9, 22.3, 28.2, 28.9, 29.1, 32.0, 37.4, 39.5, 52.4, 55.8, 65.4,
80.3, 112.5, 128.6, 130.3, 131.4, 134.4, 155.9, 168.6, 170.7,
199.3. Anal. (C24H36O7N2) C, H.
Refer en ces
(1) Kusari, J .; Kenner, K. A.; Suh, K.-I.; Hill, D. E.; Henry, R. R.
Skeletal Muscle Protein Tyrosine Phosphatase Activity and
Tyrosine Phosphatase 1B Protein Content Are Associated with
Insulin Action and Resistance. J . Clin. Invest. 1994, 93, 1156-
1162.
(2) Ahmad, F.; Azevedo, J . L.; Cortright, R.; Dohm, G. L.; Goldstein,
B. J . Alterations in Skeletal Muscle Protein-Tyrosine Phos-
phatase Activity and Expression in Insulin-Resistant Human
Obesity and Diabetes. J . Clin. Invest. 1997, 100, 449-458.
(3) (a) DeFronzo, R. A.; Bonadonna, R. C.; Ferrannini, E. Patho-
genesis of NIDDM. Diabetes Care 1992, 15, 318-368. (b) Reaven,
G. M. Role of insulin resistance in the pathophysiology of
noninsulin dependent diabetes mellitus. Diabetes/ Metab. Rev.
1993, 9 (Suppl. 1), 5S-12S.
(4) Kahn, C. R. Insulin action, diabetogenes, and the cause of type
II diabetes. Diabetes 1994, 43, 1066-1084.
(5) Evans, J . L.; J allal, B. Protein Tyrosine Phosphatases; Their Role
in Insulin Action and Potential as Drug Targets. Exp. Opin.
Invest. Drugs 1999, 8, 139-160.
(6) Bandyopadhyay, D.; Kusari, A.; Kenner, K. A.; Liu, F.; Chernoff,
J .; Gustafson, T. A.; Kusari, J . Protein-Tyrosine Phosphatase
1B Complexes with the Insulin Receptor in vivo and Is Tyrosine-
Phosphorylated in the Presence of Insulin. J . Biol. Chem. 1997,
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N-[(1S)-2-(3-Acetyl-4-m eth oxyca r bon ylm eth oxy-p h en -
yl)-1-p en tylca r ba m oyl-eth yl]su ccin a m ic Acid (41). Trif-
luoroacetic acid (0.16 mL, 2.1 mmol) was carefully added to a
stirring solution of 40 (116 mg, 0.25 mmol) in CH2Cl2 (3 mL)
at 0 °C. The mixture was stirred for 3 h allowing the solution
to warm to ambient temperature. The volatiles were removed
in vacuo, and the residue was partitioned between EtOAc (3
mL) and saturated aqueous NaHCO3 (3 mL). The organic layer
was dried (MgSO4) and concentrated to dryness to afford 94
mg (>100%) of the crude amine as a colorless oil. The amine
was dissolved in CH2Cl2 (3 mL) and cooled with ice to 0 °C.
Succinic anhydride (25 mg, 0.25 mmol) and triethylamine (77
µL, 0.55 mmol) were added, and the mixture was stirred for
16 h allowing the solution to warm to ambient temperature.
The mixture was diluted with CH2Cl2 (5 mL), and the organic
phase was washed with 10% aqueous HCl (2 × 3 mL), dried
(MgSO4), and concentrated. The residue was purified by flash
chromatography (SiO2, mobile impurities were eluted with 5%
MeOH in CH2Cl2 and then 5% MeOH/1% HOAc in CH2Cl2 to
bring the product). The collected fractions were concentrated,
and the remaining HOAc was removed by azeotroping with
toluene on a rotavapor and then drying overnight under high
vacuum, which furnished 75 mg (64%) of 41 as a white/yellow
solid. 1H NMR 400 MHz (MeOH-d4): δ 0.91 (t, 3H, J ) 7.1,
14.4), 1.23 (m, 2H), 1.31 (m, 2H), 1.43 (m, 2H), 2.38-2.59 (m,
4H), 2.68 (s, 3H), 2.86 (dd, 1H, J ) 8.7, 13.8), 3.09-3.17 (m,
3H), 3.80 (s, 3H), 4.51 (dd, 1H, J ) 6.1, 8.7), 4.87 (s, 2H), 6.97
(d, 1H, J ) 8.6), 7.40 (dd, 1H, J ) 2.4, 8.6), 7.59 (d, 1H, J )
2.4), 7.93 (br t, 1H), 8.25 (br d, 1H). 13C NMR (MeOH-d4): δ
14.7, 23.8, 30.3, 30.5, 30.6, 31.8, 32.5, 38.2, 41.0, 53.0, 56.6,
66.6, 114.4, 129.8, 132.2, 132.4, 136.1, 158.0, 170.9, 173.5,
175.0, 176.8, 202.3. MS (ESI) 463 (M - H). Anal. (C23H32O8N2)
C, H.
N-[(1S)-2-(3-Acetyl-4-ca r boxym eth oxy-p h en yl)-1-p en -
tylca r ba m oyl-eth yl]su ccin a m ic Acid (42). A solution of 41
(65 mg, 0.14 mmol) and 2.5 M aqueous LiOH (223 µL, 0.56
mmol) in THF/MeOH/H2O 3:1:1 (3 mL) was stirred at ambient
temperature for 16 h. The reaction mixture was acidified with
10% aqueous HCl and extracted with EtOAc (4 × 2 mL). The
organic layer was dried (MgSO4) and concentrated to dryness,
which furnished 37 mg (59%) of 42. 1H NMR 500 MHz (MeOH-
d4): δ 0.88 (t, 3H, J ) 7.2, 14.5), 1.22 (m, 2H), 1.29 (m, 2H),
1.40 (m, 2H), 2.38-2.58 (m, 4H), 2.66 (s, 3H), 2.84 (dd, 1H, J
) 8.5, 13.9), 3.08-3.17 (m, 3H), 4.49 (dd, 1H, J ) 6.3, 8.5),
4.77 (s, 2H), 6.95 (d, 1H, J ) 8.5), 7.37 (dd, 1H, J ) 2.3, 8.5),
7.56 (d, 1H, J ) 2.3). 13C NMR (MeOH-d4): δ 14.3, 23.4, 29.9,
30.11, 30.14, 31.4, 32.1, 37.9, 40.5, 56.2, 66.3, 114.1, 129.4,
131.6, 132.0, 135.7, 157.8, 171.9, 173.1, 174.5, 176.3, 202.1.
MS (ESI) 449 (M - H). Anal. (C22H30O8N2) C, H.
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J M011100Y