Structure-based optimization of protein tyrosine phosphatase 1B inhibitors: From the active site to the second phosphotyrosine binding site

J. Med. Chem. 2007, 50, 4681-4698

4681

Structure-Based Optimization of Protein Tyrosine Phosphatase 1B Inhibitors: From the Active

Site to the Second Phosphotyrosine Binding Site

Douglas P. Wilson,^†Zhao-Kui Wan,*^,†Wei-Xin Xu,^†Steven J. Kirincich,^†Bruce C. Follows,^†Diane Joseph-McCarthy,^†

Kenneth Foreman,^†,‡Alessandro Moretto,^†Junjun Wu,^†Min Zhu,^†Eva Binnun,^†Yan-Ling Zhang,^§,|May Tam,^§David V. Erbe,^§

James Tobin,^§Xin Xu, Louis Leung,^∞Adam Shilling,^∞Steve Y. Tam,^†Tarek S. Mansour,^†,∇and Jinbo Lee^†,#

Chemical and Screening Sciences, and CardioVascular and Metabolic Diseases, Wyeth Research, 200 Cambridge Park DriVe, Cambridge,

Massachusetts 02140, Chemical and Screening Sciences, Wyeth Research, 401 Middletown Road, Pearl RiVer, New York 10965,

DiscoVery PK, Wyeth Research, One Burtt Rd, AndoVer, Massachusetts 01810, and Biotransformation, Wyeth Research, 500 Arcola Road,

CollegeVille, PennsylVania 19426

ReceiVed March 5, 2007

Protein tyrosine phosphatase 1B (PTP1B) is a negative regulator of the insulin and leptin receptor pathways

and thus an attractive therapeutic target for diabetes and obesity. Starting with a high micromolar lead

compound, structure-based optimization of novel PTP1B inhibitors by extension of the molecule from the

enzyme active site into the second phosphotyrosine binding site is described. Medicinal chemistry, guided

by X-ray complex structure and molecular modeling, has yielded low nanomolar PTP1B inhibitors in an

efficient manner. Compounds from this chemical series were found to be actively transported into hepatocytes.

This active uptake into target tissues could be one of the possible avenues to overcome the poor membrane

permeability of PTP1B inhibitors.

1. Introduction

Protein tyrosine phosphatase 1B (PTP1B^a) is a negative

regulator of the insulin and the leptin receptor pathways.^1-4Two

independent studies of PTP1B-deficient mice have revealed

phenotypes of enhanced insulin sensitivity, improved glycemic

control, and resistance to high fat diet induced obesity.^5,6

Furthermore, treatment of diabetic mice with PTP1B antisense

oligonucleotides reduced the expression level of the enzyme

and subsequently normalized blood glucose and improved

insulin sensitivity.^7,8Thus PTP1B has been considered as an

attractive therapeutic target for type 2 diabetes and obesity.^9-11

Small molecule inhibitors of this enzyme have been pursued

extensively by both industry and academia.^12-21However,

compounds that have good potency and are readily accessible

to target tissues remain elusive.^22,23

Our PTP1B small molecule inhibitor program started with a

weak but well characterized lead compound, 1, which binds to

the enzyme active site (site A) and has a K_ivalue of 230 µM

(Figure 1).²⁴Early exploration of the inhibitor scaffolds resulted

in the discovery of a tricyclic compound, 2 (K_i) 9.2 µM), which

was ∼25 fold more potent than 1.²⁴This result suggested that

the bicyclic thienopyridine template was not required for enzyme

inhibition. Upon further exploration, it was later discovered that

the pyridyl ring could be completely removed and resulted in

the design and synthesis of 3, a compound that was 70-fold

Figure 1. Comparison of bicyclic, tricyclic, and monocyclic thiophenes

as PTP1B inhibitors.

more potent than our starting lead 1.²⁵An X-ray cocrystal

structure of PTP1B and 3 revealed that the 3′-position was

pointing toward the second phosphotyrosine binding site (site

B), a site that has been shown to offer opportunities for both

potency and selectivity improvement.^26,27Attempts to bridge a

fragment for site A and a fragment for site B using flexible

linkers have produce mixed results.²⁸Although notable success

was achieved by scientists from Abbott Laboratories,^16,29the

use of flexible linkers may have incurred a high degree of

rotational freedom. This in turn may be responsible for the poor

physical-chemical properties for the overall molecules. Our

approach was to build our PTP1B inhibitors from site A toward

site B using rigid functionality in a stepwise manner, in which

atom efficiency could be optimized along the way as guided

by X-ray complex structures. Our structure-based effort resulted

in significant improvements in potency and selectivity and is

discussed herein.

* Corresponding author. Phone: 617-665-5635. Fax: 617-665-5682.

E-mail: zwan@wyeth.com.

^†Chemical and Screening Sciences, Wyeth Research, Cambridge.

^‡Present address: OSI Pharmaceuticals, Inc., 1 Bioscience Park Dr.,

Farmingdale, NY 11735.

2. Chemistry

The synthesis of PTP1B inhibitors started with alkylation of

4 with ethyl bromoacetate or tert-butyl bromoacetate, followed

by Suzuki coupling with various boronic acids to provide 6a-c

in good yields (Scheme 1). The Suzuki coupling reaction was

highly regioselective toward the C5-position when Pd(PPh₃)₄

was used as the catalyst and THF was used as the solvent in

the reactions.²⁵Intermediate 6a was further derivatized according

to standard procedures to give various amides, ureas, sulfona-

mides, carbamates, and alkylamino analogs 7.^30-32Intermediate

^§Cardiovascular and Metabolic Diseases, Wyeth Research, Cambridge.

^|Present address: Merck Research Laboratories, 33 Avenue Louis

Pasteur, Boston, MA 02115.

Discovery PK, Wyeth Research, Andover.

^∞Biotransformation, Wyeth Research, Collegeville.

^∇Chemical and Screening Sciences, Wyeth Research, Pearl River.

^#Present address: Ensemble Discovery Corp., 99 Erie St., Cambridge,

MA 02139.

^aAbbreviations: PTP1B, protein tyrosine phosphatase 1B; TCPTP, T-cell

protein tyrosine phosphatase 1B.

Published on Web 08/17/2007

4682 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19

Scheme 1. Synthesis of Compounds 8-23

Wilson et al.

Table 1. Inhibition Constants of Compounds 8-17 against PTP1B

Enzyme

compd

R

K_i(µM)

8

9

NH₂

OH

1.7

1.0

10

11

12

13

14

15

16

17

OMe

NHC(O)CH₂CH₃

NHC(O)Ph

NHC(O)NHCH(CH₃)₂

NHC(O)OCH₃

NHS(O)₂(4-F-Ph)

NH-benzyl

4.0

2.0

2.1

2.0

0.44

0.47

0.68

O-benzyl

Reagents and conditions: (a) ethyl (or tert-butyl) bromoacetate, K₂CO₃,

DMF, 60 °C (R₁) Et or tBu); (b) Pd(PPh₃)₄, arylboronic acid, KF, THF,

100 °C; (c) X ) NH₂, (i) acid chloride (isocyanate, chloroformate, or

sulfonyl chloride), DIPEA, CH₂Cl₂, rt; (ii) ketone (or aldehyde), NaB-

H(OAc)₃, HOAc, DCE, rt; X ) OH, benzyl bromide, K₂CO₃, DMF,

60 °C; (d) LiOH·H₂O, THF/H₂O, rt.

Final deprotection using LiOH in THF/water completed the

synthesis of compounds 26-55.

3. Results and Discussion

Scheme 2. Synthesis of Compounds 26-55

3.1. Structure-Activity Relationship. The 3′-position of 3

offers a favorable trajectory toward the second phosphotyrosine

binding site, as revealed by previously reported X-ray complexes

of 3 and PTP1B.²⁵To effect the extension of the molecule from

the active site toward the second phosphotyrosine binding site,

an amino or a hydroxyl group was introduced as a functional

handle. Compared to compound 3, a hydrogen bond donor such

as an amino (8) or a hydroxyl group (9) at the 3′ position

provided slight improvements in potency, 2-3-fold, respectively

(Table 1). Removal of the hydrogen-bond donor on compound

9 and replacing it with a methoxy group (10) caused a 4-fold

decrease in inhibitory activity. Furthermore, attempts to increase

the acidity of the NH on compound 8 using amides (e.g., 11,

12), ureas (e.g., 13), and carbamates (e.g., 14) did not translate

into better inhibitory activity. The structure-activity relationship

remained flat and the K_iof these compounds remained around

2 µM. These results suggested that a hydrogen-bond donor,

presumably interacting with Asp48, might be favorable at the

3′-position. However, the directionality of the hydrogen bond

was not as optimal as that between a hydrogen-bond donor (e.g.,

OH or NH) at the 4′-position and the Asp48 residue, which has

been reported previously.²⁵

Some sulfonamide analogs did provide modest improvements

in potency (e.g., 15 vs 8). However, it was not clear whether

the acidity of the NH group or the hydrogen-bonding played a

significant role because the NH-benzyl (16) or O-benzyl (17)

groups offered similar improvement in inhibitory activity. In

the case of compound 17, there is no hydrogen-bond donor at

the 3′-position. Thus, van der Waals interactions between the

inhibitors and the enzyme might be more important. The X-ray

complex structure of 16 and PTP1B provided the first hint of

our success in extending the inhibitor into the second phospho-

tyrosine-binding site (Figure 2). The benzyl side chain of 16

has passed through the “gateway” between the active site and

the second phosphotyroine binding site, which is lined with the

Gly259 residue. Consistent with the structure-activity relation-

ship (SAR) in Table 1, the benzyl group of 16 bends toward

Met258 and forms a van der Waals contact with its side chain.

This is the key contributing factor for the potency improvement

in the case of 16 and 17 (the X-ray complex structure of 17

and PTP1B has also been solved, but is not shown here; the

benzyl group of 17 overlays nicely with that of 16).

Reagents and conditions: (a) tert-butyl 4-oxopiperidine-1-carboxylate

(or tert-butyl 4-formylpiperidine-1-carboxylate), NaBH(OAc)₃, AcOH, DCE,

rt; (b) HCl/EtOAc/MeOH, rt; (c) method A, chloroformate, pyridine, rt;

method B, isocyanate, DMF, rt; method C, sulfonyl chloride, pyridine, rt;

(d) LiOH·H₂O, THF/H₂O, rt.

6b was alkylated with benzyl bromide to give the precursor of

compound 17. Hydrolysis of 6a-c and 7 using LiOH in THF/

water provided the compounds 8-23 in good to excellent yields.

Compounds 26-55 were prepared by following the reaction

steps in Scheme 2. The synthesis started with intermediate 6a

or 6d-f, which were prepared according to the procedures in

Scheme 1. In the synthesis of 6d, methyl 4,5-dichloro-3-

hydroxythiophene-2-carboxylate was prepared by following

literature procedures³³and was subsequently used in the Suzuki

coupling reaction. Reductive amination of 6a (or 6d-f) and

tert-butyl 4-oxopiperidine-1-carboxylate (or tert-butyl 4-formylpi-

peridine-1-carboxylate) gave intermediate 24. Deprotection with

TFA in CH₂Cl₂and subsequent reactions with acid chlorides,

chloroformates, isocyanates, or sulfonyl chlorides in the presence

of DIPEA provided intermediates 25 in good to excellent yields.

NoVel Thiophenes as PTP1B Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4683

Figure 2. X-ray complex structure of 16 and PTP1B. The inhibitor

(colored by element with carbons in purple) and key active site residues

(colored by element with carbons in gray) are shown in a capped stick

representation.

Table 2. Inhibition Constants of Compounds 18-23 against PTP1B

Enzyme

compd

R

K_i(µM)

18

19

20

21

22

23

cyclohexylmethyl

cyclohexyl

cyclopentyl

cycloheptyl

4-tetrahydro-2H-pyranyl

3,3,5,5-tetramethylcyclohexyl

0.20

0.21

0.39

0.12

0.31

0.036

The SAR at the 3′-position has been expanded to include a

variety of cycloalkyl groups (Table 2), in order to optimize van

der Waals interaction with the Met258 side chain. Indeed,

unsaturated cycloalkyl groups (18-21) also increased inhibitory

activity significantly, with K_ivalues 2-4-fold better than that

of the corresponding benzyl analog (16). Cyclohexylmethyl (18)

and cyclohexyl (19) analogs have similar potency (K_i∼ 200

nM), but the latter have better atom efficiency due to the lack

of a methylene spacer. Incorporation of an oxygen atom on the

cyclohexyl ring as in the case of compound 22 resulted in little

change in potency against the PTP1B enzyme.

The X-ray complex structure of 19 and PTP1B revealed the

orientation of the cyclohexyl ring, which lies flat along the

tunnel wall between the active site and the second phosphoty-

rosine binding site (Figure 3A). The side chain of Arg24 adopts

a folded-down conformation to line the outer wall of this tunnel.

In contrast, the Arg24 side chain generally adopts a fold-up

conformation in the apo protein^34,35and in the complex

structures of inhibitors without a side chain into the second

phosphotyrosine binding site (e.g., 3 and PTP1B).²⁵Consistent

with that of compound 16 (Figure 2), van der Waals interactions

between the cyclohexyl group of 19 and the Met258 side chain

play an important role in binding affinity. In addition, the

cocrystal structure revealed opportunities for further improve-

ment of these van der Waals interactions (Figure 3A). There is

a small hydrophobic “hole” next to Met258, a residue against

which the cyclohexyl side chain of 19 packs. Molecular

modeling suggested that a methyl group extending out from

the 3′′-position of the cyclohexyl ring could provide a good fit

into this hydrophobic cavity (Figure 3A,B). To avoid introducing

chirality into the molecule, compound 23 was designed and

synthesized with a symmetric 3,3,5,5-tetramethylcyclohexy-

Figure 3. (A) X-ray structure of 19 (carbons in pink) bound to PTP1B

superimposed with a model for a compound with an additional methyl

(carbons in purple) bound to PTP1B. The small molecules are shown

in capped stick representation, and a molecular surface is shown on

the protein in gray. (B) Model of the compound with the additional

methyl bound to PTP1B. A molecular surface is shown on the protein

in gray with the ligand in a space-filling model colored by element

with carbons in purple. (C) X-ray structure of 23 bound to PTP1B with

a transparent molecular surface in gray on the protein. The inhibitor

and key active site residues (colored by element with carbons in green)

are shown in a capped stick representation.

4684 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19

Wilson et al.

Table 3. Inhibition Constants of Compounds 26-35 against PTP1B

Enzyme

Table 4. Inhibition Constants of Compounds 36-40 against PTP1B

Enzyme

compd

X

R

K_i(µM)

compd

X

R

K_i(µM)

36

37

38

39

40

O

S(O)₂CH₂Ph

S(O)₂Ph

C(O)NHPh

0.024

0.074

0.31

0.007

0.013

26

27

28

29

30

31

32

33

34

35

Br

H

3.8

0.19

0.39

0.30

0.044

0.055

0.004

2.0

0.008

0.00068

NHCH₂

OCH₂

C(O)CH₃

C(O)OCH₃

C(O)NHCH₂Ph

S(O)₂CH₃

S(O)₂Ph

S(O)₂CH₂Ph

Me

Cl

lamino group introduced at the 3′-position of 3. Confirming the

molecular modeling prediction, the presence of the methyl

groups at the 3′′-position of the cyclohexyl ring significantly

improved inhibitory activity against PTP1B. Compound 23 has

a K_iof 36 nM, almost an order of magnitude better than that of

19. More interestingly, the binding mode of 23 to PTP1B was

exactly as predicted by molecular modeling, with a methyl group

nicely filling the hydrophobic “hole” next to Met258 side chain

(Figure 3C).

The design and synthesis of 23 was inspired by structural

information from X-ray complexes. Though a potent and

efficient inhibitor of PTP1B enzyme, compound 23 does not

fully occupy the second phosphotyrosine binding site (Figure

3C). Its 3,3,5,5-tetramethylcyclohexyl side chain is still some

distance from Phe52 and Ala27, the residues that are situated

at the far end of this site. These residues are different in PTP1B

vs in T-cell protein tyrosine phosphatase (TCPTP),^27,36which

may offer an opportunity for selectivity. To extend the molecule

further into the second phosphotyrosine binding site, an ami-

nopiperidine group was introduced at the 3′-position of 3 as a

functional handle (Scheme 2). Compound 26 (K_i) 3.8 µM),

with the piperidine group, was much less active than 19 (K_i)

0.21 µM), which might due to the proximity of its basic nitrogen

to residues Arg24 and Arg254 (Table 3). Neutralizing the basic

nitrogen of 26 with amide (e.g., 27), carbamate (e.g., 28), and

urea (e.g., 29) recovered the inhibitory potency against PTP1B

(K_i∼ 0.2-0.3 µM). What really stood out among the various

modifications was the methylsulfonamide analog 30, with a K_i

of 44 nM, even though it lacked the methyl groups of compound

23. Replacing the methylsulfonamide (30) with a phenylsul-

fonamide (31) did not offer any added benefit. However,

inserting a methylene spacer between the sulfonyl group and

phenyl group resulted in a significant improvement in potency.

Compound 32, the first single digit nanomolar compound in

this chemical series (K_i) 4 nM) was thus obtained. The potency

of this chemical series could be further improved by fine-tuning

the substitution at the 4-position of the thiophene template,

which presumably adjusts the positioning of the benzyl sul-

fonamide side chain in the second phosphotyrosine binding site.

The attempt to synthesize a tetramethylpiperidine sulfonamide

was not successful. Replacing the bromo group (32) with a

hydrogen (33) also resulted in significant loss in potency (almost

3 orders of magnitude). While a methyl group (34) was tolerated

here, a chloro group (35) provided the best potency with

subnanomolar activity against PTP1B (K_i) 0.68 nM, Table

3). In general, replacing the 4-bromo with a 4-chloro group

Figure 4. X-ray structure of 32 bound to PTP1B shown with molecular

surface on the protein colored by electrostatic potential. The inhibitor

(colored by element with carbons in purple) and key active site residues

(colored by element with carbons in gray) are shown in a capped stick

representation.

offered about 4-6-fold improvement in potency (analogs of 23

showed the similar trend; data not shown). Compared to the

original lead (1), this structure-based optimization effort pro-

duced a compound (35) that is >300 000-fold more potent.

Fine-tuning the linker region of 32 was also attempted with

representative examples shown in Table 4. Replacing the NH

linker (32) with an oxygen atom (36) at the 3′-position resulted

in a slight loss of potency (∼6-fold). Inserting a methylene

between the NH and the piperidine benzyl sulfonamide (37)

was not favorable. In the case of compound 37, shortening the

side chain by removing the methylene on the sulfonamide side

(38) resulted in further loss of activity. Surprisingly, replacing

the sulfonamide side chains with a phenyl urea group (39)

regained single-digit nanomolar potency. The NH linker of 39

could be interchanged with an oxygen linker (40) without a

significant loss in potency (Table 4).

The X-ray complex structure of 32 and PTP1B clearly

indicates how this compound interacts with the enzyme (Figure

4). Compound 32 fully occupies both the active site and the

second phosphotyrosine binding site, with the thiophene head

group at the active site and the benzylsulfonamide group at the

second site. One of the sulfonyl oxygens forms a hydrogen bond

with a backbone amide, and the other oxygen interacts with

Arg24 and Arg254 via water-mediated hydrogen bonds. These

hydrogen bond interactions may compensate for the loss of the

van der Waals interaction between the methyl group of 23 and

Met258. This likely explains why compounds 30 and 23 have

similar potency. The benzyl group caps two water molecules

at the second phosphotyroine binding site and interacts with

NoVel Thiophenes as PTP1B Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4685

Table 5. Inhibition Constant of Compounds 3, 23, and 32 against

PTP1B, CD45, LAR, and TCPTP

Table 6. Inhibition Constant of Compounds 41-55 against PTP1B and

TCPTP

K_i(µM)

compd

PTP1B

CD45

LAR

TCPTP

3

23

32

3.2

0.036

0.004

280

151

77

>500

>1,000

>500

1.3

0.026

0.005

K_i(µM)

Phe52 in a edge-to-edge fashion. All these interactions combined

in an additive fashion result in the high affinity of 32 to PTP1B

enzyme.

compd

series

R

PTP1B

TCPTP

41

42

43

44

45

46

47

48

49

50

51

52

53

54

A

B

4-Cl-Ph

3-Cl-Ph

2-Cl-Ph

0.038

0.010

0.005

0.006

0.004

0.006

0.002

0.014

0.001

0.004

0.003

0.010

0.009

0.004

0.002

0.006

0.008

0.002

0.012

0.002

0.006

0.005

0.009

3.2. Selectivity against Other Phosphatases. In addition to

potency improvements, one added benefit for extending PTP1B

inhibitors from the active site into the second phosphotyrosine

binding site could be improving selectivity against other protein

tyrosine phosphatases (PTPases). Assay results of three repre-

sentative compounds, 3, 23, and 32, against four different

phosphatases (PTP1B, CD45, LAR, and TCPTP) are shown in

Table 5. Compound 3, which resides only at the active site,

has excellent selectivity against LAR (>150 fold), good

selectivity against CD45 (∼87 fold), but is more active against

TCPTP (∼2-3-fold more active against TCPTP vs PTP1B).

Compound 23, the side chain of which has entered the second

phosphotyrosine binding site, improves selectivity against CD45

(∼4200-fold). The improved selectivity between PTP1B and

CD45 may be partially due to the differences at the gateway

between the active site and the second phosphotyrosine binding

site of these two enzymes.²⁶PTP1B has a glycine (Gly259)

lining the bottom of the gateway, while CD45 has the bigger

amino acid, leucine. In addition to steric hindrance at the

gateway, other interactions between the inhibitor and the second

phosphotyrosine binding site may also contribute to selectivity.

Compound 32, which fully occupies both the active site and

the second phosphotyrosine binding site, shows the best

selectivity against CD45 (∼19 000-fold). Furthermore, com-

pound 32 has shifted the relative selectivity between PTP1B

and TCPTP when compared to compound 3 (Table 5).

To further probe the second phosphotyrosine binding site and

the relative selectivity between PTP1B and TCPTP, various

substitutions on the phenyl groups of 32 (series A) and 39 (series

B) were examined. Some representative examples are shown

in Table 6. In general, the potency and relative selectivity of

these analogs has the following order: ortho > meta > para

(e.g., 41 vs 42 vs 43). This is consistent with the X-ray complex

structure of 32 and PTP1B (Figure 4), in which the para-position

of the benzyl group packs tightly against the protein surface

and thus does not allow further substitution. Therefore, effort

was concentrated on exploring the effect of ortho-substituents

on potency and relative selectivity between PTP1B and TCPTP.

For analogs of 32 (series A), various substituents are tolerated

(44-50). The best potency (∼1 nM) and relative selectivity (∼2-

fold) was seen in compound 50, with a methylsulfonamide group

at the ortho-position. Analogs of 39 (series B) show a similar

trend (ortho > meta > para, data not shown). Again, relative

selectivity between PTP1B and TCPTP ranges from 2- to 3-fold

(e.g., 51-54). In conclusion, extending the PTP1B inhibitor into

the second phosphotyrosine binding site does improve selectivity

against other PTPases (e.g., CD45). However, the potential of

this site for significantly distinguishing PTP1B from TCPTP

remains a challenge, due to the high homology of these two

enzymes.¹⁵

2-CF₃-Ph

2-Me-Ph

2,6-di-Me-Ph

2-NH₂-Ph

2-NHC(O)Me-Ph

2-NHC(O)NHEt-Ph

2-NHSO₂Me-Ph

2-Cl-Ph

2-OMe-Ph

2-Me-Ph

2,6-di-Me-Ph

Table 7. Concentration of 32 in Plasma, Liver, and Muscle Tissues

from PK Studies (10 mg/kg, ip)

concn (µM)

time (h)

plasma

liver

muscle

1

7

15.2^a

0.37

6.9

0.17

3.5

0.26

^aC_max

.

has good aqueous solubility (>100 µg/mL at pH 7.4 buffer).

However, permeability across biological membranes is limited

due to its polar head group. Its Caco-2 permeability coefficient

(Pe) is less than 1 × 10^-6cm/s. The stability of 32 in liver

microsomes has been profiled across three different species. It

has good stability in human (T_1/2>60 min), mouse (T_1/2>60

min), and rat (T_1/2∼46 min) liver microsomes. Furthermore,

compound 32 shows little inhibition of various cytochrome P450

enzymes (IC₅₀> 500 µM for CYP1A2, CYP2D6, CYP3A4,

CYP2A6, CYP2C8, CYP2C19, and 40 µM for CYP2C9).

Therefore, there appears to be little concern about potential

clinical drug-drug interactions for this compound.

The pharmacokinetics (PK) of 32 has been examined in two

species: male Sprague-Dawley rats and C57/B6 mice. In rats

receiving a single iv (intravenous) bolus injection of 2 mg/kg,

compound 32 has a low plasma clearance (CL ∼ 7 mL/min/

kg) and a long elimination half-life (t_1/2∼ 10 h). This is in good

agreement with the excellent in vitro stability of this compound

in liver microsomes. Further PK studies were carried out in mice

where compound 32 was administered into mice at 10 mg/kg

via ip (intraperitoneal) injection. Compound concentrations in

plasma were evaluated at various time points, and the tissue

concentrations in liver and muscle were measured at 1 and 7 h

after ip injection (Table 7). The C_maxof 32 in plasma was

observed at 1 h after ip administration, with a mean concentra-

tion of 15.2 µM (N ) 3). Surprisingly, a substantial amount of

32 was observed in both liver and muscle tissues at the 1 h

time point, despite the low membrane permeability of this

compound. The corresponding mean ( SD concentration in the

liver and muscle was 6.9 ( 2.1 and 3.5 ( 2.7 µM, respectively.

At the 7 h time point, compound concentrations deceased

significantly in all tissues including plasma to a range of 0.2-

0.4 µM.

3.3. Physical-Chemical Properties, Metabolic Stability,

and Phamacokinetics. The physical-chemical properties of

compound 32 have been evaluated extensively. This compound

4686 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19

Wilson et al.

of these PTP1B inhibitors for better delivery in vivo will be

presented in due course.

5. Experimental Section

5.1. Chemistry. Commercial reagents and solvents were used

as received without further purification. ¹H NMR spectra were

recorded on a Bruker 400 MHz spectrometer. LC-MS data were

collected using a Micromass LCT mass spectrometer with electro-

spray ionization in conjunction with a Waters 2795 LC system.

Liquid chromatography (LC-MS) was performed using a Phe-

nomenex C18 column (Mercury MS Luna 5µ C18(2), 20 × 2 mm)

with mobile phase of 0.1% formic acid (FA) in H₂O (A) and 0.1%

formic acid in MeCN (B) and a gradient of 15-100% B in 3 min

followed by 1.5 min at 100% B. HRMS data were recorded on a

Bruker APEXIII-7T FTMS spectrometer with electrospray ioniza-

tion. Purity of compounds was also analyzed by RF-HPLC using

the following two conditions: (1) H₂O/MeCN/0.1% FA; (2) H₂O/

MeOH/0.1% FA (a gradient of 5-100% B in 7 min followed by 2

min at 100% B).

Figure 5. Uptake of 32 and fexofenadine into rat hepatocytes in the

presence and absence of verapamil. Significant differences in uptake

in the presence of verapamil (p < 0.05, t-test) are denoted with “*”.

3.4. Cellular Uptake. Compound 32 showed low membrane

permeability in the Caco2 assay and thus was expected to have

low accessibility to target tissues such as liver and muscle in

vivo. However, a significant amount of 32 was detected in liver

tissue during the ip PK study in C57/B6 mice (Table 7). This

surprising observation was suggestive of a possible active uptake

mechanism. Since compound 32 exists as an anion under

physiological conditions, it was suspected that one or more

organic anion transporting polypeptides (OATPs) may be

involved in its delivery into liver tissues. To evaluate this

possibility, an experiment was performed to study the uptake

of 32 into primary rat hepatocytes in the absence and presence

of an OATP inhibitor, verapamil.³⁷Fexofenadine, a known

substrate of OATPs, was used as a positive control in this

experiment.^37,38The results of this study are summarized in

Figure 5. The OATP inhibitor, verapamil, caused an 87%

decrease in the uptake of fexofenadine into hepatocytes.

Comparably, a 70% decrease in the uptake of 32 was observed

in the presence of verapamil. This supports the hypothesis that

32 is a substrate of organic anion transporting polypeptides and

is indeed actively transported into hepatocytes. Thus, the high

concentration of 32 in liver tissues during PK studies could be

attributed to its active uptake. In vitro systems such as OATP-

expressing or transfected cells (e.g., HeLa, MDCK or Xenopus

laeVis ooctyes)³⁹may help to further assess the involvement of

different rat (OATP1, 2 or 4) or human (OATP1B1, 1B3 or

2B1) OATPs in the hepatic uptake of 32 or analogs. Results of

in vivo experiments following ip dosing in ob/ob mice were

not reproducible and therefore cannot be used as an in vivo

proof of concept. Further work on the use of prodrugs of these

potent inhibitors for demonstration of in vivo efficacy will be

reported separately.

4,5-Dibromo-3-ethoxycarbonylmethoxythiophene-2-carboxy-

lic Acid Methyl Ester (5). General Procedure of Alkylation.

Potassium carbonate (13.07 g, 94.7 mmol) and ethyl bromoacetate

(10.5 mL, 94.7 mmol) were added to a 150 mL of a DMF solution

of 4,5-dibromo-3-hydroxythiophene-2-carboxylic acid methyl ester

(20 g, 63.1 mmol). The resulting suspension was stirred at 50 °C

overnight. The reaction mixture was cooled to room temperature,

followed by addition of 200 mL of Et₂O. The mixture was filtered

through a pad of Celite to remove any solid materials. Solvents

were evaporated under reduced pressure, and the resulting solid

was dissolved in CH₂Cl₂and loaded onto a pad of silica. The crude

product was purified by flash column chromatography using 1/6

EtOAc/hexane as eluent. 4,5-Dibromo-3-ethoxycarbonylmethox-

ythiophene-2-carboxylic acid methyl ester (24.9 g, 99%) was

1

obtained as a white solid. H NMR (400 MHz, CDCl₃): δ ppm

1.30 (t, J ) 7.07 Hz, 3 H), 3.85 (s, 3 H), 4.27 (q, J ) 7.07 Hz, 2

H), 4.90 (s, 2 H).

4,5-Dibromo-3-tert-butoxycarbonylmethoxylthiophene-2-carbox-

ylic acid methyl ester (4.31 g, 95%) was prepared from 4,5-

dibromo-3-hydroxythiophene-2-carboxylic acid methyl ester (3.17

g, 10 mmol), tert-butyl bromoacetate (1.77 mL, 12 mmol), and K₂-

CO₃(2.76 g, 20 mmol) according to the same alkylation procedure.

¹H NMR (400 MHz, CDCl₃): δ ppm 1.42 (s, 9 H), 3.78 (s, 3 H),

4.75 (s, 2 H). Anal. Calcd for C₁₂H₁₄Br₂O₅S: C, 33.51; H, 3.28;

N, 0. Found: C, 33.60; H, 3.08; N, 0.

5-(3-Aminophenyl)-4-bromo-3-tert-butoxycarbonylmethox-

ythiophene-2-carboxylic Acid Methyl Ester (6a). General Pro-

cedure for Suzuki Coupling Reaction. 4,5-Dibromo-3-tert-

butoxycarbonylmethoxylthiophene-2-carboxylic acid methyl ester

(130 mg, 0.3 mmol), 3-aminophenylboronic acid (66 mg, 0.42

mmol), KF (52.2 mg, 0.9 mmol), and Pd(PPh₃)₄(17.3 mg, 0.015

mmol) in THF (4 mL) were mixed in a sealed tube. The reaction

mixture was heated at 120 °C in a microwave reactor (Personal

Chemistry) for 20 min. The precipitate was removed by filtering

through a pad of Celite and then washed with EtOAc. The crude

product was purified by flash column chromatography using 1/10

EtOAc/hexane as eluent. 5-(4-Aminophenyl)-4-bromo-3-tert-bu-

toxycarbonylmethoxythiophene-2-carboxylic acid methyl ester (93

4. Conclusion

PTP1B is an attractive therapeutic target for type 2 diabetes

and obesity. The nature of its active site, which favors highly

polar molecules, has created difficulties for drug discovery. The

present report demonstrates a successful use of structural

information in guiding the extension of PTP1B inhibitors from

the active site to the second phosphotyrosine binding site to

improve potency and selectivity in an efficient manner. A more

than 300 000-fold improvement in potency was achieved using

this approach (1 vs 32). Furthermore, careful examination of

PK data led to the discovery of an active uptake mechanism of

this class of compounds (e.g., 32) into hepatocytes. This could

be a possible avenue to overcome the low passive diffusion and

thus poor tissue accessibility of polar PTP1B inhibitors. Future

effort in further improving the physical-chemical properties

1

mg, 70%) was obtained as a white solid. H NMR (400 MHz,

CDCl₃): δ ppm 1.50 (s, 9 H), 3.79 (br, s, 2 H), 3.87 (s, 3 H), 4.82

(s, 2 H), 6.75 (dd, J ) 7.96, 2.15 Hz, 1 H), 6.96 (t, J ) 2.02 Hz,

1 H), 7.03 (d, J ) 7.58 Hz, 1 H), 7.23 (t, J ) 7.83 Hz, 1 H), 7.26

(s, 1 H). Anal. Calcd for C₁₈H₂₀BrNO₅S: C, 48.88; H, 4.56; N,

3.17. Found: C, 48.84; H, 4.45; N, 3.10.

4-Bromo-3-ethoxycarbonylmethoxy-5-(3-hydroxyphenyl)th-

iophene-2-carboxylic Acid Methyl Ester (6b). Compound 6b (334

mg, 65%) was prepared by following the general procedure of

Suzuki coupling, using 4,5-dibromo-3-ethoxycarbonylmethox-

ythiophene-2-carboxylic acid methyl ester (500 mg, 1.24 mmol),

Pd(PPh₃)₄(143 mg, 0.12 mmol), KF (216 mg, 3.74 mmol) and

3-hydroxyphenylboronic acid (171 mg, 1.24 mmol) as starting

NoVel Thiophenes as PTP1B Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4687

1

materials. H NMR (400 MHz, CDCl₃): δ ppm 1.32 (t, J ) 7.20

(t, J ) 7.20 Hz, 3 H), 3.87 (s, 3 H), 4.30 (q, J ) 7.07 Hz, 2 H),

4.90 (s, 2 H), 7.14 (m, 3 H), 7.20 (m, 1 H), 7.37 (m, 3 H), 7.85 (m,

2 H).

Hz, 3 H), 3.85 (s, 3 H), 4.30 (q, J ) 7.07 Hz, 2 H), 4.89 (s, 2 H),

6.04 (s, 1 H), 7.18 (m, 2 H), 7.30 (m, 1 H). MS [(+)ESI, m/z]:

437.42 [M + Na]⁺.

5-(3-Benzylaminophenyl)-4-bromo-3-ethoxycarbonylmethox-

ythiophene-2-carboxylic Acid Methyl Ester (7e). General Pro-

cedure for Reductive Amination. To a 2 mL DCE solution of

5-(3-aminophenyl)-4-bromo-3-ethoxycarbonylmethoxythiophene-

2-carboxylic acid methyl ester (83 mg, 0.2 mmol) was added

benzaldehyde (24 µL, 0.24 mmol) followed by acetic acid (20 µL,

0.3 mmol) and sodium triacetoxyborohydride (106 mg, 0.5 mmol).

The reaction mixture was stirred at room temperature overnight.

The reaction mixture was then diluted with CH₂Cl₂, washed with

saturated NaHCO₃, and dried over MgSO₄. The crude product was

purified by flash column chromatography using a gradient of

EtOAc/hexane (5-25% gradient) as eluent. Pure fractions were

combined and evaporated to give 5-(3-benzylaminophenyl)-4-

bromo-3-ethoxycarbonylmethoxythiophene-2-carbox ylic acid meth-

4-Bromo-3-ethoxycarbonylmethoxy-5-(3-propionylaminophe-

nyl)thiophene-2-carboxylic Acid Methyl Ester (7a). General

Procedure for Acylation. To a 1.5 mL CH₂Cl₂solution of 5-(3-

aminophenyl)-4-bromo-3-ethoxycarbonylmethoxythiophene-2-car-

boxylic acid methyl ester (44 mg, 0.1 mmol) were added diiso-

propylethyl amine (27 µL, 0.15 mmol) and propionyl chloride (11

µL, 0.12 mmol). The reaction mixture was stirred at room

temperature for 1 h. The reaction mixture was then diluted with

CH₂Cl₂; washed with dilute aqueous HCl, saturated NaHCO₃, and

brine; and dried over MgSO₄. 4-Bromo-3-ethoxycarbonylmethoxy-

5-(3-propionylaminophenyl)thiophene-2-carboxylic acid methyl

1

ester (45 mg, 90%) was obtained as a yellow oil. H NMR (400

MHz, CDCl₃): δ ppm 1.26 (t, J ) 7.58 Hz, 3 H), 1.51 (s, 9 H),

2.43 (q, J ) 7.58 Hz, 2 H), 3.87 (s, 3 H), 4.82 (s, 2 H), 7.38 (m,

2 H), 7.43 (s, 1 H), 7.63 (m, 1 H), 7.83 (s, 1 H).

1

yl ester (65 mg, 64%) as a yellow solid. H NMR (400 MHz,

CDCl₃): δ ppm 1.31 (t, J ) 7.07 Hz, 3 H), 3.86 (s, 3 H), 4.24 (s,

1 H), 4.29 (q, J ) 7.16 Hz, 2 H), 4.37 (s, 2 H), 4.88 (s, 2 H), 6.70

(m, 1 H), 6.92 (m, 1 H), 6.97 (dd, J ) 7.58, 1.01 Hz, 1 H), 7.23

(t, J ) 7.83 Hz, 1 H), 7.29 (m, 1 H), 7.37 (m, 4 H).

4-Bromo-3-tert-butoxycarbonylmethoxy-5-(3-methoxycarbo-

nylaminophenyl)thiophene-2-carboxylic Acid Methyl Ester (7b).

General Procedure for Carbamate Formation. To a 1.5 mL CH₂-

Cl₂solution of 5-(3-amino-phenyl)-4-bromo-3-tert-butoxycarbon-

ylmethoxythiophene-2-carboxylic acid methyl ester (42 mg, 0.095

mmol) was added diisopropylethylamine (34 µL, 0.19 mmol)

followed by methyl chloroformate (13 µL, 0.17 mmol) and the

reaction mixture was stirred at room temperature overnight. The

reaction mixture was then diluted with CH₂Cl₂, washed with dilute

aqueous HCl and saturated NaHCO₃, and dried over MgSO₄.

Filtration and evaporation provided 4-bromo-3-tert-butoxycarbo-

nylmethoxy-5-(3-methoxycarbonylaminophenyl)thiophene-2-car-

5-(3-Benzyloxyphenyl)-4-bromo-3-ethoxycarbonylmethox-

ythiophene-2-carboxylic Acid Methyl Ester (7f). To a 4 mL

solution of 4-bromo-3-ethoxycarbonylmethoxy-5-(3-hydroxyphe-

nyl)thiophene-2-carboxylic acid methyl ester (50 mg, 0.12 mmol)

in DMF were added benzyl bromide (22 µL, 0.18 mmol) and K₂-

CO₃(33 mg, 0.24 mmol). The resulting suspension was stirred at

60 °C overnight. DMF was evaporated under reduced pressure. CH₂-

Cl₂(20 mL) was then added, and the resulting organic solution

was washed with water twice and brine once and then dried over

anhydrous Na₂SO₄. The crude product was purified by flash column

chromatography to give 5-(3-benzyloxyphenyl)-4-bromo-3-ethoxy-

carbonylmethoxythiophene-2-carboxylic acid methyl ester (52 mg,

86% yield). ¹H NMR (400 MHz, CDCl₃): δ ppm 1.32 (t, J ) 7.20

Hz, 3 H), 3.87 (s, 3 H), 4.29 (q, J ) 7.24 Hz, 2 H), 4.91 (s, 2 H),

5.12 (s, 2 H), 7.06 (m, 1 H), 7.24 (m, 1 H), 7.29 (m, 1 H), 7.39 (m,

6 H).

5-(3-Aminophenyl)-4-bromo-3-carboxymethoxythiophene-2-

carboxylic Acid (8). General Procedure for Hydrolysis. To a 2

mL THF solution of 5-(3-amino-phenyl)-4-bromo-3-tert-butoxy-

carbonylmethoxythiophene-2-carboxylic acid methyl ester (6a; 16

mg, 0.036 mmol) was added 1 mL of 1 N LiOH solution. The

mixture was stirred at room temperature overnight, and then THF

was removed under reduced pressure. Aqueous HCl (1 N) was

added slowly until the acidity of the solution reached pH ∼1. The

precipitate was filtered, washed with water, and dried to give 5-(3-

aminophenyl)-4-bromo-3-carboxymethoxythiophene-2-carboxylic

acid (9.5 mg, 71%) as a white solid. ¹H NMR (400 MHz, CDCl₃):

δ ppm 4.88 (s, 2 H), 6.75 (d, J ) 7.07 Hz, 1 H), 6.88 (d, J ) 7.07

Hz, 1 H), 6.94 (s, 1 H), 7.20 (t, J ) 8.08 Hz, 1 H).

1

boxylic acid methyl ester (40 mg, 84%) as a pale yellow oil. H

NMR (400 MHz, CDCl₃): δ ppm 1.51 (s, 9 H), 3.80 (s, 3 H), 3.87

(s, 3 H), 4.82 (s, 2 H), 6.77 (s, 1 H), 7.35 (dt, J ) 7.64, 1.61 Hz,

1 H), 7.39 (t, J ) 7.83 Hz, 1 H), 7.47 (m, 1 H), 7.73 (s, 1 H).

4-Bromo-3-tert-butoxycarbonylmethoxy-5-[3-(3-isopropylure-

ido)phenyl]thiophene-2-carboxylic Acid Methyl Ester (7c).

General Procedure for Urea Formation. To a 1 mL DMF solution

of 5-(3-aminophenyl)-4-bromo-3-tert-butoxycarbonylmethoxy-

thiophene-2-carboxylic acid methyl ester (35 mg, 0.079 mmol) was

added isopropyl isocyanate (38 µL, 0.4 mmol), and the reaction

mixture was stirred at 60 °C overnight. The reaction mixture was

then cooled to room temperature, diluted with water, and extracted

with EtOAc. The organic phase was washed with water and brine

and dried over MgSO₄. The crude product was purified by flash

column chromatography using a gradient of hexane/EtOAc (0-

30% gradient) as eluent. Pure fractions were combined and

evaporated to give 4-bromo-3-tert-butoxycarbonylmethoxy-5-[3-

(3-isopropylureido)phenyl]thiophene-2-carboxylic acid methyl ester

1

(21 mg, 50%) as a pale yellow oil. H NMR (400 MHz, CDCl₃):

4-Bromo-3-carboxymethoxy-5-(3-hydroxyphenyl)thiophene-

2-carboxylic Acid (9). 4-Bromo-3-ethoxycarbonylmethoxy-5-(3-

hydroxyphenyl)thiophene-2-carboxylic acid methyl ester (6b; 39

mg, 0.09 mmol) was hydrolyzed according to the general procedure

for hydrolysis to give 4-bromo-3-carboxymethoxy-5-(3-hydroxy-

phenyl)-thiophene-2-carboxylic acid (26 mg, 73%) as a white solid.

¹H NMR (400 MHz, DMSO-d₆): δ ppm 4.88 (s, 2 H), 6.89 (m, 1

H), 7.07 (m, 2 H), 7.32 (t, J ) 8.08 Hz, 1 H), 9.84 (s, 1 H).

HRMS: calcd for C₁₃H₉BrO₆S + H⁺, 372.937 60; found (ESI-

FTMS, [M + H]⁺), 372.937 85. RF-HPLC: H₂O/MeCN/0.1%

formic acid, 99%; H₂O/MeCN/0.1% formic acid, 99%.

4-Bromo-3-carboxymethoxy-5-(3-methoxyphenyl)thiophene-

2-carboxylic Acid (10). 4-Bromo-3-ethoxycarbonylmethoxy-5-(3-

methoxyphenyl)thiophene-2-carboxylic acid methyl ester (38 mg,

59%) was synthesized according to the general procedure for Suzuki

coupling, using 3-methoxyphenylboronic acid as the coupling

partner in the reaction. ¹H NMR (400 MHz, CDCl₃): δ ppm 1.33

(m, 3 H) 3.86 (s, 3 H) 3.88 (s, 3 H) 4.30 (q, J ) 7.16 Hz, 2 H)

4.92 (s, 2 H) 6.98 (m, 1 H) 7.22 (m, 2 H) 7.37 (m, 1 H). MS [(+)-

ESI, m/z]: 429.45 [M + H]⁺.

δ ppm 1.08 (d, J ) 6.57 Hz, 6 H), 1.44 (s, 9 H), 3.75 (s, 3 H), 3.93

(m, 1 H), 4.70 (s, 2 H), 5.13 (d, J ) 7.58 Hz, 1 H), 7.14 (m, 2 H),

7.20 (t, J ) 7.83 Hz, 1 H), 7.40 (m, 2 H).

4-Bromo-3-ethoxycarbonylmethoxy-5-[3-(4-fluorobenzene-

sulfonylamino)phenyl]thiophene-2-carboxylic Acid Methyl Ester

(7d). General Procedure for Sulfonamide Formation. To a 1

mL pyridine solution of 5-(3-aminophenyl)-4-bromo-3-ethoxycar-

bonylmethoxythiophene-2-carboxylic acid methyl ester (30 mg,

0.072 mmol) was added 4-fluorobenzenesulfonyl chloride (15 mg,

0.079 mmol), and the reaction mixture was stirred at room

temperature overnight. The reaction mixture was then diluted with

15 mL of 1 N HCl and extracted with EtOAc. The organic layer

was washed with dilute aqueous HCl, saturated NaHCO₃, and brine

and dried over MgSO₄. Filtration and evaporation gave the crude

product, which was purified by preparative TLC (35% EtOAc/

hexane). The product band was isolated to give 4-bromo-3-

ethoxycarbonylmethoxy-5-[3-(4-fluoro-benzenesulfonylamino)phenyl]-

thiophene-2-carboxylic acid methyl ester (48 mg, 86%) as a

1

colorless glassy solid. H NMR (400 MHz, CDCl₃): δ ppm 1.32

4688 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19

Wilson et al.

4-Bromo-3-ethoxycarbonylmethoxy-5-(3-methoxyphenyl)th-

iophene-2-carboxylic acid methyl ester (38 mg, 0.088 mmol) was

then hydrolyzed according to the general procedure for hydrolysis

to give 4-bromo-3-carboxymethoxy-5-(3-methoxy-phenyl)-thiophene-

4-Bromo-3-carboxymethoxy-5-[3-(4-fluorobenzenesulfony-

lamino)phenyl]thiophene-2-carboxylic Acid (15). 4-Bromo-3-

ethoxycarbonylmethoxy-5-[3-(4-fluorobenzenesulfonylamino)phenyl]-

thiophene-2-carboxylic acid methyl ester (7d; 48 mg, 0.084 mmol)

was hydrolyzed according to the general procedure for hydrolysis

to give 4-bromo-3-carboxymethoxy-5-[3-(4-fluorobenzenesulfony-

lamino)phenyl]thiophene-2-carboxylic acid (43 mg, 96%) as a white

solid. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 4.89 (s, 2 H), 7.21

(m, 1 H), 7.32 (m, 1 H), 7.42 (m, 4 H), 7.86 (m, 2 H), 10.61 (s, 1

H). HRMS: calcd for C₁₉H₁₃BrFNO₇S₂+ H⁺, 529.937 36; found

(ESI-FTMS, [M + H]⁺), 529.9384. RF-HPLC: H₂O/MeCN/0.1%

formic acid, 96%; H₂O/MeCN/0.1% formic acid, 95%.

1

2-carboxylic acid (25 mg, 71%) as a white solid. H NMR (400

MHz, DMSO-d₆): δ ppm 3.82 (s, 3 H), 4.88 (s, 2 H), 7.08 (dd, J

) 8.34, 2.27 Hz, 1 H), 7.22 (m, 2 H), 7.45 (t, J ) 7.96 Hz, 1 H).

HRMS: calcd for C₁₄H₁₁BrO₆S + H⁺, 386.953 25; found (ESI-

FTMS, [M + H]⁺), 386.953 45. RF-HPLC: H₂O/MeCN/0.1%

formic acid, 99%; H₂O/MeCN/0.1% formic acid, 99%.

4-Bromo-3-carboxymethoxy-5-(3-propionylaminophenyl)th-

iophene-2-carboxylic Acid (11). 4-Bromo-3-ethoxycarbonylmethoxy-

5-(3-propionylaminophenyl)thiophene-2-carboxylic acid methyl

ester (7a; 45 mg, 0.096 mmol) was hydrolyzed according to the

general procedure for hydrolysis to give 4-bromo-3-carboxymethoxy-

5-(3-propionylaminophenyl)thiophene-2-carboxylic acid (32 mg,

5-(3-Benzylaminophenyl)-4-bromo-3-carboxymethox-

ythiophene-2-carboxylic Acid (16). 5-(3-Benzylaminophenyl)-4-

bromo-3-ethoxycarbonylmethoxythiophene-2-carboxylic acid meth-

yl ester (7e; 40 mg, 0.079 mmol) was hydrolyzed according to the

general procedure for hydrolysis to give 5-(3-benzylaminophenyl)-

4-bromo-3-carboxymethoxythiophene-2-carboxylic acid (28 mg,

1

83%) as an off-white solid. H NMR (400 MHz, DMSO-d₆): δ

ppm 1.09 (t, J ) 7.58 Hz, 3 H), 2.34 (m, 2 H), 4.89 (s, 2 H), 7.33

(s, 1 H), 7.44 (t, J ) 7.83 Hz, 1 H), 7.67 (s, 1 H), 8.03 (s, 1 H),

10.08 (s, 1 H). HRMS: calcd for C₁₆H₁₄BrNO₆S + H⁺, 427.979 80;

found (ESI-FTMS, [M + H]⁺), 427.980 16. RF-HPLC: H₂O/

MeCN/0.1% formic acid, 98%; H₂O/MeCN/0.1% formic acid, 99%.

5-(3-Benzoylaminophenyl)-4-bromo-3-carboxymethox-

ythiophene-2-carboxylic Acid (12). 5-(3-Benzoylaminophenyl)-

4-bromo-3-ethoxycarbonylmethoxythiophene-2-carboxylic acid meth-

yl ester (27 mg, 60%) was prepared as a colorless glassy solid

according to the general procedure of acylation using 5-(3-

aminophenyl)-4-bromo-3-ethoxycarbonylmethoxythiophene-2-car-

boxylic acid methyl ester (36 mg, 0.087 mmol) and benzoyl chloride

1

77%) as a pale yellow solid. H NMR (400 MHz, DMSO-d₆): δ

ppm 4.31 (s, 2 H), 4.85 (s, 2 H), 6.70 (d, J ) 8.84 Hz, 1 H), 6.79

(d, J ) 7.07 Hz, 1 H), 6.83 (s, 1 H), 7.20 (m, 2 H), 7.35 (m, 4 H).

HRMS: calcd for C₂₀H₁₆BrNO₅S + H⁺, 462.000 53; found (ESI-

FTMS, [M + H]⁺), 462.000 91. RF-HPLC: H₂O/MeCN/0.1%

formic acid, 97%; H₂O/MeCN/0.1% formic acid, 97%.

5-(3-Benzyloxyphenyl)-4-bromo-3-carboxymethoxythiophene-

2-carboxylic Acid (17). 5-(3-Benzyloxyphenyl)-4-bromo-3-ethoxy-

carbonylmethoxythiophene-2-carboxylic acid methyl ester (7f; 52

mg, 0.10 mmol) was hydrolyzed according to the general procedure

for hydrolysis to give 5-(3-benzyloxyphenyl)-4-bromo-3-car-

boxymethoxythiophene-2-carboxylic acid (49 mg, >95%) as a white

solid. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 4.86 (s, 2 H), 5.18

(s, 2 H), 7.16 (dd, J ) 7.96, 2.15 Hz, 1 H), 7.23 (dd, J ) 6.44,

1.39 Hz, 1 H), 7.28 (m, 1 H), 7.35 (m, J ) 7.33 Hz, 1 H), 7.44 (m,

5 H). RF-HPLC: H₂O/MeCN/0.1% formic acid, 100%; H₂O/

MeCN/0.1% formic acid, 100%.

1

(14 µL, 0.12 mmol) as starting materials. H NMR (400 MHz,

CDCl₃): δ ppm 1.32 (t, J ) 7.07 Hz, 3 H), 3.88 (s, 3 H), 4.29 (q,

J ) 7.16 Hz, 2 H), 4.91 (s, 2 H), 7.48 (m, 4 H), 7.56 (m, 1 H),

7.76 (m, 1 H), 7.89 (m, 2 H), 7.98 (m, 2 H).

5-(3-Benzoylaminophenyl)-4-bromo-3-ethoxycarbonylmethoxy-

thiophene-2-carboxylic acid methyl ester (27 mg, 0.052 mmol) was

then hydrolyzed according to the general procedure for hydrolysis

to give 5-(3-benzoylaminophenyl)-4-bromo-3-carboxymethox-

ythiophene-2-carboxylic acid (27 mg, >95%) as a pale yellow solid.

¹H NMR (400 MHz, DMSO-d₆): δ ppm 4.90 (s, 2 H) 7.42 (m, 1

H) 7.53 (m, 3 H) 7.61 (m, 1 H) 7.91 (m, 1 H) 7.97 (m, 2 H) 8.21

(t, J ) 1.77 Hz, 1 H) 10.46 (s, 1 H). HRMS: calcd for C₂₀H₁₄-

BrNO₆S + H⁺, 475.979 80; found (ESI-FTMS, [M + H]⁺),

475.980 43. RF-HPLC: H₂O/MeCN/0.1% formic acid, 95%; H₂O/

MeCN/0.1% formic acid, 99%.

4-Bromo-3-carboxymethoxy-5-[3-(cyclohexylmethylamino)-

phenyl]thiophene-2-carboxylic Acid (18). 4-Bromo-5-[3-(cyclo-

hexylmethylamino)phenyl]-3-ethoxycarbonylmethoxythiophene-2-

carboxylic acid methyl ester was obtained in 69% yield as a pale

yellow oil, by following the general procedure for reductive

1

amination. H NMR (400 MHz, CDCl₃): δ ppm 0.99 (m, 2 H),

1.21 (m, 4 H), 1.32 (t, J ) 7.20 Hz, 3 H), 1.70 (m, 5 H), 2.98 (d,

J ) 6.82 Hz, 2 H), 3.87 (s, 3 H), 4.30 (q, J ) 7.24 Hz, 2 H), 4.90

(s, 2 H), 6.65 (m, 1 H), 6.85 (m, 1 H), 6.93 (d, J ) 7.58 Hz, 1 H),

7.22 (t, J ) 7.83 Hz, 1 H).

4-Bromo-3-carboxymethoxy-5-[3-(3-isopropylureido)phenyl]-

thiophene-2-carboxylic Acid (13). 4-Bromo-3-tert-butoxycarbo-

nylmethoxy-5-(3-methoxycarbonylaminophenyl)thiophene-2-car-

boxylic acid methyl ester (7c; 21 mg, 0.04 mmol) was hydrolyzed

according to the general procedure for hydrolysis to give 4-bromo-

3-carboxymethoxy-5-[3-(3-isopropylureido)phenyl]thiophene-2-car-

4-Bromo-5-[3-(cyclohexylmethylamino)phenyl]-3-ethoxycarbo-

nylmethoxythiophene-2-carboxylic acid methyl ester was then

hydrolyzed according to the general procedure for hydrolysis to

give 4-bromo-3-carboxymethoxy-5-[3-(cyclohexylmethylamino)-

phenyl]thiophene-2-carboxylic acid in 59% yield as a pale yellow

solid. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 0.94 (m, 2 H), 1.20

(m, 4 H), 1.67 (m, 5 H), 2.87 (d, J ) 6.57 Hz, 2 H), 4.87 (s, 2 H),

6.66 (dd, J ) 8.21, 1.64 Hz, 1 H), 6.76 (d, J ) 7.58 Hz, 1 H), 6.81

(m, 1 H), 7.17 (t, J ) 7.96 Hz, 1 H). HRMS: calcd for C₂₀H₂₂-

BrNO₅S + H⁺, 468.047 49; found (ESI-FTMS, [M + H]⁺),

468.048 21. RF-HPLC: H₂O/MeCN/0.1% formic acid, 100%; H₂O/

MeCN/0.1% formic acid, 100%.

1

boxylic acid (14 mg, 77%) as a white solid. H NMR (400 MHz,

DMSO-d₆): δ ppm 1.10 (d, J ) 6.57 Hz, 6 H), 3.75 (m, 1 H),

4.88 (s, 2 H), 6.07 (d, J ) 7.83 Hz, 1 H), 7.18 (dd, J ) 6.19, 1.64

Hz, 1 H), 7.35 (t, J ) 7.83 Hz, 1 H), 7.42 (m, 1 H), 7.81 (t, J )

1.77 Hz, 1 H), 8.54 (s, 1 H). HRMS: calcd for C₁₇H₁₇BrN₂O₆S +

H⁺, 457.006 35; found (ESI-FTMS, [M + H]⁺), 457.007 08. RF-

HPLC: H₂O/MeCN/0.1% formic acid, 96%; H₂O/MeCN/0.1%

formic acid, 96%.

4-Bromo-3-carboxymethoxy-5-(3-cyclohexylaminophenyl)th-

iophene-2-carboxylic Acid (19). 4-Bromo-5-(3-cyclohexylami-

nophenyl)-3-ethoxycarbonylmethoxythiophene-2-carboxylic acid

methyl ester (35 mg, 70%) was prepared as a yellow oil, according

to the general procedure for reductive amination, from 5-(3-

aminophenyl)-4-bromo-3-ethoxycarbonylmethoxythiophene-2-car-

boxylic acid methyl ester (41 mg, 0.1 mmol) and cyclohexanone

4-Bromo-3-carboxymethoxy-5-(3-methoxycarbonylaminophe-

nyl)thiophene-2-carboxylic Acid (14). 4-Bromo-3-tert-butoxycar-

bonylmethoxy-5-(3-methoxycarbonylaminophenyl)thiophene-2-

carboxylic acid methyl ester (7b; 40 mg, 0.08 mmol) was

hydrolyzed according to the general procedure for hydrolysis to

give 4-bromo-3-carboxymethoxy-5-(3-methoxycarbonylaminophe-

nyl)thiophene-2-carboxylic acid (9 mg, 26%) as an off-white solid.

¹H NMR (400 MHz, DMSO-d₆): δ ppm 3.69 (s, 3 H), 4.89 (s, 2

H), 7.29 (m, 1 H), 7.43 (t, J ) 7.96 Hz, 1 H), 7.55 (d, J ) 9.60

Hz, 1 H), 7.86 (s, 1 H), 9.90 (s, 1 H). HRMS: calcd for C₁₅H₁₂-

BrNO₇S + H⁺, 429.959 06; found (ESI-FTMS, [M + H]⁺),

429.959 73. RF-HPLC: H₂O/MeCN/0.1% formic acid, 96%; H₂O/

MeCN/0.1% formic acid, 97%.

1

(13 µL, 0.12 mmol). H NMR (400 MHz, CDCl₃): δ ppm 1.21

(m, 3 H), 1.32 (m, 3 H), 1.39 (m, 2 H), 1.66 (m, 1 H), 1.77 (m, 2

H), 2.08 (dd, J ) 12.76, 3.16 Hz, 2 H), 3.28 (m, 1 H), 3.70 (s, 1

H), 3.87 (s, 3 H), 4.30 (q, J ) 7.07 Hz, 2 H), 4.90 (s, 2 H), 6.64

(dd, J ) 7.71, 1.89 Hz, 1 H), 6.85 (m, 1 H), 6.90 (m, 1 H) 7.21 (t,

J ) 7.83 Hz, 1 H).

NoVel Thiophenes as PTP1B Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4689

4-Bromo-5-(3-cyclohexylaminophenyl)-3-ethoxycarbonylmethox-

ythiophene-2-carboxylic acid methyl ester (35 mg, 0.07 mmol) was

then hydrolyzed according to the general procedure for hydrolysis

to give 4-bromo-3-carboxymethoxy-5-(3-cyclohexylaminophenyl)-

thiophene-2-carboxylic acid (26 mg, 83%) as a pale yellow solid.

¹H NMR (400 MHz, DMSO-d₆): δ ppm 1.18 (m, 5 H), 1.34 (m,

2 H), 1.60 (m, 1 H), 1.73 (m, 2 H), 1.95 (m, 1 H), 4.88 (s, 2 H),

6.70 (m, 1 H), 6.77 (m, 1 H), 6.84 (s, 1 H), 7.19 (t, J ) 7.71 Hz,

1 H). HRMS: calcd for C₁₉H₂₀BrNO₅S + H⁺, 454.031 83; found

(ESI-FTMS, [M + H]⁺), 454.0323. RF-HPLC: H₂O/MeCN/0.1%

formic acid, 100%; H₂O/MeCN/0.1% formic acid, 100%.

456.011 53. RF-HPLC: H₂O/MeCN/0.1% formic acid, 98%; H₂O/

MeCN/0.1% formic acid, 99%.

4-Bromo-3-carboxymethoxy-5-[3-(3,3,5,5-tetramethylcyclo-

hexylamino)phenyl]thiophene-2-carboxylic Acid (23). 4-Bromo-

3-ethoxycarbonylmethoxy-5-[3-(3,3,5,5-tetramethylcyclohexylamino)-

phenyl]thiophene-2-carboxylic acid methyl ester was obtained in

56% yield as a pale yellow glassy solid, by following the general

1

procedure for reductive amination. H NMR (400 MHz, CDCl₃):

δ ppm 0.90 (m, 2 H), 0.94 (s, 6 H), 1.05 (m, 2 H), 1.12 (s, 6 H),

1.29 (s, 1 H), 1.32 (t, J ) 7.20 Hz, 3 H), 1.89 (d, J ) 11.87 Hz,

1 H), 3.61 (m, 2 H), 3.87 (s, 3 H), 4.30 (q, J ) 7.16 Hz, 2 H), 4.89

(s, 2 H), 6.63 (dd, J ) 7.07, 1.26 Hz, 1 H), 6.90 (m, 2 H), 7.21 (t,

J ) 7.96 Hz, 1 H).

4-Bromo-3-carboxymethoxy-5-(3-cyclopentylaminophenyl)th-

iophene-2-carboxylic Acid (20). 4-Bromo-5-(3-cyclopentylami-

nophenyl)-3-ethoxycarbonylmethoxythiophene-2-carboxylic acid

methyl ester was obtained in 75% yield as a yellow glassy solid,

4-Bromo-3-ethoxycarbonylmethoxy-5-[3-(3,3,5,5-tetramethylcy-

clohexylamino)phenyl]thiophene-2-carboxylic acid methyl ester was

then hydrolyzed according to the general procedure for hydrolysis

to give 4-bromo-3-carboxymethoxy-5-[3-(3,3,5,5-tetramethylcy-

clohexylamino)phenyl]thiophene-2-carboxylic acid in 66% yield as

a pale yellow solid. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 0.91

(s, 6 H), 0.95 (m, 2 H), 1.09 (m, 7 H), 1.26 (m, 1 H), 1.75 (d, J )

12.38 Hz, 2 H), 3.51 (m, 1 H), 4.87 (s, 2 H), 6.68 (dd, J ) 8.21,

1.64 Hz, 1 H), 6.76 (d, J ) 7.33 Hz, 1 H), 6.84 (s, 1 H), 7.18 (t,

J ) 7.96 Hz, 1 H). Anal. Calcd for C₂₃H₂₈BrNO₅S: C, 54.12; H,

5.53; N, 2.74. Found: C, 54.41; H, 5.21; N, 2.60. RF-HPLC: H₂O/

MeCN/0.1% formic acid, 100%; H₂O/MeCN/0.1% formic acid,

100%.

4-Bromo-3-tert-butoxycarbonylmethoxy-5-[3-(piperidin-4-

ylamino)phenyl]thiophene-2-carboxylic Acid Methyl Ester Hy-

drochloride (24). To a 12 mL DCE solution of 5-(3-amino-phenyl)-

4-bromo-3-tert-butoxycarbonylmethoxythiophene-2-carboxylic acid

methyl ester (1.33 g, 3.0 mmol) were added tert-butyl-4-oxo-1-

piperidinecarboxylate (0.897 g, 4.5 mmol), acetic acid (0.25 mL,

4.5 mmol), and sodium triacetoxyborohydride (1.48 g, 7.0 mmol),

and the resulting mixture was stirred at room temperature overnight.

The reaction mixture was then diluted with CH₂Cl₂, washed twice

with saturated NaHCO₃, and dried over MgSO₄. Filtration and

evaporation gave the crude product, which was purified by flash

column chromatography using a gradient of hexane/EtOAc (5-

35% gradient) as eluent. Pure fractions were combined and

evaporated to give 4-[3-(3-bromo-4-tert-butoxycarbonylmethoxy-

5-methoxycarbonylthiophen-2-yl)phenylamino]piperidine-1-car-

boxylic acid tert-butyl ester (1.62 g, 86%) as a pale yellow foam.

¹H NMR (400 MHz, CDCl₃): δ ppm 1.37 (m, 2 H), 1.47 (s, 9 H),

1.51 (s, 9 H), 2.07 (m, 2 H), 2.94 (t, J ) 12.13 Hz, 2 H), 3.47 (m,

1 H), 3.69 (m, 1 H), 3.87 (s, 3 H), 4.06 (m, 2 H), 4.82 (s, 2 H),

6.65 (m, 1 H), 6.87 (m, 1 H), 6.94 (ddd, J ) 7.64, 1.58, 0.88 Hz,

1 H), 7.23 (t, J ) 7.96 Hz, 1 H).

1

by following the general procedure for reductive amination. H

NMR (400 MHz, CDCl₃): δ ppm 1.32 (t, J ) 7.07 Hz, 3 H), 1.50

(m, 2 H), 1.69 (m, 4 H), 2.05 (m, 2 H), 3.81 (m, 1 H), 3.87 (s, 3

H), 4.30 (q, J ) 7.16 Hz, 2 H), 4.90 (s, 2 H), 6.65 (m, 1 H), 6.87

(m, 1 H), 6.93 (m, 1 H), 7.22 (t, J ) 7.96 Hz, 1 H).

4-Bromo-5-(3-cyclopentylaminophenyl)-3-ethoxycarbonylmethox-

ythiophene-2-carboxylic acid methyl ester was then hydrolyzed

according to the general procedure for hydrolysis to give 4-bromo-

3-carboxymethoxy-5-(3-cyclopentylaminophenyl)thiophene-2-car-

1

boxylic acid in 79% yield as a pale yellow solid. H NMR (400

MHz, DMSO-d₆): δ ppm 1.47 (m, 2 H), 1.56 (m, 2 H), 1.67 (m,

2 H), 1.93 (m, 2 H), 3.71 (m, 1 H), 4.88 (s, 2 H), 6.67 (m, 1 H),

6.77 (d, J ) 1.26 Hz, 1 H), 6.82 (d, J ) 2.02 Hz, 1 H), 7.17 (m,

1 H). HRMS: calcd for C₁₈H₁₈BrNO₅S + H⁺, 440.016 19; found

(ESI-FTMS, [M + H]⁺), 440.017 24. RF-HPLC: H₂O/MeCN/0.1%

formic acid, 100%; H₂O/MeCN/0.1% formic acid, 100%.

4-Bromo-3-carboxymethoxy-5-(3-cycloheptylaminophenyl)th-

iophene-2-carboxylic Acid (21). 4-Bromo-5-(3-cycloheptylami-

nophenyl)-3-ethoxycarbonylmethoxythiophene-2-carboxylic acid

methyl ester was obtained in 72% yield as a pale yellow glassy

solid, by following the general procedure for reductive amination.

¹H NMR (400 MHz, CDCl₃): δ ppm 1.32 (t, J ) 7.07 Hz, 3 H),

1.57 (m, 10 H), 2.03 (m, 2 H), 3.47 (m, 1 H), 3.76 (s, 1 H), 3.87

(s, 3 H), 4.30 (q, J ) 7.16 Hz, 2 H), 4.90 (s, 2 H), 6.60 (dd, J )

8.21, 2.40 Hz, 1 H), 6.81 (t, J ) 1.89 Hz, 1 H), 6.90 (d, J ) 7.58

Hz, 1 H), 7.21 (t, J ) 7.96 Hz, 1 H).

4-Bromo-5-(3-cycloheptylaminophenyl)-3-ethoxycarbonylmethox-

ythiophene-2-carboxylic acid methyl ester was then hydrolyzed

according to the general procedure for hydrolysis to give 4-bromo-

3-carboxymethoxy-5-(3-cycloheptylaminophenyl)thiophene-2-car-

1

boxylic acid in 71% yield as a pale yellow solid. H NMR (400

MHz, DMSO-d₆): δ ppm 1.53 (m, 10 H), 1.90 (t, J ) 6.82 Hz, 2

H), 4.88 (s, 2 H), 6.65 (dd, J ) 8.59, 2.27 Hz, 1 H), 6.77 (m, 2 H),

7.19 (t, J ) 7.83 Hz, 1 H). HRMS: calcd for C₂₀H₂₂BrNO₅S +

H⁺, 468.047 48; found (ESI-FTMS, [M + H]⁺), 468.048 17. RF-

HPLC: H₂O/MeCN/0.1% formic acid, 96%; H₂O/MeCN/0.1%

formic acid, 98%.

4-Bromo-3-carboxymethoxy-5-[3-(tetrahydropyran-4-ylami-

no)phenyl]thiophene-2-carboxylic Acid (22). 4-Bromo-3-tert-

butoxycarbonylmethoxy-5-[3-(tetrahydropyran-4-ylamino)phenyl]-

thiophene-2-carboxylic acid methyl ester was obtained in 83% yield

as a pale yellow glassy solid, by following the general procedure

for reductive amination. ¹H NMR (400 MHz, CDCl₃): δ ppm 1.51

(m, 13 H), 2.06 (m, 2 H), 3.53 (m, 3 H), 3.72 (s, 1 H), 3.87 (s, 3

H), 4.82 (s, 2 H), 6.66 (dd, J ) 7.83, 2.02 Hz, 1 H), 6.88 (m, 1 H),

6.95 (m, 1 H), 7.23 (t, J ) 7.96 Hz, 1 H).

4-[3-(3-Bromo-4-tert-butoxycarbonylmethoxy-5-methoxycarbo-

nylthiophen-2-yl)phenylamino]piperidine-1-carboxylic acid tert-

butyl ester (1.33 g, 2.1 mmol) was dissolved in 10.5 mL of 1 N

HCl in EtOAc (prepared by bubbling dry HCl gas into dry EtOAc)

and 2 mL of MeOH. The reaction mixture was stirred at room

temperature overnight. The reaction mixture was then evaporated

exhaustively to give 4-bromo-3-tert-butoxycarbonylmethoxy-5-[3-

(piperidin-4-ylamino)phenyl]thiophene-2-carboxylic acid methyl

ester hydrochloride in quantitative yield as a white solid. ¹H NMR

(400 MHz, MeOD): δ ppm 1.46 (s, 9 H), 2.00 (m, 2 H), 2.26 (m,

2 H), 3.09 (td, J ) 12.95, 2.65 Hz, 1 H), 3.51 (m, 2 H), 3.84 (s, 3

H), 3.89 (m, 2 H), 4.82 (s, 2 H), 7.47 (m, 1 H), 7.61 (m, 2 H), 7.67

(m, 1 H).

4-Bromo-3-ethoxycarbonylmethoxy-5-[3-(piperidin-4-ylamino)-

phenyl]thiophene-2-carboxylic acid methyl ester hydrochloride salt

was prepared by following the same procedure, except that 5-(3-

aminophenyl)-4-bromo-3-ethoxycarbonylmethoxythiophene-2-car-

boxylic acid methyl ester was used as starting material in the

4-Bromo-3-tert-butoxycarbonylmethoxy-5-[3-(tetrahydropyran-

4-ylamino)phenyl]thiophene-2-carboxylic acid methyl ester was

then hydrolyzed according to the general procedure for hydrolysis

to give 4-bromo-3-carboxymethoxy-5-[3-(tetrahydropyran-4-ylami-

no)phenyl]thiophene-2-carboxylic acid in 84% yield as a pale

1

reductive amination step. H NMR (400 MHz, CD₃OD): δ ppm

1

yellow solid. H NMR (400 MHz, DMSO-d₆): δ ppm 1.39 (m, 2

1.48 (t, J ) 7.20 Hz, 3 H), 1.81-1.94 (m, 2 H), 2.41-2.49 (m, 2

H), 3.30-3.38 (m, 2 H), 3.59-3.66 (m, 2 H), 3.82-3.89 (m, 1 H),

4.03 (s, 3 H), 4.44 (q, J ) 7.07 Hz, 2 H), 5.08 (s, 2 H), 6.94-6.98

(m, 1 H), 7.06-7.09 (m, 1 H), 7.11-7.13 (m, 1 H), 7.39-7.44

(m, 1 H).

H), 1.90 (d, J ) 11.37 Hz, 2 H), 3.44 (m, 4 H), 3.86 (m, 2 H), 4.87

(s, 2 H), 6.72 (dd, J ) 8.34, 2.27 Hz, 1 H), 6.77 (d, J ) 7.58 Hz,

1 H), 6.85 (s, 1 H), 7.19 (t, J ) 7.96 Hz, 1 H). HRMS: calcd for

C₁₈H₁₈BrNO₆S + H⁺, 456.011 10; found (ESI-FTMS, [M + H]⁺),

4690 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19

Wilson et al.

4-[3-(3-Bromo-4-ethoxycarbonylmethoxy-5-methoxycarbon-

ylthiophen-2-yl)phenylamino]piperidine-1-carboxylic Acid Meth-

yl Ester (25a). General Procedure for Carbamate Formation

at the Piperidine Nitrogen. To a 1 mL pyridine solution of

4-bromo-3-ethoxycarbonylmethoxy-5-[3-(piperidin-4-ylamino)phe-

nyl]thiophene-2-carboxylic acid methyl ester hydrochloride salt (60

mg, 0.1 mmol) was added methyl chloroformate (18 µL, 0.24

mmol). The reaction mixture was stirred at room temperature for

5 h, and then 10 mL of 2 N HCl was added and extracted with

EtOAc. The organic layer was washed with saturated NaHCO₃and

brine and dried over MgSO₄. Filtration and evaporation gave the

crude product, which was purified by flash column chromatography

using a gradient of EtOAc/hexane (20-60% gradient) as eluent.

Pure fractions were combined and evaporated to give 4-[3-(3-

bromo-4-ethoxycarbonylmethoxy-5-methoxycarbonylthiophen-2-

yl)phenylamino]piperidine-1-carboxylic acid methyl ester (15 mg,

27%) as a pale glassy solid. ¹H NMR (400 MHz, CDCl₃): δ ppm

1.32 (t, J ) 7.07 Hz, 3 H), 1.41 (m, 2 H), 2.09 (m, 2 H), 3.02 (m,

2 H), 3.48 (m, 1 H), 3.71 (s, 3 H), 3.87 (s, 3 H), 4.11 (m, 2 H),

4.30 (q, J ) 7.07 Hz, 2 H), 4.90 (s, 2 H), 6.66 (m, 1 H), 6.87 (m,

1 H), 6.95 (m, 1 H), 7.23 (t, J ) 7.83 Hz, 1 H).

generalprocedureforhydrolysistogive4-bromo-3-(carboxymethoxy)-

5-[3-(piperidin-4-ylamino)phenyl]thiophene-2-carboxylic acid (46

mg, 49%) as a white solid. H NMR (400 MHz, DMSO-d₆): δ

1

ppm 1.58 (m, 2 H), 2.05 (m, 2 H), 3.03 (m, 2 H), 3.38 (m, 2 H),

3.57 (m, 1 H), 4.77 (s, 2 H), 6.03 (d, J ) 7.58 Hz, 1 H), 6.72 (dd,

J ) 8.08, 1.77 Hz, 1 H), 6.80 (d, J ) 7.58 Hz, 1 H), 6.84 (s, 1 H),

7.20 (t, J ) 7.96 Hz, 1 H), 8.45 (s, 1 H), 8.60 (s, 1 H). RF-HPLC:

H₂O/MeCN/0.1% formic acid, 100%; H₂O/MeCN/0.1% formic

acid, 100%.

5-[3-(1-Acetylpiperidin-4-ylamino)phenyl]-4-bromo-3-car-

boxymethoxythiophene-2-carboxylic Acid (27). 5-[3-(1-Acetyl-

piperidin-4-ylamino)phenyl]-4-bromo-3-ethoxycarbonylmethoxy-

thiophene-2-carboxylic acid methyl ester was obtained in 66% yield

as a yellow oil, by following the general procedure for reductive

amination except that 1-acetylpiperidin-4-one was used as starting

material in the reaction. ¹H NMR (400 MHz, CDCl₃): δ ppm 1.32

(t, J ) 7.20 Hz, 2 H), 1.40 (m, 2 H), 2.17 (m, 6 H), 2.49 (dd, J )

6.57, 2.27 Hz, 1 H), 2.88 (m, 1 H), 3.23 (m, 1 H), 3.55 (s, 1 H),

3.74 (m, 1 H), 3.83 (m, 1 H), 3.88 (s, 3 H), 4.30 (q, J ) 7.16 Hz,

1 H), 4.50 (m, 1 H), 4.91 (s, 2 H), 6.67 (m, 1 H), 6.88 (m, 1 H),

6.96 (m, 1 H), 7.24 (t, J ) 7.83 Hz, 1 H).

5-[3-(1-Benzylcarbamoylpiperidin-4-ylamino)phenyl]-4-bromo-

3-ethoxycarbonylmethoxythiophene-2-carboxylic Acid Methyl

Ester (25b). General Procedure for Urea Formation at the

Piperidine Nitrogen. To a 1 mL DMF solution of 4-bromo-3-

ethoxycarbonylmethoxy-5-[3-(piperidin-4-ylamino)phenyl]thiophene-

2-carboxylic acid methyl ester (50 mg, 0.1 mmol) was added benzyl

isocyanate (14 µL, 0.11 mmol) and the reaction mixture was stirred

at room temperature. After 3 h, the reaction mixture was diluted

with water, acidified with 1 N HCl, and extracted with EtOAc.

The organic layer was washed with saturated NaHCO₃and brine

and dried over MgSO₄. Filtration and evaporation gave the crude

product, which was then purified by flash column chromatography

using a gradient of EtOAc/hexane (20-100% gradient) as eluent.

Pure fractions were combined and evaporated to give 5-[3-(1-

benzylcarbamoylpiperidin-4-ylamino)phenyl]-4-bromo-3-ethoxycar-

bonylmethoxythiophene-2-carboxylic acid methyl ester (18 mg,

5-[3-(1-Acetylpiperidin-4-ylamino)phenyl]-4-bromo-3-ethoxycar-

bonylmethoxythiophene-2-carboxylic acid methyl ester was then

hydrolyzed according to the general procedure for hydrolysis to

give 5-[3-(1-acetylpiperidin-4-ylamino)phenyl]-4-bromo-3-car-

boxymethoxythiophene-2-carboxylic acid in 51% yield as a yellow

solid. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 1.25 (m, 2 H), 1.94

(m, 2 H), 2.00 (s, 3 H), 2.82 (m, 1 H), 3.18 (m, 2 H), 3.52 (s, 1 H),

3.78 (m, 1 H), 4.21 (m, 1 H), 4.84 (s, 2 H), 6.72 (m, 1 H), 6.77 (d,

J ) 7.58 Hz, 1 H), 6.85 (s, 1 H), 7.19 (t, J ) 7.83 Hz, 1 H).

HRMS: calcd for C₂₀H₂₁BrN₂O₆S + H⁺, 497.037 65; found (ESI-

FTMS, [M + H]⁺), 497.038 93. RF-HPLC: H₂O/MeCN/0.1%

formic acid, 96%; H₂O/MeCN/0.1% formic acid, 98%.

4-Bromo-3-(carboxymethoxy)-5-(3-(1-(methoxycarbonyl)pi-

peridin-4-ylamino)phenyl)thiophene-2-carboxylic Acid (28). Com-

pound 25a (15 mg, 0.027 mmol) was hydrolyzed according to the

generalprocedureforhydrolysistogive4-bromo-3-(carboxymethoxy)-

5-(3-(1-(methoxycarbonyl)piperidin-4-ylamino)phenyl)thiophene-

1

29%) as a pale yellow glassy solid. H NMR (400 MHz, CDCl₃):

δ ppm 1.32 (t, J ) 7.07 Hz, 3 H), 1.42 (dd, J ) 13.64, 2.27 Hz,

2 H), 2.10 (m, 2 H), 3.01 (m, 2 H), 3.49 (m, 1 H), 3.74 (s, 1 H),

3.87 (s, 3 H), 3.94 (m, 2 H), 4.29 (q, J ) 7.07 Hz, 2 H), 4.43 (d,

J ) 5.31 Hz, 2 H), 4.80 (t, J ) 5.31 Hz, 1 H), 4.90 (s, 2 H), 6.65

(m, 1 H), 6.87 (m, 1 H), 6.95 (d, J ) 8.34 Hz, 1 H), 7.23 (t, J )

7.96 Hz, 1 H), 7.31 (m, 5 H).

1

2-carboxylic acid (10 mg, 72%) as a pale yellow solid. H NMR

(400 MHz, DMSO-d₆): δ ppm 1.27 (m, 2 H), 1.92 (m, 2 H), 3.01

(m, 2 H), 3.46 (m, 1 H), 3.59 (s, 3 H), 3.91 (m, 2 H), 4.87 (s, 2 H),

6.71 (dd, J ) 8.21, 2.40 Hz, 1 H), 6.78 (d, J ) 7.58 Hz, 1 H), 6.85

(s, 1 H), 7.19 (t, J ) 7.83 Hz, 1 H). HRMS: calcd for C₂₀H₂₁-

BrN₂O₇S + H⁺, 513.032 56; found (ESI-FTMS, [M + H]⁺),

513.033 14. RF-HPLC: H₂O/MeCN/0.1% formic acid, 97%; H₂O/

MeCN/0.1% formic acid, 98%.

5-[3-(1-Benzylcarbamoylpiperidin-4-ylamino)phenyl]-4-bromo-

3-carboxymethoxythiophene-2-carboxylic Acid (29). Compound

25b (18 mg, 0.029 mmol) was hydrolyzed according to the general

procedure for hydrolysis to give 5-[3-(1-benzylcarbamoylpiperidin-

4-ylamino)phenyl]-4-bromo-3-carboxymethoxythiophene-2-carbox-

ylic acid (16 mg, 94%) as an off-white solid. ¹H NMR (400 MHz,

DMSO-d₆): δ ppm 1.29 (m, 2 H), 1.90 (m, 2 H), 2.91 (m, 2 H),

3.47 (m, 1 H), 3.93 (dd, J ) 12.63, 1.52 Hz, 2 H), 4.24 (d, J )

5.81 Hz, 2 H), 4.88 (s, 2 H), 6.72 (dd, J ) 8.21, 2.15 Hz, 1 H),

6.78 (d, J ) 8.59 Hz, 1 H), 6.85 (m, 1 H), 7.08 (t, J ) 6.32 Hz, 1

H), 7.24 (m, 5 H). HRMS: calcd for C₂₆H₂₆BrN₃O₆S + H⁺,

588.079 84; found (ESI-FTMS, [M + H]⁺), 588.080 57. RF-

HPLC: H₂O/MeCN/0.1% formic acid, 95%; H₂O/MeCN/0.1%

formic acid, 95%.

4-Bromo-3-ethoxycarbonylmethoxy-5-[3-(1-methanesulfo-

nylpiperidin-4-ylamino)phenyl]thiophene-2-carboxylic Acid Meth-

yl Ester (25c). General Procedure for Sulfonamide Formation

at the Piperidine Nitrogen. To a 1 mL pyridine solution of

4-bromo-3-tert-butoxycarbonylmethoxy-5-[3-(piperidin-4-ylamino)-

phenyl]thiophene-2-carboxylic acid methyl ester hydrochloride (50

mg, 0.1 mmol) was added methanesulfonyl chloride (9 µL, 0.12

mmol), and the reaction mixture was stirred at room temperature

overnight. The reaction was quenched with 8 mL of 2 N HCl and

extracted with EtOAc. The organic layer was washed with saturated

NaHCO₃and brine and dried over MgSO₄. Filtration and evapora-

tion gave the crude product, which was purified by flash column

chromatography using a gradient of hexane/EtOAc (20-60%

gradient) as eluent. Pure fractions were combined and evaporated

to give 4-bromo-3-ethoxycarbonylmethoxy-5-[3-(1-methanesulfo-

nylpiperidin-4-ylamino)phenyl]thiophene-2-carboxylic acid methyl

1

ester (12 mg, 21%) as a pale yellow glassy solid. H NMR (400

MHz, CDCl₃): δ ppm 1.32 (t, J ) 7.20 Hz, 3 H), 1.59 (m, 2 H),

2.20 (m, 2 H), 2.82 (s, 3 H), 2.92 (m, 2 H), 3.46 (dd, J ) 11.87,

8.34 Hz, 1 H), 3.78 (m, 2 H), 3.88 (s, 3 H), 4.30 (q, J ) 7.16 Hz,

2 H), 4.90 (s, 2 H), 6.66 (ddd, J ) 8.15, 2.46, 0.76 Hz, 1 H), 6.87

(m, 1 H), 6.97 (ddd, J ) 7.58, 1.64, 0.88 Hz, 1 H), 7.24 (t, J )

7.96 Hz, 1 H).

4-Bromo-3-(carboxymethoxy)-5-[3-(piperidin-4-ylamino)phe-

nyl]thiophene-2-carboxylic Acid (26). 4-Bromo-3-ethoxycarbo-

nylmethoxy-5-[3-(piperidin-4-ylamino)phenyl]thiophene-2-carbox-

ylic acid methyl ester HCl salt was hydrolyzed according to the

4-Bromo-3-carboxymethoxy-5-[3-(1-methanesulfonylpiperi-

din-4-ylamino)phenyl]thiophene-2-carboxylic Acid (30). Com-

pound 25c (12 mg, 0.021 mmol) was hydrolyzed according to the

general procedure for hydrolysis to give 4-bromo-3-carboxymethoxy-

5-[3-(1-methanesulfonylpiperidin-4-ylamino)phenyl]thiophene-2-

carboxylic acid (6 mg, 54%) as a pale yellow solid. ¹H NMR (400

MHz, DMSO-d₆): δ ppm 3.53 (d, J ) 11.37 Hz, 2 H), 4.87 (s, 2

H), 6.72 (dd, J ) 8.21, 1.64 Hz, 1 H), 6.79 (d, J ) 8.08 Hz, 1 H),

6.85 (s, 1 H), 7.20 (t, J ) 8.08 Hz, 1 H). HRMS: calcd for C₁₉H₂₁-

BrN₂O₇S₂+ H⁺, 533.004 64; found (ESI-FTMS, [M + H]⁺),

NoVel Thiophenes as PTP1B Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4691

533.005 55. RF-HPLC: H₂O/MeCN/0.1% formic acid, 100%; H₂O/

MeCN/0.1% formic acid, 100%.

mL × 2). The crude product was purified by preparative HPLC to

give two products, {4-bromo-2-hydroxymethyl-5-[3-(1-phenyl-

methanesulfonylpiperidin-4-ylamino)phenyl]thiophen-3-yloxy}-

acetic acid (100 mg, 64%) and 3-carboxymethoxy-5-[3-(1-

phenylmethanesulfonylpiperidin-4-ylamino)phenyl]thiophene-2-

carboxylic acid methyl ester (30 mg, 21%).

3-Carboxymethoxy-5-[3-(1-phenylmethanesulfonylpiperidin-4-

ylamino)phenyl]thiophene-2-carboxylic acid methyl ester (30 mg,

0.055 mmol) was hydrolyzed according to the general procedure

for hydrolysis to give 3-carboxymethoxy-5-[3-(1-phenylmethane-

sulfonylpiperidin-4-ylamino)phenyl]thiophene-2-carboxylic acid (16

mg, 55%) as an off-white solid after preparative HPLC purification.

¹H NMR (400 MHz, MeOD): δ ppm 1.55 (m, 2 H), 2.03 (m, 2

H), 2.84 (m, 2 H), 3.57 (m, 1 H), 3.69 (d, J ) 12.63 Hz, 2 H), 4.35

(s, 2 H), 4.89 (s, 2 H), 7.01 (d, J ) 7.33 Hz, 1 H), 7.26 (s, 2 H),

7.40 (m, 7 H). HRMS: calcd for C₂₅H₂₆N₂O₇S₂+ H⁺, 531.125 42;

found (ESI-FTMS, [M + H]⁺), 531.125 45. RF-HPLC: H₂O/

MeCN/0.1% formic acid, 100%; H₂O/MeCN/0.1% formic acid,

100%.

5-(3-{[1-(Benzylsulfonyl)piperidin-4-yl]amino}phenyl)-3-(car-

boxymethoxy)-4-methylthiophene-2-carboxylic Acid (34). A

round-bottom flask equipped with a distillation head was charged

with 3-hydroxy-4-methylthiophene-2-carboxylic acid methyl ester³⁹

(1.5 g, 8.7 mmol) and acetic acid (22 mL). The stirred reaction

was treated with Br₂(540 µL, 11 mmol) and then heated to 70 °C.

After 18 h of heating, the reaction was cooled to room temperature

and the acetic acid was removed under reduced pressure to provide

5-bromo-3-hydroxy-4-methylthiophene-2-carboxylic acid methyl

ester (2.19 g, 99%) as a light red solid. ¹H NMR (400 MHz,

CDCl₃): δ ppm 2.08 (s, 3 H), 3.88 (s, 3 H).

5-Bromo-3-ethoxycarbonylmethoxy-4-methylthiophene-2-car-

boxylic acid methyl ester was prepared from methyl 5-bromo-3-

hydroxy-4-methylthiophene-2-carboxylate by following the general

procedure for alkylation to give an orange solid (2.94 g, 74%). ¹H

NMR (400 MHz, CDCl₃): δ ppm 1.29 (t, J ) 7.20 Hz, 3 H), 2.16

(m, 3 H), 3.83 (m, 3 H), 4.24 (q, J ) 7.16 Hz, 2 H), 4.89 (s, 2 H).

5-[3-(1-Benzenesulfonylpiperidin-4-ylamino)phenyl]-4-bromo-

3-carboxymethoxythiophene-2-carboxylic Acid (31). 5-[3-(1-

Benzenesulfonylpiperidin-4-ylamino)phenyl]-4-bromo-3-tert-bu-

toxycarbonylmethoxythiophene-2-carboxylic acid methyl ester was

obtained as a colorless glassy solid in 55% yield by following the

general procedure for sulfonamide formation at the piperidine

nitrogen, except that phenylsulfonyl chloride was used as starting

material. ¹H NMR (400 MHz, CDCl₃): δ ppm 1.50 (s, 9 H), 1.56

(m, 2 H), 2.14 (m, 2 H), 2.54 (td, J ) 11.62, 2.53 Hz, 2 H), 3.28

(m, 1 H), 3.65 (s, 1 H), 3.74 (m, 2 H), 3.86 (s, 3 H), 4.80 (s, 2 H),

6.59 (dd, J ) 7.83, 2.02 Hz, 1 H), 6.78 (m, 1 H), 6.93 (d, J ) 7.58

Hz, 1 H), 7.19 (t, J ) 7.83 Hz, 1 H), 7.60 (m, 3 H), 7.78 (m, 2 H).

5-[3-(1-Benzenesulfonylpiperidin-4-ylamino)phenyl]-4-bromo-

3-tert-butoxycarbonylmethoxythiophene-2-carboxylic acid methyl

ester was then hydrolyzed according to the general procedure for

hydrolysis to give 5-[3-(1-benzenesulfonylpiperidin-4-ylamino)-

phenyl]-4-bromo-3-carboxymethoxythiophene-2-carboxylic acid in

82% yield as a pale yellow solid. ¹H NMR (400 MHz, DMSO-d₆):

δ ppm 1.44 (m, 2 H), 1.98 (m, 2 H), 3.56 (d, J ) 11.37 Hz, 2 H),

4.85 (s, 2 H), 6.66 (dd, J ) 7.71, 1.89 Hz, 1 H), 6.76 (m, 2 H),

7.15 (t, J ) 7.71 Hz, 1 H), 7.66 (t, J ) 7.07 Hz, 2 H), 7.75 (m, 3

H). HRMS: calcd for C₂₄H₂₃BrN₂O₇S₂+ H⁺, 595.020 28; found

(ESI-FTMS, [M + H]⁺), 595.020 88. RF-HPLC: H₂O/MeCN/0.1%

formic acid, 95%; H₂O/MeCN/0.1% formic acid, 97%.

4-Bromo-3-carboxymethoxy-5-[3-(1-phenylmethanesulfonylpi-

peridin-4-ylamino)phenyl]thiophene-2-carboxylic Acid (32). 4-Bro-

mo-3-tert-butoxycarbonylmethoxy-5-[3-(piperidin-4-ylamino)phenyl]-

thiophene-2-carboxylic acid methyl ester hydrochloride (56 mg, 0.1

mmol) was dissolved in a vigorously stirred mixture of 0.5 mL of

CH₂Cl₂and 0.5 mL of saturated sodium bicarbonate solution.

R-Toluenesulfonyl chloride (23 mg, 0.12 mmol) was then added

and the biphasic mixture was stirred at room temperature for 3 h.

The reaction mixture was diluted with CH₂Cl₂, washed with

saturated sodium bicarbonate solution, and dried over MgSO₄.

Filtration and evaporation gave the crude product, which was

purified by flash column chromatography using a gradient of

EtOAc/hexane (5-35% gradient) as eluent. Pure fractions were

combined and evaporated to give 4-bromo-3-tert-butoxycarbonyl-

methoxy-5-[3-(1-phenylmethanesulfonylpiperidin-4-ylamino)phe-

nyl]thiophene-2-carboxylic acid methyl ester (47 mg, 69%) as a

5-(3-Aminophenyl)-3-ethoxycarbonylmethoxy-4-methylthiophene-

2-carboxylic acid methyl ester was prepared from 5-bromo-3-

ethoxycarbonylmethoxy-4-methylthiophene-2-carboxylic acid meth-

yl ester by following the general procedure for Suzuki coupling to

1

give a white solid (350 mg, 82%). H NMR (400 MHz, CDCl₃):

δ ppm 1.30 (t, J ) 7.07 Hz, 3 H), 2.26 (s, 3 H), 3.76 (s, 2 H), 3.85

(s, 3 H), 4.26 (q, J ) 7.07 Hz, 2 H), 4.91 (s, 2 H), 6.70 (m, 1 H),

6.76 (m, J ) 1.89, 1.89 Hz, 1 H), 6.85 (m, 1 H), 7.21 (t, J ) 7.83

Hz, 1 H).

tert-Butyl 4-(3-(4-(2-tert-butoxy-2-oxoethoxy)-5-(methoxycar-

bonyl)-3-methylthiophen-2-yl)phenylamino)piperidine-1-carboxy-

late was prepared by following the general procedure for reductive

amination to give a yellow solid (2.17 g, 97%).

tert-Butyl-4-(3-(4-(2-tert-butoxy-2-oxoethoxy)-5-(methoxycarbo-

nyl)-3-methylthiophen-2-yl)phenylamino)piperidine-1-carboxy-

late was dissolved in 1 N HCl in EtOAc and MeOH. The reaction

was stirred at room temperature for overnight. The solvent was

removed under reduced pressure to give methyl-3-(2-tert-butoxy-

2-oxoethoxy)-4-methyl-5-(3-(piperidin-4-ylamino)phenyl)thiophene-

2-carboxylate hydrochloride as a white solid (1.5 g, 83%).

Methyl-5-(3-(1-(benzylsulfonyl)piperidin-4-ylamino)phenyl)-3-

(2-tert-butoxy-2-oxoethoxy)-4-methylthiophene-2-carboxylate was

prepared by following the general procedure for sulfonamide

formation at the piperidine nitrogen.

Methyl-5-(3-(1-(benzylsulfonyl)piperidin-4-ylamino)phenyl)-3-

(2-tert-butoxy-2-oxoethoxy)-4-methylthiophene-2-carboxylate was

hydrolyzed by following the general procedure for hydrolysis to

give compound 34 (96 mg, 71%) a pale beige solid. ¹H NMR (400

MHz, DMSO-d₆): δ ppm 1.36 (m, 2 H), 1.91 (m, 2 H), 2.18 (s, 3

H), 2.92 (s, 2 H), 3.42 (m, 1 H), 3.56 (m, 2 H), 4.40 (s, 2 H), 4.86

(s, 2 H), 6.69 (s, 2 H), 7.18 (m, 1 H), 7.40 (m, 6 H). HRMS: calcd

for C₂₆H₂₈N₂O₇S₂+ H⁺, 545.141 07; found (ESI-FTMS, [M +

H]⁺), 545.1423. RF-HPLC: H₂O/MeCN/0.1% formic acid, 96%;

H₂O/MeCN/0.1% formic acid, 100%.

1

colorless glassy solid. H NMR (400 MHz, CDCl₃): δ ppm 1.41

(m, 2 H), 1.51 (s, 9 H), 2.03 (m, 2 H), 2.78 (m, 2 H), 3.35 (m, 1

H), 3.62 (m, 2 H), 3.87 (s, 3 H), 4.24 (s, 2 H), 4.82 (s, 2 H), 6.61

(dd, J ) 7.83, 2.02 Hz, 1 H), 6.83 (m, 1 H), 6.94 (d, J ) 8.08 Hz,

1 H), 7.21 (t, J ) 7.83 Hz, 1 H), 7.40 (m, 5 H).

4-Bromo-3-tert-butoxycarbonylmethoxy-5-[3-(1-phenylmethane-

sulfonylpiperidin-4-ylamino)phenyl]thiophene-2-carboxylic acid meth-

yl ester (47 mg, 0.069 mmol) was hydrolyzed according to the

general procedure for hydrolysis to give 4-bromo-3-carboxymethoxy-

5-[3-(1-phenylmethanesulfonylpiperidin-4-ylamino)phenyl]thiophene-

1

2-carboxylic acid (37 mg, 88%) as a pale yellow solid. H NMR

(400 MHz, DMSO-d₆): δ ppm 1.36 (m, 2 H), 1.96 (m, 2 H), 2.92

(m, 2 H), 3.53 (m, 2 H), 4.40 (s, 2 H), 4.87 (s, 2 H), 6.71 (dd, J )

8.08, 1.52 Hz, 1 H), 6.79 (d, J ) 7.58 Hz, 1 H), 6.84 (t, J ) 1.89

Hz, 1 H), 7.20 (t, J ) 7.83 Hz, 1 H), 7.40 (m, 5 H). Anal. Calcd

for C₂₅H₂₅BrN₂O₇S₂·H₂O: C, 47.85; H, 4.34; N, 4.46. Found: C,

48.16; H, 4.43; N, 4.32. RF-HPLC: H₂O/MeCN/0.1% formic acid,

99%; H₂O/MeCN/0.1% formic acid, 98%.

5-(3-{[1-(Benzylsulfonyl)piperidin-4-yl]amino}phenyl)-3-(car-

boxymethoxy)thiophene-2-carboxylic Acid (33). To a round-

bottomed flask were added 4-bromo-3-carboxymethoxy-5-[3-(1-

phenylmethanesulfonylpiperidin-4-ylamino)phenyl]thiophene-2-

carboxylic acid methyl ester (160 mg, 0.26 mmol) and ethanol (20

mL). The solution was cooled in a water bath and then excess

sodium borohydride was added. The mixture was stirred for

overnight at room temperature and water (20 mL) was then added.

The pH of the solution was adjusted to ∼4 with 1 N aqueous HCl.

The reaction mixture was extracted with ethyl acetate (20 mL ×

3). The organic layers were combined and washed with brine (10

4692 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19

Wilson et al.

5-(3-{[1-(Benzylsulfonyl)piperidin-4-yl]amino}phenyl)-3-(car-

boxymethoxy)-4-chlorothiophene-2-carboxylic Acid (35). Methyl

4,5-dichloro-3-hydroxythiophene-2-carboxylate was prepared ac-

cording to a published procedure.³³A solution of methyl 4,5-

dichloro-3-hydroxythiophene-2-carboxylate (6.3 g, 20 mmol), tert-

butyl bromoacetate (4.9 mL, 24 mmol), and K₂CO₃(4.6 g, 24

mmol) in DMF (50 mL) was heated at 60 °C for 1 h. The cooled

solution was diluted with ethyl acetate (400 mL), extracted with

water (4 × 100 mL), washed with brine solution, dried over

magnesium sulfate, filtered, and evaporated. The crude product was

purified by flash column chromatography (20% EtOAc/hexane as

eluent) to provide methyl 3-(2-tert-butoxy-2-oxoethoxy)-4,5-dichlo-

rothiophene-2-carboxylate (8.4 g, 88%) as a white solid. ¹H NMR

(400 MHz, CDCl₃): δ ppm 1.48 (s, 9 H), 3.85 (s, 3 H), 4.84 (s, 2

H).

temperature for 36 h. The solvent was removed and the crude

product was purified by flash column chromatography to give 4-[3-

(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)phenoxy]piperidine-1-

carboxylic acid tert-butyl ester (635 mg, 79%) as a colorless

1

crystalline. H NMR (400 MHz, CDCl₃): δ ppm 1.33 (m, 12 H),

1.48 (m, 9 H), 1.75 (m, 2 H), 1.89 (m, 2 H), 3.37 (m, 2 H), 3.67

(m, 2 H), 4.53 (m, 1 H), 7.01 (m, 1 H), 7.29 (m, 1 H), 7.34 (d, J

) 2.78 Hz, 1 H), 7.40 (m, 1 H).

4-[3-(3-Bromo-4-ethoxycarbonylmethoxy-5-methoxycarbonylth-

iophen-2-yl)phenoxy]piperidine-1-carboxylic acid tert-butyl ester

(125 mg, 51%) was prepared according to the general procedure

for Suzuki coupling, using 4,5-dibromo-3-ethoxycarbonylmethox-

ythiophene-2-carboxylic acid methyl ester (212 mg, 0.53 mmol)

and 4-[3-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)phenoxy]pi-

peridine-1-carboxylic acid tert-butyl ester (165 mg, 0.41 mmol) as

the starting materials. ¹H NMR (400 MHz, CDCl₃): δ ppm 1.5 (s,

9 H), 1.8 (m, 2 H), 2.0 (m, 2 H), 3.4 (m, 2 H), 3.7 (m, 2 H), 4.5

(m, 1 H), 6.9 (dd, J ) 7.7, 2.1 Hz, 1 H), 7.1 (m, 1 H), 7.2 (m, 1

H), 7.3 (t, J ) 8.0 Hz, 1 H).

4-Bromo-3-ethoxycarbonylmethoxy-5-[3-(piperidin-4-yloxy)phe-

nyl]thiophene-2-carboxylic acid methyl ester (TFA salt) was

obtained by treating 4-[3-(3-bromo-4-ethoxycarbonylmethoxy-5-

methoxycarbonylthiophen-2-yl)phenoxy]piperidine-1-carboxylic acid

tert-butyl ester (210 mg, 0.35 mmol) with TFA/CH₂Cl₂(v/v 10:1)

at room temperature overnight.

4-Bromo-3-ethoxycarbonylmethoxy-5-[3-(1-phenylmethanesulfo-

nylpiperidin-4-yloxy)phenyl]thiophene-2-carboxylic acid methyl

ester (62 mg, 81%) was prepared as colorless oil according to the

general procedure for sulfonamide formation at the piperidine

nitrogen, using 4-bromo-3-ethoxycarbonylmethoxy-5-[3-(piperidin-

4-yloxy)phenyl]thiophene-2-carboxylic acid methyl ester TFA salt

(70 mg, 0.117 mmol) as the starting material.

A solution of methyl 3-(2-tert-butoxy-2-oxoethoxy)-4,5-dichlo-

rothiophene-2-carboxylate (3.0 g, 8.8 mmol), 3-aminophenylboronic

acid (1.29 g, 10.5 mmol), Pd₂(dba)₃(400 mg, 0.44 mmol), KF (1.5

g, 26.4 mmol), and tri-tert-butylphosphine tetrafluoroborate (255

mg, 0.88 mmol) in dioxane (30 mL) was heated in a sealed tube at

70 °C for 48 h. The cooled solution was absorbed onto silica gel

and solvent was evaporated. The crude product was purified by

flash column chromatography (20-30% EtOAc/hexane as eluent)

to provide methyl 5-(3-aminophenyl)-3-(2-tert-butoxy-2-oxoet-

hoxy)-4-chlorothiophene-2-carboxylate (2.10 g, 60%) as a yellow

1

oil. H NMR (400 MHz, CDCl₃): δ ppm 1.49 (s, 9 H), 3.87 (s, 3

H), 4.83 (s, 2 H), 6.73 (dd, J ) 8.08, 2.27 Hz, 1 H), 6.99 (bs, 1 H),

7.05 (d, J ) 7.58 Hz, 1 H), 7.22 (t, J ) 7.96 Hz, 1 H).

A solution of methyl 5-(3-aminophenyl)-3-(2-tert-butoxy-2-

oxoethoxy)-4-chlorothiophene-2-carboxylate (212 mg, 0.53 mmol)

and 1-(benzylsulfonyl)piperidin-4-one (190 mg, 0.74 mmol) in 1,2-

dichloroethane (5 mL) was diluted with AcOH (50 mL). Sodium

triacetoxyborohydride (340 mg, 1.59 mmol) was added to the

solution, and the reaction was stirred at room temperature for 3 h.

Solvents were evaporated, and the crude product was dissolved with

EtOAc. The organic layer was washed with aqueous NaHCO₃and

brine, dried over Na₂SO₄, and filtered. The crude product was

purified by flash column chromatography (30% EtOAc/hexane as

eluent) to provide methyl 5-(3-{[1-(benzylsulfonyl)piperidin-4-yl]-

amino}phenyl)-3-(2-tert-butoxy-2-oxoethoxy)-4-chlorothiophene-

Compound 36, 5-(3-{[1-(benzylsulfonyl)piperidin-4-yl]oxy}-

phenyl)-4-bromo-3-(carboxymethoxy)thiophene-2-carboxylic acid

(43 mg, 57%), was prepared as a white solid according to the

general procedure for hydrolysis, using 4-bromo-3-ethoxycarbon-

ylmethoxy-5-[3-(1-phenylmethanesulfonylpiperidin-4-yloxy)phenyl]-

thiophene-2-carboxylic acid methyl ester (60 mg, 0.092 mmol) as

the starting material. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 1.7

(m, 2 H), 1.9 (m, 2 H), 3.1 (m, 2 H), 3.4 (m, 2 H), 4.4 (s, 2 H), 4.6

(m, 1 H), 4.8 (s, 2 H), 7.1 (m, 1 H), 7.2 (m, 2 H), 7.4 (m, 6 H).

5-[3-({[1-(Benzylsulfonyl)piperidin-4-yl]methyl}amino)phenyl]-

4-bromo-3-(carboxymethoxy)thiophene-2-carboxylic Acid (37).

tert-Butyl 4-((3-(3-bromo-4-(2-tert-butoxy-2-oxoethoxy)-5-(meth-

oxycarbonyl)thiophen-2-yl)phenylamino)methyl)piperidine-1-car-

boxylate was prepared by following the general procedure of

reductive amination, using 5-(3-aminophenyl)-4-bromo-3-ethoxy-

carbonylmethoxythiophene-2-carboxylic acid methyl ester (830 mg,

1.9 mmol), tert-butyl 4-formylpiperidine-1-carboxylate (400 mg,

1.9 mmol), acetic acid (0.16 mL, 2.9 mmol), and NaBH(OAc)₃as

1

2-carboxylate (201 mg, 60%) as a yellow foam. H NMR (400

MHz, CDCl₃): δ ppm 1.50 (s, 9 H), 1.95-2.09 (m, 2 H), 2.70-

2.87 (m, 2 H), 3.35 (br. s., 1 H), 3.54-3.68 (m, J ) 12.88 Hz, 3

H), 3.88 (bs, 3 H), 4.24 (s, 2 H), 4.83 (s, 2 H), 6.60 (dd, J ) 7.83,

2.02 Hz, 1 H), 6.83-6.87 (m, J ) 1.77 Hz, 1 H), 6.97 (d, J ) 8.34

Hz, 1 H), 7.22 (t, J ) 7.83 Hz, 1 H), 7.37-7.43 (m, 5 H).

A solution of methyl 5-(3-{[1-(benzylsulfonyl)piperidin-4-yl]-

amino}phenyl)-3-(2-tert-butoxy-2-oxoethoxy)-4-chlorothiophene-

2-carboxylate (201 mg, 0.31 mmol) and LiOH monohydrate (44

mg, 0.93 mmol) in THF (2 mL), MeOH (2 mL), and water (1 mL)

was stirred at room temperature for 6 h. The volatiles were removed

under vacuum, and the residue was diluted with water (10 mL)

and acidified with 10% aqueous HCl. The resulting precipitate was

collected by filtration and dried under vacuum to provide 5-(3-{-

[1-(benzylsulfonyl)piperidin-4-yl]amino}phenyl)-3-(carboxymethoxy)-

4-chlorothiophene-2-carboxylic acid (82 mg, 46%) as a tan solid.

¹H NMR (400 MHz, DMSO-d₆): δ ppm 1.27-1.46 (m, 2 H), 1.94

(d, J ) 9.85 Hz, 2 H), 2.92 (t, J ) 10.99 Hz, 2 H), 3.53 (d, J )

12.88 Hz, 2 H), 4.40 (s, 2 H), 4.92 (s, 2 H), 6.71 (d, J ) 8.84 Hz,

1 H), 6.82 (d, J ) 7.83 Hz, 1 H), 6.87 (s, 1 H), 7.21 (t, J ) 7.96

Hz, 1 H), 7.32-7.46 (m, 5 H). HRMS: calcd for C₂₅H₂₅ClN₂O₇S₂

+ H⁺, 565.086 45; found (ESI-FTMS, [M + H]⁺), 565.087 23. RF-

HPLC: H₂O/MeCN/0.1% formic acid, 94%; H₂O/MeCN/0.1%

formic acid, 95%.

1

starting materials. H NMR (400 MHz, CDCl₃): δ ppm 1.13-

1.22 (m, 3 H), 1.46 (s, 9 H), 1.51 (s, 9 H), 1.74-1.83 (m, 4 H),

2.70 (t, J ) 13.52 Hz, 2 H), 3.07 (t, J ) 6.82 Hz, 2 H), 3.87 (s, 3

H), 4.82 (s, 2 H), 6.65 (ddd, J ) 7.07, 1.52, 1.26 Hz, 1 H), 6.85-

6.87 (m, 1 H), 6.93-6.97 (m, 1 H), 7.23 (t, J ) 7.83 Hz, 1 H).

tert-Butyl 4-((3-(3-bromo-4-(2-tert-butoxy-2-oxoethoxy)-5-(meth-

oxycarbonyl)thiophen-2-yl) phenylamino)methyl)piperidine-1-car-

boxylate (973 mg, 1.5 mmol) was dissolved in 1 N HCl/EtOAc

(7.6 mmol) and MeOH (1.5 mL) and the reaction mixture was

stirred at room temperature. A thick slurry resulted, which required

the addition of 5 mL of EtOAc to aid in stirring. The mixture was

allowed to stir overnight and then evaporated to give methyl

4-bromo-3-(2-tert-butoxy-2-oxoethoxy)-5-(3-(piperidin-4-ylmethy-

lamino)phenyl)thiophene-2-carboxylate hydrochloride (837 mg,

1

97%) as a white solid. H NMR (400 MHz, CDCl₃): δ ppm 1.49

5-(3-{[1-(Benzylsulfonyl)piperidin-4-yl]oxy}phenyl)-4-bromo-

3-(carboxymethoxy)thiophene-2-carboxylic Acid (36). To a solu-

tion of 3-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)phenol (441

mg, 2.0 mmol), 4-hydroxypiperidine-1-carboxylic acid tert-butyl

ester (603.8 mg, 3.0 mmol), and triphenylphosphine (788 mg, 3.0

mmol) in THF (5 mL) was added DIAD (0.59 mL, 3.0 mmol)

dropwise at 0 °C. The resulting solution was stirred at room

(s, 9 H), 1.53-1.64 (m, 2 H), 2.06-2.20 (m, 3 H), 3.03 (td, J )

12.95, 2.40 Hz, 2 H), 3.37 (d, J ) 6.57 Hz, 2 H), 3.41-3.48 (m,

2 H), 3.86 (s, 3 H), 4.86 (s, 2 H), 7.37-7.43 (m, 1 H), 7.50-7.61

(m, 3 H).

4-Bromo-3-tert-butoxycarbonylmethoxy-5-{3-[(1-phenylmethane-

sulfonylpiperidin-4-ylmethyl)amino]phenyl}thiophene-2-carboxy-

NoVel Thiophenes as PTP1B Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4693

lic acid methyl ester was prepared in 79% yield according to the

general procedure for sulfonamide formation at the piperidine

nitrogen, using 4-bromo-3-(2-tert-butoxy-2-oxoethoxy)-5-(3-(pip-

eridin-4-ylmethylamino)phenyl)thiophene-2-carboxylate hydrochlo-

ride (57 mg, 0.1 mmol) and phenylmethanesulfonyl chloride (23

mg, 0.12 mmol) as starting materials. ¹H NMR (400 MHz,

CDCl₃): δ ppm 1.24 (m, 2 H), 1.51 (s, 9 H), 1.67 (m, 1 H), 1.76

(m, 2 H), 2.54 (td, J ) 12.32, 2.40 Hz, 2 H), 3.03 (d, J ) 6.82 Hz,

2 H), 3.69 (d, J ) 12.38 Hz, 2 H), 3.87 (s, 3 H), 4.21 (s, 2 H), 4.82

(s, 2 H), 6.62 (dd, J ) 7.83, 2.02 Hz, 1 H), 6.83 (m, 1 H), 6.94 (d,

J ) 8.08 Hz, 1 H), 7.22 (t, J ) 7.83 Hz, 1 H), 7.38 (m, 5 H).

4-Bromo-3-tert-butoxycarbonylmethoxy-5-{3-[(1-phenylmethane-

sulfonylpiperidin-4-ylmethyl)amino]phenyl}thiophene-2-carboxy-

lic acid methyl ester was hydrolyzed according to the general

procedure for hydrolysis to give compound 37 in 75% yield as a

8.72, 1.89 Hz, 1 H), 6.78 (d, J ) 7.58 Hz, 1 H), 6.84 (t, J ) 1.77

Hz, 1 H), 6.91 (t, J ) 7.33 Hz, 1 H), 7.20 (m, 3 H), 7.45 (dd, J )

8.72, 1.14 Hz, 2 H), 8.44 (s, 1 H). HRMS: calcd for C₂₆H₂₆BrN₃O₆S

+ H⁺, 588.079 84; found (ESI-FTMS, [M + H]⁺), 588.079 91.

5-(3-{[1-(Anilinocarbonyl)piperidin-4-yl]methoxy}phenyl)-4-

bromo-3-(carboxymethoxy)thiophene-2-carboxylic Acid (40). To

a solution of 4-bromo-3-ethoxycarbonylmethoxy-5-(3-hydroxyphe-

nyl)thiophene-2-carboxylic acid methyl ester in DMF (4 mL) was

added 4-methanesulfonyloxypiperidine-1-carboxylic acid tert-butyl

ester (2 equiv) and K₂CO₃(excess). The mixture was stirred at

80 °C overnight. The solution was then washed with saturated

NaHCO₃and dried over Na₂SO₄. The crude product was purified

by flash column chromatography to give 4-[3-(3-bromo-4-tert-

butoxycarbonylmethoxy-5-methoxycarbonylthiophen-2-yl)phenoxym-

ethyl]piperidine-1-carboxylic acid tert-butyl ester (84 mg, 39%).

¹H NMR (400 MHz, CDCl₃): δ ppm 1.3 (m, 2 H), 1.5 (s, 9 H),

1.5 (s, 9 H), 1.8 (m, 2 H), 2.0 (m, 1 H), 2.8 (t, J ) 12.3 Hz, 2 H),

3.8 (d, J ) 6.6 Hz, 2 H), 3.9 (s, 3 H), 4.8 (s, 2 H), 7.0 (dd, 1 H),

7.2 (t, 1 H), 7.2 (dt, J ) 8.3 Hz, 1 H), 7.3 (t, J ) 8.0 Hz, 1 H).

4-Bromo-3-ethoxycarbonylmethoxy-5-[3-(piperidin-4-ylmethox-

y)phenyl]thiophene-2-carboxylic acid methyl ester TFA salt was

obtained by treating 4-[3-(3-bromo-4-tert-butoxycarbonylmethoxy-

5-methoxycarbonylthiophen-2-yl)phenoxymethyl]piperidine-1-car-

boxylic acid tert-butyl ester with 30% TFA in DCM at room

temperature.

4-Bromo-3-ethoxycarbonylmethoxy-5-[3-(1-phenylcarbamoylpi-

peridin-4-ylmethoxy)phenyl]thiophene-2-carboxylic acid methyl

ester (90% yield) was prepared according to the general procedure

for urea formation at the piperidine nitrogen. ¹H NMR (400 MHz,

CDCl₃): δ ppm 1.2 (t, J ) 7.2 Hz, 3 H), 1.4 (m, 2 H), 1.7 (m, 1

H), 1.8 (m, 2 H), 2.3 (td, J ) 12.0, 2.5 Hz, 2 H), 3.7 (d, J ) 6.3

Hz, 2 H), 3.8 (s, 3 H), 4.2 (q, J ) 7.3 Hz, 2 H), 4.8 (s, 2 H), 6.8

(dd, 1 H), 7.1 (t, 1 H), 7.1 (m, 1 H), 7.3 (t, J ) 8.0 Hz, 1 H), 7.5

(m, 3 H), 7.7 (m, 2 H).

4-Bromo-3-ethoxycarbonylmethoxy-5-[3-(1-phenylcarbamoylpi-

peridin-4-ylmethoxy)phenyl]thiophene-2-carboxylic acid methyl

ester was hydrolyzed according to the general procedure for

hydrolysis to give compound 40 in 75% yield. ¹H NMR (400 MHz,

DMSO-d₆): δ ppm 1.0 (d, 2 H), 2.0 (td, 2 H), 2.8 (m, 2 H), 3.0 (d,

2 H), 3.3 (d, 2 H), 4.5 (m, 2 H), 6.1 (m, 2 H), 6.3 (m, 4 H), 6.4 (m,

3 H). HRMS: calcd for C₂₆H₂₅BrN₂O₇S + H⁺, 589.063 86; found

(ESI-FTMS, [M + H]⁺), 589.064 26.

1

pale yellow solid. H NMR (400 MHz, DMSO-d₆): δ ppm 1.14

(m, 2 H), 1.66 (m, 1 H), 1.79 (d, J ) 12.38 Hz, 2 H), 2.67 (m, 2

H), 2.94 (d, J ) 6.57 Hz, 2 H), 3.57 (d, J ) 12.38 Hz, 2 H), 4.38

(s, 2 H), 4.88 (s, 2 H), 6.68 (dd, J ) 8.97, 2.40 Hz, 1 H), 6.77 (d,

J ) 6.82 Hz, 1 H), 6.81 (s, 1 H), 7.18 (t, J ) 7.96 Hz, 1 H), 7.39

(m, 5 H). HRMS: calcd for C₂₆H₂₇BrN₂O₇S₂+ H⁺, 623.051 59;

found (ESI-FTMS, [M + H]⁺), 623.052 43. RF-HPLC: H₂O/

MeCN/0.1% formic acid, 97%; H₂O/MeCN/0.1% formic acid, 98%.

5-{3-[(1-Benzenesulfonylpiperidin-4-ylmethyl)amino]phenyl}-

4-bromo-3-carboxymethoxythiophene-2-carboxylic Acid (38).

5-{3-[(1-Benzenesulfonylpiperidin-4-ylmethyl)amino]phenyl}-4-

bromo-3-tert-butoxycarbonylmethoxythiophene-2-carboxylic acid

methyl ester was prepared in 60% yield according to the general

procedure for sulfonamide formation at the piperidine nitrogen,

using 4-bromo-3-(2-tert-butoxy-2-oxoethoxy)-5-(3-(piperidin-4-yl-

methylamino)phenyl)thiophene-2-carboxylate hydrochloride (57 mg,

0.1 mmol) and benzenesulfonyl chloride (15 µL, 0.12 mmol) as

1

starting materials. H NMR (400 MHz, CDCl₃): δ ppm 1.38 (m,

2 H), 1.50 (s, 9 H), 1.56 (m, 1 H), 1.85 (d, J ) 10.86 Hz, 2 H),

2.26 (td, J ) 11.94, 2.40 Hz, 2 H), 3.04 (d, J ) 6.57 Hz, 2 H),

3.83 (m, 2 H), 3.87 (s, 3 H), 4.81 (s, 2 H), 6.60 (m, 1 H), 6.80 (m,

1 H), 6.93 (dd, J ) 7.96, 1.39 Hz, 1 H), 7.20 (t, J ) 7.83 Hz, 1 H),

7.52 (m, 2 H), 7.59 (m, 1 H), 7.76 (m, 2 H). 5-{3-[(1-Benzene-

sulfonylpiperidin-4-ylmethyl)amino]phenyl}-4-bromo-3-tert-butox-

ycarbonylmethoxythiophene-2-carboxylic acid methyl ester was

hydrolyzed according to the general procedure for hydrolysis to

1

give compound 38 in 82% yield as a pale yellow solid. H NMR

(400 MHz, DMSO-d₆): δ ppm 1.25 (m, 2 H), 1.52 (m, 1 H), 1.82

(m, 2 H), 2.21 (m, 2 H), 2.90 (d, J ) 6.06 Hz, 2 H), 3.67 (d, J )

11.87 Hz, 2 H), 4.86 (s, 2 H), 6.64 (dd, J ) 7.96, 1.89 Hz, 1 H),

6.77 (m, 2 H), 7.15 (t, J ) 7.83 Hz, 1 H), 7.63 (m, 2 H), 7.71 (m,

3 H). HRMS: calcd for C₂₅H₂₅BrN₂O₇S₂+ H⁺, 609.035 94; found

(ESI-FTMS, [M + H]⁺), 609.035 63. RF-HPLC: H₂O/MeCN/0.1%

formic acid, 98%; H₂O/MeCN/0.1% formic acid, 99%.

5-[3-({[1-(Anilinocarbonyl)piperidin-4-yl]methyl}amino)-

phenyl]-4-bromo-3-(carboxymethoxy)thiophene-2-carboxylic Acid

(39). 4-Bromo-3-tert-butoxycarbonylmethoxy-5-{3-[(1-phenylcar-

bamoylpiperidin-4-ylmethyl)amino]phenyl}thiophene-2-carboxyl-

ic acid methyl ester was obtained in 62% yield by following the

general procedure for urea formation at the piperidine nitrogen,

using methyl 4-bromo-3-(2-tert-butoxy-2-oxoethoxy)-5-(3-(piperi-

din-4-ylmethylamino)phenyl)thiophene-2-carboxylate hydrochloride

(57 mg, 0.1 mmol), DIPEA (27 µL, 0.15 mmol), and phenyl

isocyanate (12 µL, 0.11 mmol) as starting materials. ¹H NMR (400

MHz, CDCl₃): δ ppm 1.31 (m, 2 H), 1.51 (s, 9 H), 1.88 (m, 3 H),

2.89 (m, 2 H), 3.10 (d, J ) 6.06 Hz, 2 H), 3.87 (s, 3 H), 4.12 (m,

2 H), 4.82 (s, 2 H), 6.38 (s, 1 H), 6.66 (m, 1 H), 6.87 (m, 1 H),

6.96 (dd, J ) 6.69, 1.64 Hz, 1 H), 7.03 (m, 1 H), 7.24 (m, 1 H),

7.29 (m, 2 H), 7.35 (m, 2 H).

4-Bromo-3-(carboxymethoxy)-5-[3-({1-[(4-chlorobenzyl)sul-

fonyl]piperidin-4-yl}amino)phenyl]thiophene-2-carboxylic Acid

(41). 4-Bromo-3-tert-butoxycarbonylmethoxy-5-{3-[1-(4-chlorophe-

nylmethanesulfonyl)piperidin-4-ylamino]phenyl}thiophene-2-car-

boxylic acid methyl ester (108 mg, 77%) was prepared according

to the general procedure for sulfonamide formation at the piperidine

nitrogen, using 4-bromo-3-tert-butoxycarbonylmethoxy-5-[3-(pip-

eridin-4-ylamino)phenyl]thiophene-2-carboxylic acid methyl ester

1

HCl salt (110 mg, 0.20 mmol) as the starting material. H NMR

(400 MHz, CDCl₃): δ ppm 1.43 (m, 2 H), 1.51 (s, 9 H), 2.08 (dd,

J ) 13.52, 2.91 Hz, 2 H), 2.82 (m, 2 H), 3.39 (m, 1 H), 3.65 (m,

3 H), 3.88 (s, 3 H), 4.18 (s, 2 H), 4.82 (s, 2 H), 6.63 (m, 1 H), 6.84

(m, 1 H), 6.95 (d, J ) 7.58 Hz, 1 H), 7.22 (t, J ) 7.83 Hz, 1 H),

7.37 (m, 4 H).

Compound 41, 4-bromo-3-carbonylmethoxy-5-{3-[1-(4-chloro-

phenylmethanesulfonyl)piperidin-4-ylamino]phenyl}thiophene-2-

carboxylic acid (55 mg, 98%), was prepare as a yellow solid

according to the general procedure for hydrolysis, using 4-bromo-

3-tert-butoxycarbonylmethoxy-5-{3-[1-(4-chlorophenylmethane-

sulfonyl)piperidin-4-ylamino]phenyl}thiophene-2-carboxylic acid

methyl ester (62 mg, 0.087 mmol) as the starting material. ¹H NMR

(400 MHz, DMSO-d₆): δ ppm 1.36 (m, 2 H), 1.96 (d, J ) 13.89

Hz, 2 H), 2.93 (t, J ) 9.85 Hz, 2 H), 3.33 (s, 1 H), 3.53 (m, 2 H),

4.43 (s, 2 H), 4.87 (s, 2 H), 6.71 (dd, J ) 8.59, 2.02 Hz, 1 H), 6.79

(m, 1 H), 6.85 (m, 1 H), 7.20 (t, J ) 7.96 Hz, 1 H), 7.45 (d, J )

2.53 Hz, 4 H). HRMS: calcd for C₂₅H₂₄BrClN₂O₇S₂+ H⁺,

642.996 96; found (ESI-FTMS, [M + H]⁺), 642.997 07.

4-Bromo-3-(carboxymethoxy)-5-(3-(1-(3-chlorobenzylsulfonyl)-

piperidin-4-ylamino)phenyl)thiophene-2-carboxylic Acid (42).

4-Bromo-3-tert-butoxycarbonylmethoxy-5-{3-[(1-phenylcarbam-

oylpiperidin-4-ylmethyl)amino]phenyl}thiophene-2-carboxylic acid

methyl ester (41 mg, 0.062 mmol) was hydrolyzed according to

the general procedure for hydrolysis to give compound 39 as a pale

yellow solid in 90% yield. ¹H NMR (400 MHz, DMSO-d₆): δ ppm

1.16 (m, 2 H), 1.79 (m, 3 H), 2.77 (m, 2 H), 2.97 (d, J ) 5.81 Hz,

2 H), 4.15 (d, J ) 12.63 Hz, 2 H), 4.87 (s, 2 H), 6.70 (dd, J )

4694 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19

Wilson et al.

4-Bromo-3-tert-butoxycarbonylmethoxy-5-{3-[1-(3-chlorophenyl-

methanesulfonyl)piperidin-4-ylamino]phenyl}thiophene-2-carboxy-

lic acid methyl ester was prepared by following the general

procedure for sulfonamide formation at the piperidine nitrogen to

yl ester (0.096 g, 0.15 mmol) was hydrolyzed according to the

general procedure for hydrolysis to give 4-bromo-3-(carboxy-

methoxy)-5-[3-({1-[(2-methylbenzyl)sulfonyl]piperidin-4-yl}amino)-

phenyl]thiophene-2-carboxylic acid (0.046 g, 49%) as a white solid.

¹H NMR (400 MHz, CD₃OD): δ ppm 1.41 (m, 2 H), 2.00 (m, 2

H), 2.35 (s, 3 H), 2.93 (t, J ) 11.12 Hz, 2 H), 3.38 (m, 1 H), 3.60

(d, J ) 12.88 Hz, 2 H), 4.27 (s, 2 H), 4.82 (s, 2H), 6.65 (dd, J )

8.84, 2.78 Hz, 1 H), 6.77 (dd, J ) 6.82, 1.52 Hz, 1 H), 6.83 (t, J

) 1.89 Hz, 1 H), 7.12 (m, 4 H), 7.25 (d, J ) 7.07 Hz, 1 H).

HRMS: calcd for C₂₆H₂₇BrN₂O₇S₂+ H⁺, 623.051 58; found (ESI

(+), [M + H]⁺), 623.0529. RF-HPLC: H₂O/MeCN/0.1% formic

acid, 95%; H₂O/MeCN/0.1% formic acid, 96%.

4-Bromo-3-(carboxymethoxy)-5-[3-({1-[(2,6-dimethylbenzyl)-

sulfonyl]piperidin-4-yl}amino)phenyl]thiophene-2-carboxylic Acid

(46). 4-Bromo-3-tert-butoxycarbonylmethoxy-5-{3-[1-(2,6-dimeth-

ylphenylmethanesulfonyl)piperidin-4-ylamino]phenyl}thiophene-2-

carboxylic acid methyl ester was prepared by following the

procedure in the synthesis of 32, using 4-bromo-3-tert-butoxycar-

bonylmethoxy-5-[3-(piperidin-4-ylamino)phenyl]thiophene-2-car-

boxylic acid methyl ester (0.194 g, 0.35 mmol) and 2,6-

dimethylbenzylsulfonyl chloride (0.24 g, 1.14 mmol) as starting

materials. 4-Bromo-3-tert-butoxycarbonylmethoxy-5-{3-[1-(2,6-

dimethylphenylmethanesulfonyl)piperidin-4-ylamino]phenyl}-

thiophene-2-carboxylic acid methyl ester (0.091 g, 34%) was

obtained as a yellow oil. ¹H NMR (400 MHz, CDCl₃): δ ppm 1.52

(m, 9 H), 1.59 (m, 2 H), 2.47 (s, 6 H), 2.96 (m, 2 H), 3.45 (m, 1

H), 3.77 (m, 2 H), 3.88 (s, 3 H), 4.35 (s, 2 H), 4.82 (s, 2 H), 6.74

(d, J ) 8.08 Hz, 1 H), 6.97 (s, 1 H), 7.06 (m, J ) 12.88, 7.58 Hz,

3 H), 7.15 (m, 1 H), 7.26 (m, 1 H).

4-Bromo-3-tert-butoxycarbonylmethoxy-5-{3-[1-(2,6-dimeth-

ylphenylmethanesulfonyl)piperidin-4-ylamino]phenyl}thiophene-2-

carboxylic acid methyl ester (0.091 g, 0.15 mmol) was hydrolyzed

according to the general procedure for hydrolysis to give 4-bromo-

3-(carboxymethoxy)-5-[3-({1-[(2,6-dimethylbenzyl)sulfonyl]piperi-

din-4-yl}amino)phenyl]thiophene-2-carboxylic acid (0.043 g, 45%)

as a white solid. ¹H NMR (400 MHz, CD₃OD): δ ppm 1.48 (m, 2

H), 2.06 (d, J ) 10.36 Hz, 2 H), 2.36 (s, 6 H), 3.04 (t, J ) 10.61

Hz, 2 H), 3.43 (m, J ) 10.23, 10.23 Hz, 1 H), 3.69 (m, J ) 12.63

Hz, 2 H), 4.34 (s, 2 H), 4.78 (s, 2 H), 6.68 (dd, J ) 8.21, 1.64 Hz,

1 H), 6.79 (d, J ) 8.08 Hz, 1 H), 6.85 (m, 1 H), 6.96 (m, J ) 7.33

Hz, 2 H), 7.02 (m, 1 H), 7.12 (t, J ) 7.83 Hz, 1 H). HRMS: calcd

for C₂₇H₂₉BrN₂O₇S₂+ H⁺, 637.067 23; found (ESI/FTMS, [M +

H]⁺), 637.0677. RF-HPLC: H₂O/MeCN/0.1% formic acid, 96%;

H₂O/MeCN/0.1% formic acid, 95%.

1

give a light yellow solid (110 mg, 86%). H NMR (400 MHz,

CDCl₃): δ ppm 1.45 (m, 2 H), 1.50 (d, J ) 10.11 Hz, 9 H), 1.55

(m, 4 H), 2.09 (m, 2 H), 2.85 (m, 2 H), 3.39 (s, 1 H), 3.64 (m, 2

H), 3.88 (s, 3 H), 4.82 (s, 2 H), 6.64 (d, J ) 2.02 Hz, 1 H), 6.85

(s, 1 H), 6.96 (d, J ) 7.83 Hz, 1 H), 7.34 (m, 6 H).

4-Bromo-3-carboxymethoxy-5-{3-[1-(3-chlorophenylmethane-

sulfonyl)piperidin-4-ylamino]phenyl}thiophene-2-carboxylic acid

was prepared as a yellow solid (81 mg, 90%) by following the

general procedure for hydrolysis. ¹H NMR (400 MHz, DMSO-d₆):

δ ppm 1.39 (s, 2 H), 1.95 (d, J ) 31.83 Hz, 2 H), 2.96 (s, 1 H),

3.56 (s, 1 H), 4.45 (s, 2 H), 4.87 (s, 2 H), 6.71 (s, 1 H), 6.79 (m,

1 H), 6.85 (t, J ) 1.89 Hz, 1 H), 7.20 (t, J ) 7.83 Hz, 1 H), 7.40

(d, J ) 2.02 Hz, 1 H), 7.43 (m, 2 H), 7.50 (s, 1 H). HRMS: calcd

for C₂₅H₂₄BrClN₂O₇S₂+ H⁺, 641.989 68; found (ESI-FTMS, [M

+ H]⁺), 641.989 24.

3-Carboxymethoxy-5-{3-[1-(2-chlorobenzenesulfonyl)piperi-

din-4-ylamino]phenyl}-4-methylthiophene-2-carboxylic Acid (43).

Methyl 4-bromo-3-(2-tert-butoxy-2-oxoethoxy)-5-(3-(1-(2-chlo-

robenzylsulfonyl)piperidin-4-ylamino)phenyl)thiophene-2-carboxy-

late was prepared by following the general procedure for sulfona-

mide formation at the piperidine nitrogen to give a tan solid (112

1

mg, 80%). H NMR (400 MHz, CDCl₃): δ ppm 1.49 (m, 9 H),

1.56 (m, 2 H), 2.13 (m, 2 H), 2.24 (s, 3 H), 2.98 (m, 2 H), 3.43 (m,

1 H), 3.62 (m, 1 H), 3.85 (m, 2 H), 3.85 (m, 3 H), 4.79 (d, 2 H),

6.58 (m, 1 H), 6.62 (m, 1 H), 6.79 (m, J ) 7.58 Hz, 1 H), 7.20 (t,

J ) 7.83 Hz, 1 H), 7.41 (ddd, J ) 8.21, 6.69, 1.52 Hz, 1 H), 7.50

(m, J ) 7.58, 7.58, 1.52 Hz, 1 H), 7.53 (d, J ) 1.52 Hz, 1 H), 8.08

(dd, J ) 7.71, 1.89 Hz, 1 H).

Methyl 4-bromo-3-(2-tert-butoxy-2-oxoethoxy)-5-(3-(1-(2-chlo-

robenzylsulfonyl)piperidin-4-ylamino)phenyl)thiophene-2-carboxy-

late was hydrolyzed by following the general procedure for

hydrolysis to give compound 43 (53 mg, 43%) as a beige solid. ¹H

NMR (400 MHz, DMSO-d₆): δ ppm 1.41 (d, J ) 5.31 Hz, 2 H),

1.94 (s, 2 H), 2.17 (m, 3 H), 2.96 (s, 2 H), 3.65 (s, 2 H), 4.85 (s,

2 H), 5.87 (t, 1 H), 6.39 (m, 1 H), 6.63 (m, J ) 6.95, 6.95 Hz, 2

H), 6.68 (s, 1 H), 7.15 (t, J ) 7.83 Hz, 1 H), 7.58 (m, 1 H), 7.70

(m, 2 H), 7.99 (dd, J ) 7.83, 1.52 Hz, 1 H). HRMS: calcd for

C₂₅H₂₅ClN₂O₇S₂+ H⁺, 565.086 50; found (ESI-FTMS, [M + H]⁺),

565.086 53. RF-HPLC: H₂O/MeCN/0.1% formic acid, 98%; H₂O/

MeCN/0.1% formic acid, 98%.

4-Bromo-3-(carboxymethoxy)-5-{3-[(1-{[2-(trifluoromethyl)-

benzyl]sulfonyl}piperidin-4-yl)amino]phenyl}thiophene-2-car-

boxylic Acid (44). ¹H NMR (400 MHz, DMSO-d₆): δ ppm 1.32-

1.51 (m, 2 H), 1.93-2.09 (m, 2 H), 2.99-3.17 (m, 2 H), 3.42-

3.54 (m, 1 H), 3.55-3.68 (m, 2 H), 4.53 (s, 3 H), 4.88 (s, 2 H),

6.74 (dd, J ) 8.59, 1.77 Hz, 1 H), 6.81 (d, J ) 7.33 Hz, 1 H), 6.87

(s, 1 H), 7.21 (t, J ) 7.83 Hz, 1 H), 7.57-7.65 (m, 1 H), 7.67-

7.76 (m, 2 H), 7.80 (d, J ) 7.83 Hz, 1 H). HRMS: calcd for C₂₆H₂₄-

BrF₃N₂O₇S₂+ H⁺, 677.023 31; found (ESI-FTMS, [M + H]⁺),

677.0242.

5-[3-({1-[(2-Aminobenzyl)sulfonyl]piperidin-4-yl}amino)-

phenyl]-4-bromo-3-(carboxymethoxy)thiophene-2-carboxylic Acid

(47). 5-{3-[1-(2-Amino-phenylmethanesulfonyl)piperidin-4-ylami-

no]phenyl}-4-bromo-3-ethoxycarbonylmethoxythiophene-2-carbox-

ylic acid methyl ester was obtained in 19% yield as a pale yellow

foam, according to the general procedure for sulfonamide formation

1

at the piperidine nitrogen followed by SnCl₂reduction. H NMR

(400 MHz, CDCl₃): δ ppm 1.3 (t, J ) 7.2 Hz, 3 H), 1.5 (m, 2 H),

2.1 (m, 2 H), 2.9 (m, 2 H), 3.4 (m, 1 H), 3.7 (m, 2 H), 3.9 (s, 3 H),

4.3 (m, 4 H), 4.9 (s, 2 H), 6.6 (m, 1 H), 6.8 (m, 2 H), 6.8 (m, 1 H),

6.9 (m, 1 H), 7.1 (m, 2 H), 7.2 (t, J ) 7.8 Hz, 1 H), 7.3 (s, 1 H).

5-{3-[1-(2-Amino-phenylmethanesulfonyl)piperidin-4-ylamino]-

phenyl}-4-bromo-3-ethoxycarbonylmethoxythiophene-2-carboxy-

lic acid methyl ester was hydrolyzed according to the general

procedure for hydrolysis to give compound 47 in 36% yield as a

pale brown solid. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 1.4 (m,

2 H), 1.8 (m, J ) 6.7, 6.7 Hz, 1 H), 2.0 (m, 2 H), 3.0 (m, 2 H), 3.6

(m, 2 H), 4.3 (s, 2 H), 4.9 (s, 2 H), 6.6 (m, 1 H), 6.8 (m, 3 H), 6.9

(m, 1 H), 7.1 (m, 1 H), 7.1 (dd, J ) 7.7, 1.6 Hz, 1 H), 7.2 (t, J )

8.0 Hz, 1 H). HRMS: calcd for C₂₅H₂₆BrN₃O₇S₂+ H⁺, 624.046 83;

found (ESI-FTMS, [M + H]⁺), 624.047 53.

5-{3-[(1-{[2-(Acetylamino)benzyl]sulfonyl}piperidin-4-yl)ami-

no]phenyl}-4-bromo-3-(carboxymethoxy)thiophene-2-carboxy-

lic Acid (48). ¹H NMR (400 MHz, DMSO-d₆): δ ppm 1.27-1.49

(m, 2 H), 1.86-2.01 (m, 2 H), 2.08 (s, 3 H), 2.80-2.96 (m, 2 H),

3.32-3.44 (m, 1 H), 3.46-3.55 (m, 2 H), 4.50 (s, 2 H), 6.74 (d, J

) 6.82 Hz, 1 H), 6.83 (d, J ) 8.34 Hz, 1 H), 6.88 (s, 1 H), 7.14-

4-Bromo-3-(carboxymethoxy)-5-[3-({1-[(2-methylbenzyl)sul-

fonyl]piperidin-4-yl}amino)phenyl]thiophene-2-carboxylic Acid

(45). 4-Bromo-3-tert-butoxycarbonylmethoxy-5-[3-(1-o-tolylmethane-

sulfonylpiperidin-4-ylamino)phenyl]thiophene-2-carboxylic acid meth-

yl ester was prepared by following the procedure in the synthesis

of 32, using 4-bromo-3-tert-butoxycarbonylmethoxy-5-[3-(piperi-

din-4-ylamino)phenyl]thiophene-2-carboxylic acid methyl ester (0.2

g, 0.38 mmol) and o-tolyl-methanesulfonyl chloride (0.24 g, 1.14

mmol) as starting materials. 4-Bromo-3-tert-butoxycarbonylmethoxy-

5-[3-(1-o-tolylmethanesulfonylpiperidin-4-ylamino)phenyl]thiophene-

2-carboxylic acid methyl ester (0.096 g, 40%) was obtained as a

1

yellow oil. H NMR (400 MHz, CDCl₃): δ ppm 1.51 (s, 9 H),

2.09 (dd, J ) 12.88, 4.04 Hz, 2 H), 2.47 (s, 3 H), 2.89 (m, 2 H),

3.41 (m, 1 H), 3.67 (m, 4 H), 3.88 (s, 3 H), 4.28 (s, 2 H), 4.82 (s,

2 H), 6.63 (m, 1 H), 6.85 (m, 1 H), 6.95 (m, 1 H), 7.26 (m, 5 H).

4-Bromo-3-tert-butoxycarbonylmethoxy-5-[3-(1-o-tolylmethane-

sulfonylpiperidin-4-ylamino)phenyl]thiophene-2-carboxylic acid meth-

NoVel Thiophenes as PTP1B Inhibitors

Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19 4695

7.28 (m, 2 H), 7.29-7.38 (m, 1 H), 7.43 (dd, J ) 7.58, 1.52 Hz,

1 H), 7.50 (d, J ) 8.34 Hz, 1 H), 9.44 (s, 1 H). HRMS: calcd for

C₂₇H₂₈BrN₃O₈S₂- H⁺, 664.042 84; found (ESI-FTMS, [M -

H]-), 664.0423. RF-HPLC: H₂O/MeCN/0.1% formic acid, 95%;

H₂O/MeCN/0.1% formic acid, 96%.

oxyphenyl isocyanate (22 µL, 0.17 mmol), and DIPEA (67 µL,

0.38 mmol) as starting materials. ¹H NMR (400 MHz, CDCl₃): δ

ppm 1.33 (m, 2 H), 1.51 (s, 9 H), 1.87 (dd, J ) 11.37, 8.59 Hz, 3

H), 2.90 (m, 2 H), 3.10 (d, J ) 6.32 Hz, 2 H), 3.87 (s, 3 H), 3.88

(s, 3 H), 4.14 (d, J ) 13.14 Hz, 2 H), 4.82 (s, 2 H), 6.66 (m, 1 H),

6.86 (m, 2 H), 6.94 (m, 3 H), 7.13 (s, 1 H), 7.24 (t, J ) 7.96 Hz,

1 H), 8.14 (m, 1 H).

4-Bromo-3-(carboxymethoxy)-5-[3-({1-[(2-{[(ethylamino)-

carbonyl]amino}benzyl)sulfonyl]piperidin-4-yl}amino)phenyl]-

1

thiophene-2-carboxylic Acid (49). H NMR (400 MHz, DMSO-

4-Bromo-3-tert-butoxycarbonylmethoxy-5-(3-{[1-(2-methoxy-

phenylcarbamoyl)piperidin-4-ylmethyl]amino}phenyl)thiophene-2-

carboxylic acid methyl ester (58 mg, 0.084 mmol) was hydrolyzed

according to the general procedure for hydrolysis to give 4-bromo-

3-carboxymethoxy-5-(3-{[1-(2-methoxyphenylcarbamoyl)piperidin-

4-ylmethyl]amino}phenyl)thiophene-2-carboxylic acid (48 mg,

d₆): δ ppm 1.05 (t, J ) 7.20 Hz, 3 H), 1.26-1.46 (m, 2 H), 1.93

(d, J ) 9.85 Hz, 2 H), 2.91 (t, J ) 10.61 Hz, 2 H), 3.06-3.18 (m,

J ) 6.95, 4.42 Hz, 2 H), 3.29-3.44 (m, 1 H), 3.44-3.58 (m, 2 H),

4.42 (s, 2 H), 6.63 (t, J ) 4.42 Hz, 1 H), 6.71 (dd, J ) 8.34, 1.77

Hz, 1 H), 6.79 (d, J ) 7.58 Hz, 1 H), 6.84 (d, J ) 1.77 Hz, 1 H),

6.99-7.09 (m, 1 H), 7.20 (t, J ) 7.83 Hz, 1 H), 7.24-7.30 (m, 1

H), 7.35 (dd, J ) 7.83, 1.52 Hz, 1 H), 7.70 (dd, J ) 8.21, 1.14 Hz,

1 H), 7.77 (s, 1 H). HRMS: calcd for C₂₈H₃₁BrN₄O₈S₂- H⁺,

693.069 39; found (ESI-FTMS, [M - H]-), 693.071. RF-HPLC:

H₂O/MeCN/0.1% formic acid, 99%; H₂O/MeCN/0.1% formic acid,

99%.

1

92%) as a pale yellow solid. H NMR (400 MHz, DMSO-d₆): δ

ppm 1.16 (m, 2 H), 1.80 (m, 3 H), 2.81 (m, 2 H), 2.97 (d, J ) 5.81

Hz, 2 H), 3.80 (s, 3 H), 4.07 (d, J ) 13.39 Hz, 2 H), 4.87 (s, 2 H),

6.70 (m, 1 H), 6.77 (d, J ) 7.83 Hz, 1 H), 6.85 (m, 2 H), 6.98 (m,

2 H), 7.19 (t, J ) 7.83 Hz, 1 H), 7.57 (s, 1 H), 7.66 (d, J ) 7.58

Hz, 1 H). HRMS: calcd for C₂₇H₂₈BrN₃O₇S - H⁺, 618.090 41;

found (ESI-FTMS, [M - H]-), 618.091 51. RF-HPLC: H₂O/

MeCN/0.1% formic acid, 96%; H₂O/MeCN/0.1% formic acid, 95%.

4-Bromo-3-(carboxymethoxy)-5-(3-{[1-({2-[(methylsulfonyl)-

amino]benzyl}sulfonyl)piperidin-4-yl]amino}phenyl)thiophene-

1

2-carboxylic Acid (50). H NMR (400 MHz, DMSO-d₆): δ ppm

4-Bromo-3-carboxymethoxy-5-{3-[(1-o-tolylcarbamoylpiperi-

din-4-ylmethyl)amino]phenyl}thiophene-2-carboxylic Acid (53).

4-Bromo-3-tert-butoxycarbonylmethoxy-5-{3-[(1-o-tolylcarbam-

oylpiperidin-4-ylmethyl)amino]phenyl}thiophene-2-carboxylic acid

methyl ester was obtained in 41% yield by following the general

procedure for urea formation at the piperidine nitrogen, using

4-bromo-3-(2-tert-butoxy-2-oxoethoxy)-5-(3-(piperidin-4-ylmethy-

lamino)phenyl)thiophene-2-carboxylate hydrochloride (86 mg, 0.15

mmol), o-tolyl isocyanate (20 µL, 0.17 mmol), and DIPEA (67

µL, 0.38 mmol) as starting materials. ¹H NMR (400 MHz,

CDCl₃): δ ppm 1.33 (m, 2 H), 1.51 (s, 9 H), 1.88 (m, 3 H), 2.25

(s, 3 H), 2.91 (td, J ) 12.88, 2.02 Hz, 2 H), 3.10 (d, J ) 5.81 Hz,

2 H), 3.87 (s, 3 H), 3.95 (s, 1 H), 4.11 (d, J ) 13.14 Hz, 2 H), 4.82

(s, 2 H), 6.15 (s, 1 H), 6.66 (m, 1 H), 6.87 (m, 1 H), 6.96 (m, 1 H),

7.01 (td, J ) 7.45, 1.26 Hz, 1 H), 7.18 (m, 2 H), 7.24 (t, J ) 7.96

Hz, 1 H), 7.62 (d, J ) 8.08 Hz, 1 H).

4-Bromo-3-tert-butoxycarbonylmethoxy-5-{3-[(1-o-tolylcarbam-

oylpiperidin-4-ylmethyl)amino]phenyl}thiophene-2-carboxylic acid

methyl ester (43 mg, 0.064 mmol) was hydrolyzed according to

the general procedure for hydrolysis to give 4-bromo-3-car-

boxymethoxy-5-{3-[(1-o-tolylcarbamoylpiperidin-4-ylmethyl)amino]-

phenyl}thiophene-2-carboxylic acid (34 mg, 88%) as a pale yellow

solid. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 1.17 (m, 2 H), 1.78

(m, 3 H), 2.15 (s, 3 H), 2.78 (m, 2 H), 2.97 (d, J ) 6.06 Hz, 2 H),

4.11 (d, J ) 13.39 Hz, 2 H), 4.88 (s, 2 H), 6.70 (dd, J ) 8.21, 1.64

Hz, 1 H), 6.78 (d, J ) 8.34 Hz, 1 H), 6.84 (t, J ) 1.89 Hz, 1 H),

7.02 (td, J ) 7.26, 1.64 Hz, 1 H), 7.10 (m, 1 H), 7.18 (m, 3 H),

7.97 (s, 1 H). HRMS: calcd for C₂₇H₂₈BrN₃O₆S - H⁺, 602.0955;

found (ESI-FTMS, [M - H]-), 602.097 88. RF-HPLC: H₂O/

MeCN/0.1% formic acid, 93%; H₂O/MeCN/0.1% formic acid,

94%.

1.27-1.48 (m, 2 H), 1.88-2.05 (m, 2 H), 2.86-3.01 (m, 2 H),

3.02 (s, 3 H), 3.35-3.48 (m, 1 H), 3.48-3.58 (m, 2 H), 4.60 (s, 2

H), 6.72 (d, J ) 7.58 Hz, 1 H), 6.80 (d, J ) 7.58 Hz, 1 H), 6.86

(s, 1 H), 7.21 (t, J ) 7.96 Hz, 1 H), 7.27-7.35 (m, 1 H), 7.36-

7.47 (m, 3 H), 7.51 (dd, J ) 7.83, 1.52 Hz, 1 H), 9.14 (s, 1 H).

HRMS: calcd for C₂₆H₂₈BrN₃O₉S₃- H⁺, 700.009 83; found (ESI-

FTMS, [M - H]-), 700.0084. RF-HPLC: H₂O/MeCN/0.1% formic

acid, 98%; H₂O/MeCN/0.1% formic acid, 99%.

4-Bromo-3-carboxymethoxy-5-(3-{[1-(2-chlorophenylcarbam-

oyl)-piperidin-4-ylmethyl]amino}phenyl)thiophene-2-carboxy-

lic Acid (51). 4-Bromo-3-tert-butoxycarbonylmethoxy-5-(3-{[1-(2-

chlorophenylcarbamoyl)piperidin-4-ylmethyl]amino}phenyl)thiophene-

2-carboxylic acid methyl ester was obtained in 56% yield by

following the general procedure for urea formation at the piperidine

nitrogen, using 4-bromo-3-(2-tert-butoxy-2-oxoethoxy)-5-(3-(pip-

eridin-4-ylmethylamino)phenyl)thiophene-2-carboxylate hydrochlo-

ride (86 mg, 0.15 mmol), 2-chlorophenyl isocyanate (20 µL, 0.17

mmol), and DIPEA (67 µL, 0.38 mmol) as starting materials.

¹H NMR (400 MHz, CDCl₃): δ ppm 1.33 (dd, J ) 12.25, 2.91

Hz, 2 H), 1.51 (s, 9 H), 1.91 (m, 3 H), 2.94 (m, 2 H), 3.10 (d, J )

6.06 Hz, 2 H), 3.87 (s, 3 H), 4.15 (m, 2 H), 4.82 (s, 2 H), 6.66 (m,

1 H), 6.87 (m, 1 H), 6.94 (m, 2 H), 7.04 (s, 1 H), 7.23 (m, 2 H),

7.33 (dd, J ) 8.08, 1.52 Hz, 1 H), 8.18 (dd, J ) 8.34, 1.52 Hz, 1

H).

4-Bromo-3-tert-butoxycarbonylmethoxy-5-(3-{[1-(2-chlorophe-

nylcarbamoyl)piperidin-4-ylmethyl]amino}phenyl)thiophene-2-car-

boxylic acid methyl ester (58 mg, 0.084 mmol) was hydrolyzed

according to the general procedure for hydrolysis to give 4-bromo-

3-carboxymethoxy-5-(3-{[1-(2-chlorophenylcarbamoyl)piperidin-4-

ylmethyl]amino}phenyl)thiophene-2-carboxylic acid (46 mg, 88%)

1

as a pale yellow solid. H NMR (400 MHz, DMSO-d₆): δ ppm

4-Bromo-3-carboxymethoxy-5-(3-{[1-(2,6-dimethylphenylcar-

bamoyl)piperidin-4-ylmethyl]amino}phenyl)thiophene-2-car-

boxylic Acid (54). 4-Bromo-3-tert-butoxycarbonylmethoxy-5-(3-

{ [ 1 - ( 2 , 6 - d i m e t h y l p h e n y l c a r b a m o y l ) p i p e r i d i n - 4 -

ylmethyl]amino}phenyl)thiophene-2-carboxylic acid methyl ester

was obtained in 69% yield by following the general procedure for

urea formation at the piperidine nitrogen, using 4-bromo-3-(2-tert-

butoxy-2-oxoethoxy)-5-(3-(piperidin-4-ylmethylamino)phenyl)th-

iophene-2-carboxylate hydrochloride (57 mg, 0.1 mmol), 2,6-

dimethylphenyl isocyanate (17 µL, 0.12 mmol), and DIPEA (45

µL, 0.25 mmol) as starting materials. ¹H NMR (400 MHz,

CDCl₃): δ ppm 1.30 (m, 3 H), 1.51 (s, 9 H), 1.86 (d, J ) 9.60 Hz,

2 H), 2.24 (s, 6 H), 2.90 (m, 2 H), 3.10 (d, J ) 6.32 Hz, 2 H), 3.87

(s, 3 H), 4.10 (m, 2 H), 4.82 (s, 2 H), 5.82 (s, 1 H), 6.66 (m, 1 H),

6.87 (m, 1 H), 6.96 (d, J ) 8.34 Hz, 1 H), 7.24 (m, 1 H).

1.16 (m, 2 H), 1.79 (m, 3 H), 2.82 (m, 2 H), 2.97 (d, J ) 6.82 Hz,

2 H), 4.11 (d, J ) 13.39 Hz, 2 H), 4.87 (s, 2 H), 6.70 (dd, J )

8.21, 2.15 Hz, 1 H), 6.78 (d, J ) 7.58 Hz, 1 H), 6.84 (t, J ) 1.77

Hz, 1 H), 7.12 (td, J ) 7.64, 1.64 Hz, 1 H), 7.19 (t, J ) 7.83 Hz,

1 H), 7.27 (td, J ) 7.71, 1.52 Hz, 1 H), 7.43 (dd, J ) 7.96, 1.39

Hz, 1 H), 7.49 (dd, J ) 7.96, 1.39 Hz, 1 H), 8.10 (s, 1 H). HRMS:

calcd for C₂₆H₂₅BrClN₃O₆S - H⁺, 622.040 88; found (ESI-FTMS,

[M - H]-), 622.042 17. RF-HPLC: H₂O/MeCN/0.1% formic acid,

96%; H₂O/MeCN/0.1% formic acid, 96%.

4-Bromo-3-carboxymethoxy-5-(3-{[1-(2-methoxyphenylcar-

bamoyl)piperidin-4-ylmethyl]amino}phenyl)thiophene-2-car-

boxylic Acid (52). 4-Bromo-3-tert-butoxycarbonylmethoxy-5-(3-

{ [ 1 - ( 2 - m e t h o x y p h e n y l c a r b a m o y l ) p i p e r i d i n - 4 -

ylmethyl]amino}phenyl)thiophene-2-carboxylic acid methyl ester

was obtained in 56% yield by following the general procedure for

urea formation at the piperidine nitrogen, using 4-bromo-3-(2-tert-

butoxy-2-oxoethoxy)-5-(3-(piperidin-4-ylmethylamino)phenyl)th-

iophene-2-carboxylate hydrochloride (86 mg, 0.15 mmol), 2-meth-

4-Bromo-3-tert-butoxycarbonylmethoxy-5-(3-{[1-(2,6-dimeth-

ylphenylcarbamoyl)piperidin-4-ylmethyl]amino}phenyl)thiophene-

2-carboxylic acid methyl ester was hydrolyzed according to the

4696 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 19

Wilson et al.

Table 8. Refinement Statistics of PTP1B-Inhibitor Complex Structures

general procedure for hydrolysis to give 4-bromo-3-carboxymethoxy-

5-(3-{[1-(2,6-dimethylphenylcarbamoyl)piperidin-4-ylmethyl]amino}-

phenyl)thiophene-2-carboxylic acid (35 mg, 82%) as a pale yellow

solid. ¹H NMR (400 MHz, DMSO-d₆): δ ppm 1.14 (m, 2 H), 1.80

(m, 3 H), 2.13 (s, 6 H), 2.79 (m, 2 H), 2.97 (d, J ) 6.06 Hz, 2 H),

4.12 (d, J ) 12.63 Hz, 2 H), 4.88 (s, 2 H), 6.70 (dd, J ) 8.34, 1.52

Hz, 1 H), 6.78 (d, J ) 7.58 Hz, 1 H), 6.84 (t, J ) 1.89 Hz, 1 H),

7.03 (m, 3 H), 7.19 (t, J ) 7.96 Hz, 1 H). HRMS: calcd for C₂₈H₃₀-

BrN₃O₆S - H⁺, 616.111 15; found (ESI-FTMS, [M - H]-),

616.112 83. RF-HPLC: H₂O/MeCN/0.1% formic acid, 92%; H₂O/

MeCN/0.1% formic acid, 93%.

5.2. Enzymatic Assays. The enzymatic assays were carried out

at room temperature in 96-well plates. The assay buffer contained

50 mM 3,3-dimethyl glutarate, 1 mM EDTA, 1 mM TCEP, and

0.01% Triton (pH 7.0 with ionic strength of 0.15 M adjusted by

sodium chloride). The reaction was initiated by addition of the

enzyme at a final concentration of 10 or 100 nM for PTP1B, 20

nM for TCPTP, 20 nM for CD45, and 270 nM for LAR,

respectively. The initial rate of the PTPase-catalyzed hydrolysis of

p-nitrophenol phosphate (pNPP) was measured by following the

absorbance change at 405 nm. IC₅₀values were determined under

a fixed pNPP concentration of 1 mM. All the assays were carried

out in duplicate or triplicate, and the average results are presented.

Inhibition constants (K_i) were derived from each IC₅₀based on

competitive inhibition K_i) IC₅₀K_m/(K_m+ [substrate]). For tight

binding inhibitors, K_iis calculated from nonlinear fitting based on

the tight binding equation

compd

resolution (Å)

R-factor

16

2.3

.1997

.2315

0.007

1.34

19

2.1

.1927

.2287

0.006

1.25

23

2.1

.1942

.2255

0.006

1.41

32

2.5

.2089

.2451

0.009

1.47

R_free

rms^a(b) (Å)

rms^b(a) (deg)

^aRoot-mean-square bond deviation from standard. ^bRoot-mean-square

angle deviation from standard.

The structures were solved by molecular replacement with the

available h-PTP1B catalytic domain structure model. Cycles of

model rebuilding and refinement were carried out with CNS

system for X-ray crystallography; NMR; and QUANTA (Accelrys

Inc.). Inhibitor was built into the difference density after the

first cycle of refinement. Water molecules were assigned accord-

ing to F_o- F_cmaps. The refinement statistics are summarized in

Table 8.

5.4. Molecular Modeling. Analogs were initially docked into

the active site of the PTP1b protein structure from the X-ray

complex with compound 19. As additional protein-inhibitor

complex structures were determined, the binding site model was

refined. Typically 1000 Monte Carlo cycles were carried out to

dock each analog into the site, while also allowing selected protein

residues to undergo constrained movement.⁴²Figures 2-4 were

generated using Benchware3D (Tripos, 2006, St. Louis, MO) and

rendered with the Persistance of Vision Raytracer (POV-RAY)

software (www.povray.org).

5.5. Pharmacokinetic and Metabolic Stability Studies. Phar-

macokinetics of PTP1B inhibitors was determined in male C57/

B6 mice (20-30 g, Taconic Farm, NY) after iv or ip administration.

The compounds were prepared in DSM/PEG-200/saline solution

for the iv administration or in 0.5% MC/2% TW/water as a

suspension for the ip administration. Blood samples were collected

over a period of 24 h, and plasma samples were harvested and stored

at -80 °C until assay. The target tissues (liver and muscle) were

also collected at selected time points. Tissues were homogenized

in saline and the drug concentrations in the homogenates were

analyzed. Quantization of PTP1B inhibitors in plasma and tissue

homogenate was carried with a verified LC/MS/MS method.

In vitro metabolic half-life (T_1/2) and metabolic pathways were

determined in liver microsomes from rats, mice, and humans using

an NADPH-regenerating system consisting of MgCl₂(10 mM),

glucose-6-phosphate (3.6 mM), NADP⁺(1.3 mM) and glucose-6-

phosphate dehydrogenase (0.4 units/mL) in sodium phosphate buffer

(0.1 M, pH 7.4), UDPGA (4 mM), and substrate (1 mM for

metabolic stability and 10 mM for metabolite profiling). Incubations

were initiated by the addition of the NADPH-generating system

and conducted for up to 30 min in a shaker-water bath at 37 °C.

For the determination of in vitro metabolic half-life, aliquots of

the incubation mixture were removed at 0, 10, 20, and 30 min and

added to acetonitrile containing the appropriate internal standard.

Following centrifugation and evaporation of the supernatant liquid,

the samples were reconstituted in 20% methanol in water for

analysis by LC/MS.

5.6. Studies of Compound Active Uptake into Hepatocytes.

Rat primary hepatocytes (from a pool of seven or more livers) plated

as a monolayer on 6-well collagen-coated plates were obtained from

In Vitro Technologies, Inc. (Baltimore, MD). Incubations were

based on a modification of previous methods (Kouzuki, H.; Suzuki,

H.; Ito, K.; Ohashi, R.; Sugiyama, Y. Contribution of sodium

taurocholate co-transporting polypeptide to the uptake of its possible

substrates into rat hepatocytes. J. Pharmacol. Exp. Ther. 1998, 286,

1043-1050). Cells were washed three times with 2 mL of Krebs-

Heinsleit buffer (KH; 142 mM NaCl, 23.8 mM Na₂CO₃, 4.83 mM

KCl, 0.96 mM KH₂PO₄, 1.2 mM MgSO₄, 12.5 mM HEPES, 5 mM

glucose, and 1.53 mM CaCl₂, pH 7.3) and preincubated for 5 min

at 37 °C. Viability was assessed by microscopic examination. Fresh

KH (1 mL) prewarmed to 37 °C containing compound 32 (10 µM

final concentration) with and without 100 µM verapamil in DMSO/

V₀

2E₀

V )

× [ (E - I - K_app)²+ 4E₀K_app+ (E₀- I₀- K_app)]

x

0

K_m

K_i) K_app

(

)

K_m+ S

IC₅₀) K_i

+ E₀/2

(

)

K_m

where V and V₀are initial activity in the presence and the absence

of inhibitors, E₀is enzyme concentration, I₀is inhibitor concentra-

tion, K_mis the Michaelis-Menten constant, and S is the substrate

concentration. Data shown are representative of at least two

independent determinations with a less than 2-fold difference

between measurements.

PTPases used in the assays were recombinant human PTP1B

(hPTP1B catalytic domain, residues 1-299, expressed and purified

according to literature procedures⁴¹), recombinant human TCPTP

(residues 1-299, expressed and purified in house), recombinant

human CD45 (cytoplasmic domain, residues 484-1281, purchased

from Biomol), and recombinant human LAR (soluble catalytic

LAR-D1 domain, residues 1275-1613, purchased from Biomol).

5.3. X-ray Crystallographic Studies. Human recombinant

PTP1B catalytic domain (residues 1-299) was prepared as

described.⁴¹The complex solution was made by mixing an excess

of inhibitor dissolved in DMSO with protein (protein:inhibitor )

1:1.5-3.0), incubating on ice for 1 h, and then filtering with 0.1

µm filters. Crystals of h-PTP1B-inhibitor complexes were obtained

by vapor diffusion at 4 °C using 0.1 M HEPES (pH 7.0), 100-

250 mM MgCl₂, 14-19% PEG 3350, 3 mM DTT, and 10-15 mg/

mL protein. Typically, rodlike crystals appeared overnight and

continued to grow to a maximum size of 0.2 mm × 0.2 mm × 0.7

mm within 10 days. The crystals belonged to space group P3(1)-

21, a ) b ) 88 Å, c ) 104 Å, R ) â ) 90, γ ) 120°. The crystals

were cryoprotected by transferring into the crystallization solution

25% glycerol. Cryoprotected crystals were flash-cooled in liquid

nitrogen prior to data collection.

Diffraction data of the h-PTP1B-inhibitor complex crystals were

collected by either in-house conventional X-ray generator using

Raxis-IV image detectors or using synchrotron radiation with CCD

at ALS (Advanced Light Source in Lawrence National Laboratory

at Berkeley, CA). All data were collected at 100 K and processed

with DENZO/SCALPACK.

NoVel Thiophenes as PTP1B Inhibitors

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MeOH (0.01/0.09% v/v) was added to each well and incubated at

37 °C in 5% CO₂atmosphere for 5 min and terminated with the

addition of 2 mL of ice-cold KH. Cells were washed three times

with ice-cold KH, scraped, and homogenized with 200 µL of

incubation media. Samples were extracted with acetonitrile contain-

ing internal standard, vortexed, and centrifuged at 21 000 g for 8

min at 4 °C. The supernatant was analyzed by LC/MS/MS with

concentrations of compound 32 quantitated on the basis of

interpolation from a six-point standard curve. Activity in the

presence of verapamil, normalized for protein content (Bradford,

M. A rapid and sensitive method for the quantitation of microgram

quantities of protein utilizing the principle of protein-dye binding.

Anal. Biochem. 1976, 72, 248-254), was reported as a percentage

of activity in the absence of verapamil. Uptake of 10 µM

fexofenadine was used as a positive control for OATP uptake.

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Acknowledgment. The authors would like to acknowledge

Nelson Huang, Ning Pan, and Walter Massefski for their role

in the characterization of the small molecules published herein.

The authors would also like to acknowledge the contributions

to our PTP1B project by Rajeev Hotchandani, Michael J. Smith,

Michelle Markus, Lydia Mosyak, Desiree Tsao, William S.

Somers, and George Vlasuk.

Supporting Information Available: A table of HPLC purity.

This material is available free of charge via the Internet at http://

pubs.acs.org.

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JM0702478

Article Doi

Structure-based optimization of protein tyrosine phosphatase 1B inhibitors: From the active site to the second phosphotyrosine binding site

DOI: 10.1021/jm0702478

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